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Department of Integrative Biology and Physiology, University of Minnesota Medical School, Minneapolis, Minnesota
ABSTRACT I. INTRODUCTION A. Perspective B. Scope of Review II. CARDIAC GENE TRANSFER TOOLS AND PRINCIPLES A. Viral Vectors 1. Retroviral vectors 2. Adenoviral vectors 3. AAV B. Nonviral Vectors 1. Naked DNA C. In Vivo Vector Delivery Techniques 1. Direct myocardial injection 2. Intravascular delivery methods D. In Vitro Gene Transfer/Acute Genetic Engineering 1. Cardiac myocyte culturing systems III. Ca2+ HANDLING PROTEINS A. Regulators of Ca2+ Release From the SR 1. FKBP12.6/calstabin2 2. Junctional SR proteins: CSQ, triadin, junctin, and histidine-rich Ca2+-binding protein A) CSQ. B) JUNCTIN. C) TRIADIN. D) HRC. B. Regulators of Cytoplasmic Ca2+ Removal 1. SERCA2A and phospholamban 2. Sarcolipin 3. NCX 4. Phospholemman C. Ca2+ Binding Proteins That Modulate Cardiac Performance 1. Parvalbumin 2. Sorcin 3. S100 proteins IV. SARCOMERIC TARGETS AND TEMPLATES A. Protein Turnover and Stoichiometry B. Thin Filament Proteins, Isoforms, Mutants, and Chimeras 1. Cardiac TnI 2. Gene transfer of TnI isoforms 3. TnI phosphorylation by PKA 4. Cardiac TnT 5. Cardiac TnC 6. Tropomyosin 7. Actin 8. Capping proteins and molecular rulers C. Thick Filament Proteins 1. Cardiac myosin 2. The myosin essential (MLC-1/ELC) and regulatory (MLC-2/ RLC) light chains 3. Thick filament accessory proteins: C-protein (MyBP-C) 4. Thick filament accessory proteins: titin V. CYTOSKELETAL PROTEINS A. Dystrophin and Dystrophin-Associated Proteins 1. Dystrophin 2. Dystrophin gene transfer 3. Sarcoglycans 4. alpha-Sarcoglycan 5. β-Sarcoglycan 6. gamma-Sarcoglycan 7. delta-Sarcoglycan B. Intermediate Filaments (desmin) C. Microtubules VI. CARDIAC SIGNALING PATHWAYS A. Gene Transfer Influencing the β-Adrenergic Signaling Pathway 1. β-Adrenergic receptors and G proteins 2. Cycling of β-ARs 3. Downstream β-AR signaling A) ADENYLYL CYCLASE. B) GS-COUPLED RECEPTORS. C) AKAPS. D) END-TARGET PROTEINS. 4. Myosin binding protein C 5. Heat shock proteins, p20 6. Protein phosphatase 1 B. Gene Transfer of Ca2+/Calmodulin Kinase C. Gene Transfer and PKC Signaling 1. Signaling upstream from PKC A) RECEPTORS AND PLC. B) PKC ISOFORMS. 2. Downstream translocation and other signaling pathways A) MYOFILAMENT TARGET PROTEIN, TROPONIN I. 3. Other targets A) PICOT. D. Gene Transfer of Protein Phosphatases 1. PP1 and inhibitor 1 and 2 2. Calcineurin E. Gene Transfer and MAPK Signaling F. Myocardial Nitric Oxide Synthase Signaling and Contractile Function 1. NOS1 or neuronal NOS 2. NOS2 or inducible NOS signaling 3. NOS3 or endothelial NOS G. Other Signaling Proteins of Interest 1. Superoxide dismutase 2. Other signaling targets VII. FUTURE DIRECTIONS GRANTS ACKNOWLEDGMENTS REFERENCES
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I. INTRODUCTION |
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Heart disease is the leading cause of combined morbidity and mortality in the western world. Globally, it is estimated that over the course of the next three decades heart disease will be the leading cause of death worldwide, including both high- and low-income countries (970). The past years have seen tremendous insights into the molecular underpinnings of cardiac performance leading to clinically relevant therapeutics to treat heart disease. Nonetheless, the growing burden of cardiovascular disease in this country and throughout the world necessitates continued vigor directed at the mechanistic basis of heart disease with the goal of identifying new therapeutic targets and implementing effective treatment modalities. The elucidation of the human genome, together with a growing appreciation of the complexities of cardiac gene expression and proteome, lends hope that new discoveries will be forthcoming in treating acquired and inherited diseases of the heart. Cardiac gene transfer presents a unique strategy to design cardiac performance by tailoring specific physiological outcomes in the heart.
This review is focused on the application of recombinant viral vector systems as gene delivery vehicles to the normal and diseased heart. One could argue that the birth of molecular cardiology was ushered in during the early 1990s by the development and implementation of genetic strategies for targeted engineering of gene expression in cardiac muscle. Since that time, there have been a number of excellent reviews focused on the application of transgenesis and ES cell gene targeting in mammals relating to the heart (151, 400, 517, 746, 999). This review concentrates on another avenue of gene-based engineering by featuring emergent viral vector technologies. First, the origins and applications of gene transfer technologies for the heart are reviewed, with an emphasis on the strengths and limitations of recombinant viral vector and nonviral systems for cardiac gene delivery. Next, cardiac muscle targets and the applications of vector technology to the heart are highlighted by discussing gene transfer of key elements of cardiac excitation-contraction (EC) coupling, with an emphasis on intracellular Ca2+ handling and contractile/regulatory proteins of the sarcomere (Fig. 1). In concert, new discoveries in the cytoskeletal matrix as applied to inherited and acquired cardiac disease are discussed with an eye towards new targets for acute and long-term genetic engineering in the heart. Finally, we review vector-based approaches for modifying essential components of the cell signaling network in the heart. Recent advances in gene transfer of cardiac membrane channels and biological pacemakers are addressed elsewhere (17, 184, 185, 524).
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II. CARDIAC GENE TRANSFER TOOLS AND PRINCIPLES |
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The field of gene therapy and gene transfer in general has utilized an array of unique vector systems. As a thorough review of all of these systems could easily fill a textbook, this section only examines commonly used gene transfer vectors (Table 1) for myocardial and isolated myocyte transduction and briefly describes their major advantages and drawbacks. The field of immunology, as it pertains to delivered transgenes, could also constitute an entire review by itself. As such, discussions of immunology here are focused only on responses to a particular gene transfer vector. Gene transfer vectors are generally classified as viral or nonviral based, and this distinction provides the framework for the following discussion.
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Retroviral vectors are derived from a variety of wild-type retroviruses. Moloney murine leukemia virus (MLV) and lentivirus are two common examples, but employing less common retroviruses, such as foamy virus, for gene transfer is also gaining in popularity (39, 555). Despite many differences, these vectors all share common traits. One is the storage of genetic information in the form of single-stranded RNA that is reverse-transcribed into double-stranded DNA (provirus) once the virus enters the host cell (636). A vital characteristic of retroviral infection is its integration as double-stranded DNA into the host's genome (636). Unlike some retroviruses, lentivirus can efficiently transduce nondividing cells such as cardiac myocytes (633, 1009), and therefore, this section will focus on the use of lentiviral vectors (503). As lentivirus integrates into the host genome, lifetime transduction could potentially be achieved following a single transduction event. This is not without risk, however, as any integrating vector holds the potential for serious insertional mutagenesis events. Like many integrating vectors, lentivirus shows preference for inserting into active chromatin (123, 162, 472).
Lentivirus is a retrovirus related to the HIV virus (443). It contains a proteinaceous capsid surrounded by an envelope derived from the host plasma membrane. Production of the vector is an ever-evolving field, with improvements being made in production efficiency, purity, and safety (481). Production generally involves either a stably transduced cell line, which buds off lentivirus vectors into the supernatant and is collected and concentrated, or a plasmid cotransfection system that introduces a genome coding plasmid and helper virus into cells (103, 418, 515, 529). The plasmid cotransfection system is widely used to produce lentiviral vectors and has undergone a number of generations of development. Each generation was aimed at increasing growth efficiency while reducing the chance of generating replication-competent viruses. Currently, lentiviral vectors cannot be grown to titers on par with adenoviral or adeno-associated viral vectors (AAV) (294, 529, 807). Also, unlike vectors such as AAV, highly purified, large-scale, lentiviral vector production is extremely challenging. Lentivirus vectors are mostly concentrated but with some concomitant impurities. However, technologies such as high-performance liquid chromatography (HPLC) offer the possibility of truly purifying vector stocks (796). A final disadvantage is that lentiviral vectors are less stable than other vectors and are more difficult to manipulate due to this lability (529).
Currently, relatively few studies have been reported using lentivirus vectors to transduce the myocardium in vivo (71, 238). Although these vectors are capable of transducing 80–100% of cardiac myocytes in vitro (71, 588, 779, 780), in vivo efficiencies rarely achieve a transduction efficiency of above 30% (71, 238). For cardiac expression, direct injection is by far the most efficient means of delivery, with little expression seen after vascular delivery (996). However, in cardiac transplant rejection studies, this relatively low level of expression has yielded significant results (1008). The immunology/toxicology of lentivirus vectors when used for cardiac delivery is not yet well understood, but data will undoubtedly be available in the future.
Lentiviral vectors represent an appealing method for cardiac transduction. They offer the ability to stably transduce nondividing cardiac myocytes, a potentially useful experimental/therapeutic characteristic. As the use of lentiviral vectors to transduce the myocardium is a relatively young field, many questions remain unanswered. Future studies will have to focus on increasing transduction efficiencies, determining optimal administration methods, and elucidating the host immune response following vector administration/transduction. Advances in production and purification of these vectors will also expand the potential applications and ease of use of this vector.
Adenovirus-based vectors (Ad) have been a workhorse for a variety of gene transfer studies spanning several decades. Despite its limitations, this experimental approach will likely continue to be a valuable tool in the future. Along with AAV vectors, Ad vectors represent one of the most efficient means of both in vivo and in vitro cardiac transduction. Adenovirus has been an invaluable reagent for cardiac muscle gene transfer (170, 245, 294, 439, 813, 858, 966).
Adenovirus is a member of the Adenoviridae class of viruses. It is a double-stranded DNA virus (dsDNA) with a 36-kb genome capped with inverted terminal repeats (ITRs). The ITRs function as origins of viral genome replication. The genome encodes dozens of protein products divided into early and late transcriptional events using a variety of space-efficient internal promoters and splice sites. Adenovirus has a nonenveloped icosahedron capsid (60–90 nm in diameter) surrounding the dsDNA virion (119, 267). Its proteinaceous capsid consists of many different proteins with functions ranging from providing structure to docking and infection. The 12 vertices each contain a penton base and fibrous "spike" that is used to attach the virus to coxsackievirus (CAR) or adenovirus receptors on the host cell membrane (Fig. 2) (46). Once attached, the adenovirus is endocytosed and transported to the endosome where the virion is thought to escape through a pH-dependent endoplasmolysis (291, 971). These events occur over an estimated 15–20 min time span (291, 971), and subsequently the dsDNA migrates to the nucleus for transcription (466).
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7 kb, Table 1) for exogenous expression cassettes. Vector DNA is transfected into permissive packaging cells (commonly HEK 293), which are eventually lysed as a result of viral production. The viral lysate is plaque purified, and a pure clone is used to seed more packaging cells. The viral lysate/cell debris is collected, and the Ad vector is purified/concentrated using a variety of methods such as centrifugation or chromatography. Gutted adenovirus has large portions of the adenoviral genome deleted, yielding vectors with a much larger cloning capacity (30–40 kb, Table 1) relative to Ad and AAV (35, 196, 264, 265, 807). Gutted adenovirus can be grown concurrently with a helper virus (which resembles first-generation Ad vectors) to provide critical functions in trans. After production, the gutted vector is separated from the helper virus by density using equilibrium centrifugation. Commonly, the packaging signal of the helper virus is flanked by recombinase targets resulting in excision of the packaging signal in the presence of the proper recombinase. The recombinase is expressed in the packaging cells, thus limiting the amount of packaged helper that needs to be removed by differential centrifugation. Adenovirus represents some of the largest viral vectors available for cardiac gene transfer. Most gene transfer to adult cardiac myocytes has been performed with second- and third-generation serotype 5 adenovirus (439, 965).
Adenoviral vectors are extremely efficient at transducing the myocardium. This is true with a variety of injection methods and experimental animal models. After direct injection into the myocardium (Fig. 3, Table 2), 60–80% of the exposed cardiac cells can be transduced (170, 307, 439, 568, 858). Peak expression is generally seen within 3 days and is typically very strong. In addition to its use as an experimental tool, adenoviral delivery has been used to improve cardiac function in a number of studies and as such makes this vector a potential candidate for clinical applications (583, 801, 858, 968). Injection of the vector directly into the intravascular space results in poor transduction of the myocardium, and large amounts of Ad vector are acutely toxic, especially to the liver (720). However, when the cardiac circulation is isolated, either during transplantation studies or during heart-lung bypass, global transduction efficiencies of 30–50% can be achieved (83, 84, 187, 188, 801, 845). These techniques frequently make use of elevated hydrostatic pressure and permeabilizing adjuvants (187, 188). In vitro adenoviral transduction is even more efficient, and a preferred gene delivery vector for cultured cardiac myocytes will be discussed in subsequent sections. Reporter assays in which adenoviral delivery of chloramphenicol acetyltransferase (CAT), green fluorescent protein (GFP), or Lac Z (β-galactosidase) genes to isolated adult cardiac myocytes have shown a transduction efficiency approaching 100%, which occurs rapidly between days 1–2 in culture (132, 424, 439, 490, 765, 965).
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Recombinant AAVs are generating considerable interest in the field of cardiac gene transfer (294, 390, 668, 893, 997). AAV is a Dependovirus member of Parvoviridae (63, 821) with a particle size of 20 nm. As a Dependovirus, AAV is incapable of replicating in host cells under most physiological circumstances and requires coinfection with a helper virus for replication. Adenovirus or herpesvirus is the most frequently used helper virus, but others including human papillimo virus have also been effective helper viruses (63). The AAV capsid is a nonenveloped proteinaceous capsid made of three proteins termed VP1, VP2, and VP3, and the capsid seems to be devoid of most posttranslational modifications. AAV has a single-stranded DNA (ssDNA) genome where both Watson and Crick strands appear to be packaged equally. The AAV genome is relatively small (4.5 kb, Table 1). Several promoters and alternate splicing control the expression of the capsid and Rep proteins. Rep proteins are critical elements involved in genome replication, integration, and packaging. The AAV genome also contains two ITRs, which are important for genome packaging, replication, and stability (47, 516, 836, 842, 984). Several distinct serotypes of AAV have been identified (Table 3), with serotype 2 being the most commonly used for gene transfer vectors. (For reviews on AAV, see Ref. 443.)
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Integration of gene transfer vectors into the host genome is a safety concern that can be seen as both a benefit and a potential limitation. Integrating gene transfer vectors offer the possibility of permanent transduction avoiding the need for repeat gene delivery. Integrating vectors, aside from AAV, have occasionally caused human disease through insertional mutagenesis (309, 310, 800). For AAV vectors, this phenomenon must be interpreted in light of several facts. Classically, wild-type AAV integrates preferentially into two specific sites in the human genome, perhaps guided by the homology between the ITRs and genomic sequence. This integration is mediated by Rep proteins (785, 935). As the Rep proteins are deleted in AAV vectors, the rate of integration is much lower than that of the wild-type virus. Also, much of the site specificity of integration appears to be lost in recombinant AAV vectors (700). Recombinant AAV vectors typically have a low rate and random pattern of integration and appear to favor integration into transcriptionally active and open chromatin (628–630). Even in the absence of integration, expression from AAV vectors can persist in striated musculature for many years (21).
AAV vectors can be produced to high purity and titer (63). Early methods used adenovirus as a helper virus for AAV vector production, but adenoviral contamination was often found in these preparations. Current protocols now employ a helper-free plasmid cotransfection system to avoid an Ad contamination (299, 300, 983). Here a plasmid containing the AAV genome is transfected into cells, frequently HEK293 cells, along with a plasmid containing helper genes to function in trans. Vector purification and concentration are dependent on the specific capsid serotype. Capsids from some serotypes, such as serotypes 2 and 6, bind heparin avidly enough to be purified over a heparin sulfate-Sepharose column (124, 321, 716). Vector production of all AAV serotypes can use serial centrifugation methods. Of note, the capsids of most serotypes can cross-package the ITRs from genomes of other virus serotypes (commonly serotype 2) in a process termed pseudotyping (63, 716). This phenomenon permits easy manipulation of the vector capsid, with altered production and tissue tropism characteristics (Table 3) without having to clone new vector genomes. In the last several years methods using baculovirus expression to produce AAV vectors have also been described (831, 910, 911).
One of the main disadvantages of AAV vectors is their relatively limited cloning capacity (Table 1). The entire expression cassette of interest, including the open reading frame and transcriptional regulatory sequence, must not exceed 4.5 kb, allowing for 300 bp of ITR sequence (836). This cloning capacity severely limits the potential for gene transfer cassettes. Strategies to overcome this limitation include cotransduction with two vectors containing one-half of an expression cassette. The two genomes then rearrange in vivo to yield a larger, intact expression cassette (261, 262, 469). Also, many large genes have been modified into "micro" versions that still retain functionality, yet meet the size requirements for AAV vectors (329, 893, 997). Initial reports using this approach have been very encouraging, although a broad range of transduction efficiencies have been reported. Another concern is that the general human population has an antibody titer to AAV. This titer generally has a reasonable amount of cross-reactivity with several other serotypes (116, 322), raising the possibility that a neutralizing titer may limit AAV's therapeutic potential in vivo (522).
AAV vectors have been widely used for cardiac gene transfer. Serotypes 1, 6, 7, 8, and 9 (Table 3) appear to be the most efficient for transduction, although many of the newer less described serotypes may also efficiently transduce myocardial tissue (293, 294, 672, 844). Depending on the exact capsid serotype and delivery method, AAV vectors have transduced nearly 100% of cardiac tissues (294, 893). Various studies utilizing intravenous delivery demonstrate global cardiac transduction, an exciting finding in terms of therapeutic strategies as well as experimental manipulations (294, 390, 893). More focused delivery to the heart by direct injection results in strong local expression of the gene product (997). Using AAV vectors, expression levels do not peak as fast as with adenovirus and generally take between 10 days and 2 mo to reach their peak. Once present, AAV expression can last for years (21, 63).
AAV vectors are currently one of the most promising vectors for genetic manipulation of the heart in vivo. Studies using AAV for cardiac transduction have been very successful in delivering a variety of genes to several mammalian species including mice, rats, rabbits, dogs, pigs, and nonhuman primates (21, 422, 668, 672, 893). Additionally, several clinical trials have demonstrated clear human transduction with AAV vectors (432, 522, 604). Studies in heart kinetics and energetics, transplant rejection, and structural abnormalities have all been successfully performed using AAV vectors (113, 293, 778). These include several very promising therapeutic reports where a dramatic reduction of disease morbidity and mortality was demonstrated in several animal models of muscular dystrophy (293, 294, 893). The future use of AAV for clinical application will likely require further investigation of the immune response to AAV, development of smaller regulatory sequences for use in AAV vectors, and elucidation of the intracellular handling of the AAV capsid and genome, a complicated and poorly understood topic not covered in this review.
Finally, other viral-based vectors such as Epstein-Barr, foamy, and simian virus 40 (SV40) (555, 843, 890) have also been reported for use in gene transfer studies but have not been included in this discussion for reasons of space, frequency of use, and a lack of a rich literature in cardiac transduction. This does not preclude these vectors, however, as viable options for cardiac gene transfer in future studies.
Pure DNA carrying an expression cassette is perhaps one of the most basic transfer vectors. In this situation, clonal DNA that is generally derived from a bacterial plasmid (pDNA) is introduced to the tissue of interest. Through means not completely understood, the cells take up the pDNA, transport it to the nucleus, and express the exogenous gene. This type of striated musculature transduction has been known for approximately two decades (974, 975).
pDNA vectors have many potential advantages. pDNA can be readily produced in large amounts to very high purity through a variety of methods available commercially and for the laboratory environment. Common laboratory methods for purifying pDNA include alkaline lysis and phenol-chloroform extraction and DEA-dextran (diethylaminoethyl) binding columns. Huge fermentors are commercially available to produce and HPLC purify gram quantities of extremely pure plasmid under good manufacturing practice (GMP) conditions. Because there are no true "infection particles" with pDNA, the resultant material can also be stored easily for long periods of time without loss of potency. Additionally, recombinant manipulation of the pDNA is much easier. Unlike recombinant viral genomes, expression cassette size is much less of a concern. Although smaller genomes are generally propagated more efficiently, bacterial strains can easily maintain plasmids of 30 kb and above. This allows for the cloning of extremely large expression cassettes with larger portions of native regulatory sequence compared with viral vectors. This is advantageous in terms of having more natural transcriptional activity and possible tissue-specific expression patterns, a challenge with viral vectors such as AAV.
pDNA vectors are also attractive gene transfer reagents because they lack a viral proteinaceous or membranous component. This contributes to both the stability and small immune response associated with pDNA vectors. Because organisms have evolved ways of neutralizing viral transduction events, virally delivered genes can elicit very potent humoral and cellular immune responses against the genetic vectors. Overall pDNA does not tend to elicit a potent immune reaction, but "bacterial" DNA sequences are recognized by the body and can spark a small immune reaction. In a potentially related protective mechanism, a DNA sequence that is covalently attached to bacterial sequences can be silenced in vivo. In a series of experiments, delivery of circular DNA with excised bacterial sequence resulted in prolonged expression of the transgene.
There are many potential benefits of using pDNA vectors to transduce the myocardium, but at least two major obstacles remain: low efficiencies of transduction and persistence of transduction. Despite attempts with a variety of administration techniques, pDNA vectors have very low cardiac transduction efficiency compared with their viral counterparts (975). Nonetheless, pDNA vectors are still being explored for cardiac transduction due to their many advantageous qualities, and they are still considered useful experimental and therapeutic reagents for studies that do not require global and persistent expression. Continued investigation into pDNA vectors may result in gains in both transduction efficiencies and persistence of expression.
C. In Vivo Vector Delivery Techniques
Delivery of foreign expression cassettes to the myocardium has a range of possible experimental and therapeutic applications. Regardless of the experimental or therapeutic objective, these genetic manipulations will likely rely on efficient transduction of the myocardium. Transduction efficiency requires the marriage of safe, efficient, and producible gene transfer vectors with a delivery system that is technically efficient, feasible, and well tolerated. The following two sections describe the current state of vector delivery technologies used for in vivo transduction of the myocardium.
In general, the ideal gene delivery method should be technically simple, inexpensive, safe, and only transduce specified regions of the targeted tissue. At present, delivery techniques meeting all of these criteria do not exist. Current delivery methods can be classified into two broad categories: direct delivery to the myocardium and intravascular systemic delivery (Fig. 3, Table 2). As so many specific methods have been developed for vector delivery within each category, the following discussion will highlight the basic permutations of each delivery method. While this section is separated into vectors and delivery methods for the purpose of discussion, the pairing of vector and delivery system can have specific advantages and disadvantages as they pertain to transduction efficiency. For instance, intravascular delivery is often combined with AAV vectors (Table 2), while AAV is rarely if ever delivered by electroporation.
1. Direct myocardial injection
Direct injection of the gene transfer vector to the myocardium is perhaps conceptually the most obvious approach. This method has been extensively used with most available gene transfer vectors and involves the introduction of an injectant directly into the heart musculature by a syringe or similar device (858) (Fig. 3). The vector gains direct access to the myocardial cells through the interstitial space and enters via the specific vector's entry mechanisms. This method has been utilized in studies addressing a variety of disease models including ischemic heart disease, heart failure, and muscular dystrophies. Additionally, several different animal models ranging from common laboratory animals (rodents and rabbits) to larger mammals (canines and pigs) are amenable to this type of manipulation. The exact method of injection varies by the experimental system and objectives.
Overall, direct injection has been associated with excellent survival rates. Injections can be done blind by targeting the heart through the chest wall or trans-diaphragm from the abdominal cavity. While this method may seem rather unreliable, it has been used with effectiveness by a number of investigators, especially in small animals (858). The injection can also be done through the chest wall, under the guidance of ultrasound technology. Ultrasound guidance improves the accuracy of direct injection by allowing some visualization of the heart itself. Despite the technical simplicity of the direct injection method, it is difficult to reproducibly transduce the same area of the heart to similar magnitudes between animals and studies, even with ultrasound guidance. Direct injection can also be done following surgeries that expose the heart, permitting direct visualization for delivering injectant to the target tissue (858). This technique has been used both on beating hearts and after a transient cardiac arrest with similar effectiveness. Either ultrasound guidance or injection based on coronary vascular anatomy permits reasonably comprehensive transduction of the vascular walls of rodents (320). Although this necessitates a surgery and all of the associated complications, it heightens investigator confidence in the site of injection(s).
With either blind or visualized direct injection technique, one major limitation is the targeted musculature must be physically large enough and easily accessible for the procedure. Thus, in most rodent studies, the sites of injection to nonseptal walls of the ventricles are limited. It is possible that in larger animals with thicker atrial walls the technique could also be applied to atrial tissue. Another limitation of this delivery route is the poor accessibility of the septal wall, trabeculae, and papillary muscles, but ultrasound guidance has been used to improve gene transfer to the septal wall by direct injection.
Transduction efficiency not only depends on the method of direct injection but also appears to be highly dependent on the vector and vector dosage. The use of adenovirus and AAV vectors with direct injection has resulted in strong transduction of much of the ventricular tissue (858). In contrast, injection with retrovirus-based vectors, such as lentivirus, results in comparatively lower expression and amount of transduced cardiac tissue (71, 779, 780, 1009). The delivery of naked or complexed pDNA with this technique results in patchy and lower intensity expression throughout the injected wall relative to the transduction efficiency of viral-based vectors.
One permutation of the direct injection method is to inject the vector into the pericardial space rather than the myocardium itself. The injection of adenoviral vectors into the pericardial space resulted in moderate to weak expression in 30–40% of the rodent heart 6 wk after injection (247). As might be expected, the pattern of expression appeared to have a graded intensity with the strongest areas of expression neighboring the pericardial space. In this case, transduction was observed after including enzymes such as collagenase in the injectant to disrupt the extracellular matrix and to allow for more diffuse dissemination of the vector.
The vast majority of studies have introduced the injectant with a syringe of some type; however, vectors have also been introduced using "gene gun" technology (537, 645, 907, 908). In this instance, the vector consists of gold particles complexed with pDNA. Transduction involves surgically exposing the heart of the animal, commonly a rat, and bombarding the cardiac musculature with the gold particles under gas pressure with a device such as the Helios Gene Gun. Although this technique is not dependent on biologically active particles as when using virally derived vectors, it has several limitations. These studies have reported a reasonably high death rate from the gene gun procedure that seems related mostly to complications from the thoracotomy rather than the use of the gene gun itself. Heart expression levels with this technique are not robust (537, 645, 907, 908). The transduced cells are limited to a rather superficial layer of the myocardium, and expression within this layer is not strong. Given the nature of the technique, it is unlikely that many deeper cells could be transduced without killing the more superficial muscle. Presently, this approach is not significantly advantageous relative to direct injection with viral or nonviral vectors.
Most injectants contain the vector and a physiological salt solution, but some studies have reported the addition of enzymes like proteases or hyaluronidase to degrade the extracellular matrix (ECM) (247, 462, 694, 695). The idea behind adding enzymes to the injectant is to facilitate vector diffusion through the tissue by degrading the ECM, thus yielding greater transduction efficiency. In fact, such techniques have improved the transduction efficiency of direct injections targeted for tumors and the pericardial space. Whether these compounds have a place in direct injection of the cardiac musculature is unclear as degradation of the cardiac ECM is fraught with potential complications and needs careful control. Adjuvants have a clearer role in intravascular delivery to the heart, which will be described shortly.
Direct injection of vectors to the myocardium is a useful technique due to its relative simplicity and application to a variety of vectors, animal models, and experimental systems. It is, however, limited by transduction patterns and the requirement for directly visualizing the heart through surgery to achieve significant levels of transduction. For experimental and therapeutic manipulations that only require transduction of a limited amount of the myocardium (e.g., focal revascularization), direct injection remains an attractive technique. It is likely that any experiment or therapy requiring global transduction of the myocardium will be unable to use direct injection methods.
2. Intravascular delivery methods
The vasculature offers an attractive portal for delivering gene transfer vectors as most cells, including those in the myocardium, lie in close proximity to capillary beds. Vectors placed in the venous space travel to the heart and throughout the circulation, including the coronary circulation (Fig. 3, Table 2). By using the heart's vast capillary network in this way, gene tranfer vectors gain access to a majority of the heart's myocytes. However, a number of imposing roadblocks complicate this scenario. The blood itself may contain neutralizing antibodies to the vector of choice, especially when they are viral-based. The blood also contains proteins, like albumin and platelets, which may absorb or inactivate the vector. There are also physical barriers to transduction including the endocardial cells and the capillary endothelium. Once out of the vascular lumen, the vector must also cross the ECM-filled interstitial space to transduce the target cell. The lung also tends to act as a sponge for many gene transfer vectors (833–835), and any vectors placed in the venous space have to pass through the lung before reaching the left heart for ejection into the systemic circulation. Finally, there is the response of the host itself. Large intravascular amounts of a foreign material, especially from viral delivery, can lead to organ toxicity and/or an allergic reaction (720). Despite early difficulties, many groups have developed several vascular delivery methods that can result in high-efficiency transduction.
Historically, direct injection of pDNA and adenovirus vectors into the bloodstream has resulted in poor transduction of the myocardium, although some tissues such as the liver transduced well (383, 743, 884). With intravenous injection of pDNA, transduction of the heart was sparse. Viral vectors, such as adenovirus, are not tolerated well when intravenously injected, and they have inefficient expression. These results likely reflect the difficulty in overcoming the physical barriers to transduction at levels of vector that are readily prepared and tolerated by the experimental animal. Recently this approach has been revisited using a high-pressure/high-volume technique sometimes called hydroporation (359, 974, 1001). In this technique a viral or nonviral vector is delivered in a very high volume of injectant (approximately blood volume in some cases) resulting in high vascular pressures. Frequently, this is restricted to a specific limb by occlusion of the local vasculature but has been applied body-wide to rodents. Large volumes and pressures of injectant are thought to physically disrupt barriers formed by the endothelium and ECM, which in turn permit greater escape of the vector from the vascular lumen and wider dissemination throughout the tissue. This may result in a mild and temporary disruption of the plasma membranes themselves, which would further aid transduction. This high pressure/volume technique can result in moderate-high transduction of the skeletal but not the cardiac musculature (292). Also, it is unclear how this technique would be tolerated by various animals and how easily it can be adapted to the heart.
In light of the initial disappointing cardiac transduction obtained after the administration of vectors to the vascular space, many groups began developing intravascular delivery techniques aimed at overcoming these barriers to cardiac transduction. These techniques mainly focused on increasing local cardiac vector dose and/or dwell time and increasing the permeability of the cardiac microvasculature. For instance, percutaneous catheters have been used to deliver vectors directly to the heart (346, 373) as this technique should increase the local vector dosage in the heart, thereby increasing transduction efficiency. Catheters have been used to deliver a variety of vectors (pDNA, adenovirus, and AAV) to several areas of the heart including the right atrium and in the root of the aorta just above the sinuses for access to the coronary arteries. A guiding modality such as ultrasound or fluoroscopy is often used (377). The technique has also proven effective in a range of animal models including rodents, canines, and sheep (84, 377, 658). Some studies have used balloon catheters to occlude the vascular outflow of the heart in an attempt to further increase dwell time and pressure of the injectant (346). Adjuvants such as nitroprusside, substance P, adenosine, and histamine have been added to the injectant to increase microvascular permeability to aid in vector extravasation (186, 188, 294, 504, 505). Overall, these techniques have resulted in impressive levels of myocardial transduction. Additionally techniques that use permeabilizing agents and viral vectors tend to be highly efficient. Recently, this technique has been combined with others such as sonoporation to increase the efficiency of complexed DNA transduction. This technique is mildly invasive, requiring vascular catheters that are associated with risks such as bleeding and infection. Some of the adjuvants used are toxic at high levels and can expose multiple tissues to the gene transfer vector, resulting in nonspecific transduction. Expertise in catheter manipulation and access to appropriate equipment are also required. Despite these drawbacks, percutaneous catheters represent an attractive and efficient means of cardiac vector gene delivery.
A method with similar goals to the percutaneous catheters has been termed cardiac isolation or cross-clamping (83, 84, 292, 318). This is a widely varied method with many individual techniques. In general, the animal is subjected to open-chest surgery where the heart circulation (inflow, outflow, or both) is isolated with clamping or balloon angioplasty. The animal is placed on heart-lung bypass with induced cardioplegia. Frequently, the blood is washed out and replaced with a buffer containing permeabilizing agents such as adenosine, histamine, and papaverine. The vector is then introduced under pressure and commonly allowed to have a dwell time of up to 15 min. Vectors are most frequently an adenovirus, though AAV and plasmid vectors have been used. The cardioplegia is reversed, and the animal is taken off heart-lung bypass (83, 84). In another variation, the heart is excised and manipulated in situ before transplant (662). This technique is comparable to the catheter-based method, it is highly invasive and has surgical risks, that can include infection or aortic dissection, commensurate with any open chest manipulation of the heart. Obviously, this technique also requires tremendous surgical expertise and an operating suite capable of supporting the procedures. One potential advantage of this technique is that it can limit exposure of the vector and potentially toxic adjuvants, such as papaverine, to the heart only (83, 84). Thus systemic toxicity and transduction of noncardiac tissues may be avoided.
Recently, some promising results were reported with AAV vectors. AAV vectors pseudotyped with capsid proteins from some of the less commonly used serotypes, such as 1, 6, 8, and 9 among others, are known to transduce muscle cells much more efficiently than the commonly used serotype 2 capsid (Table 3). Several reports have been published demonstrating global cardiac transduction, with both marker and therapeutic genes, in mice after a single injection of AAV into the tail vein (Fig. 3) (262, 293, 294, 390, 893, 944, 1014). Surprisingly, tail vein injection does not require permeabilizing adjuvants, but at suboptimal vector doses the permeabilizing agent vascular endothelial growth factor (VEGF) has been advantageous (294). Potential drawbacks of tail vein injection include the requirement of high vector doses and the possibility of vector expression in noncardiac tissues. This technique is capable of transducing other tissues, such as skeletal muscle, although the development of cardiac specific expression cassettes may be able to overcome this possible problem (783). Nonetheless, cardiac restricted expression has been achieved (893). Despite the use of high AAV doses, tail vein injection was tolerated in mice and did not result in early morbidity, obvious toxicity, or immune responses directed at the vector capsid itself (294). At present, tail vein injection represents the only method available to transduce virtually every cardiac myocyte in small rodents, such as mice and hamsters. Future studies are needed to demonstrate if this delivery method can be efficiently translated to larger animals. One canine has been manipulated by systemic injection with a suboptimal vector dose (based on mouse data). Encouragingly, this canine demonstrated 60% cardiac transduction, similar to the mouse model, with very little expression in other tissues (61). Another limitation is that systemic delivery currently achieves high expression levels only with AAV vectors, which have a limited packaging capacity (Table 1). Similar experiments with other vectors, such as adenovirus, fail to transduce the myocardium to the same extent. These intravascular delivery methods represent the best current technology for achieving global cardiac transduction.
D. In Vitro Gene Transfer/Acute Genetic Engineering
A complementary approach to in vivo gene transfer and transgenic animal models is gene transfer to isolated cardiac myocytes in vitro (Figs. 2 and 4). Acute gene transfer in vitro offers a powerful experimental approach for understanding the direct effects of a genetic manipulation on cardiac myocyte structure and function. Traditional transfection methods have been partially successful in understanding the mechanisms of cardiac growth and differentiation in neonatal and fetal cardiac myocytes as these myocytes are easily cultured and are amenable to transfection by foreign DNA (306, 674, 676, 886, 900). Extrapolation of these data to the adult cardiac myocyte has been difficult due to several key differences between neonatal and adult cardiac myocytes that include features like morphological and sarcomeric organization, contractile and Ca2+ handling protein isoform expression, signaling molecule and transcription factor expression, and the dynamic versus quiescient nature of the culturing system (397, 624, 803). Compared with neonatal myocytes, acute genetic modification of adult cardiac myocytes is more challenging, because differentiated cardiac myocytes are more difficult to maintain in primary culture, and they are not amenable to traditional transfection techniques (DEA-dextran, electroporation, Ca2+ phosphate, and lipofection; Refs. 424, 439, 765). Alternative methods, like direct injection of foreign DNA into the myocardium, are equally inefficient (<0.02%) as the foreign DNA tends to localize to the injection site (93, 94, 494). Thus the aforementioned approaches are unsuitable for comprehensively manipulating cardiac gene expression in a controlled environment.
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The first successful reports of adenoviral gene transfer to isolated adult cardiac myocytes were published in the early 1990s (424, 439). Isolated adult rodent cardiac myocytes cultured in serum-free conditions were adenovirally transduced with various reporter gene constructs driven by powerful, constitutively active promoters. In both studies nearly 100% of the adult cardiac myocytes were uniformly transduced, which is in stark contrast to the low transduction efficiency seen with pDNA transfection to neonatal cells (424, 439). Kass-Eisler et al. (424) also performed direct injection of the adenoviral reporter gene construct into the myocardium and found a 5,000-fold increase in transduction efficiency relative to direct pDNA injection. Viral-based reporter gene activity was first measured 4 h posttransduction (424), and there was a dose-dependent increase in reporter activity which was assessed across time in culture (424, 439). These studies showed great promise for adenoviral gene transfer as a tool for understanding cardiac function, but questions still remained about transgene stability, the effects of adenovirus on myocyte morphology, contractile protein expression, myocyte contractile function, and the stability of cardiac myocytes in serum-free primary culture. Rust et al. (765) answered several of these questions by reporting culturing methods in which adult cardiac myocytes were stable and retained their differentiated state for 1 wk in serum-free culture conditions (765). In cardiac myocyte primary culture, adenovirus-mediated gene expression achieved nearly 100% transduction efficiency (765). Furthermore, adenoviral transduction did not affect the normal rod-shaped adult cardiac myocyte morphology, contractile protein isoform expression, or the isometric tension-pCa relationship for the duration of the culturing period providing evidence that the adult cardiac myocyte phenotype is truly retained (765).
1. Cardiac myocyte culturing systems
Isolating adult cardiac myocytes for study in short-term primary culture has a reputation for being challenging as they can be unstable in the presence of physiological extracellular Ca2+ and they readily dedifferentiate in the presence of serum (928). Nonetheless, cell culture experiments are important for uncovering the primary molecular and cellular effects of an experimental manipulation in a controlled environment. It is debatable whether there are any true immortal cardiac cell lines that retain both the genetic and protein profile, and morphological and contractile characteristics of bonafide adult cardiac myocytes. For instance, the SV40 large T transformed atrial cells (125) and ventricular tumor cells (770) possess some adult cardiac myocyte features but are largely inadequate both structurally and functionally compared with isolated adult cardiac myocytes. For this reason, many laboratories have become quite adept at cardiac myocyte isolation for primary culture and gene transfer (231, 317, 319, 347, 606, 956–967, 1012). Historically, the rat has been the preferred species for cardiac myocyte culture mainly because of availability and size (582). Isolation and culturing methods have now been expanded to include mouse and larger mammals such as rabbit, cat, dog, and human (109, 156, 160, 169, 355, 361). For contractile structure-function studies, it is imperative that cultured cardiac myocytes meet the following criteria: 1) be tolerant of physiologic Ca2+ (1.2–1.8 mM), 2) retain a functional metabolic system, 3) create a homogeneous population of myocytes absent of contaminating and proliferating cells, 4) maintain their ultrastructure, cellular morphology and Ca2+ handling systems, and 5) remain quiescent and stable in culture. These criteria ensure that myocyte preparations are repeatable and yield a standard for "healthy" and physiologically relevant myocytes.
Early reports of Ca2+-tolerant isolated cardiac myocytes were published more than 30 years ago (397). There were two main cardiac myocyte culturing approaches, one requiring serum and the other termed the "cell reattached" method which used serum-free conditions. Serum-based cardiac myocyte culturing protocols were successful and could maintain cells for weeks and even months (125, 384, 396, 397), but the serum contained enough growth and miscellaneous factors that within days these cultured myocytes "dedifferentiated" and lost the adult myocyte phenotype. In contrast, serum-free culture conditions permitted adult cardiac myocytes to retain their highly differentiated rod-shaped morphology, myofilament and metabolic ultrastructure, and intact Ca2+ handling and transverse-tubule density (581, 693, 765). Many of the current protocols have been derived from the original work on serum-free rat myocyte culturing methods of Haworth et al. (343, 344) and Jacobson and Piper (397). Although isolation and culturing procedures differ slightly between laboratories and across species, they generally require certain fundamental elements described below. Once the heart is excised or tissue fragments obtained (as in human heart isolation) and cannulated, the hearts/myocardial tissue undergoes retrograde perfusion on a modified Langendorff apparatus (Fig. 4) such that a low Ca2+ enzymatic solution travels from the aorta through the coronaries for global exposure of the myocardium to the enzyme mixture. Most enzymatic solutions contain collagenase, hyaluronidase, protease, or a combination of these enzymes. Enzymatic perfusion is followed by gentle mechanical digestion and a slow titration of Ca2+ to bring the concentration back up to physiological levels. For some species, such as mouse or human hearts, Ca2+ titration is performed in the presence of an EC coupling inhibitor like 2,3-butanedione monoxime (BDM) as these myocytes tend to be ultrasensitive to extracellular Ca2+ after isolation.
There are numerous publications documenting the successful isolation and use of adenoviral gene transfer to cardiac myocytes from failing and nonfailing rodents (158, 319, 355, 378, 564, 764, 956–967), rabbits (109, 156, 355, 810, 878), canines (361), felines (166, 527), and humans (160, 168, 169). In all cases, adenoviral gene transfer is reported to be highly efficient and efficacious. Adenoviral gene transfer to myocytes isolated from a variety of species is complementary to making multiple transgenic lines in both rodent and larger mammals without the added cost, time, or larger mammal limitations to transgenesis. In addition, the availability of adenoviral gene transfer is tremendously important as intact Ca2+ handling is a critical component of both physiological and pathophysiological processes. Rodent EC coupling is quite different from that of larger mammals (rabbit, dog, and human; Ref. 49), which can impact the extrapolation of results from rodent models to the human.
Acute genetic engineering has become a valuable experimental approach for elucidating the physiological role of normal and disease-related Ca2+ handling, myofilament, cytoskeletal, and signaling proteins in cardiac muscle. The following sections report the physiological insights gained from a rich and growing body of literature involving vector-mediated gene transfer to cardiac myocytes in vitro and in vivo.
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III. Ca2+ HANDLING PROTEINS |
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100 nM at diastole to 500 nM to 1 µM during systole binds to troponin C and induces myofilament activation initiating cross-bridge cycling. The removal of Ca2+ from the cytoplasm during relaxation is carried out by the ATP-dependent Ca2+ pump, SERCA2a, and the sarcolemmal sodium-calcium exchanger (NCX) (50) (Fig. 5).
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High-resolution biophysical and genetic approaches including transgenesis and acute gene transfer have been used to elucidate the mechanistic role of important Ca2+ cycling modulators in the heart under normal and diseased conditions (49, 55, 121, 140, 451, 517, 952). Acute genetic engineering provides a means for directly studying gene dosage effects. Titration of the transgene expression to obtain "physiological" versus "pharmacological" levels of expression is an important consideration when interpreting the functional outcomes of genetically manipulating Ca2+ handling proteins. Acute gene transfer studies are not limited to overexpression strategies as dominant negative mutants or delivery of knockdown molecules offer alternative approaches for understanding the direct role of Ca2+ handling proteins in adult cardiac myocyte function. This section focuses on acute genetic engineering approaches to modulate Ca2+ cycling and cardiac function. This will be accomplished by highlighting the following three components of EC coupling: 1) molecules involved in Ca2+ release from the SR, 2) molecules involved in Ca2+ sequestration, and 3) additional Ca2+ binding proteins that alter cardiac performance.
A. Regulators of Ca2+ Release From the SR
RyR (RYR2 isoform) is the Ca2+ release channel in cardiac muscle SR. RyR is a homotetramer composed of four 565-kDa monomeric subunits (667). RyR is extensively regulated via several associated proteins including protein kinase A (PKA), anchoring protein (mAKAP), protein phosphatases (PP1 and PP2A), sorcin, calmodulin, S100 proteins, and FKBP12.6 (Calstabin2) (531). RyR is also a substrate for posttranslational modification, and its functionality is altered by several small molecules including Ca2+, ATP, and Mg2+ (551). In the junctional SR, RyR interacts with calsequestrin (CSQ), triadin, and junctin (610). The interactions between these proteins contribute to the CICR mechanism of EC coupling (Fig. 5). RyR structure-function has not yet been studied using acute genetic engineering techniques. The large open reading frame of RyR raises challenges for viral-mediated gene transfer strategies (Table 1). Additional challenges to studying the physiological role of RyR through gene transfer technology includes finding physiologically relevant doses of RyR subunit overexpression and the incorporation of exogenous RyR subunits into the SR membrane. Nonetheless, several laboratories have addressed the molecular mechanisms of CICR and RyR function through modulating the RyR's regulatory proteins (FKBP12.6, CSQ, junctin, and triadin) as reviewed in the following sections.
FKBP12.6 is a cis-trans isomerase protein that binds tightly to the cardiac ryanodine receptor (RYR2). It is a 12.6-kDa protein that is thought to bind to each subunit of the channel in a 1:1 ratio on the cytosolic side of RyR (478, 951). FKBP12.6 is considered a RyR channel stabilizer in the closed state during diastole, and it appears to faciltitate the functional coupling of RyR channels (534, 981). Elegant biophysical, transgenesis, and acute gene transfer strategies have yielded important insights into the physiological role of FKBP12.6 (478, 531, 952). Adenoviral-mediated gene transfer of FKBP12.6 in both rat and rabbit cardiac myocytes revealed consistent gains in cardiac myocyte function. A five- to sixfold overexpression of FKBP12.6 in rat and rabbit myocytes directly reduced SR Ca2+ leak and Ca2+ spark frequency and amplitude (280, 513, 710), corroborating biophysical evidence for FKBP12.6's role in stabilizing channel gating during diastole (534, 981). The reduction in SR Ca2+ leak in turn elevated SR Ca2+ load and increased myocyte fractional shortening (280, 710). Unique to the rat myocyte was the finding that FKBP12.6 overexpression hastened Ca2+ transient decay (280, 710). FKBP12.6 gene transfer and overexpression appears to synchronize RyR channel opening during systole, which in turn stabilizes the RyR and reduces random RyR openings and Ca2+ leak during diastole. It should be noted that the effects of RyR phosphorylation on FKBP12.6-RyR interactions and the subsequent functional outcome still requires further clarification as there is some discrepancy in the literature (376, 410, 534, 727, 837, 951). Nonetheless, FKBP12.6 represents an excellent candidate molecule for further gene transfer studies that could be used to explore the role of FKBP12.6 in heart failure, in posttranslational modification of the RyR, and as a potential therapeutic agent.
A related FKBP isoform, FKBP12.0, is also expressed in the heart, and in some mammals the concentration of FKBP12.0 is higher than FKBP12.6 (409). Both FKBP12.0 and 12.6 have highly homologous structure and function, yet their affinities for the cardiac RyR (RYR2) differ significantly (409, 710). FKBP12.0 has a high affinity for the skeletal muscle RyR (RYR1) and can interact with RYR2, but FKBP12.6 has a much higher affinity and seemingly preferential interaction with the cardiac RYR2 (409, 710). Interestingly, knocking out FKBP12.0 in a transgenic mouse is lethal early in development due to severe congenital cardiac defects in the absence of skeletal muscle pathology (819). This mouse model implicates a key role for FKBP12.0 in the early functioning myocardium, but the physiological function of FKBP12.0 versus FKBP12.6 in the adult heart remains unclear. Recently, FKBP12.0 was acutely overexpressed in isolated adult rabbit cardiac myocytes by adenoviral gene transfer (809). A threefold FKBP12.0 overexpression increased SR Ca2+ content similar to FKBP12.6 (809). This acute gene transfer model also uncovered several functional differences between FKBP isoforms. Overexpression of FKBP12.0 (809) had opposing effects on Ca2+ spark amplitude, duration, and frequency relative to acutely transduced FKBP12.6 myocytes (513, 710). Additionally, FKBP12.0 reduced the sensitivity of the cardiac RyR to the CICR mechanism causing a decrease in EC coupling gain (809), a result not seen in FKBP12.6 transduced rabbit myocytes (513, 710). These acute gene transfer studies suggest that FKBP12.0 has distinct and possibly reciprocal effects on RyR function relative to FKBP12.6 in adult cardiac myocytes, although ultimately both FKBP proteins caused an increase in SR Ca2+ content.
2. Junctional SR proteins: CSQ, triadin, junctin, and histidine-rich Ca2+-binding protein
RyR has several binding partners located on the luminal side of the channel within the SR (Fig. 5). Calsequestrin (CSQ, 46 kDa) is the most abundant SR Ca2+ binding protein. It binds 20 Ca2+/molecule (cardiac isoform) with moderate affinity (Kd = 1–100 µM), and it actively participates in Ca2+ cycling by regulating the SR's luminal Ca2+ concentration (825). CSQ's binding partners triadin (isoform 1, 35 kDa) and junctin (26 kDa) act together to tether CSQ to the RyR complex, permitting a physical coupling of CSQ to RyR (1003). The interaction between these proteins is thought to play a vital role in CICR. In addition, histidine-rich Ca2+-binding protein (HRC; 170 kDa) is a moderate-affinity, high-capacity SR Ca2+ binding protein that interacts with SR luminal proteins (e.g., triadin) and is considered a secondary intra-SR Ca2+ storage source other than CSQ.
A) CSQ. Physiological insights into CSQ's role in cardiac muscle function have been obtained from transgenic mouse (414, 792) and acute genetic manipulation studies (571, 877), but the focus here will be placed primarily on results obtained from acute gene transfer. Acute two- to fourfold overexpression of CSQ in adult rat and rabbit cardiac myocytes directly increased SR Ca2+ content as assessed by rapid caffeine application (571, 877). Rat cardiac myocytes acutely overexpressing CSQ had increased amplitude and duration of Ca2+ sparks and waves in the absence of changes in the frequency of these Ca2+ release events (458, 877), providing evidence that CSQ overexpression delays the closure of RyR. Imperatoxin A, a pharmacological activator of local RyR-mediated Ca2+ release events, was also used to assess the direct effects of CSQ overexpression on RyR refractory period (877). Results from this experiment demonstrated that imperatoxin A-induced Ca2+ spark frequency was reduced with CSQ overexpression in rat cardiac myocytes, suggesting that CSQ influences the RyR's Ca2+-dependent refractory period through its buffering of luminal Ca2+ in the SR (877). Targeted knockdown of CSQ by adenoviral delivery of antisense CSQ to isolated adult rat cardiac myocytes caused the opposite effects of CSQ overexpression (458, 877) in which SR Ca2+ content and Ca2+ current (ICa)-triggered Ca2+ transient amplitude were significantly reduced. CSQ knockdown also reduced the periodicity of Ca2+ sparks as well as increased the probability of Ca2+ wave propagation (458, 877). The combined approach of acute CSQ overexpression and knockdown provides evidence that CSQ plays a vital role in determining SR Ca2+ storage capacity and in modulating RyR function through its influence on SR luminal [Ca2+] (458, 877). The effects of acute CSQ overexpression on EC coupling appeared species dependent, as differences in CICR and Ca2+ wave and spark generation varied between rodents and larger mammals. In contrast to rat myocytes overexpressing CSQ, Ca2+ transient amplitude, when triggered by ICa, was reduced in rabbit myocytes overexpressing CSQ (571, 877). Additional species-dependent differences were seen with Ca2+ spark measurements in which CSQ overexpression did not alter the amplitude, duration, or frequency of Ca2+ sparks in rabbit myocytes. These discrepancies on the direct effects of CSQ on EC coupling may be attributed to several factors including 1) species-dependent differences in ICa (49), 2) gene dosage effects in which rat had 4-fold overexpression versus rabbit myocyte transduction which had 1.5-fold overexpression, or 3) the difference in CSQ backbone used for mutagenesis as the rat studies utilized the canine CSQ cDNA while the rabbit studies used rabbit CSQ sequence.
In comparing complementary genetic models of acute in vitro and chronic in vivo overexpression of CSQ, data from both models suggest that CSQ is a vital mediator of Ca2+ storage in the SR. Functional findings in transgenic mouse models (414, 792), however, differed from acute gene transfer studies as 10- to 20-fold overexpression of CSQ caused a reduction in SR Ca2+ release, which attenuated CICR mechanisms. These transgenic mice also showed signs of cardiac hypertrophy (414) and a transition to the fetal gene program (792). Interpreting the results from CSQ overexpression in transgenic mouse models compared with those of adenoviral mediated acute gene transfer is challenging as the transgenic mouse models had adaptive responses to the transgene that resulted in hypertrophy, altered cellular morphology, and changes in the expression of Ca2+ release and Ca2+ reuptake proteins. All of these alterations could contribute to the difference in functional outcomes measured in acute versus long-term gene transfer models. Additionally, the pharmacological levels of CSQ overexpression obtained in the transgenic models relative to the lower level of CSQ overexpression obtained with acute adenoviral gene transfer likely contribute to the differential findings and should be considered when performing a comparative analysis between models.
CSQ dysregulation has been associated with arrhythmic disorders. Seven different allelic variants in the CSQ locus have been linked to inherited forms of catecholaminergic polymorphic ventricular tachycardia (CPVT), a disorder that is associated with stress-induced sudden cardiac death (http://www.fsm.it/cardmoc/, inherited arrhythmias database). To date, only two of the mutant CSQ alleles, D307H and R33Q, have been studied using acute genetic engineering (876, 925). Acute, fourfold overexpression of the CSQ mutant, D307H, in adult rat cardiac myocytes had a dominant negative effect to decrease SR Ca2+ content and ICa-induced Ca2+ transient amplitude (925). These results demonstrated that the D307H mutant directly decreases the sensitivity of the SR to CICR mechanisms. Additionally, isoproterenol and escalations in pacing frequency induced extra nonrhythmic Ca2+ transients, a cellular mimetic of delayed afterdepolarizations (DAD) that are characteristic of CPVT. In contrast, threefold overexpression of the R33Q CSQ mutant in isolated rat cardiac myocytes did not affect SR Ca2+ content and had a dominant effect to increase Ca2+ transient amplitude in response to a triggering ICa (876). The R33Q mutant also increased spontaneous Ca2+ spark and wave frequency, suggesting this mutant enhances Ca2+ leak and thus RyR activity (925). The physiological consequences of expressing D307H versus R33Q mutant CSQ were clearly different, yet both resulted in CPVT. In the case of the D307H mutant, the arrhythmic phenotype was ascribed to the inability of this mutant to bind Ca2+, thus causing a misregulation of Ca2+ release (876) similar to the results from acute CSQ knockdown (877). The R33Q CSQ mutant, however, had Ca2+ binding properties similar to wild type, but the heightened Ca2+ leak was attributed to altered CSQ-RyR interactions that caused overactivation of RyR (876).
B) JUNCTIN. Junctin has been identified as an important tethering component in CSQ's interaction with RyR. To date, there is only one report of acute gene manipulation of junctin in the heart (260). Canine junctin was adenovirally delivered to isolated adult cardiac myocytes. An acute twofold overexpression of junctin directly decreased SR Ca2+ content as well as decreased the Ca2+ transient amplitude but did not alter cellular fractional shortening (260). Overexpression of junctin also increased myocyte contractility and accelerated relaxation kinetics (260). The mechanistic basis for this disconnect between contractile function and Ca2+ transient parameters in junctin overexpressing myocytes remains unclear. It is possible that junctin may affect additional aspects of EC coupling beyond that of Ca2+ release. Interestingly, complementary transgenic mouse models in which pharmacological doses of junctin were achieved also demonstrated reduced SR Ca2+ content, but the Ca2+ transient amplitude was preserved (369, 1002). The differences between acute and long-term genetic engineering in this case were likely due to the compensatory changes in Ca2+ handling proteins that were detected in the transgenic mouse models (369, 1002). Additionally, junctin overexpressing mouse models demonstrated remodeling of the SR and t-tubule system with 10-fold junctin overexpression (1002) and hypertrophy and histopathology with 30-fold overexpression (369).
C) TRIADIN. There are three cardiac triadin isoforms, triadins 1, 2 and 3, with the dominant isoform being triadin 1 (304). Triadin and junctin are the products of different genes but have highly homologous sequences and structures that are postulated to play redundant roles in regulating Ca2+ release through RyR. The anchoring role of triadin, like junctin, has been fairly well defined, but to date, triadin's functional role in the Ca2+ release process has remained elusive. To address these issues, adenoviral-mediated acute overexpression of triadin in cultured adult rat ventricular myocytes revealed a direct ability of triadin to activate RyR and promote Ca2+ release (875). With threefold triadin overexpression, Ca2+ release from RyR was enhanced as Ca2+ spark frequency increased, but spark amplitude was lowered. Triadin overexpression also increased RyR open probability during single-channel recording and increased Ca2+ transient amplitude at smaller trigger ICa (875). Consequently, SR Ca2+ content was decreased due to heightened spontaneous Ca2+ release at rest. Transduced myocytes were also arrhythmogenic when stimulated in the presence of isoproterenol (875). Triadin's direct activation of RyR may be through its interaction with CSQ and RyR, since a truncated triadin mutation lacking the domain important for CSQ interaction showed no effect on RyR Ca2+ release (875). An acute genetic approach showed consistent results supporting a direct regulatory role of triadin on RyR. The discrepancy between results from acute versus long-term triadin overexpression in transgenic mice (438) was postulated to involve compensatory adaptations by other key Ca2+ handling proteins, culture-related changes of myocyte structure and function, the level of overexpression, and/or location of these overexpressed triadin molecules (600). The transgenic mouse models have also shown that pharmacological doses of triadin can negatively affect EC coupling and lead to maladaptive cardiac hypertrophy (438).
D) HRC.
HRC is a luminal SR binding protein that has a histidine-rich repeat region located at the center of the molecule and is responsible for both Ca2+ binding and interactions with other junctional proteins. The Ca2+ binding ability of HRC and its interaction with junctional proteins suggest a potentially important physiological role of HRC in cardiac function. Adenoviral-mediated acute gene transfer of HRC demonstrated a direct and significant inhibitory effect on the Ca2+ transient and myocyte contractility when overexpressed at low levels of 1.7-fold (223). In this study, HRC overexpression slowed Ca2+ transient decay and as a consequence myocyte relaxation (223). Additionally, HRC overexpression increased SR Ca2+ load but blunted Ca2+ transient amplitude and fractional shortening, suggesting that HRC not only affects SR Ca2+ storage but also modulates Ca2+ release (223). Unexpectedly, acute HRC overexpression induced an upregulation of triadin and junctin in the absence of changes in other SR Ca2+ release (CSQ and RyR) and Ca2+ reuptake (SERCA2a, PLN) proteins (223). The increased protein levels of triadin and junctin contribute to the difficulty of understanding HRC's direct role in regulating both Ca2+ storage and release. It is likely that the combined effects of the changes in these luminal SR proteins are affecting myocyte physiology reported here. Interestingly, acute gene transfer with triadin and junctin, when expressed at levels similar to those measured in acutely engineered HRC myocytes, had opposite effects on SR Ca2+ load and Ca2+ release (260, 875). These findings further underscore the complex interactions between HRC, triadin, and junctin and their effects on myocyte physiology. While HRC affects SR Ca2+ content, similar to that demonstrated with the acute expression of CSQ (877), HRC has divergent effects on SR Ca2+ release, suggesting that CSQ and HRC are modulating RyR Ca2+ release via different mechanisms. To date, the direct effects of HRC still remain unresolved, possibly necessitating the use of other acute genetic engineering approaches including adenoviral-mediated HRC downregulation or dominant negative mutagenesis strategies.
Interestingly, HRC null and overexpression transgenic mouse models have been generated and showed an increase in triadin expression with no change in SR load (297, 398). These findings confound our understanding of HRC's role in cardiac muscle function as both downregulation and overexpression of HRC produced similar effects on SR Ca2+ storage. Results from the HRC overexpression mouse model (297) show important differences compared with results from acute gene transfer (223). In contrast to the acute HRC overexpression in rat myocytes, transgenic mice had slow Ca2+ reuptake by SERCA2a and slow Ca2+ extrusion by NCX. This in turn slowed the Ca2+ transient decay rate but not cellular contractile function (297). Chronic HRC overexpression also caused the development of cardiac hypertrophy and an age-dependent transition to congestive heart failure (297). Remodeling of the heart was not apparent in transgenic lines with less than fourfold overexpression, suggesting that high levels of HRC are not tolerated at the organismal level (297). Perhaps gene dosage is partially responsible for the disparity between the complementary overexpression models as are the compensatory changes in other Ca2+ handling proteins including SERCA and NCX that occurred with chronic overexpression of HRC (297).
B. Regulators of Cytoplasmic Ca2+ Removal
Cardiac cytosolic Ca2+ content is highly regulated via transport proteins that either sequester Ca2+ into the SR or extrude Ca2+ across the sarcolemma. The mechanisms for extruding Ca2+ from the cytosol in mammalian cardiac muscle include the following: SERCA2a, sarcolemmal NCX, mitochondrial Ca2+ uniporter, and sarcolemmal Ca2+-ATPase (50). SERCA2a sequesters cytosolic Ca2+ into the SR by transporting two Ca2+ per molecule of hydrolyzed ATP against a steep Ca2+ gradient. There are five SERCA isoforms that are encoded by three genes, with SERCA2a being the predominant isoform expressed in the cardiac muscle (263). SERCA2a is regulated by the closely associated phosphoprotein, phospholamban (PLN), although sarcolipin, described below, can also contribute to SERCA regulation (55, 539). PLN, a small pentameric protein complex comprised of 6-kDa monomers (55), dynamically regulates SERCA2a function on a beat-to-beat basis by its phosphorylation state. It is the interplay between kinases and phosphatases that determines PLN's phosphorylation status (539). In the unphosphorylated state, PLN lowers the affinity of SERCA2a for Ca2+, thereby inhibiting Ca2+ transport (25, 451). Phosphorylation of PLN by PKA at Ser-16 or by CAMKII at Thr-17 reverses the PLN-mediated inhibition of SERCA2a (see also sect. VI) (539). PLN phosphorylation is an important contributor to the hastening of myocardial relaxation during β-adrenergic stimulation as this PLN modification increases Ca2+ reuptake into the SR (950). PLN protein levels are generally unchanged in failing human hearts; however, a reduction in the extent of PKA-mediated Ser-16 phosphorylation of PLN and/or an increase in PLN to SERCA2a ratio are often noted in heart failure. Consequently, SERCA2a function is decreased in heart failure (517). Thus both SERCA2a and PLN are considered attractive gene therapy candidates for improving Ca2+ handling in heart failure patients.
PLN phosphorylation status is contingent on the activity of PP1 (106, 539), the phosphatase involved in counteracting PKA-mediated phosphorylation of PLN. PP1 dephosphorylates PLN which in turn inhibits SERCA2a activity. An additional level of regulation is due to the actions of inhibitor 1 (I-1) protein (539). When activated by PKA, I-1 negatively controls the phosphatase activity of PP1 (539). The resulting I-1-dependent inhibition of PP1 maximizes the phosphorylation state of PLN, thereby increasing SERCA2a function. I-1 plays a vital role in the positive inotropic effects of β-adrenergic stimulation as it assists in maximizing PKA activity in a cardiac myocyte (678, 850). The roles of PP1 and I-1 have been studied using acute gene transfer and are fully reviewed in section VI.
Important insights into the physiological role of SERCA2a and PLN in cardiac muscle have come from transgenic mouse models and are reviewed elsewhere (517, 558). This section highlights experimental results obtained through the use of acute genetic engineering. Several of these gene transfer studies have been performed with the eventual goal of restoring SR Ca2+ uptake and SERCA2a activity (263, 337, 478) which become diminished in the failing heart. The reduction in Ca2+ reuptake in the failing heart is attributed to reduced expression of SERCA2a or a decreased pump activity, which can also be associated with an increase in the PLN/SERCA2a ratio (18, 559). Acute genetic engineering strategies have been used to increase SERCA2a expression and/or activity to restore cardiac function in models of heart failure. Studies employing adenoviral gene transfer of SERCA2a have shown that overexpression of SERCA2a can significantly enhance Ca2+ release, hasten relaxation, and decrease diastolic [Ca2+] (167, 169, 268, 317, 319, 559, 583, 801). Acute overexpression of PLN increases the PLN/SERCA2a ratio, which functionally results in decreased Ca2+ transient amplitude, prolonged Ca2+ transient decay time, and increased diastolic [Ca2+], characteristics that are similar to myocytes from failing hearts (320). Overexpressing SERCA2a can rescue the "failing myocyte" phenotype that is created when the PLN/SERCA2a ratio is increased (168, 169, 317, 319, 583, 801). Importantly, aberrant Ca2+ cycling and contractile deficits characteristic of failing human myocytes can be corrected by restoring the level of SERCA2a expression. This functional correction of the failing myocyte by SERCA2a gene transfer is manifest in increased shortening and relaxation velocity, heightened peak systolic Ca2+, and lower diastolic [Ca2+], in addition to a corrected cell shortening-frequency response (169).
Hirsch et al. (361) demonstrated the effects of adenoviral-mediated overexpression of SERCA2a in cardiac myocytes isolated from a canine model of diastolic heart failure. This model was generated by a descending thoracic aortic coarctation resulting in left ventricular (LV) pressure overload over a year. Cardiac myocytes isolated from canines with diastolic dysfunction were transduced with SERCA2a Ad5 vectors. Acute expression of SERCA2a enhanced relaxation in the failing canine myocytes. In this model, SERCA2a overexpression unexpectedly resulted in a loss of isoproterenol-mediated inotropy during cardiac myocyte contractile measurements in vitro (361). The mechanism of this effect is unknown but is postulated to be due to a diminished PLN/SERCA2a ratio in SERCA2a transduced myocytes. Considering that cardiac reserve is critical to global cardiac performance, a full understanding of the effects of SERCA2a overexpression on β-adrenergic molecular inotropy is important to ascertain.
In vivo adenoviral gene transfer of SERCA2a has been performed in several animal models of heart failure. In a rat model of heart failure induced by aortic banding, Miyamoto et al. (583) used intracoronary gene delivery of SERCA2a at the time of transition from compensated hypertrophy to heart failure. SERCA2a expression restored systolic and diastolic function in this model (583). In a subsequent study, the same group demonstrated that in vivo SERCA2a gene transfer normalized SERCA2a expression levels in the failing myocardium which in turn improved cardiac function, energetics, and survivability (170). Appropriate titration of SERCA2a expression in failing cardiac muscle can restore the normal stoichiometry between PLN and SERCA, which prevents cytosolic Ca2+ overload and left ventricular dysfunction. Taken together, these studies implicate SERCA2a gene transfer as a potential treatment for contractile dysfunction in failing hearts.
PLN gene knockdown and targeted mutant gene delivery have also been used successfully to modulate SERCA2a activity and consequently myocardial physiology (206, 227, 347, 1018). Acute expression of a dominant negative PLN construct, K3E/R14E, significantly increased fractional shortening and hastened Ca2+ transient decay and relaxation times in isolated rabbit ventricular myocytes (347). Gene transfer of PLN K3E/R14E to myocytes isolated from a rabbit HF model directly increased SR Ca2+ content, which in turn corrected the contractile dysfunction in the failing rabbit myocytes (1018). A V49A PLN mutant also acted as a dominant negative form of PLN to enhance myocyte contractility and relaxation (577). Both sets of dominant negative PLN variants identified critical sites within PLN that have dominant functional consequences over native PLN to reverse its normal inhibition of SERCA2a activity.
An alternative gene transfer strategy to enhance SERCA2a activity involves using a constitutively phosphorylated PLN mimetic developed by substituting the serine residue at codon 16 with glutamic acid (PLN S16E) (372). Acute adenoviral delivery of this PLN phosphomimetic to neonatal rat cardiac myocytes increased contractility and had positive lusitropic effects in the absence of any β-adrenergic stimulation (372), suggesting that the PLN S16E enhances Ca2+ cycling and thus contractile function relative to baseline. The efficacy of PLN S16E to halt heart failure progression was also examined using in vivo transcoronary delivery of recombinant AAV serotype 2 to BIO14.6 cardiomyopathic hamsters (372). Transcoronary delivery of an AAV2 reporter construct had 79% transduction efficiency, and the transgene was stable for at least 7 mo (372). In this model, PLN S16E increased SR Ca2+ cycling and slowed the loss of systolic and diastolic function characteristic of the cardiomyopathic hamster (372). Additionally, PLN S16E prevented thinning of the posterior LV wall (372). In a complementary rodent heart failure model, AAV in vivo delivery of PLN S16E blocked the transition to ventricular dilation observed in the nontransduced infarcted rodent model (394). Hemodynamically, PLN S16E improved LV contractility and diastolic function as well as lowered end diastolic pressures and prevented the transition to LV dilation in this HF rat model (394). Together, these studies highlight the potential for in vivo AAV-mediated delivery of PLN S16E as a therapy for progressive dilated cardiomyopathy (DCM) and associated heart failure.
Several acute PLN gene knock-down strategies have also been used to alter SERCA2a function. Adenoviral gene transfer of antisense RNA directed towards PLN in neonatal adult rat (206, 347) and human failing cardiac myocytes (168) demonstrated decreased PLN expression and consequently increased rates of Ca2+ transient decay and enhanced myocyte contractility. In another knockdown approach, AAV-mediated gene transfer of a short hairpin RNA (shRNA) to neonatal rat myocytes silenced PLN but did not significantly influence the levels of other Ca2+ cycling or cellular PKA targets (227). This silencing of PLN significantly increased basal SR Ca2+ uptake, but reduced PKA-directed Ca2+ uptake for up to 7 days after gene transfer. While delivery of antisense PLN significantly decreased PLN levels in neonatal rat myocytes, it failed to decrease PLN levels in adult cardiac myocytes (347), causing some disagreement about using an antisense PLN approach to knockdown PLN. In a separate study, the expression of PLN was significantly decreased after adenoviral delivery of antisense PLN to failing human myocytes (168).
Viral-mediated gene delivery of an antibody targeted to the cytoplasmic portion of PLN has also been used to acutely alter SERCA2a activity (557). Expression of this antibody did not affect the expression or localization of PLN and affected the Ca2+ transient similar to phosphorylated PLN when expressed in adult mouse myocytes (557). In vivo gene delivery of this antibody also acutely improved basal contractility and relaxation in mice with diabetic cardiomyopathy. However, β-adrenergic modulation of contractile function was muted in myocytes expressing this antibody, which may ultimately be detrimental for long-term survival. Although the increases in basal Ca2+ uptake observed with knockdown strategies are beneficial in terms of improved relaxation rates, the loss of adrenergic modulation may also be important for contractile function in failing hearts. In a subsequent study, silencing PLN by adenovirally delivering an antibody directed towards PLN's cytoplasmic domain restored contractile function and Ca2+ handling both at the myocyte and organ level in cardiomyopathic hamsters (178). Gene transfer of PLN antibodies represents a new approach for modifying PLN-SERCA interactions and provides a novel therapeutic strategy for improving failing cardiac myocyte contractile function.
Sarcolipin (SLN) is a 31-amino acid SR membrane protein (23, 25, 660) with close structural similarity to PLN (660). The SLN and PLN sequences diverge significantly at the NH2 and COOH termini (55). SLN does not contain the Ser-16 or Thr-17 phosphorylation sites but has the potential for phosphorylation at Thr-5 (55). The sequence variation in the COOH terminus may be responsible for the differential regulatory function of the proteins at high [Ca2+] (24, 921). SLN is predominantly expressed in the atria and fast skeletal muscle, and it can be found in the ventricle but at much lower concentrations than PLN (25, 55). Similar to PLN, SLN inhibits SERCA activity in the basal state, but the mechanism of reversal of SLN-mediated inhibition by β-adrenergic activation is unclear (23, 24).
SLN expression is highly species dependent. In rodents, both SLN mRNA and protein are expressed in the atria but are almost negligible in the ventricle. In contrast, in larger mammals including humans, SLN mRNA is highly expressed in fast-twitch skeletal muscle compared with atria and ventricle (920). SLN expression is developmentally regulated and has a pathophysiological component as patients with atrial arrhythmias have low levels of SLN transcript (578, 920).
Adenoviral (31) or transgenic (25, 30) overexpression of SLN has been performed to identify the role of SLN in cardiac physiology. Isolated adult rat cardiac myocytes acutely engineered with SLN had decreased myocyte shortening in the absence of any changes in Ca2+ transient amplitude relative to control myocytes (31). Acute SLN overexpression also slowed myocyte relaxation and Ca2+ transient decay time, suggesting that SLN depresses SERCA2a activity in ventricular myocytes (31). Immunofluorescence assays showed an overlap of vector-derived SLN with the native SERCA2a and PLN providing evidence of SLN colocalization with SERCA2a and PLN (31). This was further supported in PLN pull-down assays with SLN (31). In two different complementary transgenic mouse models (25, 30), chronic SLN overexpression in the ventricle caused a loss of function similar to the acute gene transfer model in which a reduction in rate of shortening and relaxation was observed at the organ (25) and myocyte (30) level. One SLN transgenic mouse model revealed diminished Ca2+ transient and myocyte shortening amplitude (30), which was ascribed to SLN-dependent changes in SERCA2a's Ca2+ affinity. Stimulating β-adrenergic pathways corrected the loss in contractility reported in SLN overexpressing mice (25, 30) and in PLN knockouts with SLN overexpression (289). These findings suggest that SLN inhibition of SERCA may be reversed by β-adrenergic signaling, with the caveat that compensatory changes in SLN mouse models may also be contributing to the isoproterenol-dependent enhancement of contractility and Ca2+ dynamics. These studies provide evidence that SLN is another potential therapeutic target, although additional work is required to identify the in vivo mechanism of SLN-mediated SERCA2a inhibition.
The NCX transports 3 Na+ for 1 Ca2+ and is an important regulator of Ca2+ homeostasis and contractility in cardiac myocytes, especially in larger mammals. NCX can operate in both Ca2+ efflux (forward) and Ca2+ influx (reverse) modes, depending on the internal and external concentration of both Na+ and Ca2+ and membrane potential (53). During diastole, SERCA2a and NCX both contribute to cytosolic Ca2+ removal. The competition between SERCA2a and NCX for Ca2+ consequently determines the Ca2+ content of the SR per cardiac cycle (49). NCX has also been postulated as an important component in heart failure as several animal models of hypertrophy and heart failure have shown elevated NCX expression and/or activity (53, 824), a characteristic that has also been reported in tissue from failing human hearts (53, 824). There are, however, conflicting reports of heart failure-induced decreases or unchanged NCX content. Hassenfuss and Pieske (337) found that in failing human heart samples the ratio of NCX to SERCA protein expression consistently increased two- to fourfold relative to nonfailing hearts (824).
Insights into NCX function under normal and diseased conditions have been gained through acute gene transfer studies. Adenoviral-mediated gene transfer of NCX to isolated cardiac myocytes has shown somewhat conflicting results. Several studies have demonstrated that gene transfer of NCX causes contractile dysfunction in rabbit ventricular myocytes due to a reduced SR Ca2+ load (719, 798, 799). Bölck et al. (69) reported that NCX gene transfer enhanced both systolic Ca2+ amplitude and myocyte fractional shortening in rat cardiac myocytes at low stimulation rates, but these functional parameters became reduced at higher pacing frequencies. In a recent study, Münch et al. (611) investigated the functional consequences of NCX overexpression using both in vitro and in vivo adenoviral gene transfer to nonfailing and failing rabbit hearts. With in vitro adenoviral gene transfer, NCX overexpression in isolated nonfailing cardiac myocytes depressed contractility at all pacing frequencies, while NCX overexpression in failing myocytes had an even greater frequency-independent reduction in contractility. In contrast, in vivo NCX overexpression in failing rabbit hearts improved contractility and contractile reserve as the hearts had increased responsiveness to β-adrenergic stimulation and, therefore, enhanced inotropic and lusitropic responses (611). Long-term NCX overexpression in nonfailing rabbits in vivo had only minor effects on myocardial contractility and led to myocardial hypertrophy, in accordance with previous findings in transgenic mice (8, 736, 879, 992). The mixed results obtained from rat versus rabbit NCX gene transfer models might be attributed to the higher intracellular Na+ concentration in rodent cardiac myocytes, which would favor reverse-mode NCX action (51). Elevated intracellular Na+ concentrations have been found in human (692) and rabbit (177) failing hearts, in which case overexpression of NCX may be beneficial for heart failure by improving SR Ca2+ load; however, this benefit may be offset by heightened arrhythmogenicity.
As NCX is the primary mechanism of Ca2+ efflux from cardiac myocytes, it has been postulated that loss of this extrusion mechanism should cause significant Ca2+ overload and thus contribute to heart failure (52, 54, 379, 948). Several laboratories have demonstrated that global NCX1 knockout in mice results in embryonic lethality (117, 452, 934). Interestingly, a recently engineered cardiac specific NCX1 knockout mouse survived well into adulthood with no signs of cardiomyopathy (354, 703–705, 737, 738). Pharmacological inhibitors of NCX function have been utilized, but the results are more difficult to interpret owing to drug specificity issues (739). To better understand NCX's role in cardiac myocyte physiology in the absence of complex influences from adaptive responses in transgenic mice or the pleiotropic effects of pharmacological inhibitors, transient knockdown strategies have been employed to acutely suppress NCX expression and activity. The initial reports of NCX knockdown used an antisense oligonucleotide strategy in which naked DNA was delivered to embryonic (863) or neonatal rat cardiac myocytes (497, 827). These studies showed that naked DNA delivery of antisense NCX is a viable strategy for knocking down NCX expression in neonatal (497, 827) but not adult rat cardiac myocytes (379, 859). To improve efficiency, Tadros et al. (859) used adenoviral delivery of the antisense NCX and achieved a 30 and 66% knockdown at 3 and 6 days after gene transfer, respectively. Under physiological conditions (37°C, 1.1 mM [Ca2+]o, 1–3 Hz pacing frequency), a 30% knockdown of NCX in adult rat cardiac myocytes resulted in heightened diastolic [Ca2+]i in the absence of action potential remodeling or changes in myocyte contractility/Ca2+ transient amplitude during caffeine contracture. NCX knockdown tempered the contractile and Ca2+ transient response to varying extracellular [Ca2+] in cardiac myocytes, showing the importance of extracellular [Ca2+] in addition to NCX density on Ca2+ efflux. Higher levels of NCX knockdown have been achieved with adenoviral delivery of interference RNA (RNAi) to cultured neonatal rat myocytes (379). This strategy was highly efficient and elicited >90% knockdown of NCX expression. NCX knockdown in this system depressed the Ca2+ transient amplitude and relaxation rate in addition to action potential remodeling. Taken together, these studies have demonstrated that acute genetic engineering strategies can be used to knockdown NCX protein expression. These studies, however, have not entirely resolved the direct effects of NCX knockdown on adult cardiac myocyte function as only rodent models have been tested at this point. Considering that rodent Ca2+ extrusion relies heavily on SR reuptake whereas NCX plays a greater role in larger mammals (50), further study in larger animal models is required to fully understand the physiological and pathophysiological role of NCX.
Phospholemman (PLM) is a 72-amino acid transmembrane protein localized to the sarcolemma of cardiac and skeletal muscle. It is a member of the recently discovered "FKYD" family that regulates ion transport (854) and is a substrate for 
- and β-adrenergic signaling molecules PKA and protein kinase C (PKC) (496, 709). PLM associates with Na+-K+-ATPase pump (NKA) and can regulate the pump's affinity for Na+ (143). Several studies have now demonstrated that PLM acts on NKA in a manner similar to phospholamban's regulation of SERCA2a, such that phosphorylation of PLM by PKA increases NKA pump activity through a mechanism of PLM-NKA disinhibition which concomitantly increases the pump's affinity for Na+ (176, 251, 822). Sodium homeostasis is critical under both normal cardiac function and during heart failure. There is evidence that PLM expression is decreased relative to NKA and that a higher percentage of PLM is phosphorylated in failing human and rabbit hearts (74). In contrast, PLM expression was elevated in a rat model of heart failure (115, 808, 1006). Together these studies suggested an important role for PLM in both healthy and diseased cardiac myocytes.
Song et al. (832) virally transduced isolated adult cardiac myocytes with PLM which resulted in PLM overexpression (42% greater than than wild-type) within 3 days after gene transfer. Overexpression of PLM increased maximal myocyte shortening and Ca2+ transient amplitudes at low [Ca2+]o (0.6 mM). These parameters were blunted at higher [Ca2+]o (5.0 mM) and had minimal affects at physiological [Ca2+] (832). This same model was also used to assess the direct effects of phospholemman overexpression on SR Ca2+ content, NCX current, and action potential generation (1006). In this study, 3.5-fold overexpression of PLM at 3 days after gene transfer caused similar contractile changes at high and low [Ca2+]o as demonstrated earlier (832). These contractile abnormalities, however, could be corrected by coexpressing the PLM with NCX (1006). Here, SR Ca2+ content in PLM overexpressing myocytes was unchanged, but the Ca2+ decay rate was significantly slower, suggesting that NCX and PLM are functionally interacting. Additionally, the myocyte action potential and resting membrane potential were not significantly altered, but the reverse-mode NCX current was lower at higher clamped voltages. Taken together, these studies suggest that PLM directly affects cardiac myocyte function, perhaps through NCX. Recent evidence from in vitro pull-down assays and cotransfection of PLM and NCX into HEK cells suggests there is a direct physical interaction between these proteins (941). There are a few limitations to the acute gene transfer studies presented here that may be confounding our understanding of PLM's role in cardiac myocyte physiology. For example, PLM is a target for - and β-adrenergic posttranslational modification which regulates NKA. In these published acute gene transfer studies, there is no control for phosphorylation status of PLM. Acute gene transfer of targeted amino acid substitutions to create phosphomimetics or permanently inactive PLM could yield more insight into the regulatory role of PLM in cardiac function. Furthermore, species differences in Ca2+ and Na+ homeostasis, particularly with respect to the role of NCX, may result in differential species-dependent roles for PLM.
C. Ca2+ Binding Proteins That Modulate Cardiac Performance
Parvalbumin (Parv) is a cytosolic divalent cation buffer that is a member of the EF-hand Ca2+ binding protein family (199). Parv contains two high-affinity Ca2+ binding sites (Kpvca = 107–109 M–1) that competitively bind Mg2+ but with much lower Mg2+ affinity (Kpvmg = 103–105 M–1) (199). Parv is expressed in several tissues including fast skeletal muscle but is not normally found in the mammalian heart (199). In fast skeletal muscle, Parv is thought to act as a delayed Ca2+ buffer that hastens relaxation in a dose-dependent manner (352, 374).
Although not naturally occurring in the heart, there is evidence of successful human Parv gene transfer in cardiac myocytes. The effects of ectopic Parv gene transfer on cardiac performance have been pursued both in vitro and in vivo. Parv gene transfer in isolated rodent cardiac myocytes caused a significant increase in Ca2+ sequestration rate and myocyte relaxation performance under basal conditions (931). The same acute Parv gene transfer procedure rescued diastolic function in myocytes obtained from hypothyroid rats, a model of diastolic heart failure (931). Diastolic dysfunction is also a clinical feature of hypertrophic cardiomyopathy (HCM). As such a cellular mimetic of HCM was developed by acutely expressing an HCM-linked mutant -tropomyosin in isolated cardiac myocytes. Notably, cotransduction of Parv corrected the slow relaxation, a characteristic of most HCM myocyte models, by increasing the rate of Ca2+ removal (139). In addition, acute Parv gene transfer successfully reversed the slow relaxation kinetics of senescent rodent cardiac myocytes (378). In vitro gene transfer of Parv or SERCA2a in myocytes from a canine model of diastolic heart failure showed comparative efficacy in hastening myocyte relaxation (361). In considering Parv as a potential therapeutic for the treatment of diastolic heart failure, a notable feature of Parv-transduced myocytes is their retained capacity for responding to β-adrenergic stimulation, a result not obtained with SERCA2a overexpression (361). These studies indicate that Parv gene transfer may offer unique potential as a primary treatment for diastolic dysfunction in failing hearts.
The effects of Parv in single myocytes have been translated to organ-level function. In vivo gene transfer of Parv via direct intramyocardial injection achieved significant Parv expression (858). The Parv expressing hearts showed accelerated relaxation rates as measured by a working heart preparation as well as by in vivo micromanometry and echocardiography (858). In vivo adenovirus-mediated Parv gene transfer in aged rats showed a reduced expression efficiency (568); nonetheless, Parv still redressed the slow relaxation in these aged hearts (568).
A model of Parv's role in cardiac myocyte performance has been proposed (140) (Fig. 6). Briefly, Parv binds Mg2+ during late diastole due to the high [Mg2+] relative to [Ca2+] (Fig. 6A). The voltage-dependent increase in cytosolic [Ca2+] (1 µM) triggers an exchange of bound Mg2+ to Ca2+ at Parv's metal binding sites (Fig. 6B). This process results in delayed Ca2+ binding by Parv through early diastole because the unbinding of Mg2+ is relatively slow. As intracellular [Ca2+] declines to near resting levels in mid to late diastole, Ca2+ dissociates from Parv (Fig. 6, C and D). Therefore, significant amounts of Ca2+ can be transiently buffered by Parv during early relaxation, which in turn accelerates relaxation in Parv-transduced cardiac myocytes relative to controls.
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0.01–0.10 mM for wild-type Parv (141). This critical concentration range limits the buffering of Ca2+ to the relaxation phase. Therefore, peak contraction and Ca2+ transient amplitude remain unaffected while diastolic relaxation and Ca2+ transient decay times are accelerated. Recent studies using Parv isoforms suggest that experimentally modified Parv constructs may be developed to limit the deleterious aspects of Parv function (i.e., depressing contractility) while maintaining enhanced relaxation performance (755). Sorcin, soluble resistance-related calcium-binding protein, is a highly conserved 22-kDa protein that has five EF-hand Ca2+ binding domains (519). Sorcin is expressed in several tissues, including the heart (561). Sorcin is localized to the t tubules and SR where it can interact with the RyR in cardiac myocytes (508, 561, 563). Increases in intracellular [Ca2+] initiate a Ca2+-dependent movement of Sorcin from the cytosol to the SR (563), where it can interact with specific target proteins including RyR (561), L-type Ca2+ channel (DHPR) (562), and SERCA2a (536). In lipid bilayer preparations, sorcin directly alters the open probability of single RyR units inhibiting local Ca2+ release (225, 508). This effect is abrogated upon sorcin phosphorylation by PKA (508). Sorcin also plays a role in the diseased heart, as sorcin expression is altered in several heart failure models (536, 664) and mislocalized in spontaneously hypertensive rats (560).
Both acute viral-based genetic engineering (242, 536, 810, 846) and mouse transgenesis (560) have been used to elucidate the physiological role of sorcin. Gene transfer of sorcin to isolated adult rat (242, 536) and mouse (846) cardiac myocytes caused a gain in contractility with a 3.5-fold increase in sorcin expression. In isolated rodent myocytes, sorcin overexpression increased fractional shortening, Ca2+ transient amplitude, and SR Ca2+ content (242, 536, 846). Sorcin's role as an acute positive inotrope was also confirmed at the organ level using in vivo adenoviral gene delivery to adult rodent hearts. In vivo, sorcin acutely hastened the rate of pressure development and relaxation and significantly enhanced systolic pressure (242, 846). In contrast, acute overexpression of sorcin in vitro (810) had the opposite effect on cellular function relative to in vivo gene transfer to the rodent myocardium. In rabbit myocytes, sorcin significantly blunted shortening and Ca2+ transient amplitudes (810). Additionally, sorcin overexpression in the rabbit model had reduced SR Ca2+ content and Ca2+ spark amplitude and duration, suggesting that sorcin has a negative effect on EC coupling in rabbit myocytes (810). The discrepancy between these acute gene transfer models could be related to the following factors: 1) the different levels of sorcin overexpression in rabbit versus rodent myocytes and 2) the enhanced contribution of NCX to Ca2+ handling in rabbit versus rodent myocytes (50). For instance, in rabbit myocytes overexpressing sorcin, there was an NCX-dependent slowing of Ca2+ decay time (810). In contrast, rodent myocytes that acutely overexpressed sorcin had enhanced Ca2+ transient decay time (536, 846) and SERCA2a activity (536), providing evidence of species-dependent differences in sorcin function. Notably, pharmacological levels of sorcin expression (20-fold increase) in a transgenic mouse model caused significant reductions in cardiac contractility and relaxation properties in the absence of cardiac hypertrophy or morphological remodeling (560), highlighting the importance of sorcin dosage and time of exposure to functional outcome at the organ level.
The functional role of PKA-dependent posttranslational modification of sorcin is still unclear. Valdivia et al. (912) suggest that sorcin itself might be influenced by β-adrenergic stimuli, since PKA phosphorylation of sorcin diminishes its inhibitory effect on RyRs (508). In contrast, Frank et al. (242) used β-adrenergic agonists and blockade with acute sorcin overexpression in rat cardiac myocytes and found that sorcin-mediated gains in contractile function occurred independent of β-adrenergic stimulation. These data suggest that unmodified sorcin may be regulating RyR function. Interestingly, PKA-mediated hyperphosphorylation of sorcin was identified in animal models of heart failure (536). PKA-mediated phosphorylation enhanced sorcin's Ca2+ sensitivity, which in turn may modulate the translocation of this protein to the SR during pathophysiological conditions in the heart.
Structural and genetic studies have suggested causality between a naturally occurring sorcin mutant (F112L) and inherited hypertension and hypertrophic cardiomyopathy (552, 595, 912). Currently, the sorcin F112L mutant has not been studied using acute gene transfer, but a transgenic mouse model with 20-fold overexpression of this mutation has a dilated phenotype (131). Surprisingly, isolated cardiac myocytes from sorcin F122L mice have increased SR Ca2+ load, which enhanced both Ca2+ transient and shortening amplitudes (131). These results are opposite to the results obtained with similar levels of wild-type sorcin overexpression (560). The gene dosage effects of sorcin F112L on cardiac function remain unclear and require further experiments to determine the primary dosage effects of sorcin variants on cardiac myocyte physiology.
S100 proteins constitute a highly conserved 21+ member family of EF-hand Ca2+-binding proteins (351, 525). S100 proteins are small in size (10–12 kDa) and have similar structural features that include a conventional COOH-terminal divalent cation binding domain and a lower affinity NH2-terminal unconventional EF-hand metal binding domain (525). The S100 isoforms differ in terms of their metal binding affinities, dimerization capacity, and posttranslational modifications (525). S100 isoforms are differentially expressed in a tissue-dependent manner (351, 525). In cardiac muscle, S100A1 is the predominant isoform, but S100A4, S100A6, and S100B have also been identified in the heart (315, 425). In general, the binding of Ca2+ to an S100 protein exposes a hydrophobic region of the molecule that is available to interact/modulate various intracellular target proteins (525). These Ca2+-dependent protein-protein interactions are important for various molecular processes including contraction, cell cycle regulation, cell growth, and secretion. Several of the S100 proteins can also interact with target proteins independent of binding Ca2+ (525).
Gene transfer studies performed in vitro and in vivo have contributed greatly to understanding the role of S100A1 protein on cardiac function in the healthy and diseased heart (607). Acute adenoviral-mediated gene transfer of S100A1 to healthy isolated rabbit (605) and rat (731) adult ventricular cardiac myocytes and engineered cardiac tissue (605, 732) caused a positive inotropic response in which cell shortening and Ca2+ transient amplitudes were significantly enhanced. A fivefold overexpression of S100A1 directly hastened the time to peak Ca2+ and Ca2+ transient decay velocity, which in turn increased myocyte shortening and relaxation kinetics (605). This gain in inotropy was attributed to heightened Ca2+ reuptake by SERCA2a (605). Although S100A1 is downregulated in heart failure (730), S100A1 knockout (194), and cardiac-specific S100A1 overexpressing (608) transgenic mouse hearts have normal morphology and tissue histology. Similar to the acute gene transfer studies, a fourfold overexpression of S100A1 resulted in increased contractility and Ca2+ transient amplitudes at both the cellular and organ level under basal and β-adrenergic stimulated conditions despite the Ca2+ transient kinetics remaining unchanged (608). The gain in inotropy was ascribed to an increased SR Ca2+ load that occurred without detectable changes in the expression of several key Ca2+ handling proteins (608). The increased SR Ca2+ content could be due to several factors including S100A1's interaction with RyR to decrease Ca2+ leak (927) and the enhanced Ca2+ uptake by SERCA2a when S100A1 is overexpressed (605). Target proteins of S100A1 have been reported to include titin (986), SERCA2a (435, 436), and RyR (435, 436, 608, 895, 927), which are all molecular elements that regulate intracellular Ca2+ homeostasis and/or cardiac myocyte contractility. Interestingly, S100A1 knockout mice appear to have normal cardiac function under basal conditions but have impaired cardiac contractility when challenged with hemodynamic stressors like β-adrenergic stimulation (194). Infarcted S100A1 knockout mice also have a hastened transition to heart failure, low survivability, and significant loss in contractile function relative to infarcted wild-type mice and nontransgenic littermates (609). In contrast, S100A1 overexpressing mice have improved mortality, normal cardiac function, and protection from hypertrophic remodeling after infarct (609). Taken together, these complementary genetic models highlight a potential role for S100A1 in the compromised heart and in the contractile responsiveness to β-adrenergic signaling.
S100A1 is considered an attractive prospect for heart failure gene therapy because of its positive inotropic qualities under baseline conditions. As S100A1 protein levels are substantially reduced during heart failure, S100A1's capabilities as a therapeutic transgene have been studied in the failing rodent myocardium both in vitro (606) and in vivo (606, 697, 698) using either adenoviral or adeno-associated (AAV, serotype 6) viral gene delivery. These studies utilized a cryothermic approach to induce myocardial damage leading to progressive heart failure over a 12-wk time span. In vitro adenoviral S100A1 delivery to isolated failing cardiac myocytes returned S100A1 expression to nonfailing myocyte levels and significantly enhanced failing myocyte contractility, Ca2+ transient amplitude, and SR Ca2+ content (606). S100A1 delivery to failing cardiac myocytes in vitro partially restored SERCA2a ATPase activity, decreased diastolic Ca2+ leak, and returned intracellular [Na+] back to nonfailing cardiac myocyte concentrations (606). After infarct, in vivo S100A1 delivery by adenovirus (606, 698) and AAV (697) had comparable rescuing effects on both organ level and myocyte contractility under baseline conditions and during β-adrenergic stimulation despite having inhomogeneous expression across the myocardium. Additionally, increased S100A1 expression after infarct reversed changes in gene expression classically observed during heart failure (606, 697, 698). Collectively, these reports suggest that S100A1 gene transfer could correct aberrant Ca2+ cycling and contractility common to heart failure, thereby improving the performance of the diseased heart.
S100B expression, which is normally undetectable in healthy myocardium, is induced in the diseased heart (419, 675, 902). Tsoporis and co-workers (901, 902) have shown in vitro and in vivo that the induction of S100B expression is a compensatory response to myocardial stress (901, 902). They demonstrated that the expression of S100B limited norepinephrine-induced cardiac hypertrophy structurally and genetically. It appears that S100B expression could be a component of the myocyte response to trophic stimulation that serves as a negative-feedback mechanism to limit cellular growth and associated alterations in gene expression. It is still unclear, however, whether S100B expression contributes directly to the pathogenesis of the failing heart.
Unlike S100B, S100A6 protein (calcyclin) is also normally expressed in cardiac muscle as well as in a wide distribution of tissues (463). S100A6 expression in the heart is upregulated in rodent infarct models and in neonatal myocytes that are exposed to hypertrophic agents (901). The precise function of S100A6 in the adult heart, however, is unclear at present and could be readily studied using gene transfer strategies.
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IV. SARCOMERIC TARGETS AND TEMPLATES |
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The human heart faithfully generates 3 billion contractile cycles over an average life span. During this time, the heart must regularly replace "old" sarcomeric proteins with new ones (Fig. 8). It is extraordinary that mechanisms are in place to accomplish high-fidelity myofilament replacement while preserving sarcomeric stoichiometry and while maintaining full cardiac functionality. The sarcomere is a highly ordered three-dimensional lattice, and in the living adult cardiac myocyte this complex architecture has to be maintained as the myocyte contracts. In this context, sarcomere maintenance reflects a state of dynamic equilibrium in which newly synthesized myofilament proteins incorporate into the contractile apparatus as the old myofilament proteins are replaced (Fig. 8). Insights into the process of myofilament protein dynamics have been achieved using multiple approaches. One approach has been to study myofilament assembly during development (27, 203, 205). These seminal studies uncovered the mechanistic basis for the de novo synthesis of myofibrils or what has been termed myofibrillogenesis. With the use of cultured embryonic cardiac myocytes and high-resolution imaging techniques, the formation of nascent myofibrils could be temporally and spatially visualized. Several studies used isoform-specific antibodies (27, 180, 495, 741, 942), transfection (150), and/or microinjection of recombinant fluorophore-tagged myofilament proteins to observe the time course of nascent myofibril formation and the highly ordered assembly of new myofilament proteins in the developing sarcomere (788, 789, 929). These studies and others also documented the precise regulation of myofilament protein gene expression during myofibrillogenesis (27, 203, 205, 495, 659, 741, 797, 914). Collectively, these studies demonstrated that during myofibrillogenesis sarcomere assembly is extremely dynamic as it involves orchestrated changes in myofilament gene expression, cyclical patterns of protein degradation and synthesis, and new protein incorporation.
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3.2–3.5 days, troponin C (TnC) 5.3 days, Tm 5.5 days, myosin heavy chain (MyHC) 5.5 days, actin 10 days, and the myosin light chains (MLC) 7–9 days (Fig. 8A). Studies using microinjection of recombinant epitope-tagged myofilament proteins in adult cardiac myocytes or permeabilized dedifferentiated muscle fibers soaked in labeled sarcomeric proteins further demonstrated that exogenous myofilament proteins can rapidly (on a minute time scale) incorporate and localize appropriately into the sarcomere (183, 388, 511, 788). More recent studies using gene transfer strategies that rely on the myocyte's native transcriptional/translational machinery have been highly instructive in ascertaining the mechanisms of sarcomeric maintenance in adult cardiac myocytes. Michele et al. (565) used adenoviral gene transfer of recombinant epitope-tagged tropomyosin (Tm) and cardiac troponin I (cTnI) into isolated adult rat cardiac myocytes and high-resolution confocal imaging to examine sarcomere assembly in a physiologically intact adult cardiac myocyte. They found that newly synthesized epitope-tagged thin filament proteins (cTnI and Tm) stoichiometrically incorporate into the adult sarcomere in a time-dependent manner (565). The time-dependent stoichiometric replacement revealed itself through the increase in epitope-tagged Tm and cTnI with a concomitant decrease in the targeted native sarcomeric protein, leaving total thin filament protein expression unchanged as quantified by Western blot and confocal microscopy. This result was interpreted as the native "old" thin filament protein turning over and being replaced by the newly synthesized Tm and cTnI (Fig. 8B). Notably, the rate of incorporation correlated to the previously determined biochemical half-lives of the thin filament (532). This study also demonstrated that Tm has an ordered stoichiometric replacement, where newly synthesized Tms have targeted replacement at the pointed end of the thin filament (565). In contrast, TnI replacement was stochastic (565). These results, in conjunction with previous work (532, 533), suggested a model of Tm and Tn maintenance in which newly synthesized Tms and TnIs incorporate into the thin filament in an ordered (Tm) and stochastic (TnI) fashion, directly replacing the "old" Tms/Tns (Fig. 8B) (565). Transgenic mouse models corroborate this model of stoichiometric myofilament replacement as transgenic animals also maintain myofilament protein stoichiometry despite an overabundance of sarcomeric transgene transcripts (151, 258, 403, 567, 621, 735, 872, 874, 903, 999). The mechanism that governs ordered stoichiometric replacement in the sarcomere is not well understood; however, structural constraints and free energy barriers are likely influential variables. The mechanism of Tn turnover as a unit also remains in question. One hypothesis is that Tn stays bound to actin as newly synthesized subunits replace endogenous components. Alternatively, it is possible that the entire "old" Tn complex dissociates, reassembles with new Tn constituents (i.e., TnI, TnT, and TnC), and then reattaches to actin-Tm. Studies of sarcomeric maintenance with acute gene transfer have been performed primarily in rodent models, but more recently the overexpression of sarcomeric genes and stoichiometric replacement in larger mammals including rabbit (355, 786) and human (160) has now been demonstrated.
The conservation of myofilament protein stoichiometry in adult striated muscle highlights sarcomeric proteins as unique targets for genetic manipulation. Sarcomeric proteins manipulated by acute gene transfer include isoforms, mutants, and chimeric Tm, TnT, and TnI (564, 565, 764, 956–967) as well as MyHC (355). A model system of replacement, as demonstrated by several cardiac myofilament proteins, is well suited for elucidating the fundamental contributions of the myofilaments to cardiac muscle physiology using genetic engineering strategies. With the advances in gene transfer technology, genetic manipulation of myofilament proteins has been used to study function both in vitro and in vivo. Two genetic approaches, transgenic animals and gene transfer to isolated adult cardiac myocytes, have served as the predominant methods for studying myofilament function in intact myocytes. Both model systems offer unique and complementary opportunities for advancement in understanding cardiac muscle function. Transgenic animal models have been critically important for understanding the role of sarcomeric proteins in myocardial function under normal and diseased conditions in vivo, and these models and functional outcomes have been extensively reviewed (151, 400, 744, 745, 999).
The strategy governing adenoviral gene transfer of myofilament proteins to isolated adult cardiac myocytes involves a competition between endogenous and vector-derived gene expression. Gene transfer utilizes the host cell's own transcriptional/translational machinery to express and incorporate the engineered gene product into the sarcomere (Fig. 2). As the native myofilament proteins turnover, they are replaced one-to-one by vector-derived myofilament proteins, resulting in a time-dependent and progressive increase in the percent replacement of native myofilament protein (Fig. 8B). For specific sarcomeric proteins, near full replacement can be achieved by 6–7 days in primary culture, particularly for those myofilament proteins with shorter half-lives.
B. Thin Filament Proteins, Isoforms, Mutants, and Chimeras
The use of acute genetic engineering is an attractive approach for studying the role of thin filament proteins on cardiac myocyte structure and function. Several thin filament proteins have been studied via targeted acute gene transfer technology. Mouse transgenesis and biochemical reconstitution protocols have also tremendously improved our understanding of thin filament regulation (278, 279, 871, 999), but this section will focus on key observations and insights gained from acute genetic engineering of the thin filament by gene transfer.
Cardiac TnI was the first myofilament protein targeted by acute gene transfer technology. The recent crystal structure determination in conjunction with NMR, biochemical, and functional studies has modeled cTnI as a molecular switch within the sarcomere (Fig. 7) that regulates Ca2+-dependent cross-bridge cycling (484, 556, 830, 867). During diastole, myocyte intracellular Ca2+ is low and cTnI binds tightly to actin, inhibiting strong actin-myosin interactions. The elevation of intracellular Ca2+ initiates systole by weakening the cTnI-actin interaction, causing a conformational change in cTnI that promotes strong cTnI-cTnC interaction and in turn permits active cross-bridge cycling.
Cardiac TnI (cTnI) plays a fundamental role in cardiac function under both physiological and pathophysiological conditions. cTnI is also a prominent player in the heart's developmental transition from an embryonic stage, where the dominant TnI isoform expressed is slow skeletal troponin I (ssTnI), to the adult heart where the predominant isoform is cTnI (729, 774). During physiological stress, cTnI can also be posttranslationally modified in response to β-adrenergic signaling and changes in pH (556). Pathophysiologically, cTnI has been implicated as a dominant player in both acquired and inherited cardiomyopathies (278, 279, 638), and it has been identified as a site for proteolytic cleavage resulting in an acute loss of function known as myocardial stunning (617).
2. Gene transfer of TnI isoforms
Westfall et al. (966) first determined the direct functional effects of two TnI isoforms expressed in the heart using adenoviral gene transfer of ssTnI into isolated adult cardiac myocytes. With near full replacement of native cTnI by the ssTnI isoform, there was a concomitant gain in myofilament Ca2+ sensitivity of tension and altered cooperativity demonstrating that TnI isoforms directly influence sarcomeric function. Cardiac myocytes transduced with ssTnI also showed a gain in function during a bout of acute acidosis (pH 6.2) where myofilament Ca2+ sensitivity of tension was preserved in ssTnI myocytes relative to the marked drop in sensitivity sustained by control cTnI myocytes. In confirmation, an ssTnI transgenic mouse model also demonstrated a gain in myofilament Ca2+ and pH sensitivity that resulted in delayed relaxation and Ca2+ transient decay rates in unloaded isolated myocytes (228). At the organ level, expression of the ssTnI isoform in the adult heart caused diastolic dysfunction (228).
The TnI isoform specific differences in Ca2+ and pH sensitivity suggested there were unique domains within each TnI isoform influencing sarcomeric function. To establish the relative contributions of TnI isoform specific domains on myofilament function, chimeras of the cTnI and ssTnI isoforms were engineered and expressed via adenoviral gene transfer in adult cardiac myocytes (956, 957). The N-slow/card-C chimera was developed by engineering 68 amino acids from the NH2 terminus of ssTnI onto the 110 COOH-terminal domain of cTnI. The other chimera, N-card/slow-C, was designed by joining the first 100 amino acids of cTnI with the last 120 amino acids of ssTnI. Gene transfer of N-slow/card-C chimera desensitized the myofilaments to Ca2+ in tension-pCa assays. In contrast, N-card/slow-C myocytes had a dramatic increase in myofilament Ca2+ sensitivity of tension. With these TnI variants expressed at near full replacement, the following myofilament Ca2+ sensitivity of tension hierarchy was established: N-slow/card-C < cTnI < ssTnI << N-card/slow-C. Chimeric TnI studies revealed the importance of TnI's COOH-terminal domain to myofilament function and unexpectedly showed an additive effect of cTnI's unique NH2 terminal (N-card/slow-C chimera) beyond that of ssTnI alone. Additionally, cTnI's NH2-terminal domain proved to be critical to normal cTnI function as the N-slow/card-C chimera caused a loss of function. Together, these results showed that the NH2-terminal domain of cTnI is an additional and important contributor to myofilament Ca2+ sensitivity, a result that was not predicted in earlier biochemical studies (721, 896, 915).
Complementary ssTnI and TnI chimera gene transfer approaches underscore TnI's prominent role in affecting both myofilament Ca2+ and pH sensitivity. Tension-pCa measurements at pH 6.2 showed a loss of Ca2+-activated tension generation in N-slow/card-C myocytes similar to wild-type cardiac myocytes. In contrast, N-card/slow-C myocytes had a tempered response to acidosis similar to ssTnI myocytes. These data show that the COOH-terminal region of TnI contains the pH-sensitive domain that induces a TnI isoform-dependent change in myofilament Ca2+ sensitivity during acidosis.
Gene transfer of ssTnI variants with targeted amino acid substitutions to adult cardiac myocytes identified a single amino acid that fully converted the pH sensitivity observed with ssTnI to the cTnI phenotype (160, 959, 961). Acute gene transfer experiments showed that stoichiometric replacement of native cTnI with a ssTnI variant (ssTnIQAE: R125Q, H132A, V134E) converted ssTnI to the cTnI phenotype during Ca2+-activated tension measurements at neutral and acidic pH in permeabilized myocytes (959, 961, 964). A single codon substitution, ssTnIH132A, fully converted ssTnI to the cTnI phenotype (160, 959, 961). In subsequent studies using biochemical exchange, Dargis et al. (155) reported results from the converse experiment where His was substituted for an Ala at codon 162 in human cTnI sequence. This substitution caused a conversion of the cTnI acidic phenotype to that of ssTnI in ATPase assays. This result was also demonstrated by targeted adenoviral gene transfer of the A164H rat cTnI variant in intact adult cardiac myocytes (160). Not detected in solution biochemical studies (155) was the gain in Ca2+-activated tension at neutral pH that was observed in A164H myocytes from both acute gene transfer and the A164H transgenic mouse model (160). These results highlight the importance of studying function through multiple approaches and in the context of the intact adult cardiac myocyte.
Gene transfer of the cTnI A164H variant was also shown to correct contractile dysfunction associated with isolated failing human myocytes, raising the prospect that this phenotype conversion may have protective effects in chronic heart failure. A164H transgenic mice showed that a single amino acid substitutions in cTnI can dramatically alter function, as these mice achieved better myocardial performance with greater energetic economy during bouts of ischemia and postmyocardial infarction (160). Collectively, these studies show that a unique histidine residue in ssTnI is responsible for TnI isoform differences in myofilament Ca2+ and pH sensitivity. Complementary gene transfer approaches have aided in understanding isoform-specific functional outcomes, and they highlight the mechanistic influence of sarcomeric molecules on myocardial performance under physiological and pathophysiological conditions (964).
Acute gene transfer with ssTnI, TnI chimeras, and the A164H variant of cTnI demonstrate the functional importance of cTnI's COOH-terminal region in determining cardiac myofilament Ca2+ sensitivity. The A164H transgenic mouse did not show histopathology or myocyte disarray at the organ level (160, 228). This is interesting given that other Ca2+-sensitizing TnI molecules can cause disease, including inherited HCM. Several publications have linked HCM alleles to mutant sarcomeric gene products that act as dominant alleles by causing a gain of myofilament Ca2+ sensitivity (258, 278, 279, 403, 811, 871, 960). Mutations in the cTnI gene, TNNI3, cosegregated with clinical cases of inherited cardiomyopathy. The HCM-linked cTnI mutation R146G increased Ca2+-activated tension generation in detergent-skinned papillary muscles from the R146G transgenic mice and rabbits (403, 786). Unlike A164H mice, the R146G transgenic animals developed overt histopathology and myocyte disarray (403, 786). Both ssTnI and R146G mutant cTnI caused similar gains in myofilament Ca2+ sensitivity, but at the organ level these two TnI alleles had very different effects on cardiac morphology and life span. The mechanistic basis for these divergent outcomes, in lieu of similar gains in Ca2+ sensitivity, remains unknown. Gene transfer of R146G cTnI to adult rat cardiac myocytes showed that this HCM linked mutant cTnI behaves quite differently from ssTnI or A164H in the context of the intact myocyte. R146G mutant cTnI had poor incorporation efficiency into the sarcomere relative to wild-type cTnI, cTnI flag, and ssTnI (960). R146G cTnI also yielded a lower total replacement (45%), but it still caused a gain in myofilament Ca2+ sensitivity of tension at neutral pH. When the analogous mutation was engineered into the ssTnI backbone (R115G), Ca2+-activated tension was similar to R146G cTnI and ssTnI. Thus the R115G ssTnI mutation did not additively increase myofilament Ca2+ sensitivity beyond that of ssTnI alone, suggesting that the R146G and ssTnI may affect Ca2+ sensitivity via the same mechanism. Tension-pCa relationships were measured during acidosis (pH 6.2) and showed that, unlike ssTnI, the R146G mutant cTnI and R115G ssTnI responded similarly without having a protective functional effect during acidosis. R115G ssTnI attenuated the protective pH effect of ssTnI, making it more similar to R146G cTnI mutant. Collectively, these results suggest that a combination of increased Ca2+ sensitivity in conjunction with increased pH sensitivity may be responsible for the overt organ level histopathology associated with some HCM cases but not seen in other Ca2+-sensitive models.
The R146G cTnI mutation is only one of a number of cardiomyopathy-linked mutations identified in cTnI which now total over 20 (278, 279, 638). Recently, six new mutations in cTnI were identified and linked to a highly malignant and clinically distinct disease entity, restrictive cardiomyopathy (RCM) (590). The six identified RCM-linked mutant TNNI3 alleles result in the following amino acid substitutions L145Q, R146W, A172T, K179E, D191G, and R193H, which occupy several of cTnI's critical functional domains. Interestingly, mutations at cTnI codon R146 can result in HCM (R146G or R146Q) or RCM (R146W). These shared domains suggest that the location of the mutation is important to the functional outcome, but they do not address the phenotypic divergence seen at the organ level. Biochemical reconstitution studies showed that RCM-linked mutations in cTnI hypersensitize the myofilaments to Ca2+ relative to the already sensitized HCM counterparts (277, 445, 998). Acute gene transfer experiments demonstrated that RCM-linked mutant R193H cTnI myocytes are hypersensitive to Ca2+ (158). Unexpectedly, R193H mutant cTnI also disinhibited the thin filament at resting Ca2+ concentrations and caused an acute cellular remodeling from normal rod-shaped myocyte morphology to a "short-squat" phenotype. This acute cellular remodeling occurred with the progressive stoichiometric incorporation of RCM-linked mutant cTnI into the cardiac sarcomere and could not be explained by altered Ca2+ sensitivity alone.
The 193 codon in cTnI is not only the site of an RCM-linked de novo point mutation but also a site of proteolytic cleavage during myocardial infarction (MI). MI can cause an acute but reversible disease state called myocardial stunning. The pathogenesis of myocardial stunning still remains elusive. It has been investigated with a transgenic mouse model (618) and biochemical reconstitution studies (239) but with conflicting results. The "stunned" transgenic mouse model had a desensitization of the myofilaments to Ca2+-activated tension (618), while reconstitution studies found the opposite effect on ATPase activity (239). These divergent results highlight the importance of delineating the primary versus secondary effects of the stunned cTnI variant for understanding the direct effects of truncated cTnI on myocyte function.
Studies using gene transfer of TnI isoforms and chimeras constructed from the fetal, slow skeletal isoform and the adult, cardiac isoform provided insight into the functional TnI domains that contribute to the decrease in myofilament Ca2+ sensitivity observed with PKA phosphorylation (967). PKA phosphorylates the Ser-23/24 cluster, which is located within the 32-amino acid extension of cTnI, but gene transfer studies demonstrated that it does not phosphorylate ssTnI (967). In permeabilized myocytes, PKA significantly decreases myofilament Ca2+ sensitivity of tension (967). However, expression of ssTnI or a TnI chimera lacking the 32-amino acid cTnI extension prevented this rightward shift in Ca2+ sensitivity. Phosphorylation of the TnI chimera with the 32-amino acid extension and the COOH terminus of ssTnI resulted in a shift that was comparable to cTnI. Together, these results indicated that the functional domain responsible for the phosphorylation-dependent Ca2+ shift is located within the NH2 terminus of cTnI. This conclusion is further supported by a recent study that used both viral-based gene transfer and transgenic mouse models in which tandem serine codons (23/24) in cTnI's NH2 terminus were converted to aspartic acid residues to mimic PKA-mediated cTnI phosphorylation. Myocytes from both genetic models had decreased myofilament Ca2+ sensitivity and significantly faster relaxation times relative to controls, while the addition of isoproterenol only minimally hastened relaxation beyond that of the cTnI phosphomimetic (993). This study underscores cTnI's key contribution to cardiac myocyte relaxation during β-adrenergic stimulation.
As a component of the troponin complex, TnT holds both a structural and functional role in Ca2+-mediated regulation of cross-bridge cycling (Fig. 7). The TnT molecule can be separated into two distinct functional domains: the NH2 terminus, which is responsible for anchoring the entire troponin complex to tropomyosin and mediating the cooperative thin filament transitions, while the COOH terminus plays an important role in the Ca2+-dependent interactions between TnI and TnC (282, 444).
Cardiac TnT (cTnT) was the second thin filament regulatory protein to be manipulated by gene transfer technology. The impetus for studying cTnT with a gene transfer strategy was likely motivated by the percentage (15%) of reported inherited cardiomyopathy cases that have been attributed to mutations in the gene that encodes for cTnT, TnnT2 (946). Gene transfer strategies have been critical for understanding cTnT's role both physiologically and during the HCM pathogenic processes (528, 765, 855, 947). The first acute gene transfer study utilized Ca2+ phosphate transfection of embryonic quail skeletal myotubes to express recombinant wild-type cTnT and several HCM-linked mutant cTnTs: I79N, R92Q, 
E160, and a truncated cTnT resulting from a premature splice donor site in intron 15 (855, 947). This model system had high transfection efficiency, but at least 10% of the myotube's sarcomeres were structurally dysfunctional, which could result from the dynamic nature of avian myotubes in culture. Functionally, all cTnT mutants decreased maximum Ca2+-activated tension and tended to desensitize the myofilaments to Ca2+ in tension-pCa assays (855, 947).
Adenoviral gene transfer of these cTnT mutants into stable, intact, adult cardiac myocytes was also used to study HCM-linked mutations (528, 764, 765). Marian et al. (528) transduced feline cardiac myocytes with the human cTnT mutant R92Q and showed a dose-dependent loss of contractile function starting 48 h after gene transfer. This group also performed direct injection of these recombinant adenoviruses into adult rabbit hearts. Due to the wide range in transduction efficiency (2–60%), they were unable to show any myofibrillar morphology changes and thus did not examine function. Rust et al. (764) transduced isolated adult rat cardiac myocytes with recombinant adenoviral vectors harboring TnT mutants R92Q and I79N mutations. These HCM-linked mutations caused a significant desensitization of the myofilaments to Ca2+ in the absence of a concomitant change in maximum tension generation. Acute gene transfer results (764) are in apparent conflict with those obtained from quail myotube system (855, 947). Adult cardiac myocyte primary culture and avian embryonic skeletal myotubes are very different cellular systems in terms of the dynamic nature and sarcomeric components characteristic of skeletal myotubes. Despite conflicting results, these studies have shown that disease-linked mutant cTnTs play a direct role in determining the myofilament's Ca2+ sensitivity and contractile function.
Mutations in the gene that encodes for slow skeletal muscle TnT, TNNT1, have been linked to nemaline myopathy (276). The pathogenic process associated with nemaline myopathy (described below) is widely unknown and is another potential gene transfer target. Disease-linked single point mutations in TnT can result in a loss or gain of sarcomeric function showing the relative importance of TnT to the regulation of cross-bridge cycling. Many TnT mutants have been studied using biochemical and reconstitution preparations, which goes beyond the scope of this review but are no less important in understanding TnT's role in thin filament function (276, 279, 871).
TnC is the Ca2+ sensor of the thin filament regulatory system (Fig. 7). The crystal structure of TnC in both the Ca2+-bound and unbound state has been elucidated (224, 282, 888), and the atomic structure of Tn's core domain has recently been published (867). The crystal structure in addition to NMR and fluorescence resonance energy transfer (FRET) has identified TnC as a dumbbell-shaped protein that contains a central -helical core with a globular domain on either end. There are two isoforms of TnC, cardiac or slow TnC (cTnC) and fast skeletal TnC (sTnC), which are the products of two different genes, TnnC1 and TnnC2. TnC is highly conserved across isoforms and vertebrate species (>70% similarity). Cardiac/slow TnC (cTnC) is exclusively expressed in cardiac and slow skeletal muscles while sTnC is expressed exclusively in fast skeletal muscles. Structurally, cTnC and sTnC differ at the low-affinity NH2-terminal Ca2+ binding sites (I/II). Three divalent cation binding sites are dispersed throughout the globular domains of cTnC with one low-affinity binding site (II) in the NH2 terminus and two high-affinity Ca2+/Mg2+ binding sites (III/IV) in the COOH-terminal region. At rest, when cytosolic [Ca2+] is low, Mg2+ binds the high-affinity divalent cation sites III/IV until Mg2+ becomes displaced by Ca2+ during the systolic rise in intracellular Ca2+. It is the binding of Ca2+ to site II, however, that initiates the cascade of structural events permissive of cross-bridge cycling (282, 444, 888).
Most of what is known about TnC isoform structure and function has come from solution biochemistry and replacement studies (224, 282, 888). In 1988, the first cloning and mutagenesis of avian sTnC cDNA was used by Reinach and Karlsson to examine structure function relationships in sTnC (728). This opened the door for further mutagenesis studies in both sTnC and cTnC from different species to elucidate the mechanism of Ca2+-induced regulation of contraction (28, 29, 286, 483, 715, 815, 816). Currently, there are no published reports describing the use of acute genetic engineering for understanding TnC's role in cardiac physiology. Work by Edwards et al. (202a) demonstrated that adenoviral-mediated acute gene transfer of TnC variants to isolated adult cardiac myocytes caused direct changes in cardiac myocyte function. This study sets the stage for using gene transfer as an effective method for understanding TnC's physiological role in cardiac muscle. Mutations in cTnC have been linked to both HCM and DCM (56, 364, 591, 711, 802). Critical to understanding the pathogenic process of HCM and DCM is determining the primary effects of disease-linked mutant TnCs on cardiac muscle physiology, an aim that would benefit from acute genetic engineering technology. One potential limitation is the slower turnover rate of cTnC (5 days) versus cTnI or cTnT (3 days) (532). This could constrain experimental outcomes due to limits in the amount of exogenous TnC replacement that can be achieved within the time frame of myocyte viability in serum-free primary culture. Nonetheless, elucidating the role of TnC in an intact myocyte and at the myocyte and whole organ level is still an under researched area that could be pursued through acute genetic engineering strategies.
Tropomyosin (Tm) is an extended coiled-coil peptide that spans seven actin monomers in the thin filament (Fig. 7). Tm interacts with neighboring Tm molecules in a head to tail fashion, providing stability to overlapping tropomyosins and promoting binding to sarcomeric actin. Aside from binding actin, Tm is tethered to Tn via TnT. During diastole, when [Ca2+] is low, Tm blocks strong cross-bridge binding sites on actin. As cytosolic [Ca2+] increases during systole, Tm moves from a closed to an open position, exposing sites on actin for stereospecific myosin binding. Thus Tm can be modeled as a relay switch that promotes strong cross-bridge binding in response to the Ca2+ signal (282, 283).
There are four isoforms of Tm (, β, 
, and 
), and Tm is the predominant adult cardiac isoform. There is a developmental transition from 20% βTm expression in the embryonic heart to less than 10% in a newborn, and it becomes almost negligible in the adult myocardium (622). Tropomyosin can exist as either a homo- or heterodimer in the adult heart (, β, or ββ but is mostly limited to homodimers). Tropomyosin has been the subject of a range of structure-function studies that utilized elegant biochemical methodologies (282, 283, 353, 360, 362, 550, 888, 889). A better understanding of Tm's role under both physiological and pathophysiological conditions in intact cardiac myocytes has been gained through the use of gene transfer technology. Over the past 10 years, several studies have successfully used acute genetic engineering to elucidate the direct effects of diseased linked mutant Tm on cardiac myocyte function.
Mutations in Tm (TPM1) have been linked to inherited cardiomyopathy and nemaline myopathy (skeletal muscle Tm gene, TPM3). Michele et al. (564) used acute adenoviral gene transfer of HCM-linked Tm mutations D175N, E180G, K70T, and A63V to isolated adult rat cardiac myocytes to directly elucidate the structure-function effects of these mutant Tms. The incorporation of mutant Tm closely followed the turnover of native Tm, yielding 40% stoichiometric replacement. Isometric tension-pCa assays revealed that E180G, K70T, and A63V increased myofilament Ca2+ sensitivity and had no effect on maximal tension generation. The D175N Tm mutant had no effect on Ca2+-activated tension even when myocytes were exposed to higher viral titers. The heightened Ca2+ sensitivity of E180G and A63V Tm mutations slowed relaxation in unloaded intact myocytes (566). The D175N and E180G mutations are in a region thought to be important for Tm-TnT interactions (969), while the NH2 terminus mutations K70T and A63V are in a region important for actin binding (360). Despite these distinct regional differences, three of the mutations had similar effects on myofilament Ca2+ sensitivity, suggesting that these mutations cause similar changes to Tm structure. K70T, A63V, and E180G are located in regions of the heptad repeat structure of Tm where charge is critical for maintaining salt bridge interactions that stabilize the Tm dimer heptad positions e and g (546, 841). It was postulated that the loss of charge caused by K70T and E180G would destabilize the coiled-coil Tm structure causing an increase in myofilament Ca2+ sensitivity. Biochemical reconstitution preparations in conjunction with circular dichroism analysis have shown a loss of thermal stability in Tm NH2 terminus with K70T and A63V mutated Tm, confirming the loss of stability created by these mutations (353).
The E180G and D175N mutant Tms have been expressed in transgenic mice (567, 623, 706, 707) and more recently in rats (955). The direct effects of the E180G Tm mutation in transgenic mice closely paralleled that of acute gene transfer, but the compensatory changes varied between E180G transgenic mouse models. Compensatory changes in Ca2+ handling proteins in myocytes from the E180G-FVB/N strain mouse model contributed to a marked change in diastolic function (623, 706, 707). Additionally, E180G Tm, when expressed on an FVB/N mouse genetic background, resulted in marked histopathology and hypertrophy closely mimicking HCM (706). In contrast, E180G Tm when expressed on C57BL/6 background did not show altered myocyte morphology or any histopathology (567). These reported compensatory adaptations in E180G transgenic mice underscore the important difference between acute and long-term genetic strategies and the contribution of background strain effects on organ-level phenotype. With transgenic models alone, it would be difficult to differentiate the direct effects of the E180G mutation in Tm as there were changes in myofilament Ca2+ sensitivity, Ca2+ handling function, and hypertrophic remodeling. However, coupling acute gene transfer with a transgenic mouse model pinpoints the primary versus adaptive changes that result from the transgene expression.
The D175N transgenic mouse model was also engineered (FVB/N strain) producing several lines with varying replacement. To see any changes in working heart performance or myofilament Ca2+ sensitivity, assays had to be performed on lines with >40% replacement. In higher replacement lines, the D175N Tm mutation increased myofilament Ca2+ sensitivity of tension and slowed myocardium relaxation times (218, 623). Contrasting the D175N transgenic mouse results with those of acute gene transfer illustrates the importance of the amount of replacement achieved by the transgene. Acute gene transfer of D175N mutant Tm yielded 40% replacement with no change in myofilament Ca2+ sensitivity. The ceiling for percent replacement of native Tm with mutant Tm is set by the endogenous turnover of myofilament proteins and the time-dependent viability for myocytes in serum-free primary culture. Therefore, a transgenic mouse model in this case permits higher levels of replacement because it is not constrained by primary culture conditions. The different functional phenotypes reported for the D175N mouse model and acute gene transfer myocytes could be explained in part by the heightened percent Tm replacement reported in the D175N transgenic mouse. This underscores the importance of gene dosage effects on cardiac function, which is particularly relevant in inherited cardiomyopathy cases where replacement in human heterozygotes is estimated to be at or near 50% (811).
Notably, the development of E180G and D175N rat transgenic models (955) yielded different results from those described previously in mouse (567, 623) or acute gene transfer studies (564). Extremely low levels of E180G or D175N Tm replacement had no effect on myofilament Ca2+ sensitivity or relaxation function in E180G rat myocytes, while D175N Tm desensitized the myofilaments to Ca2+ and accelerated relaxation rates (955). The divergent transgenic rat phenotype could be due to expressing a human Tm sequence in the rat (955), but Michele et al. (564) used the human Tm cDNA as the backbone for mutagenesis in the previously described acute gene transfer studies which closely paralleled the work done in transgenic mouse models. Alternative explanations are the potential differential contributions of rat versus mouse genetic modifiers and/or low levels of replacement obtained in the transgenic rat.
Mutations in Tm alleles are also causal for nemaline myopathy (NM). NM is considered a clinically heterogeneous group of congenital skeletal muscle myopathies with a histopathological marker of nemaline rod formation in skeletal muscle fibers. With few exceptions, the clinical NM phenotype is characterized by generalized muscle weakness and respiratory insufficiency with early morbidity (386, 387, 654, 656, 657). Identified NM mutations include the slow skeletal muscle gene TPM3, which is 90% homologous to the cardiac Tm gene TPM1, as well as slow skeletal TnT and -skeletal actin. It is notable that mutations in genes that are nearly identical in both sequence and protein structure could result in such divergent clinical presentation as seen in HCM versus NM. To understand the divergent pathogenic processes, Michele et al. (566) used adenoviral gene transfer of the NM-linked M9R Tm and HCM-linked A63V mutant Tm to isolated adult cardiac myocytes. The functional effects of the NM M9R mutation were indistinguishable from wild-type myocytes at core body temperature (37°C) (566). The M9R mutant's phenotype became apparent at lower temperatures (30°C) that have been reported in vivo temperatures for limb muscles. At these temperatures the M9R mutant myocytes had increased relaxation speeds relative to wild-type. This is opposite of the slowed relaxation measured in the A63V HCM mutant myocytes (566). Biochemical studies showed that the M9R mutant decreases Tm's affinity for actin with no significant changes in Ca2+-activated ATPase activity relative to wild-type Tm (362). These results were obtained at room temperature, which may have affected the Ca2+ sensitivity of the myofilaments. In comparison, a transgenic mouse overexpressing the M9R mutant Tm in skeletal muscle resulted in histopathology and muscle weakness similar to the human NM phenotype (137). In situ measurements of force and power from the gastrocnemius muscle of M9R Tm transgenic mice were indistinguishable from wild-type skeletal muscles (161). Transgenic mouse models of NM are very difficult to use for extrapolation to the human condition as few murine muscles are considered purely slow twitch. Furthermore, these studies did not exclusively examine slow-twitch muscles like the soleus or measure mutant protein expression, which adds to the difficulty in extrapolating these results to the human disease condition.
Actin is a globular protein that spontaneously polymerizes and forms F-actin, the backbone of the thin filament (Fig. 7). F-actin is visualized as a double-stranded helical filament and is a structurally polar molecule with a barbed end at the Z-line and one pointed end at the middle of the sarcomere (699). F-actin contains strong and weak myosin binding sites that are exposed when Tm slides from a blocked/closed to an open position in response to cyclical [Ca2+]i fluctuations (282, 283). The polar arrangement of actin is critical as it directs myosin heads to slide actin filaments in the appropriate direction, resulting in myocyte shortening (913).
Much of what is known about actin's functional role in cardiac muscle physiology has come from various biophysical and biochemical techniques including X-ray diffraction, cryoelectron microscopy, and in vitro motility (367, 417, 457, 510, 546, 547, 572–574, 724, 814). The physiological role of actin isoforms and disease-linked actin mutants is a field in its infancy. Acute genetic engineering involving actin may be limited by its endogenous half-life of 10 days. Actin is the slowest myofilament protein to turnover, which is the biggest hurdle to overcome within the 1-wk window of viable myocytes in serum-free cell culture. This temporal constraint may not provide ample time for incorporation and a measurable functional phenotype. Additionally, it is not well known whether actin will replace and incorporate in the same manner that has been described for other thin filament proteins given the tight regulation of its length by capping proteins and tropomodulin, as well as its interaction with Z-line proteins.
Further understanding of actin's role in cardiac muscle function will likely come from assessing the direct functional effects of diseased-linked mutations in the actin gene. Missense mutations in alpha-cardiac actin gene (ACTC, nine identified to date) and -skeletal muscle actin gene (ACTA1) have been linked to cases of DCM, HCM (589, 592, 665), and NM (386, 387, 654, 656, 657; described above), respectively. The clinical phenotype associated with ACTC mutations ranges from asymptomatic to severe cardiac dysfunction. Mutations identified in actin within Z-line anchoring domains (e.g., R312H and E361G) are associated with DCM. Other actin mutations within myosin binding domains (e.g., A331P and A295S) are associated with HCM. One actin HCM mutation (E99K) has been evaluated in vitro by a series of biochemical assays, which demonstrated a weakened interaction with myosin and reduced force production (73). Several other actin mutations have been expressed in vitro and found to demonstrate variable defects in protein folding which correlated with impaired incorporation into the cytoskeleton of mammalian (noncardiac) cell lines (919). To date, there are no published studies using acute gene transfer of cardiac or skeletal muscle actin variants. Unpulished results have shown that four representative cardiac actin HCM and DCM mutations can be expressed in isolated adult rat cardiac myocytes with efficient sarcomeric incorporation by gene transfer (177a), presenting proof of principle that acute gene transfer of actin is a feasible approach for assessing actin's role in cardiac muscle function.
Myofilament turnover in the adult myocyte is another area that might benefit from the use of acute genetic engineering. Actin thin filament length is highly uniform in muscle sarcomeres (1 µm) (750). Isolated myofibrils are very stable in vitro, and fluorescently labeled actin monomers can be incorporated only after removal of the capping proteins CapZ or tropomodulin (500). Despite these observations, in vivo thin filament maintenance is a dynamic process. In living cells, fluorescent actin rapidly incorporates into thin filaments, and monomer exchange is relatively quick compared with protein turnover, suggesting that thin filament capping does not restrict monomer addition (499). Injection of fluorescently labeled actin into thin filaments of living myocytes has been reported by several groups (183, 270, 336, 545, 817), but the site (pointed end, barbed end, or both) of actin incorporation remains debatable (499).
8. Capping proteins and molecular rulers
Actin capping proteins such as CapZ and tropomodulin (Tmod) contribute to the actin monomer dynamics at the ends of the thin filament (499, 500) and regulate the length uniformity of actin filaments within the sarcomere (241). Specifically, CapZ appears to nucleate the developing actin filament at the barbed end within the Z-line during myocyte fibrillogenesis. The capping of actin by Tmod at the pointed end is a critical regulator of thin filament assembly, length regulation, and function (793). The importance of these capping proteins has been demonstrated by several transgenic knock-out models that had early morbidity (122, 246, 332), and with overexpression models that had a DCM phenotype (853). The giant molecular ruler protein nebulin is another important regulator of actin filament length albeit in skeletal muscle (371, 543). Its role in cardiac muscle is not well understood. Compared with skeletal muscle, in which there are approximately two nebulin molecules per thin filament, nebulin is expressed at significantly substoichiometric levels in cardiac muscle. Despite these low levels, knockdown of nebulin in neonatal rat cardiac myocytes using RNAi transfection caused dramatic elongation of thin filaments from the pointed end without affecting the barbed end (544), suggesting a potential role of nebulin in thin filament assembly.
Given the complexity of actin dynamics and observed effects in transgenic mouse models, maintenance of thin filament length clearly requires precise regulation. To date, the precise role of actin capping proteins and molecular rulers in adult cardiac myofibrillar assembly and myocyte function are not fully resolved. Much of what is known about myofibrillar assembly comes from direct injection or plasmid transfection of highly dynamic cell culture systems (i.e., chick embryonic myocytes, neonatal myocytes, and serum-induced differentiating myocytes) (233, 240, 295). Concerns have been raised about microinjection techniques as they result in high levels of physical trauma to cultured myocytes (820). Additionally, most myofibrillar assembly studies have focused on morphology and histological analysis with very little analysis of the resulting functional effects of modulating capping proteins. Studying myofilament assembly and the functional role of capping proteins in the adult cardiac myocyte presents another opportunity for the use of acute genetic engineering. The major limitation of an acute genetic engineering approach will again rely on the turnover time of the capping proteins, which is thought to be fairly long (499). It is well known that the stoichiometry of capping proteins to actin is critically important (241), and studies suggest that, unlike other myofilament proteins, capping proteins can be overexpressed (852). At this time there has only been one report of the use of adenoviral gene transfer for studying the role of capping protein, Tmod, in embryonic cardiac myocytes from chicken (852). Adenoviral delivery of sense or antisense Tmod to this culture system was highly efficient, and significant overexpression/knockdown of the Tmod protein was seen by 24 h after gene transfer (852). In this setting, Tmod overexpression yielded shortened thin filaments, while Tmod downregulation caused longer thin filaments. This study showed that Tmod is important for maintaining actin stability and provides a basis for using genetic engineering as an approach for studying capping protein function.
The thick filament is predominantly composed of the molecular motor protein myosin (Fig. 7) that cyclically interacts with actin to produce force and sarcomere shortening. Cardiac myosin is a hexamer consisting of two myosin heavy chains (200 kDa each, discussed above), two essential light chains (light chain 1, MLC-1), and two regulatory light chains (light chain 2, MLC-2) (797). The thick filament also contains other myosin binding proteins such as C, H, X, and M proteins and the large elastic protein titin. Regulation of cardiac muscle contraction involves complex interactions between Ca2+ and proteins of the thin and thick filaments. While much is known about the contribution of Ca2+ and the thin filament to regulation of contractile function, much less is known about modulation by thick filament accessory proteins and their posttranslational modification. Acute gene transfer has not been extensively employed for studying thick filament proteins, but it offers an intriguing approach for directly assessing the thick filament's role in cardiac muscle physiology under normal and diseased conditions. Notably, the genes that encode for myosin heavy chain, myosin light chain, and the myosin binding proteins have identified mutant alleles that are causative for inherited cardiomyopathy. In addition, there are known changes in myosin and myosin light chain isoform expression during heart failure (26, 585, 794). Detailed transgenic and biophysical studies have been performed on thick filament mutant alleles (151, 258, 456, 723, 724, 905, 999); however, understanding the direct effects of these mutant proteins on intact cardiac muscle function is incomplete.
A potential limitation of utilizing acute gene transfer for modulating the thick filament is the turnover rate of thick filament proteins. Some of the longest myofilament half-lives are related to constituents of the thick filament. The half-life of myosin heavy chain is estimated at 5.5 days, and the light chains are even longer (7–9 days) (533), which could limit the amount of replacement or dose-response assessment garnered through acute gene transfer in serum-free myocyte cultures.
Cardiac myosin is the most abundant protein in cardiac muscle and is the primary consumer of cellular energy (ATP). Two structurally and functionally distinct isoforms of myosin, - and β-myosin heavy chain (MyHC), result from two separate genes (MYH6 and MYH7, respectively) that are differentially expressed in cardiac muscle. The transcription of each gene is independently controlled but coordinately regulated (313). Cardiac myosin isoforms can form homodimers or heterodimers and have been electrophoretically separated leading to the following nomenclature: myosin V1 (- homodimer), V2 (-β heterodimer), and V3 (β-β homodimer) (366, 701).
Despite sharing >90% amino acid homology, cardiac myosin isoforms are functionally quite distinct. For example, β-MyHC, the "slow" molecular motor, hydrolyzes ATP three to seven times slower than -MyHC (330, 918). The "fast" motor, -MyHC, represents >90% of the total myosin expressed in the normal adult rodent heart (235, 356, 553). Expression of the slow β-MyHC motor increases relative to -MyHC in rodent models of cardiovascular disease including diabetes (762), hypothyroidism, cardiac hypertrophy (553), and aging (99, 236, 932). Therefore, increased β-MyHC motor expression is one biomarker of cardiac disease in rodents. The adult cardiac ventricles of healthy larger mammals have a different myosin isoform profile where 90% of the total myosin content consists of β-MyHC (26, 284, 498, 585).
In humans, mutations in each cardiac myosin gene can cause familial cardiomyopathy (100, 226, 811), and alterations in myosin isoform expression are associated with heart failure (514, 585, 632). The most commonly affected sarcomeric protein in inherited cardiomyopathy patients is β-MyHC. In the heart, mutations in the β-MyHC gene MYH7 have been associated with both HCM and DCM (638, 811). Although the human heart expresses predominantly β-MyHC, mutations of the -MyHC gene MYH6 have recently been linked to either HCM or DCM (100). The clinical importance of β-MyHC mutations is further underscored by the finding that β-MyHC mutations have been linked to skeletal muscle myopathies including Laing distal myopathy (554), myosin storage myopathy (861), and hyaline body myopathy (68). The clinical importance of cardiac myosin makes it an attractive target for acute gene transfer strategies; however, only two studies to date have utilized such technology for studying cardiac myosin structure and function.
The first HCM-causing myosin mutation was discovered in the motor domain of the myosin molecule (258). This missense mutation results in an arginine to glutamine substitution at amino acid position 403 (R403Q), which is located in the actin-binding interface (723). Since the discovery of this myosin mutation, much effort has been devoted to determining how it affects myosin motor function and triggers cardiomyopathy (60, 146, 147, 671, 856, 905, 987). Extensive studies performed on a transgenic mouse model of the R403Q mutation demonstrated delayed cardiac relaxation and slowed chamber filling with a progressive transition to hypertrophy and myocyte disarray common to HCM (258, 259). In these studies, the mutation was engineered in the -MyHC gene, which differs from the human HCM-linked mutation that occurs in the β-MyHC gene (258). The primary effects of the R403Q substitution in β-MyHC on myosin motor and cardiac function are therefore important to determine.
A recombinant adenovirus has been used to study the structural effects of mutant β-MyHC expression on sarcomere assembly and myofibrillar organization (527). Marian et al. (527) used acute gene transfer to study the effects of the HCM-associated R403Q mutation of human MYH7 on cardiac sarcomere structure. This study used acute gene transfer in adult feline cardiac myocytes in vitro, which had an efficiency >95%. In the adult feline cardiac myocytes, expression of mutant and normal β-MyHC by viral gene transfer was determined by reverse transcription-PCR. Electron micrographs and fluorescent imaging only showed subtle alterations of sarcomere structure in all experimental groups. The R403Q mutant resulted in sarcomeric disarray in 50% of the myocytes that were examined, while no sarcomere disruption was observed in either control (no virus) or wild-type β-MyHC transduced myocytes. This result was found in both electron micrographs and in experiments using indirect immunofluorescence and fluorescent imaging. The physiological consequence of long-term expression R403Q β-MyHC mutation was also examined using a transgenic rabbit model (526). R403Q transgenic rabbits with 40% β-MyHC replacement had significant septal and posterior wall hypertrophy with myocyte disarray and heightened collagen content without changes in fractional shortening (526). Additionally, R403Q rabbits had markedly reduced survival rates (526). These results suggest that the primary effect of this mutant myosin is to disrupt sarcomere assembly and myofilament organization and perhaps is a constituent in the pathogenic process leading to HCM.
Most studies that have examined the effect of myosin isoform switching on cardiac muscle function have used hyper- or hypothyroid animals (235, 356) and transgenic mice (455, 873) or rabbits (401). It is difficult to determine the direct effect of myosin isoform shifts in these models because the phenotype represents the combined effects of myosin isoform and the compensatory response to the hormonal or genetic manipulation. Recently, a recombinant adenovirus was used to determine the functional consequence of increased relative β-MyHC expression in rodent cardiac myocytes (355). β-MyHC gene transfer demonstrated nearly 100% efficiency at the level of the isolated adult cardiac myocytes. Functionally, adenoviral gene transfer of β-MyHC attenuated sarcomere shortening compared with control myocytes (AdGFP or no viral treatment) while Ca2+ transients were unaltered. These results suggest that β-MyHC expression represents a Ca2+-independent negative inotrope among the cardiac myofilament proteins and demonstrate the feasibility of using recombinant adenoviruses to study structure-function relationships of the heart's molecular motor.
2. The myosin essential (MLC-1/ELC) and regulatory (MLC-2/ RLC) light chains
Ventricular muscle myosin is comprised of two unique accessory proteins, the essential (MLC-1) and regulatory (MLC-2) light chains. Both MLCs are bound to the neck region of myosin where they are thought to modulate myosin motor function. MLC-1 is a 17-kDa protein product of the MYL3 gene. It contains six functional domains, an actin binding site, a proline-rich region, and four helix-loop-helix regions and belongs to the superfamily of EF-hand Ca2+ binding proteins (899). The regulatory light chain, MLC-2, is a 22-kDa protein product of a different gene, MYL2. MLC-2 has several important structural regions including a phosphorylation site on serine-15 and four EF-hand domains (149). Like MLC-1, it also belongs to the superfamily of EF-hand Ca2+ binding proteins. Mutations in the myosin light chains have been linked to cardiomyopathy (226, 638). Collectively, these data suggest that both light chains play a critical role in normal cardiac physiology, particularly in cross-bridge kinetics.
To date, there are no reports of utilizing acute genetic engineering for understanding the role of myosin light chains in cardiac function. However, three different transgenic mouse models that overexpressed either the ventricular isoform of RLC (RLC2v) or ELC (ELC1v) in the atria or the atrial isoform of ELC (ELC1a) in the ventricle showed that protein stoichiometry is tightly conserved despite overexpressing light-chain transcripts (230, 402, 670). Furthermore, multiple transgenic lines have been generated with varying levels of replacement that ranged from 0 to 95%. Taken together, these data show that like other myofilament proteins, MLCs have tightly regulated stoichiometric replacement and offer titratable gene dosing. Permeabilized fiber studies from these mice showed a change in cardiac fiber function such that the incorporation of either ELC1v or RLC2v into atrial fibers caused decreased unloaded shortening velocity (787). Interestingly, myosin light-chain isoform switching, where the ventricular isoforms convert to the atrial light chains, has been documented in various human heart failure cases such as in congestive heart failure or in DCM (26, 601, 794), suggesting important and divergent physiological roles in cardiac muscle for the ventricular versus atrial myosin light chains. The precise role of the different cardiac muscle MLC isoforms in cardiac function is not fully known. A potential limitation to MLC gene transfer is the long turnover time of the light chains. The half-lives of the MLCs have been estimated at 7–9 days, which may limit the amount of replacement one can achieve with recombinant light chains delivered to myocytes in primary culture (532, 533). Modest levels of replacement (30%) in the transgenic mouse model with ECL1a overexpression in the ventricle resulted in a change in function (230), suggesting that the high levels of replacement may not be necessary to affect cardiac myocyte physiology.
3. Thick filament accessory proteins: C-protein (MyBP-C)
Myosin binding protein C (MyBP-C) is a myofilament protein of the intracellular immunoglobulin/ fibronectin superfamily that is located in the "C-zone" of the A-band in seven to nine transverse stripes spaced 43 nm apart (142, 661, 757). Distinct genes encode for the three isoforms of MyBP-C that are expressed in adult mammalian striated muscle and include fast skeletal, slow skeletal, and cardiac MyBP-C. MYBP3 is the gene that encodes for the cardiac isoform (cMyBP-C) (257), and it is expressed exclusively in cardiac muscle throughout development (256). Cardiac MyBP-C is organized similarly to the skeletal isoforms with 10 globular domains (C1-C10) and a highly conserved linker region, the MyBP-C motif (257). Cardiac MyBP-C has several unique features including an extra IgI-like NH2-terminal domain (termed C0), phosphorylation sites within the MyBP-C motif, and a 28-amino acid insertion within the C5 domain. All isoforms of MyBP-C interact with the light meromyosin (LMM) region of the myosin rod, the region that forms the thick filament backbone (599). The COOH-terminal C10 domain contains the LMM binding site of all MyBP-C isoforms (14, 663). In addition to binding the myosin rod, MyBP-C isoforms also bind to the thick filament region of the massive structural protein titin (252, 449). The interactions between MyBP-C, myosin, and titin likely contribute to the highly ordered structure of the sarcomere.
The precise role of cMyBP-C in cardiac function still remains unclear, but there is evidence suggesting that MyBP-C contributes to thick filament formation and sarcomere assembly (252, 449) and plays a regulatory role in cardiac muscle contraction. MyBP-C's role in cardiac function is still widely unknown but is suspected to contribute to β-adrenergic-mediated gains in function (105, 357, 771, 772, 839, 840), myofilament Ca2+ sensitivity (102, 331, 365, 542), and length-dependent changes in myofilament Ca2+ sensitivity (105, 358). Additionally, MyBP-C must play a critical role in normal cardiac function as 20–30% of inherited HCM cases are attributed to mutations in the gene that encodes for MyBP-C (226, 638). To date, the role of MyBP-C in cardiac muscle function has not been studied using acute genetic engineering. Despite being a fairly sizable gene (21 kb) with 35 exons, the MyBP-C transcript is 4.5 kb (257) and can be accommodated by common adenoviral vectors (Table 1). Gene transfer of MyBP-C may be limited by its turnover time, which at present is unknown, and acute gene transfer may be further complicated by its stoichiometry. To date, it is still debatable whether MyBP-C stoichiometry is maintained. This debate is largely fueled by conflicting evidence from both human and transgenic mouse models that have various levels of MyBP-C expression. For instance, a clinical study reported no detectable levels of MyBP-C in HCM patients with MyBP-C mutant alleles (760), suggesting that the mutated MyBP-C is unstable with a pathogenic mechanism leaning towards one of haploinsufficiency. A heterozygous MyBP-C null mouse model also lends some support to the mechanism of haploinsufficiency as a knockout of one MyBP-C allele resulted in a slight but significant decrease in total MyBP-C content (102). In contrast, the development of transgenic mouse models that have two- to eightfold overexpression of either wild-type MyBP-C or an HCM-linked truncated MyBP-C demonstrated that MyBP-C stoichiometry is conserved and that the truncated MyBP-C is indeed stable (990). This result is further supported by ES cell-derived transgenic mouse models with various COOH-terminal truncations that retained total MyBP-C stoichiometry (542).
The cardiac isoform of MyBP-C is the only isoform that contains phosphorylation sites. Phosphorylation of MyBP-C prevents myosin S2 and MyBP-C binding (301). The phosphorylation state of various C-protein sites alters thick filament structure and function. Ser-273, Ser-282, and Ser-302 of cMyBP-C are key substrates for PKA (237, 972), Ca2+/calmodulin-dependent kinase CAM kinase; Ref. 335), and PKC (924). Importantly, in addition to cTnI, phospholamban and other Ca2+ handling proteins, cMyBP-C is phosphorylated in response to β-adrenergic stimulation (e.g., isoproterenol) by PKA, thereby suggesting a role for cMyBP-C in the regulation of cardiac muscle contractility. Studies using gene transfer to investigate the contribution of posttranslationally modified MyBP-C on contractile function and/or studies to develop therapeutic strategies utilizing the phosphorylated versions of this protein have not been published. However, MyBP-C knockout and phosphorylation mimetic transgenic mouse models have been used to examine the role of MyBP-C phosphorylation as it pertains to the regulation of cardiac muscle contractility. Knockout models suggest that PKA-mediated phosphorylation of MyBP-C contributes to both the magnitude of desensitization of the myofilaments to Ca2+ (102) and enhanced cross-bridge cycling during β-adrenergic stimulation (345, 357, 434). A phosphomimetic transgenic mouse was designed using aspartic acid substitutions and showed that constitutive phosphorylation of MyBP-C conferred cardioprotection during ischemia-reperfusion injury (772). The precise mechanism whereby cMyBP-C and its phosphorylation status affect cardiac contractility and cardioprotective behavior during ischemia is unclear, but acute genetic engineering of cMyBP-C may offer a new tool to further uncover the role of MyBP-C phosphorylation in the fine-tuning of cardiac muscle contractility.
4. Thick filament accessory proteins: titin
Titin is the largest mammalian protein currently identified. There are several titin isoforms ranging from 2.9 to 3.7 MDa in size that are all splice variants of the same gene, TTN. The titin gene is made up of 363 exons (33) and has a cDNA of 82 kb (465). Elegant biophysical studies have shown titin's structural and functional role in cardiac muscle (290). Titin is largely responsible for the viscoelastic characteristic of striated muscle cells. Structurally, titin plays a key role in the maintenance of thick filament length and construction of Z and M-lines (296). Titin connects the Z-line to the M-line and has been modeled as a molecular spring as its structure contains an extensible region, the Ig-segment (immunoglobulin-like domain), and PEVK region (rich in proline, glutamate, valine, and lysine) that generates passive tension as sarcomeres become stretched. The relationship between passive tension and resting sarcomere length turns hyperbolic beyond that of optimal length. The different titin isoforms confer distinct variations in passive stiffness as demonstrated by the cardiac N2B isoform which elicits higher levels of passive tension per increase in sarcomere length relative to the N2A isoform due to N2B's shorter extensible region (104, 244, 465, 898). The heightened stiffness of the N2B isoform is thought to promote rapid return to diastolic filling during the cardiac cycle. Furthermore, titin can be posttranslationally modified and may directly interact with Ca2+ (290). Titin's large size presents the biggest limitation to overcome when trying to utilize gene transfer for titin manipulation. Perhaps the use of titin fragments or truncations for acute gene transfer may be a way of overcoming the packaging size limitations but with the caveat that truncated titins cannot entirely extrapolate to physiological titin function. Gutted adenoviral vectors may also be a promising candidate for titin gene delivery. Furthermore, titin's turnover time is unknown and may be rather lengthy, offering an additional limitation for using acute gene transfer in vitro.
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V. CYTOSKELETAL PROTEINS |
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Dystrophin and its associated proteins have been linked to many forms of inherited muscular dystrophy, several of which present as cardiomyopathies. The nidus of this protein complex is dystrophin (Fig. 9), a 427-kDa protein that binds to the submembranous actin lattice and bridges the gap to the membrane. At the membrane, dystrophin interacts with the integral membrane protein β-dystroglycan, which binds to the laminin receptor -dystroglycan, sarcospan, and the sarcoglycan complex (Fig. 9). This latter group of membrane proteins consists of -, β-, -, and -sarcoglycan. In addition to these interactions with membrane-bound proteins, dystrophin binds to a variety of adaptor and signaling proteins. The following sections will discuss how gene transfer has been used to understand and treat diseases resulting from the disruption of the dystrophin glycoprotein complex.
The dystrophin protein is responsible for Duchenne and Becker forms of muscular dystrophy (209). In the more severe Duchenne muscular dystrophy, the dystrophin protein is completely absent from all tissues in the body, while in the milder Becker muscular dystrophy, a truncated, but partially functional, dystrophin protein is expressed in striated muscles. The dystrophin gene spans 2.4 million base pairs of the short arm of the X-chromosome. The resulting 14-kb transcript consists of 79 exons and produces a protein with 3,685 amino acids and a molecular mass of 427 kDa, making it one of the largest known proteins (363). Dystrophin can be divided into four distinct functional regions (2). The NH2-terminal domain contains an actin binding region demonstrating homology with the actin binding proteins -actinin and β-spectrin. A second actin binding domain is located in the central rod domain (766). These actin binding domains do not bind to sarcomeric -actin in vivo, but rather interact with G-actin present just below the membrane (326). It has been proposed that this submembranous actin network is connected to the sarcomeres by an interaction with desmin (97). The bulk of dystrophin consists of a central rod domain that contains 24 spectrin-like repeats, each of which is 100 amino acids in length. Interspersed within these repeats are hinge regions that are believed to provide flexibility to the molecule. Following this rod domain is a cystine-rich domain which interacts with the transmembrane protein, β-dystroglycan, through a WW domain. β-Dystroglycan forms a heterodimer with the laminin-2 receptor -dystroglycan (Fig. 9) (215, 216). The sarcoglycan complex (see below) and sarcospan are associated with the dystroglycan heterodimer. In skeletal muscle, both the dystroglycan and sarcoglycan complex expression require functional dystrophin. In contrast, most of these proteins are present at relatively normal levels in the dystrophin-deficient heart (316, 893). The fourth domain is the COOH-terminal region containing an -dystrobrevin binding domain. Both this dystrophin domain and -dystrobrevin each contains syntrophin binding sites, and syntrophin contains a PDZ domain that may function to bring signaling molecules (such as nitric oxide synthase) into close proximity of the dystroglycan and sarcoglycan complexes (287).
One of dystrophin's proposed functions is as a stabilizer of the plasma membrane through its interactions with the intracellular cytoskeleton and extracellular matrix (153, 686). This function is particularly evident during lengthening contractions, where the extracellular matrix and intracellular cytoskeleton are moving in opposite directions (171). Another important function ascribed in part to dystrophin is the transmission of sarcomeric force to the extracellular matrix at the level of the costamere. Evidence supporting this function has been derived largely from the reduced force generation of both skeletal and cardiac muscle tissues from dystrophin null mice (171, 404, 790, 994). A third proposed function of dystrophin is related to its ability to form the nidus for the interaction and localization of many signaling molecules (287), although in skeletal muscle the contribution of this activity to the overall function of dystrophin appears to be limited (718).
Duchenne muscular dystrophy is an X-linked disorder characterized by progressive skeletal muscle weakness (209). In addition to this overt skeletal phenotype, patients are afflicted with an insidious cardiomyopathy (209). Heart disease in Duchenne muscular dystrophy is characterized by both conduction, structural, and contractile abnormalities. End-stage heart disease in this case most frequently results in DCM and significant fibrosis, especially within the posterobasal region (232). An X-linked form of DCM has been linked to alterations within the 5'-end of the dystrophin gene (892). These patients have normal levels of dystrophin expressed in skeletal muscles, but no dystrophin present in the heart (570, 615, 892). Many of these patients have disruptions in the promoter driving dystrophin expression in muscle. In the heart, the inactivation of this promoter results in the absence of dystrophin (37, 38). In contrast, there is an upregulation of alternative dystrophin isoforms in skeletal muscle which functionally replace the muscle dystrophin (614, 631). In Becker muscular dystrophy (437, 460), truncated dystrophin molecules resulting from in-frame mutations are expressed (597). These truncated dystrophin molecules have varying levels of function, which are not associated solely with the size of the deletion (43). Information gained from analyzing genotype-phenotype relationships in Becker muscular dystrophy patients provided critical information for the design of functional truncated dystrophin molecules that are amenable to gene transfer.
The vast majority of studies utilizing dystrophin gene transfer have been centered on replacing dystrophin in skeletal muscle for the treatment of Duchenne muscular dystrophy. Many of these advances are also critical to implementing dystrophin gene transfer within the heart. The most straightforward gene transfer approach is the direct injection of full-length dystrophin pDNA (Fig. 3). Unfortunately, this method results in extremely low levels of dystrophin expression in skeletal muscle (7). Although inclusion of additional transfection agents may improve the efficiency of transduction (897, 988), the levels achieved are likely too low to have physiological/therapeutic significance. The low efficiency of these methods raises the question of what are therapeutic levels of dystrophin. Studies involving female carriers of mutated dystrophin alleles suggested that levels slightly greater than 50% of normal are protective against skeletal muscle disease (127, 538), although other studies of X-linked DCM patients report that levels as low as 30% of normal dystrophin expression can prevent significant skeletal muscle disease (637). The therapeutic threshold in the heart is complicated by the inability to obtain cardiac biopsies to determine the level of expression and by variability in the manifestation of cardiac disease in these patients (288, 370, 580, 649).
The disappointing results from direct plasmid injections underscored the need for a more efficient method of gene delivery. Viral-based gene delivery vectors have been shown to be the most effective and efficient methods of introducing exogenous DNA into living cells (see sect. IIA). The only current viral vector capable of delivering full-length dystrophin is gutted adenoviral vectors (Table 1) (314, 447, 459). Direct injection of this vector into skeletal muscle resulted in an inefficient transfer of full-length dystrophin to muscle fibers local to the site of injection (172, 264, 535), although even these modest levels of transduction improved muscle function (172, 535). Clearance of transduced cells is a significant problem with adenoviral-based vector systems (see sect. IIA2), but expression can be prolonged by performing injections in neonates, which do not develop a strong immune response to the vector (110, 126, 196).
One of the most promising gene therapy vectors for striated muscle is recombinant AAV. Significantly truncated dystrophin molecules that retain functionality were first described in patients with Becker muscular dystrophy (212), and additional studies mapped the critical domains required for dystrophin function (144, 329, 687, 718, 777). These studies culminated in several functional dystrophin constructs small enough for AAV delivery (219, 329, 893, 937). Most of these truncated dystrophin constructs improved muscle pathology and membrane integrity following intramuscular injection into skeletal muscle. Only one of these constructs has been accessed to any degree in the myocardium (293, 893, 997). Dystrophin-deficient hearts expressing this truncated "micro-dystrophin" showed a significant improvement in ventricular geometry (893). The expression of micro-dystrophin protected dystrophin-deficient mice from acute heart pump failure during a dobutamine stress test (893). This same construct also significantly extended the life span of mice deficient in both dystrophin and utrophin, a dystrophin homolog upregulated in the dystrophin-deficient mouse (293). Despite these significant improvements in global cardiac function, detailed physiological assessment of this truncated dystrophin molecule revealed that deficits remain, including a reduced transmission of force by striated muscle expressing micro-dystrophin (293, 893). These studies indicate that there is room for improvement in the design of these micro-dystrophins.
While truncated dystrophin molecules are widely expressed in the heart when delivered by a single AAV (293, 294, 893), they are not fully functional. Other approaches are being explored, including the use of two AAVs, each containing half of a fully-functional dystrophin protein with splice sites engineered between them. After transduction of the cell, the AAV genomes concatamerize. Following transcription of these AAV genomes, the resulting RNA is processed at the engineered splice sites. The result is a dystrophin molecule that is larger and more functional than the micro-dystrophin delivered by a single AAV (469). The efficiency of this splicing event, however, appears somewhat less than single vector alone (262, 469). Further studies are necessary to determine the functional effects of this "trans-splicing" approach. Another promising use of cardiac gene transfer in the treatment of Duchenne muscular dystrophy is the use of AAV to introduce small RNA molecules that modulate splicing activity (174). In many patients with Duchenne muscular dystrophy, skipping a few exons yielded a highly functional dystrophin molecule. This approach has been shown to be effective in the heart and shows therapeutic promise for a significant subset of Duchenne muscular dystrophy patients.
Shortly after its initial characterization, dystrophin was found to interact with a group of membrane-bound glycoproteins (217). These proteins formed a complex, which functionally links dystrophin to the extracellular matrix by binding to laminin (215, 216, 381). This complex of dystrophin-associated proteins consists of two groups of proteins: the dystroglycan and sarcoglycan complexes (995). The laminin binding component of the dystroglycan complex consists of - and β-dystroglycan (Fig. 9), which are cleaved components of a common precursor (381). The sarcoglycan complex (Fig. 9) consists of four subunits: -, β-, -, and -sarcoglycan (587, 643). All four of these genes have been implicated in various forms of limb-girdle muscular dystrophy (LGMD), and many of them have significant cardiac phenotypes.
-Sarcoglycan (-SG) is a 387-amino acid protein with a single transmembrane domain and a large extracellular NH2-terminal domain. Extensive glycosylation of two conserved asparagine residues yields a final molecular mass of 50 kDa (747). Mutations resulting in the loss of -SG cause LGMD-2D (101, 749). Interestingly, the absence of -SG results in the concurrent loss of the other members of the sarcoglycan complex (195). -SG deficiency results in severe muscular dystrophy that is essentially localized to skeletal muscle, although there are instances of cardiac disease (195, 691). Gene transfer of -SG in the skeletal muscle of -SG-deficient mice was shown to fully restore the entire sarcoglycan complex (13, 193). Interestingly, the overexpression of -SG following gene transfer caused significant cellular toxicity. The toxic effects of -SG were independent of an immune response and seemingly secondary to alterations in the assembly of the sarcoglycan complex in the presence of excess -SG (193).
β-Sarcoglycan (β-SG) has a similar structure to that of -SG, with a single transmembrane domain and the majority of the protein present in the extracellular space. β-SG is glycosylated at three conserved asparagine residues, resulting in an increase in molecular mass from 35 kDa to a final weight of 43 kDa (72, 493). The clinical importance of this new protein became evident as genetic studies linked β-SG to LGMD-2E (72, 493). Clinically, β-SG deficiency is characterized by significant skeletal muscle disease (72, 493). Cardiomyopathy has also been reported in patients with LGMD-2E (36). Mice in which β-SG has been ablated also mirror the condition in humans, with significant pathology present in both skeletal and cardiac muscle (19, 197). β-SG functions as a nidus for the entire sarcoglycan complex, and the absence of β-SG causes a complete loss of other sarcoglycan complex members (197, 647). In contrast to -SG, β-SG is also required for the formation of the sarcoglycan complex in vascular smooth muscle, which may in part explain the greater cardiac involvement associated with β-SG deficiency (197). Gene transfer of β-SG was shown to restore the expression of the entire sarcoglycan complex and improve pathology (193, 197). Unlike -SG, overexpression of β-SG did not result in significant cellular toxicity (193).
-Sarcoglycan (-SG) is a glycosylated protein with a molecular mass of 35 kDa. Mutations within this gene have been implicated in LGMD-2C (549, 646). In contrast to other sarcoglycanopathies, the sarcoglycan complex is not completely lost in the absence of -SG (145, 549). Clinically, LGMD-2C is characterized as a severe form of muscular dystrophy, with significant cardiac disease (44, 45). Consistent with this clinical presentation, mice lacking -SG develop severe skeletal and cardiac pathology (312). Gene transfer of -SG into skeletal muscle was shown to largely reconstitute the expression of -SG and corrected the skeletal muscle pathology (138). Interestingly, similar to -SG, overexpression of -SG resulted in significant pathology, presumably secondary to disruption of normal assembly of the sarcoglycan complex (1016).
Shortly after the first description of the dystrophin-associated proteins, it was found that the cardiomyopathic hamster was lacking the sarcoglycan complex (748). The absence of mutations within the -, β-, or -SG genes strongly suggested a fourth gene may cause this disease in the hamster (548). To uncover this putative fourth sarcoglycan, a homology screen found a transcript similar to -SG (643). -SG is a 35-kDa glycoprotein with a structure similar to -SG (643). This gene was quickly linked to LGMD-2F (641) and to the cardiomyopathic hamster (642, 777). Similar to the other disorders involving sarcoglycans, patients with mutations in -SG often have severe skeletal muscle disease (643). In contrast to the other sarcoglycanopathies, mutations within -SG have been linked to cardiac disease in the absence of skeletal muscle disease (904). Mice lacking -SG also have significant cardiac and skeletal muscle pathology (136, 311). Intriguingly, the cardiac damage initiated by exercise in the -SG null mice was prevented by pretreatment with vascular relaxing agents, suggesting an important pathophysiological role of vascular smooth muscle (129, 136).
Like the other sarcoglycans, -SG gene transfer into skeletal muscle largely corrected the muscular dystrophy present in the -SG-deficient hamster (368). Similarly, direct injection of myocardium with Sendai virus-coated proteoliposomes or AAV containing -SG resulted in reconstitution of the entire sarcoglycan complex (427–429). The levels of expression obtained through direct injection sufficiently improved global cardiac function and increased the life span of the cardiomyopathic hamster (428, 429). Global cardiac transduction, through the infusion of gene therapy vectors into the coronary circulation, yielded results similar to that obtained by direct injections (385). Systemic intravascular administration of AAV containing -SG resulted in wide-spread expression of -SG in both skeletal and cardiac muscle (1014) and reconstituted the sarcoglycan complex, correcting pathological changes in striated muscle. -SG gene transfer also improved cardiac function and significantly extended the life span of treated hamsters compared with those receiving no gene therapy (1014).
B. Intermediate Filaments (desmin)
The most prominent intermediate filament in cardiac muscle is desmin (Fig. 9), which forms coiled dimers that self-assemble into homo- and heteropolymers with a number of proteins including nestin and synemin to form filaments 10 nm in diameter (249). This process is facilitated by the heat shock protein B-crystallin (639). The lattice of desmin filaments extends from the Z-line interacting with a variety of cellular structures including the nucleus and mitochondria. In addition to interactions with these organelles, many desmin filaments connect adjacent Z-lines, functioning to keep neighboring sarcomeres registered. Desmin filaments emanating from Z-lines at the periphery of the myocyte extend towards the sarcolemma. Near the membrane, these intermediate filaments interact with the proteins that make up the costameres. These proteins, including the complex of proteins associated with the integrins and dystrophin, are localized in the region of the Z-line via an interaction with desmin (576). Desmin filaments are also prominent in Purkinje fibers and at the intercalated disc (442, 887).
A variety of inherited cardiomyopathies have been defined as desmin-related myopathies and are characterized by the deposition of desmin aggregates within cardiac and/or skeletal muscle. These disorders can be categorized into two groups: primary mutations within the desmin gene and those within desmin-associated proteins (96, 679). The latter group consists of mutations of B-crystallin (392, 926). These mutations appear to result in defective assembly and subsequent aggregation of the desmin filaments (76). Expression of mutated forms of B-crystallin in adult cardiac myocytes was detrimental to contractile function (521). The myofilaments remained intact in the presence of the mutated B-crystallin, but contraction was attenuated. Further studies found a disruption of mitochondrial structure and function in myocytes expressing a mutated B-crystallin (521).
Mutations within desmin can result in pathology in skeletal muscle, cardiac muscle, or both. Most of these mutations occur in the central -helical domain of desmin. Many of these mutated proteins are unable to form filaments in vitro (34), and all result in the formation of desmin aggregates in diseased tissue (272, 285, 613, 826). Desmin mutations have been linked to both dilated (482, 584) and restrictive cardiomyopathies (20, 712, 930). Gene transfer of mutated desmin into neonatal cardiac myocytes disrupted the sarcomeric banding pattern (341). These studies demonstrate that alterations in desmin filament assembly have a dominant effect on cellular sarcomeric assembly, an observation consistent with the dominant mode of inheritance that characterizes desmin-related cardiomyopathies.
Microtubules are hollow filaments consisting of - and β-heterodimers of tubulin. Both tubulin subtypes are GTP-binding protein; however, only the GTP bound to β-tubulin is hydrolyzed during polymerization. Microtubules are dynamic structures that run longitudinally across the myocyte and are concentrated in the perinuclear regions (275). Under normal conditions, microtubules have small effects on the structural properties of cardiac myocytes (135, 644). They appear to function primarily as a means of organizing particles, vesicles, and organelles within the myocyte. Microtubules serve as the tracks for the complementary motor proteins kinesin and dynein. These proteins contribute to properly distributing macromolecules and organelles within the cell. In addition to this role in subcellular organization, microtubules also have critical roles in signal transduction within the myocyte (95).
Microtubular accumulation is implicated in the pathophysiology of ischemic cardiomyopathy, depression of function with hypertrophy, and diabetic cardiomyopathy (95). This effect is particularly apparent with the cardiac dysfunction associated with pressure overload hypertrophy. The importance of microtubule accumulation is evidenced by the remediation of the contractile deficit by agents that depolymerize microtubules (134). This accumulation of polymerized microtubules is associated with increased expression of microtubule associated protein 4 (MAP4) and β1-tubulin, but not β4-tubulin, which is the predominate isoform in the heart (634). Adenoviral-mediated overexpression of these proteins in normal adult cardiac myocytes revealed that MAP4 is sufficient for these myocytes to develop microtubular structure similar to that observed in myocytes isolated from pressure-overloaded hearts. In contrast, adenoviral-mediated expression of either β4- or β1-tubulin had no significant effect on microtubule assembly (864). The increased density of microtubules present within pressure-overloaded myocytes resulted in a significant increase in the viscous load placed on the myofilaments. This increased load significantly reduced the efficiency of the contracting cardiac myocyte and clearly contributed to the poor contractile performance of pressure-overloaded hearts (860). Microtubules play an important role in the pathophysiology of several models of heart failure secondary to pressure overload, but the importance of microtubules in other models of heart failure remains unclear (130, 163). The potential of microtubules to be manipulated by gene transfer technologies introduces the possibility of experimental or therapeutic modulation of microtubules to improve our understanding of these cytoskeletal elements in the failing heart.
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VI. CARDIAC SIGNALING PATHWAYS |
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A potential limitation of studies using in vivo adenoviral-mediated gene transfer is the difficulty in achieving long-term, homogeneous expression at the organ level. However, a new generation of vectors appears to be overcoming issues of expression duration and homogeneous expression (954). Heterogeneous and/or transient expression may be a desirable goal for studies of signaling cascades, which more likely operate under spatial and/or temporal activation. Overexpression of signaling proteins may be successful if targeted expression below pharmacological levels is achieved in the intact heart.
Many of the signaling pathways discussed below are mediated via G protein-coupled receptors. The focus of this section is on interactions between signaling proteins involved in modulating Ca2+ cycling and myofilament proteins and their overall influence on cardiac myocyte contractile function. Importantly, signaling pathways can exert both acute and chronic influences on contractile function. The signaling section will begin with reviewing the β-adrenergic signaling pathway as it pertains to the contributions to the field gleaned from gene transfer studies.
A. Gene Transfer Influencing the β-Adrenergic Signaling Pathway
1. β-Adrenergic receptors and G proteins
One of the most studied and best understood signaling pathways involved in modulating cardiac contractile function is the β-adrenergic receptor (β-AR) pathway (48, 506, 696, 883). Catecholamine binding to the β1-AR activates adenylyl cyclase via Gs to increase cAMP production, which in turn activates PKA (Fig. 10). The PKA catalytic subunit phosphorylates multiple targets, including proteins located within the sarcolemma, SR, and sarcomeres (Fig. 10) to increase cardiac contractility and relaxation rate. This basic signaling cascade is well understood based on a variety of experimental approaches including biochemical, transgenic, and knockout models (506, 883). While decreases in β-AR signaling were noted during heart failure in the 1980s (79), β-AR agonists paradoxically increased mortality in failing hearts (342, 669, 818). Gene transfer studies have been important for providing new insight into the pathway of β-AR cycling during desensitization/downregulation and the role of β-AR cycling during heart failure (340, 635, 669). Gene transfer approaches have also provided fundamental knowledge about adenylyl cyclase and A-kinase anchoring proteins (AKAPs) (324, 761). These latter proteins serve as scaffolding proteins that contribute to macromolecular assembly of activated PKA with phosphorylation targets (761). In addition, this approach has been instrumental in identifying the relative contribution of individual targets to the inotropic and lusitropic effects of β-adrenergic signaling in cardiac myocytes (178, 207, 967). Most importantly, studies on the β-AR signaling pathway have provided insight into the paradoxical observation that β-AR agonists increase mortality by demonstrating that β-agonists activate a cascade of pathophysiological events in addition to the downstream increase in contractile function. By addressing the events that directly influence contractile function, viral-mediated delivery systems have begun to provide therapeutic strategies for treating heart failure using the β-AR signaling pathway (201, 635, 881).
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s and Gi. Gi subunits negatively influence contractile function (980) (Fig. 10). In addition, β2-receptors show significant differences in their spatial activation of PKA compared with β1-ARs (15, 883). Complementary transgenic models with moderate overexpression of β2-ARs improved contractile function without significant hypertrophy (753). Gene transfer of β-ARs has also been used as a therapy to treat failing hearts (412, 464, 569, 882). Gene transfer of β2-AR during cardiopulmonary bypass in neonatal pigs increased total β-AR density and improved left ventricular function in response to β-adrenergic stimulation (412, 413). Increases in these receptors after gene transfer improved ventricular function at baseline and with β-adrenergic stimulation in loaded and unloaded rabbit hearts (540, 812) as well as in response to higher preloads (464). The beneficial effects of β2-AR overexpression are clearly dose-dependent, and high levels of overexpression are not considered beneficial (492). β2-ARs signal via multiple downstream pathways. Gene transfer of β1-AR or β2-AR into double knockout mice indicated that the protective effects of β2-AR are likely mediated via Gi and the downstream activation of phosphoinositide 3-kinase (PI3K) and Akt (477, 1015) or via functionally discrete signaling pools (506, 828).
Gene transfer approaches have also been used to understand the contribution of Gi to contractile function. Increased expression of Gi was observed with heart failure, which may influence signaling via β2 receptors (208). Atrioventricular viral-mediated gene transfer of wild-type Gi or constitutively active Gi (Q205L) significantly improved heart rate during persistent atrial fibrillation in a pig model (41, 186). Gene transfer of Gi did not significantly influence baseline isometric force in rabbit trabeculae or shortening in isolated myocytes (405). However, expression of Gi blunted the response to β-AR activation by isoproterenol in both the trabecular and myocyte preparations, and this blockade was prevented with pertussis toxin. Overall, these results indicate that signaling via Gi acts as a brake on the β-adrenergic contractile response, and it may be important for the beneficial effects of β2 receptor gene therapy.
Another strategy along these same lines has been to increase other G protein-coupled receptors, including parathyroid hormone related peptide (PTHrp) and vasopressin2 (V2) receptors, using viral-mediated gene transfer (474). Like β2-ARs, PTHrp receptors are coupled to Gs and Gi and downstream activation of phospholipase C, while V2 receptors are strongly linked only to Gs (58, 474, 806). Neither receptor, however, is expressed endogenously in cardiac myocytes. The increased expression of V2 receptors had no significant effect on basal contractile function but significantly improved the response to agonists. In contrast, gene transfer of PTHrp receptors increased basal contractile velocity in failing and nonfailing myocytes, but there was no further response to agonists. At present, it is unclear whether therapies with these receptors significantly improve contractile function at the cellular and/or organ level under pathophysiological conditions.
G protein-coupled receptor kinases (GRKs) are important for the rapid modulation of β-AR density on the myocyte cell surface (752). The most prominent of these GRKs, βARK1 (or GRK2), is elevated during pathophysiological conditions (339), such as ischemia (480), heart failure (192, 201, 752, 909), and coronary artery bypass (rabbits) (881). βARK1 is responsible for phosphorylation of agonist-occupied β-ARs, which leads to uncoupling of the receptor from downstream signaling pathways (446). Adenoviral delivery of the βARK1 inhibitor βARK-1ct, which is constructed from the COOH terminus of βARK1 and acts as a dominant negative, significantly attenuated the left ventricular dysfunction in hearts undergoing coronary artery bypass and after myocardial ischemia (813, 881, 968). Viral delivery of this inhibitor to myocytes from spontaneously hypertensive rats in heart failure also significantly improved β-AR-mediated cAMP production, peak shortening, and the rates of rat myocyte contraction as well as improved signaling in failing rabbit myocytes (9, 201). While this inhibitor restored βARK1 levels in two dilated cardiomyopathy models (200, 328), it did not consistently prevent the progression of cardiomyopathy or premature mortality. These results suggest that βARK-1 plays a critical role in modulating β-AR density on the sarcolemma, but the animal model, type of cardiac pathophysiology, and timing of gene delivery may be critical for the future development of therapeutic treatments for human heart failure. As described below, events and/or other signaling pathways may also contribute to the final progression to heart failure.
More recently, gene transfer of the βARK1ct has been compared with truncated phosducin, another GRK inhibitor (489). Both inhibitors bind to Gβ subunits, yet only βARK-1 contributes to β-AR cycling and improves cAMP accumulation in myocytes from failing rabbit hearts (489). Gene transfer of either inhibitor improved the left ventricular contractile response to isoproterenol, and it improved fractional shortening and LV end-diastolic dimension in rapidly paced, failing hearts. These results suggest that the beneficial influence of GRK inhibitors on failing hearts maybe due, at least in part, to the inhibition of Gβ subunits.
Proteins associated with βARK1 in the myocyte have also been targets for gene transfer and treatment of heart failure. PI3K binds to βARK1 in the cytosol, is targeted to β-ARs during agonist activation of β-ARs, and regulates β-AR internalization (625). Overexpression of the kinase domain, PIK, competitively displaces PI3K from βARK1 and prevents β-AR internalization and desensitization. Transient PIK expression decreased PI3K localization with β-ARs in sarcoma cells, and viral-mediated gene transfer of PIK into myocytes from failing pig hearts restored the isoproterenol-mediated enhancement of peak shortening and contraction and relaxation rates toward the nonfailing phenotype (683). The loss of β-ARs, increased norepinephrine (NE) levels, and/or chronic β-AR activation that result from heart failure are ultimately maladaptive (640, 683). Future studies may focus on gene therapy utilizing modified proteins associated with the β-AR cycling pathway, such as β-arrestin (477) and PDE4 (375, 488), which serve as important proteins in scaffolding and/or desensitization of β2-ARs.
In addition to β-ARs and associated proteins, studies have also focused on clearance of hormones involved in β-AR signaling, especially NE. Accumulation of NE during hyperstimulation of β-AR was postulated to be a major cause of structural and functional impairment during heart failure (726). Efforts to improve local NE clearance using gene transfer of uptake-1 into rabbit hearts prior to pacing-induced heart failure acutely improved NE uptake, β1-AR receptor, and SERCA2 expression, as well as diastolic and systolic contractile function, over a 2-wk time period (612). Gene transfer of uptake-1 had no significant influence on contractile function in nonfailing hearts. It is currently unclear whether the uptake-1 expression develops in cardiac myocytes or in associated neurons after gene transfer, and whether or not this strategy may be appropriate for future therapy to treat heart failure.
A) ADENYLYL CYCLASE. Gene transfer strategies are providing significant strides toward understanding signaling downstream from the β-AR. Viral-mediated gene transfer of adenylyl cyclase VI (AC-VI) was first studied in neonatal rat myocytes, which selectively increased cAMP production in response to β-AR activation but not to other agonists coupled to adenylyl cyclase (666). Gene transfer also increased the cardiac response to β-adrenergic activation 12–14 days after recombinant viral delivery in normal, ischemic, and failing hearts (324, 467, 468, 759, 865). Further work demonstrated that AC-VI delivery increased the baseline rate of contraction (dP/dtmax), and β-adrenergic responses were maintained after gene transfer into mice (758). Importantly, gene transfer of adenylyl cyclase into pigs experiencing pacing-induced heart failure improved contractile function and decreased ventricular dilatation, as well as improved cAMP production and reduced indicators of hypertrophy. These findings also agree with results obtained after transgenic expression of AC-VI, which improved mortality and ventricular function in mice with ischemic heart failure (865). Many of the beneficial effects of AC-VI can be explained by the resulting increase in cAMP production. However, increased cAMP is associated with arrhythmias, which were not observed in the transgenic model. Thus the improved mortality observed in AC-VI transgenic mice may be due to 1) the close proximity of this isoform to the sarcolemma and 2) cAMP-dependent effects of AC-VI (324, 479). It remains unclear whether gene delivery of AC-VI is capable of reversing LV morphological and functional remodeling, but this gene is garnering consideration for clinical heart failure therapy (324).
B) GS-COUPLED RECEPTORS. Gene transfer has been used for upstream activation of adenylyl cyclase using receptors for arginine vasopressin (AVP). Increased AVP expression is associated with congestive heart failure and is linked to a poor prognosis (274). The vasopressin 1 (V1) receptor is expressed in the heart while V2 receptors are expressed in renal collecting ducts but not in cardiac myocytes. The V1 receptor is coupled to phospholipase C (PLC)-β while V2 is coupled to Gs and adenylyl cyclase. Viral-mediated gene transfer of V2 in adult rat ventricular myocytes resulted in dose-dependent expression, an increase in cAMP formation, and an increased amplitude of contraction in isolated myocytes that was blocked by a V2-specific vasopressin antagonist (473). Coronary-based adenoviral gene delivery to the myocardium also improved fractional shortening and the rate of contraction in response to V2 stimulation (953). However, it remains to be determined whether gene delivery of V2 improves cardiac performance in failing hearts. In addition, the long-term benefits of this approach are controversial, as blockade of both V1 and V2 is also predicted to improve function in individuals with congestive heart failure (273). This general approach has also been utilized to determine whether delivery of other noncardiac Gs-coupled receptors to cardiac myocytes acts as a novel therapeutic strategy for bypassing the β-AR and boosting contractile function (474).
C) AKAPS. Several experiments on downstream signaling within the β-AR pathway have focused on AKAPs. AKAPs serve as cellular scaffolding for PKA by tethering the type II regulatory subunit (RII), as well as target proteins, and phosphatase/phosphodiesterases. At least 13 different AKAPs have been identified in myocardium, and the localization and function of these AKAPs was recently reviewed in detail by Reuhr et al. (761). Gene transfer and expression of Ht-31, a peptide derived from human thyroid AKAP with a similar binding affinity as AKAP for the RII domain of PKA, resulted in redistribution of the RII subunits within isolated myocytes (231). This change in PKA localization enhanced β-AR-mediated peak shortening as well as the rate of shortening and relengthening compared with controls (231). Paradoxically, the level of myofilament protein phosphorylation (e.g., cTnI, MyBP-C) in response to β-AR stimulation was significantly reduced in these myocytes compared with controls. Results from these studies provided insight into the temporal and spatial targeting of AKAPS within cardiac myocytes (231, 763). In addition, this approach has provided a better understanding of the role played by each AKAP in myocyte hypertrophy (182). Future studies are needed to investigate the influence of AKAPs on contractile function under pathophysiological conditions, as modified AKAPs could one day be used to treat heart failure.
D) END-TARGET PROTEINS. The end targets for phosphorylation have also been studied using gene transfer, and this approach has contributed significantly towards understanding β-AR signaling. These studies have focused primarily on Ca2+ cycling and sarcomeric proteins that serve as targets for β-AR signaling, with direct influences on contractile function. A high proportion of these studies have focused on phospholamban and sarcomeric troponin I, which have been described above (see sects. III and IV). Below are studies of other downstream targets.
The β-AR pathway phosphorylates a number of other proteins, including the sarcolemmal L-type Ca2+ channel (DHPR) and sarcomeric MyBP-C. Studies using gene transfer to investigate the contribution of these proteins in contractile function and/or studies to develop therapeutic strategies utilizing the phosphorylated versions of these proteins have not been addressed in detail using viral-mediated gene transfer approaches. In the case of Ca2+ channels, as well as other ion channels or transporters, there are multiple subunits. Thus cellular or organ-directed gene transfer and overexpression may not necessarily result in appropriate channel subunit organization. Experiments with modifications of MyBP-C on the PKA-targeted Ser283 (593) also may be difficult to perform, as the turnover of MyBP-C is not clear and may be too long for the typical 1–7 day time frame for myocyte contractile assays in vitro.
Proteomic analysis of the β-AR response to isoproterenol has revealed phosphorylation of the p20/pHSPB6 heat shock protein. This small heat shock protein is an -crystallin and is typically present in the cytosol of cardiac myocytes (120, 476). Phosphorylation of p20 results in its colocalization with actin (222), although recent studies suggest that p20 does not bind directly to actin (90). It has been postulated that the p20 phosphorylation state determines the translocation and the localization pattern. Viral-mediated gene transfer of p20 into adult rat myocytes increased peak shortening and Ca2+ transient amplitudes within 2 days after gene transfer without significantly influencing resting length, relaxation, or Ca2+ decay (120). Although the cellular basis for the increase in contractility and Ca2+ transient remains unknown, p20 localization with actin and its ability to bind PP1 (221) would suggest that this small heat shock protein may have direct influences on proteins within the Ca2+ cycling cascade and/or the myofilaments. It should be noted that gene transfer has been used to study the protective effect of other heat shock proteins against stresses such as heat and/or ischemia in cardiac myocytes (471). However, to date, gene transfer of these other heat shock proteins has not focused on the direct influence on contractile function.
PKA modulates PP1 activity via phosphorylation of inhibitor proteins. The role played by PP1, the inhibitor proteins, and modulation of these proteins by PKA and PKC is discussed in more detail below.
B. Gene Transfer of Ca2+/Calmodulin Kinase
The Ca2+/calmodulin-dependent protein kinase II (CAMKII) isoform phosphorylates several proteins involved in EC coupling, including the RyR and PLN. Increased CAMKII activity is observed during heart failure, and overexpression of the cytosolic variant CAMKIIc in transgenic animals causes heart failure (50, 518). Viral delivery and overexpression of wild-type CAMKIIc in rabbit myocytes altered Ca2+ cycling, but overexpression did not directly alter other proteins such as NCX or SERCA, changes that are commonly associated with heart failure (448). The major influences on Ca2+ cycling proteins included phosphorylation of RyR, without changes in the association of RyR with FKBP12.6. This phosphorylation in rabbit myocytes was associated with increased SR fractional release of Ca2+, increased diastolic leak of Ca2+ and reduced SR Ca2+ content. Despite the decrease in SR Ca2+ content, the cellular Ca2+ transient and shortening amplitude were maintained due to the increase in peak DHPR current and increased SR fractional Ca2+ release. Overexpression also enhanced the frequency-dependent acceleration of relaxation, presumably due to its phosphorylation of PLN at Ser-17 and the resulting increase in Ca2+ uptake by SERCA2. In contrast, gene transfer of wild-type, constitutively active, and dominant negative CAMKIIc into isolated rat myocytes produced quite different results (989). Here, increased SR Ca2+ content and reduced Ca2+ release in response to elevated Ca2+ were observed in myocytes overexpressing wild-type or constitutively active CAMKIIc, and these results were interpreted to indicate that CAMKIIc acts as a negative-feedback modulator of RyR. The differences between the studies with rabbit and rat myocytes have been attributed to species differences in Ca2+ handling between rat and rabbit hearts (989). Future gene transfer studies will be necessary to better understand the role played by CAMKII and may ultimately be critical for defining the direct versus compensatory adaptations that develop in transgenic mice expressing CAMKII.
C. Gene Transfer and PKC Signaling
Activation of PKC plays a key role in mediating signaling via multiple receptors (e.g., angiotensin II, endothelin, -adrenergic receptors; Fig. 11) (152, 211, 453, 598, 688, 880, 940) coupled to G proteins (e.g., primarily Gq, Gi) followed by PLC activation. PLC breaks down phosphoinositide bisphosphate (PIP2) into inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG). IP3 is translocated to the SR (Fig. 11), and binding to its receptor contributes to 5% of SR Ca2+ release (399, 603). Recently, FRET was used by labeling the binding domain of the IP3 receptor with cyan fluorescent protein, while target protein sequences were labeled with yellow. This biosensor approach demonstrated the spatiotemporal distribution of IP3 in neonatal cardiac myocytes (733). SR Ca2+ release increases in response to low levels of IP3 (433, 655), and future gene transfer approaches may prove valuable for imaging, Ca2+ cycling, and/or contractile function studies in adult cardiac myocytes.
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1. Signaling upstream from PKC
A) RECEPTORS AND PLC. Gene transfer of receptors that activate the PKC pathway has been utilized in several studies (Fig. 11). These studies have focused on the relative contribution of receptor isoforms to hypertrophy mediated via PKC and/or the ability of a specific isoform to counteract the hypertrophic phenotype produced by a dominant receptor isoform (243). With this emphasis, the acute and chronic influence of receptor isoform expression on cardiac contractile function has not yet been as thoroughly studied by gene transfer. For example, two major isoforms of the angiotensin II (ANG II) receptor (AT-1 and AT-2) are expressed in adult myocytes (82, 349). Adenoviral-mediated gene transfer of AT-1 and AT-2 each produced hypertrophy in neonatal rat myocytes, and direct lentiviral-mediated cardiac delivery of AT-2 reduced the hypertrophic response to chronic ANG II delivery (148, 220). The importance of AT receptors in the hypertrophic process is clear from these studies, but analysis of cardiac myocyte and/or cardiac contractile function would add to our understanding of these receptors.
Gene transfer of proteins/enzymes involved in hormone metabolism has been used to demonstrate the importance of cardiac PKC-related paracrine and autocrine mediators. One example is the use of angiotensin converting enzyme 2 (ACE2), which catalyzes the production of the potent vasodilator angiotensin 1-7 (ANG1-7) (229). ANG1–7 works via a non-PKC pathway (269) and counteracts the influences of the PKC-linked AT-1 receptor on cardiac hypertrophy (179, 717, 976). Lentiviral-mediated gene transfer of ACE2 produced strong cardiac expression and renal expression, albeit to a lesser extent. Functionally, ACE2 delivery also reduced hypertension and increased left ventricular end-diastolic and end-systolic dimensions in spontaneously hypertensive but not normotensive rats (179). Although the mechanism responsible for these beneficial effects is currently not clear, results from studies like these suggest that approaches designed to reduce the influence of PKC-linked signaling hormones on contractile function could be beneficial during heart failure.
Other steps in the PKC signaling pathway (Fig. 11) studied using gene transfer include PLC, heat shock proteins, and DAG kinase. PLC is critical for the production of DAG, which is critical for PKC activation/translocation in response to mediators such as endothelin, ANG II, and -adrenergic agents. Overexpression of PLC isoforms in neonatal cardiac myocytes was demonstrated after viral-based gene transfer (22). Heat shock proteins can also modulate PKC (128), and viral-mediated gene transfer has been used to investigate the influence of Hsp70 and Hsp90 on PKC- and -
expression in neonatal cardiac myocytes (128). DAG kinases breakdown DAG, and the breakdown of DAG in response to overexpression of DAG kinase 
inhibits endothelin-1-induced activation of PKC- and downstream signaling (mitogen-activated protein kinase, MAPK) of hypertrophy in neonatal cardiac myocytes (862).
B) PKC ISOFORMS.
The PKC family consists of at least 12 different isoforms, and the characteristics of the 3 subclasses have been reviewed in detail (173, 190, 619, 838). Adult cardiac myocytes in most mammalian species express four major isoforms, including classical PKC-, novel class PKC- and -, and to a lesser extent atypical PKC- (213, 713, 714). Several pathophysiological conditions and heart failure in humans are associated with increased expression of PKC- and -, plus the appearance of PKC-β, another classical isoform (77, 648). Comparable changes in these PKC isoforms were observed in rat models of pressure overload (42, 191). The gene transfer studies described below have significantly contributed to our understanding of the role played by many of these isoforms in modulating contractile function.
I) PKC-. Classical and novel PKC isoforms are activated by PLC-dependent DAG production or addition of phorbol esters (Fig. 11). The activity of classical isoforms, such as PKC-, also depend on increased cytosolic Ca2+ (173). Early studies demonstrated that peptide inhibitors directed to classical PKCs prevented phorbol ester-mediated increases in Ca2+ current (1007). Classical PKC isoforms were also found to influence hypertrophy in neonatal cells (80). Gene transfer of PKC- stimulated hypertrophy through downstream activation of MAPK and extracellular regulated kinase 1/2 (ERK1/2). This hypertrophy was also linked to signaling proteins upstream from ERK1/2, including Rho GTPases (673). These results raised questions about the role of PKC- in the development of hypertrophy related to pathophysiological events and its potential role in modulating contractile function in adult hearts. Gene transfer into adult rat cardiac myocytes demonstrated that increased PKC- expression significantly decreased the amplitude of shortening and rate of contraction, without significantly influencing resting sarcomere length or relaxation (81). Increased PP1 activity was an important contributor to these functional changes, as described below. Reductions in PKC- activity using dominant negative PKC- (dnPKC) produced the opposite effect, with significant increases in peak shortening and shortening velocity. Transgenic PKC- overexpression and knockdown of PKC- in mice produced comparable outcomes at the organ system level. The Ca2+ transient amplitude and SR Ca2+ release also increased in myocytes isolated from these mice (81). PKC- and PP1 expression increased during heart failure (77, 207), and knock-down of PKC- expression minimized contractile dysfunction in three separate models of heart failure (81). These findings were used as a basis for developing a gene therapy strategy (323). The delivery of dominant negative PKC- into rats 12 wk after cryoinfarction improved end-diastolic pressure and the positive pressure derivative within 1 wk after gene transfer (81). It should be noted that contractile function may be influenced via feedback of PKC- on the PKA pathway. Constitutively active PKC- decreased adenylyl cyclase activity in HEK cells after gene transfer, and this response was enhanced by cotransfection with β-AR1 receptors. Based on these findings, the PKC feedback response may be mediated through direct actions on β-AR1 receptors in the myocardium (302). Overall, the relationships between elevated levels of PKC agonists, increased classical PKC isoform expression, downstream target protein phosphorylation, and contractile function under pathophysiological states are only partially solved. Gene transfer may be a key approach for obtaining substantial insight into the changes in PKC signaling and their direct influence on contractile function during heart failure, in addition to further development of therapeutic targets aimed at this signaling pathway.
II) PKC-β. Expression of PKC-β1 and -β2 also increases significantly during heart failure (77). Transgenic expression of wild-type PKC-β2 or an inducible, constitutively active PKC-β2 produced decreased and increased contractile performance, respectively, in cardiac myocytes from transgenic mice (78, 868, 933). The differing functional effects may be due to the expression and activity level of PKC-β as well as the duration and developmental pattern of expression. In future studies, gene transfer could provide valuable feedback about the direct influence of PKC-β2 on contractile function in isolated, intact failing and nonfailing cardiac myocytes.
At the subcellular level, incubation of permeabilized mouse myocytes with PKC-β2 increased myofilament Ca2+ sensitivity of force, which was blunted in myocytes expressing a cTnI with Ala substitutions at phosphorylation sites (939). Gene delivery and expression of PKC-β to neonatal myocytes also increased βMyHC and MLC-2 expression (423, 939). Signaling downstream from PKC-β2 has also been studied using gene transfer. These studies suggest that p90 ribosomal S6 kinase (p90RSK) is a key downstream effector of PKC-β activated in response to reactive oxygen species, and this kinase is involved in the phosphorylation of cTnI at Ser-23/24 (393).
III) PKC-. Multiple studies have investigated the modulation of contractile function using myocardial gene delivery of PKC-. In isolated rat myocytes, basal contractile function was not significantly changed after gene transfer of GFP-tagged PKC- (420). However, phorbol ester activation of PKC resulted in a transient negative inotropic effect that was followed by a sustained positive inotropic effect after gene transfer of PKC into myocytes (420). Typically, similar doses of phorbol esters produce a sustained PKC-dependent negative inotropic effect on contraction (98, 308). The sustained increase in peak myocyte contractile function observed after PKC- gene transfer was pH dependent, and this enhanced contractile function was prevented by the PKC inhibitor bis-indolylmaleimide (bis-1). PKC- gene transfer did not change the rates of contraction or relaxation. Together, these results suggest gene transfer of PKC- produces a positive inotropic response to phorbol esters.
The role of PKC- in response to other agonists and/or cellular stimuli is less clear. For example, the PKC agonist ET-1 increased PKC- translocation (420, 530), yet expression of dominant negative PKC- did not alter the inotropic response to ET-1 (421). In contrast, in vivo gene delivery studies demonstrated that protein delivery of a PKC--selective inhibitor (V1-1), using the viral protein TAT for intracellular protein delivery (805), restored cardiac function more quickly after acute ischemia followed by reperfusion both in vivo and in vitro (389, 391). Further work is needed to determine whether PKC- activation has divergent physiological versus pathophysiological influences on function due to cellular localization.
A cellular gene transfer approach has also been utilized to gain insight into the PKC--mediated signaling cascade responsible for any changes in contractile function. Perinuclear staining of activated PKC- was observed in isolated myocytes, and there was evidence it was translocated to caveolae, which are proposed to act as a signaling platform (420, 769, 838). In addition, gene transfer studies demonstrated that constitutively active PKC- activates MAPK, JNK and p38, and the Rac-1 signaling pathways in neonatal myocytes (348, 673). As with other PKC isoforms, further cellular studies are needed to gain a more specific understanding of the downstream myofilament and/or Ca2+ cycling targets responsible for any contractile response to PKC-. An additional consideration for future work is based on the evidence supporting differential activation by cellular localization of PKC- (420) and stimulus-dependent differences in PKC--mediated modulation due to tyrosine phosphorylation (838). PKC- also cross-regulates PKC- (767), an isoform that also influences contractile function (see below). These alternative mechanisms modulating PKC- activity may play a role in influencing contractile function under physiological conditions and/or may alter the response during pathophysiological stressors (838).
IV) PKC-. A substantial body of work has focused on defining the role of PKC- in modulating cardiac contractile function. Initial work demonstrated that activation and translocation of PKC- in response to the PKC agonist ET-1 produced a significant increase in the Ca2+ transient in AT-1-derived myocytes (411). These were followed by studies in transgenic mice, and a dose-dependent effect of PKC- was observed on contractile function (677). Low-level expression and expression of dominant negative PKC- had no significant influence on basal contractile function, but higher expression produced cardiac hypertrophy, myocyte disarray, altered myofilament protein isoform expression, decreased systolic and diastolic pressure, as well as reduced positive and negative pressure derivatives. The response to the PKC agonist ANG II was also attenuated in this high expressing line. Due to the presence of hypertrophy and myocyte disarray, it became important to investigate the influence of PKC- expression on myocyte function independent of these adaptations.
To investigate the role of PKC- on contractile function independent of changes in myofilament and Ca2+ cycling expression, Baudet et al. (40) used viral-mediated gene transfer into adult rabbit myocytes. Two days after gene transfer, a 28-fold increase in PKC- activity was observed without significant changes in the expression of other PKC isoforms. However, PKC- overexpression increased peak shortening and Ca2+ by 21% and prolonged the twitch duration, which was attributed to the prolonged time to peak shortening. Relaxation was not significantly modified. In addition, the response to ET-1 was attenuated in myocytes overexpressing PKC-. Later studies in rat myocytes proposed a biphasic influence of PKC- on contractile function in response to PKC activation (420). In contrast to the earlier gene transfer studies, baseline contractile function was not significantly influenced by low or high levels of PKC- expression. A negative inotropic response to phorbol ester was observed in myocytes with 3- to 5-fold overexpression of PKC-, while myocytes having 7- to 10-fold increases showed a brief negative followed by a sustained positive inotropic response at room temperature. The differences in response to PKC agonists were attributed to the relative amount of sarcolemmal PKC- localization (420), but these results may also be agonist dependent. Additional considerations that may explain the differences between studies in rabbit versus rat myocytes include differences in Ca2+ cycling between animal models, the recording temperature during cellular function studies, and the relatively small number of cells recorded in the later study.
The targets responsible for changes in shortening in response to PKC- have not been thoroughly investigated, although work to date provides evidence that PKC- may influence contractile function more through influences on protein expression than through phosphorylation of myofilament/Ca2+ cycling proteins. For example, viral-mediated gene transfer of dominant negative PKC- or downstream activation of focal adhesion kinase (FAK) by Mansour et al. (523) demonstrated that PKC- was necessary for strain-induced recovery of sarcomere length in neonatal rat myocytes. In addition, gene transfer studies have demonstrated that PKC- downregulates SERCA2 expression in neonatal myocytes (702). Expression and phosphorylation of the gap junction protein connexin43 were also influenced by PKC- in myocytes (181).
V) PKC-. This atypical PKC is translocated from the cytoplasm to the nucleus in cardiac myocytes during ischemia (586). Future gene transfer studies are needed to determine whether this isoform plays a direct role in influencing cardiac myocyte physiology.
2. Downstream translocation and other signaling pathways
The gene transfer approach remains to be utilized for studying scaffolding proteins that bind PKC in cardiac myocytes (Fig. 11). These proteins include receptors for activated C kinases (RACKs) and substrates that interact with C kinase (STICKs). To date, at least two RACKs have been described, and these RACKs are responsible for translocation of PKC isoforms (795). Gene transfer of RACKs in other cell lines established the interrelationships between PKC and other signaling pathways, such as the MAPKs (509, 740), PLC-β2, and adenylyl cyclase (111). Future studies utilizing RACKs or STICKs may be valuable for determining their role in modulating contractile function and for understanding the role of RACKS in translocation with genetically coded fluorescent reporters within the cardiac myocyte. An important caveat with these proteins is that they may serve multiple functions within the cell (795, 838), and their influence on PKC-mediated contractile function will require consideration of the scaffolding provided for other signaling complexes.
A) MYOFILAMENT TARGET PROTEIN, TROPONIN I. Activated PKC directly phosphorylates the sarcomeric protein cTnI (Fig. 11) and alters myofilament function. PKC-dependent cTnI phosphorylation decreases myofilament Ca2+ sensitivity (92, 653) and correlates with peak PKC-dependent contractile responses using multiple agonists (152, 426, 598, 688). However, the role cTnI phosphorylation plays in the contractile response has been difficult to define, in part because PKC phosphorylation of cTnI is linked to both accelerated and decreased relaxation rates (689, 782, 958, 962). Gene transfer studies have established that PKC-mediated cTnI phosphorylation accelerates relaxation rate in response to the neurohormone ET-1. The addition of bis-1, a PKC antagonist, largely inhibited the functional effects of ET-1, suggesting that PKC was a key contributor to cTnI phosphorylation in response to ET-1. MAPKs can act as downstream effectors of activated PKC, and each pathway influences cardiac contractile function (133, 411, 491, 684), but MAPK inhibitors did not significantly influence acute ET-1-mediated cTnI phosphorylation. The PKA inhibitor H-89 did not significantly influence cTnI phosphorylation in response to ET-1 (266).
In experiments with the neonatal cardiac isoform slow skeletal TnI (ssTnI), ET-1 activation of PKC did not significantly phosphorylate the ssTnI isoform (958). All indices of relaxation were prolonged in response to ET-1 in myocytes expressing the nonphosphorylatable ssTnI. The increased peak amplitude of shortening observed in response to ET-1 was also significantly blunted in myocytes expressing ssTnI. These findings demonstrated that PKC directly phosphorylates cTnI and cTnI phosphorylation during ET-1 activation of PKC accelerates relaxation and contributes to the increased amplitude of contraction (958).
The functional contribution of cTnI phosphorylation relative to changes in Ca2+ cycling has also been investigated during ET-1 treatment. Enhanced peak shortening and modest acceleration in relaxation rate were observed in myocytes loaded with fura 2. However, the peak and rate of Ca2+ transient decay were not altered in these myocytes, which is consistent with the idea that ET-1-induced PKC activation influences peak shortening and relaxation through altered myofilament function rather than the Ca2+ transient. The contribution of phosphorylated TnI to the ET-1-mediated change in contractile function was also investigated at different stimulation frequencies, and the frequency response (0.2 to 2 Hz) remained unchanged with and without ET-1 (962).
Activation of the Na+/H+ exchange is also an important component of the contractile response to ET-1. Earlier, investigators showed ET-1 increased Na+/H+ exchange via PKC (271, 453). The ensuing alkalosis increased myofilament Ca2+ sensitivity (541) and enhanced peak shortening and the rate of contraction in myocytes (271, 453). This alkalosis could also delay relaxation (541). In the presence of the 5-N-ethyl-N-isopropylamiloride (EIPA), the relaxation response was still observed while peak shortening was attenuated. These results suggested that Na+/H+ exchange contributes to the enhanced contractile shortening, but cTnI phosphorylation plays a critical role in the relaxation response to ET.
The presence of three primary clusters (Ser-23/24, Ser-43/45, and Thr-144) in purified cTnI that are phosphorylated by PKC (652) contributes to the challenge of understanding the role of PKC-mediated TnI modifications on contractile function. These residues are Ser-23/24, which also is phosphorylated by PKA, (Fig. 10; Refs. 454, 1004), Ser-43/45, and Thr-144 (Fig. 11). In myocytes expressing ssTnI or cTnI isoform chimeras, there is evidence that the three target clusters (Ser-23/24, Ser-43/45, Thr-144) are phosphorylated in response to ET-1. ET-1 phosphorylated the N-card/slow-C TnI chimera (c/sTnI), which contains the Ser-23/24 and Ser-43/45 sites, and subsequently accelerated myocyte relaxation. These results provide evidence that at least one of the cTnI NH2-terminal clusters (Ser-23/24 and Ser-43/45) contributes to accelerated relaxation in response to ET-1. Only the Thr-144 site is present in the TnI chimera N-slow/card-C. While TnI phosphorylation in this chimera was not detected, relaxation time was intermediate between cTnI and ssTnI, with no increase in peak shortening in response to ET-1. The lack of change in peak shortening and in relaxation with N-card/slow-C TnI chimera may be caused by conformational differences which influence the ability of PKC to phosphorylate specific clusters within the myofilament. Thus the role of Thr-144 in the ET-1 response was difficult to ascertain with these TnI chimeras.
Subsequent experiments focused on the direct contribution of cTnI Thr-144 phosphorylation to ET-induced changes in myocyte contractile function (962). Thr-144 is important because it is located within residues 138-149 (1, 869), the inhibitory peptide (IP) region of TnI (338, 652, 857). The IP region of TnI is the minimum sequence needed to inhibit strong interactions between actin and myosin in the absence of Ca2+ (857, 896), and it toggles between actin (low [Ca2+]) and TnC during the Ca2+ transient. Thus cTnI Thr-144 phosphorylation may be an important modulator of the TnI "switch" between actin and TnC. Gene transfer and expression of the cTnI Thr144Pro substitution delayed relaxation in response to ET-1, providing evidence the Thr-144 residue significantly contributed to the acute PKC-mediated acceleration of relaxation in response to ET-1 (962).
The Ser-23/24 phosphorylation site is a well-documented phosphorylation site for PKA. There is now evidence that this cluster contributes to the accelerated relaxation rate in response to agonist activation of PKC (962). While a phospho-specific antibody aimed at this site failed to show significant changes in cTnI Ser-23/24 phosphorylation in response to 10 min of ET-1, this cluster was significantly phosphorylated with 60 min of ET-1. The ET-1-induced cTnI Ser-23/24 phosphorylation was blocked by the PKC inhibitor bis-1 but not by propranolol or the PKA antagonist H-89. Treatment of myocytes with other PKC agonists, including phenylephrine and phorbol 12-myristate 13-acetate, for 1 h also increased cTnI Ser-23/24 phosphorylation. Collectively, these results suggested there are temporal changes in PKC-dependent cTnI Ser-23/24 phosphorylation in response to multiple PKC agonists. This hypothesis is supported by other earlier in vitro studies (650, 653). In functional studies, longer term (60 min) activation with ET-1 continued to enhance peak shortening and relaxation rate, which were both attenuated in cTnI Ser-23/24 Ala-expressing myocytes. The ET-1 response with cTnI Ser-23/24Ala was comparable to results observed with ssTnI. The combined results from a phospho-specific antibody and cTnI Ser-23/24Ala mutant gene transfer indicated that this cluster accelerates relaxation during more prolonged PKC activation by ET (962).
The role of Ser-43/45 phosphorylation in intact myocytes is less clear. Substitution with negatively charged residues at Ser-43/45 to mimic exhaustive phosphorylation produced a decrease in maximum force and Ca2+ sensitivity in reconstituted, permeabilized, fiber bundles and slowed maximum in vitro sliding velocity (851). The decreased Ca2+ sensitivity was expected to accelerate relaxation. However, relaxation was substantially slower in cTnIAsp5 transgenic mice (781) in which Asp was substituted for all five phosphorylation sites (e.g., Ser-23/24, Ser-43/45, Thr-144). In agreement with this finding, the converse knock-in with Ala-substituted cTnI to form cTnIAla5 increased relaxation rates in response to PKC activation by ET-1. A reasonable conclusion to draw from these results is that PKC-mediated cTnI phosphorylation decreases relaxation rate. The mechanism for this slowing of relaxation is more likely due to the decrease in unloaded shortening velocity, rather than the decrease in myofilament Ca2+ sensitivity (92). Decreased cross-bridge detachment rate could account for the reduced velocity, and it would slow relaxation in intact myocytes. However, full phosphorylation of Ser-43/45 is not likely representative of physiological conditions. Further studies are needed to determine the role Ser-43/45 plays in modulating PKC-mediated alterations in contractile function in intact myocytes. A key question remaining from these studies is whether the influence of cTnI Ser-43/45 phosphorylation on cross-bridge cycling dominates the influence on myofilament Ca2+ sensitivity and exclusively slows relaxation. Alternatively, submaximal Ser-43/45 phosphorylation may decrease Ca2+ sensitivity and accelerate relaxation in intact myocytes, while more extensive phosphorylation slows cross-bridge cycling and produces a dominant decrease in relaxation rate. Another key remaining question is whether the three clusters independently and additively influence relaxation or, instead, have a hierarchical influence on relaxation. These questions can be addressed using gene transfer into adult myocytes.
Currently, it is controversial whether heart failure causes significant changes in the PKC-dependent cTnI phosphorylation state (66, 648). Decreases in overall PKC-dependent phosphorylation have been reported in failing human hearts (66), while increased Ser-43/45 phosphorylation has been correlated with decreased force in another set of failing human hearts (648). Thus, during heart failure, the downstream effects of the temporal elevation of ET-1, heightened PKC isoform expression, and altered cTnI phosphorylation on contractile function are not well understood and leave open an area of research primed for gene transfer strategies.
The strategy of studying the importance of multiple phosphorylation sites and/or the role played by other myofilament proteins and/or proteins involved in modulating Ca2+ cycling using gene transfer has not yet been thoroughly applied to PKC signaling in myocytes. Instead, biochemical techniques and transgenesis have been the predominant approach. Proteins targeted by PKC that may be of interest for future gene transfer studies include TnT, MLC2, MyBP-C, connexin43, Na+/H+ exchanger (NHE1), and DHPR.
A) PICOT. The protein interacting cousin of thioredoxin, PICOT, acts as an inducible inhibitor of hypertrophy. In addition, this protein has direct effects on contractile function (189, 408). PICOT was discovered in a yeast-2 hybrid screen and showed 30% homology with thioredoxin (973). After gene transfer into adult rat cardiac myocytes, PICOT increased peak shortening amplitude as well as the rate of contraction and relaxation. Similar results were observed in myocytes isolated from transgenic mice expressing PICOT, and comparable hemodynamic results were obtained in whole hearts. Analysis of the Ca2+ transient in myocytes indicated that more efficient Ca2+ uptake by the SR played a key role in accelerating relaxation rate after PICOT gene transfer. This increase in relaxation rate was accompanied by increased phosphorylation of phospholamban and increased myofilament Ca2+ sensitivity in these myocytes. Thus PICOT has a direct influence on contractile function, and evidence to date suggests steps in Ca2+ cycling are an important PICOT targets within myocytes.
Future studies may be expected to provide additional insight into the role played by PICOT in cardiac myocytes. For example, the peak Ca2+ amplitude and Ca2+ release rate did not contribute to the increased amplitude and rate of contraction, respectively. Thus other potential targets influenced by PICOT expression remain to be determined. It is also unclear whether the PICOT-mediated influence on contractile function results from the direct influence on one or more PKC isoforms and/or downstream signaling proteins (e.g., MAPKs; see below). Originally, PICOT was shown to associate with PKC-
rather than the endogenous cardiac PKC isoforms (973). However, PKC- protein is not detectably expressed in adult rat myocytes (714, 768), and PICOT gene transfer into neonatal cardiac myocytes did not significantly change basal phosphorylation of PKC isoforms (408). Increased phosphorylation of the major PKC isoforms, including , , and 
in response to the PKC agonists ET-1 or phenylephrine, were observed in adult myocytes, but the influence of PICOT on cellular targets involved in the contractile response has not been thoroughly investigated in adult myocytes. Expression of PICOT after gene transfer also inhibited agonist (e.g., ET-1, phenylephrine) activation of MAPKs, ERK1/2, and JNK, as well as downstream proteins such as activator protein 1 (AP-1) and nuclear factor kB (NF-
B) (305, 408, 775).
D. Gene Transfer of Protein Phosphatases
PP1 is modulated by two inhibitor proteins, inhibitor 1 (I-1) and inhibitor 2 (I-2). I-1 is a target for both PKA and PKC phosphorylation (207, 775), with PKA phosphorylating Thr-35. PKA-mediated I-1 phosphorylation on Thr-35 enhances I-1 binding to and inhibition of PP1 activity (210). The decrease in PP1 activity results in slower dephosphorylation of target proteins such as PLN and contributes to the positive inotropic and lusitropic actions of β-adrenergic agonists (207). The PKC-dependent phosphorylation site on I-1 was initially localized to Ser-67 (775), but more recently it was shown to involve Ser-65 or Thr-75 (756). Phosphorylation of Ser-67 by cdk-5 is needed in addition to Ser-65 to prevent dephosphorylation of I-1 by PP1. PKC-mediated modulation of I-1 and PP1 was worked out with a combination of gene transfer and transgenic studies (81). In these studies, overexpression of PKC- decreased peak shortening amplitude in isolated adult rat myocytes, while dominant negative PKC- (dnPKC-) or knock-down of PKC- in mice increased the amplitude of shortening. In contrast to PKA-mediated PP1 inhibition, increased PKC- expression increased PP1 activity via phosphorylation of I-1, which decreased I-1 binding to PP1. This increase in PP1 activity significantly decreased PLN phosphorylation, increased SR Ca2+ load, and slowed Ca2+ decay in myocytes. Conversely, gene transfer of dnPKC- decreased I-1 phosphorylation at the PKC site and increased the binding of I-1 to PP1, which decreased PP1 activity. The decreased PP1 activity subsequently increased PLN phosphorylation which, in turn, accelerated Ca2+ decay.
I-2 is another modulator of PP1. In vivo delivery of I-2 prevents heart failure in cardiomyopathic hamsters, as indicated by the restoration of fractional shortening and reductions in chamber size (985). Gene transfer of I-2 increased cytosolic PP1C without a change in cytosolic PP1 activity and decreased microsomal PP1C and PP1 activity, which resulted in increased PLN phosphorylation at Ser-16. Heightened PLN phosphorylation would be expected to accelerate relaxation (985). AAV delivery of I-2 to these hamsters prolonged survival time, suggesting a new treatment that does not require activating PKA.
The protein phosphatase calcineurin, or protein phosphatase 2B (PP2B), plays a significant role in myocardial hypertrophy (596, 1005). Adenoviral-mediated gene transfer of constitutively active calcineurin into neonatal rat cardiac myocytes significantly increased cell size and protected against apoptosis (165). The influences of calcineurin could be blocked by gene delivery of dominant negative nuclear factor of activated T cells (NFAT), which is a downstream transcription factor in the calcineurin hypertrophy pathway (916). These effects were mediated in part via NFAT3 and the Akt-PKB pathway. Gene transfer of the Cain/Cabin-1 inhibitory domain for calcineurin (Cain) significantly reduced acute pressure overload-induced hypertrophy in rats (7 days; Ref. 164) without changing the pressure gradient. Cardiac specific expression of Cain in transgenic mice also reduced hypertrophy. Other phosphatases, such as MAPK phosphatase (MKP-1), have been shown to inhibit ERK1/2, JNK, and p38 activation using gene transfer techniques (88). The direct and indirect influence of these phosphatases on contractile function activity has not been investigated using gene transfer.
E. Gene Transfer and MAPK Signaling
Cardiac gene transfer approaches have been used to investigate each of the three main signaling cascades within the MAPK signaling cascades. Mitogenic growth factors activate Erk1/2, while cellular stressors activate the stress-activated protein kinases, which include JNK and p38 MAPK (350, 847). Signaling for all three MAPKs is modulated by a set of two upstream kinases. Initially, a MAPKKK or MEKK is activated, followed by MAPKK or MEK activation, which modulates a specific MAPK. Several in-depth reviews have discussed the hypertrophic, apoptotic, and other transcriptional control aspects of these signaling cascades (159, 722, 848).
Currently, there is little evidence to demonstrate a direct influence of ERK1/2 on cardiac myocyte contractile function. Gene transfer has been used to demonstrate that this MAPK is a downstream target of PKC- and - in cardiac myocytes (80, 673), which in turn activate small GTPase MEKKs, including RhoA, Rac, Ras, and c-Raf-1 (722, 849). One study also has shown that excitation/contraction influences ERK1/2 activity (475). Specifically, gene transfer of Kv4.3 channels into neonatal myocytes shortened action potential duration by decreasing Ca2+ influx, which reduced ERK1/2 activation and other indices of hypertrophy (475).
More gene transfer studies have focused on JNKs, which are also referred to as stress-activated protein kinases or SAPKs. Cellular stresses such as ischemia activate the upstream MEKK1/3, which in turn activates MEK4/7 in the JNK pathway, and two isoforms of JNK are expressed in cardiac tissue (722). As with ERK1/2, studies focused on the JNK pathway indicated this cascade primarily influences cardiac function via transcriptional regulation. For example, gene transfer of a dominant negative MEKK4 (SEK-1 KR), which activates both JNK and p38, inhibited pressure overload-induced hypertrophy (118). However, it is interesting to note that gene transfer into neonatal rat myocytes of constitutively active MEK7 (MEK7D), which specifically activates JNKs, acted as a negative modulator of connexin43 expression (685). Connexin43 is a key subunit of gap junctions (917), and the cellular uncoupling observed in this study indicated a loss of gap junctions, and cellular uncoupling caused premature death in a transgenic mouse model expressing MEK7D.
The p38 MAPK has been the target of most gene transfer studies within the MAPK family, and MEK3/4/6 are the immediate upstream activators of this MAPK. Activation of p38 MAPK is observed in response to pathophysiological conditions, including increases in hemodynamic load or myocardial ischemia (67, 234). In contrast to ERK and JNK, a few studies have assessed the direct influence of p38 on contractile function (491). In earlier work using gene transfer of the constitutively active MEKKs, MKK3bE and MKK6bE (325, 742), MKK3bE or downstream p38, each increased apoptosis, and a dominant negative p38 suppressed this effect (943). In contrast, MKK6bE increased hypertrophic responses in myocytes, which was similar to the results obtained with downstream activation of p38β. The hypertrophic effects of MKK6bE were suppressed by dominant negative p38β. Increased p38 MAPK activation achieved 24 h after gene transfer of upstream MKK3bE also decreased cardiac myocyte peak shortening and diminished the rates of contraction and relaxation via a decrease in myofilament Ca2+ sensitivity (491). There were no detected changes in cellular Ca2+ cycling, and TnI phosphorylation and cellular pH did not appear to be involved in this contractile response (491). In contrast, the decay of the Ca2+ transient slowed, and SERCA2 expression decreased 3 days after gene transfer of activated MKK6 (comparable to MKK6bE) into neonatal rat cardiac myocytes (16), presumably due to activation of p38β. Diastolic Ca2+ levels also increased significantly after gene transfer of this MKK6 in electrically paced cells, and diastolic Ca2+ levels were restored after dual gene transfer of the activated MKK6 and SERCA. On the basis of these results, the combined activation of p38 and -β is expected to decrease myocyte contractile function via multiple targets. However, it is presently unclear whether their combined influence is additive, and whether their dose and temporal activation influence other Ca2+ cycling proteins. In addition, transgenic studies indicate p38 MAPK serves as a negative feedback regulator of PKA-mediated increases in contractile function (1011), yet a gene transfer approach has not been used to investigate this feedback loop in the absence of potential compensatory adaptations that may develop in transgenic animals. More detailed studies of p38-mediated influences on contractile function are likely to provide insight into these critical remaining questions.
F. Myocardial Nitric Oxide Synthase Signaling and Contractile Function
Extensive studies have focused on nitric oxide (NO) signaling in the myocardium. Nitric oxide synthase (NOS) activity is particularly important during pathophysiological conditions including myocardial ischemia, preconditioning, and heart failure. Aside from the conventional NO downstream signaling paradigm, NO regulates cardiac ion channels (i.e., DHPR and RyR) and other subcellular components through a direct signaling mechanism in which cysteine-thiol residues are posttranslationally modified in a process known as S-nitrosylation (791). NOS isoforms NOS-1 (neuronal or nNOS), NOS-2 (inducible or iNOS), and NOS-3 (endothelial or eNOS) are each capable of being expressed in cardiac myocytes. These experiments are challengeing as protein expression and activity depend on the physiological or pathophysiological state, species-dependent regional expression in hearts, and cellular localization of each NOS within myocytes. While the paracrine and autocrine influences of NOS, the downstream and feedback signaling pathways, and the role of NOS in heart failure have been extensively reviewed elsewhere (32, 70, 804), few of these investigations have utilized gene transfer to determine the direct influence of NOS or S-nitrosylation on contractile function. The lack of gene transfer studies may be due to the appearance that NO signaling plays its most important role during pathophysiological conditions and NOS influences several types of cells residing in the heart. The majority of gene transfer studies to date have investigated the direct effects of NOS on contractile function using NOS-2 or NOS-3 gene transfer (detailed below), but, in lieu of recent reports using mouse transgenesis to demonstrate a role of NOS isoforms and S-nitrosylation in Ca2+ homeostasis (198, 281), there is an additional avenue in the NOS field primed for using a gene transfer approach to further understand the role of NO-mediated posttranslational modifications in cardiac function.
Adenoviral delivery of NOS1 has focused on delivery to the neuronal compartment and/or ganglia involved in autonomic regulation of the heart. Gene transfer of this synthase to mice lacking NOS1 demonstrated the critical role for nitric oxide production in the regulation of heart rate (154, 594).
2. NOS2 or inducible NOS signaling
Inducible NOS (NOS2) activity increases in response to stress or pathophysiological conditions, such as myocardial infarction in humans (reviewed in Ref. 804). During heart failure, NOS2 is expressed by cardiac myocytes (250). Increased NOS2 expression in failing hearts had little influence on basal contractile function but significantly reduced the contractile response to β-AR stimulation (1017). The mechanism responsible for this effect was postulated to result from influences of NOS2 on Ca2+ handling proteins, including the L-type Ca2+ channel (DHPR) and the RyR (1017). In contrast to this negative influence on β-AR modulation of contractile function during heart failure, NOS2 has been shown to contribute significantly to the late phase of ischemic preconditioning. Preconditioning was first described by Murry et al. (620) as a brief ischemic event followed by reperfusion that provides protection against the development of contractile dysfunction during a later, more prolonged bout of ischemia (70). Short-term NOS2 expression after gene transfer protected against ischemia via activation of cyclooxygenase-2 (487). More significantly, NOS2 levels also increased and the myocardium was protected against myocardial infarction initiated 1–2 mo after direct cardiac adenoviral gene delivery of NOS2 to mouse hearts compared with LacZ-injected controls (486). Gene delivery of NOS2 without infarction did not cause any significant changes in baseline contractile function, although baseline heart rate was significantly depressed in this study.
There is currently disagreement about the role for NOS3 in modulating cardiac contractile function. Moderately increased levels are observed after viral-mediated gene transfer of NOS3 to isolated rat myocytes, without changes in NOS2 expression (734). This increase in NOS3 significantly increased contractility and relaxation under basal conditions. Basal and peak Ca2+ transients were also increased in fura 2-loaded myocytes. There was a slight increase in Akt phosphorylation, and the enhanced contractile function was blocked by the PI3K inhibitors LY294002 and wortmannin. On the basis of these observations, the direct effect of NOS3 was concluded to be mediated via PI3K activation of the PKB/Akt pathway, although the contractile targets for this pathway were not identified. Conflicting results were observed 3–4 days after gene delivery of myocyte-specific NOS3 into a knockout mouse model (107). Viral delivery of NOS3 with a cardiac-specific -MyHC promoter restored the increased systolic pressure and decreased relaxation rate observed in NOS3 knockout mice back toward wild type, and the enhanced β-AR-mediated contractile function observed with this knockout model also returned to wild-type levels. Similar results were observed in a NOS3 overexpression transgenic model (87). The basis for these divergent outcomes is not known, but it may result from the level of NOS3 expression or the reduced transfection efficiency in the in vivo versus in vitro models. Alternatively, these differences may result from compensatory adaptations in the whole animal compared with the myocyte, or from acute versus long-term influences of this synthase in wild-type versus knockout mice.
Under pathophysiological conditions, cardiac myocyte-specific NOS3 overexpression improved left ventricular function after infarction (64, 406). Gene delivery of NOS3 4 days before infarction significantly increased myocyte expression of NOS3 and reduced the infarct size after acute ischemia and reperfusion, although the effects on contractile function were not measured (5). One week after infarction, gene delivery of NOS3 reduced the increase in end-diastolic pressure and the ischemia-associated increases in heart weight, myocyte size, fibrotic lesions, and apoptotic cells (829). There were also significant decreases in proteins involved in oxidative stress. Some of this beneficial effect may be related to NOS3 effects on cardiac-associated neural and endothelial compartments (253, 415, 440, 441, 776). Liposome-mediated gene delivery of NOS3 also improved donor heart survival after transplantation in a rabbit model (395). In this study and in earlier work on gene therapy into the vasculature (175, 922), several beneficial effects were due to actions on endothelial cells. However, there was also significantly reduced NF-B and associated apoptosis in myocytes. In comparison, direct myocardial injection of NOS3 reportedly increased myocyte apoptosis (430), which suggests that cross-talk and dose may be important for the beneficial influences of endothelial-derived NOS3 on myocytes.
Studies have also demonstrated that heat shock protein 90 (Hsp90) acts as a scaffolding protein for NOS3, and it modulates Akt-mediated phosphorylation of Ser-1177 and calcineurin-mediated dephosphorylation of Thr-495 within eNOS (86). Interestingly, gene transfer of Hsp90 via liposomes reduced infarct size and improved end-diastolic pressure and regional contractile function during ischemia-reperfusion injury in a pig model (461). These effects were primarily mediated via the effects of Hsp90 scaffolding within the vascular compartment, and it is unclear whether a similar strategy to increase Hsp90 expression within myocytes would also prove beneficial. Gene transfer of NOS3 into NOS3 null cardiac myocytes also restored lipopolysaccharide activation of p38 MAPK and downstream activation of tumor necrosis factor-, suggesting NOS3 is an upstream modulator of this signaling cascade (680). These types of studies demonstrate the potential insights to be gained from using gene transfer approaches to understand clinically significant and complicated physiological and pathophysiologic cardiac conditions.
G. Other Signaling Proteins of Interest
Oxidative stress resulting from the generation of reactive oxygen species superoxide (O2–) followed by peroxide production decreases cardiac function during periods of stunning and ischemia-reperfusion (70; for pathway, see Ref. 89). Signaling via this pathway is closely linked to NOS signaling (520). Three isoforms of superoxide dismutase (SOD) catalyze the breakdown of superoxide into peroxide, including copper/zinc SOD (Cu/Zn-SOD) in the cytoplasm, extracellular SOD (EC-SOD) present outside the cell, and manganese SOD (Mn-SOD) in the mitochondrial matrix. Catalase detoxifies the peroxide (1013).
Woo et al. (978) initially demonstrated that delivery of EC-SOD and catalase together improved cardiac function after ischemia-reperfusion in mice. Later studies by this group demonstrated that catalase alone also restored cardiac function in response to ischemia-reperfusion (1013). Cu/Zn-SOD was used in an attempt to protect against doxorubicin cardiotoxicity in neonatal rat myocytes, although there were no significant beneficial effects (3). Gene delivery of EC-SOD alleviated cardiac stunning and improved cardiac function, as well as decreased infarct size following ischemia-reperfusion (485). However, the major mechanism of action appeared to be mediated via improved endothelial cell function derived from the increased availability of NO and H2O2, which act to normalize vasodilatation (470). These effects do not appear to be mediated by direct influences on contractile function in cardiac myocytes (382).
Modulation of Mn-SOD is critical for tolerance to oxidative stress and attenuates ischemia-reperfusion injury (4). To determine whether Mn-SOD would prove to be beneficial during cryoprotection of hearts prior to transplantation, gene delivery of Mn-SOD was followed by orthotopic transplantation into donor rats. After 4 days, hearts were removed and contractile function measured before and after 6 h of global ischemia and 1 h of reperfusion. Developed pressure, and max and min dP/dt were significantly higher after Mn-SOD delivery than with LacZ delivery. Similar improvement was previously observed using liposome gene delivery (5). Gene delivery of eNOS also restored contractile function to a similar extent, although Mn-SOD and eNOS together did not have an additive effect (6). Further work is needed to determine whether these beneficial influences are mediated directly through the myocardium or via endothelial function.
The role of several signaling proteins has been investigated using gene transfer, although relatively few investigators have directly examined the influence on cardiac and/or cardiac myocyte contractile function. For example, potentially important signaling via protein kinase D (PKD) has been studied using gene transfer. The influence of PKD on downstream phosphorylation of HDAC5 (380) and on contractile proteins (345), such as TnI and MyBP-C, has been studied using this approach. PKC activation of PKD phosphorylates and exports HDAC5 from the nucleus, and it plays a significant role in hypertrophy (923). Addition of PKD to skinned cardiac myocytes directly decreased myofilament Ca2+ sensitivity, which would be expected to accelerate relaxation. Thus this kinase pathway may play a significant role in physiological and/or pathophysiological cardiac function, as it is also a downstream target for PKC.
Examining the contractile effects of other signaling proteins like TIMP1, IB, G protein-coupled receptors, small GTPases, and annexin VI by a gene transfer approach has also been initiated. For instance, gene transfer of TIMP1 reduced matrix metalloproteinase activity 6 wk after left anterior descending artery occlusion, preserved both systolic and diastolic function, and reduced myocardial fibrosis (407). Overexpression of IB also improved end-diastolic and systolic function after infarction (894). Gene transfer of small GTPase proteins, including cdc42 (626), rhoA (906), rac1 (708), gem (616), and cyclinD1/CDK4/ras (870) has shown the importance of these proteins in transcriptional regulation and/or hypertrophic signaling within the myocardium. In addition to these transcription-based effects, viral-gene delivery of gem into guinea pig cardiac myocytes also acted as a Ca2+ channel blocker, which significantly shortened the action potential and decreased the rates of contraction and relaxation (616). Annexin VI also was shown to modulate Ca2+ cycling via modulatory influences on RyR and NCX in a transgenic model (303). Further investigation of these proteins may provide deeper insight into the direct and indirect roles of these signaling pathways in modulating contractile function.
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Address for reprint requests and other correspondence: J. M. Metzger, Dept. of Integrative Biology and Physiology, Univ. of Minnesota, Medical School, 6-125 Jackson Hall, 321 Church St. SE, Minneapolis, MN 55455 (e-mail: metzgerj@umn.edu).
Present addresses: J. Davis, Children's Hospital Medical Center, 3333 Burnet Ave., MLC7020, Cincinnati, OH 45229-3039; M. V. Westfall, M. Blankinship, T. J. Herron, G. Guerrero-Serna, E. Devaney, Univ. of Michigan, Ann Arbor, MI 48109.
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MHC403/+ mouse model of familial hypertrophic cardiomyopathy. Circ Res 84: 475–483, 1999.
