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Sports Medicine Research Unit, Department of Rheumatology, Copenhagen University Hospital at Bispebjerg, Copenhagen, Denmark
ABSTRACT I. INTRODUCTION II. CONVERSION OF MECHANICAL LOADING INTO TISSUE ADAPTATION OF TENDON AND EXTRACELLULAR MATRIX OF SKELETAL MUSCLE: THE GENERAL CONCEPT III. TENDON AND SKELETAL MUSCLE EXTRACELLULAR MATRIX CONTENT: ORGANIZATION AND PHYSIOLOGICAL FUNCTION A. Tendon Components B. Tendon Fibroblast Signaling C. Tendon Vasculature and Blood Flow Regulation D. ECM Components in Skeletal Muscle E. Functional Implications of ECM in Tendon and Muscle IV. REGULATION OF COLLAGEN AND OTHER EXTRACELLULAR MATRIX PROTEIN SYNTHESIS: INFLUENCE OF CHANGES IN MECHANICAL LOADING A. Steps of Collagen Synthesis: Methodological Considerations B. Determination of Collagen Turnover in Humans C. Responses to Increased Loading: Acute and Chronic Exercise D. Immobilization and Collagen Turnover V. DEGRADATION OF CONNECTIVE TISSUE IN TENDON AND SKELETAL MUSCLE: EFFECTS OF CHANGES IN MECHANICAL LOADING A. MMPs B. TIMPs VI. STRUCTURE OF EXTRACELLULAR MATRIX IN TENDON AND MUSCLE: RELATION TO MECHANICAL AND VISCOELASTIC PROPERTIES A. Extensibility of Tendons B. Repetitive Loading and Tendon Properties C. Aging, Disuse, and ECM D. Integrated Muscle-Tendon ECM Properties VII. LOADING AND OVERLOADING OF CONNECTIVE TISSUE STRUCTURES IN TENDON AND SKELETAL MUSCLE: ROLE OF GROWTH FACTORS A. TGF-{beta} and CTGF B. FGF C. IL-1 and IL-6 D. IGF and IGF-Binding Proteins VIII. COUPLING OF REGULATORY PATHWAYS FOR EXTRACELLULAR MATRIX TURNOVER AND SKELETAL MUSCLE CELLS TO MECHANICAL TISSUE LOADING A. Development of Skeletal Muscle and Intramuscular ECM B. ECM and Skeletal Muscle Interplay in Mature Tissue IX. CLINICAL PERSPECTIVES: PHYSIOLOGICAL UNDERSTANDING OF TISSUE OVERUSE A. Tendon Overuse B. Development of Tendon Injury: Predisposing Factors C. Tendon Rupture: Preceding Overuse D. Experimental Tendon-Overuse Models E. Tendon Overuse and Inflammation F. Tendon Overuse, Blood Flow, and Tissue Oxygenation G. Tendon Injury and Pain H. Perspectives for Treatment X. FUTURE PERSPECTIVES XI. SUMMARY
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ABSTRACT |
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I. INTRODUCTION |
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The ECM consists of a variety of substances, of which collagen fibrils and proteoglycans are truly ubiquitous (153). In addition to the proteoglycans (PG), the hydrophilic ECM includes (164, 339, 581) a variety of other proteins such as noncollagen glycoproteins (582, 583). It is known that the force transmission of the muscle-tendon complex is dependent on the structural integrity between individual muscle fibers and the ECM (48) as well as the fibrillar arrangement of the tendon and its allowance for absorption and loading of energy (15, 16). Furthermore, it is well described that the tensile strength of the matrix is based on intra- and intermolecular cross-links, the orientation, density, and length of both the collagen fibrils and fibers (57, 447, 496, 497, 630–632, 640). However, the signals triggering the connective tissue cells in response to mechanical loading, and the subsequent expression and synthesis of specific extracellular matrix proteins, as well as its coupling to the mechanical function of the tissue are only partly described (51–53, 173, 181).
This review focuses on the physiological role of the ECM, especially collagen, for the tendon-muscle interaction and the adaptation to mechanical loading. Somewhat in contrast to the classical view of the ECM tissue being relatively static and inert, evidence is evolving that tendons and intracellular connective tissue are more dynamic structures that adapt to the variety of functional demands that the musculoskeletal system is subjected to, and that this tissue adapts both in a structural and functional way to mechanical loading (53, 173, 386, 630). Recent development of refined in vivo techniques have underlined that connective tissue of skeletal muscle and tendon is a lively structure with a dynamic protein turnover and that it possesses the capacity to adapt greatly to changes in the external environment such as mechanical loading or inactivity and disuse.
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II. CONVERSION OF MECHANICAL LOADING INTO TISSUE ADAPTATION OF TENDON AND EXTRACELLULAR MATRIX OF SKELETAL MUSCLE: THE GENERAL CONCEPT |
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The ECM is a conglomerate of substances in which biochemical and biophysical properties allow for the construction of a flexible network that integrates information from loading and converts it into mechanical capacities (152, 494, 690). It serves as a scaffold for adhesion of cells mediated by integrins, dystroglycan, and proteoglycans at the cell surface and of tyrosine kinase receptors (98, 290). The interaction between the ECM and the adhesion molecules leads to activation of intracellular signaling pathways and cytoskeletal rearrangement (52, 82, 114). In combination with this, the PGs with their glycosaminoglycan side chains are able to bind and present growth factors to their relevant receptors, and furthermore, the ECM can release growth factors upon relevant mechanical stimulation. The complete signaling pathways responsible for mechanotransduction responses are yet to be described, but several candidates have been suggested from investigations on a variety of fibroblasts in dermis, vasculature, and cardiac muscle (166, 396, 667). Integrin molecules are major structural components of adhesion complexes at the cell membrane linking the ECM to the cytoskeleton (108, 128, 541). In this way integrins establish a mechanical continuum along which forces can be transmitted from the outside to the inside of the cell, and vice versa (238, 290, 291, 667, 668). It is believed that integrins are the sensors of tensile strain at the cell surface (290). Ingber et al. (291) have suggested that integrins together with the cytoskeleton form a mechanically sensitive organelle. At the myotendinous junction, lack of integrin expression will lead to structural damage during muscle contraction (442). Integrins are important structural components of the adhesion complexes at the cell membrane, and they play a crucial role in linking the ECM to the cytoskeleton (227, 412, 418, 419, 579). Thereby they provide a bridge through which forces can be transmitted between inside and outside of the cells in a two-way street principle. Further evidence for this is provided by the fact that integrins can convert mechanical signals to adaptive responses in the cell (115, 591). In addition to integrins, also the dystrophin-glycoprotein complex plays an important role in mechanotransduction of muscle and tendon tissue (107, 130, 288). The 
-subunit cytoplasmic domain of integrin is interacting with the cytoskeleton, and the demonstration of 
71-integrin linked to laminin in the ECM is important for signal transduction (81, 309a, 309b), and lack of the 2-laminin leads to muscle dystrophy. Interestingly, overexpression of 71-integrin in dystrophin-deficient mice leads to reduction in dystrophic symptoms, indicating that some substitution effects exist between integrin and laminin (107). Extracellular matrix ligands for integrins are known to be collagens, fibronectin, tenascin, and laminin (412). Several studies have demonstrated that the expression of several other ECM components are controlled by the level of mechanical loading. For example, collagen XII and tenascin-C, which are present in both tendon and other connective tissue structures like ligaments, have been shown to increase their expression and synthesis when fibroblasts are stretched in vitro and are suppressed in cells that are left in a relaxed state (127, 129). Although not yet confirmed, integrins are likely candidates for sensing tensile stress at the cell surface (290, 683, 694, 698). Thus some evidence indicates that integrin-associated proteins are involved in the signaling adaptive cellular responses to mechanical loading of the tissue, and it is likely that this takes also place in tendon and skeletal muscle ECM-related fibroblasts (531).
Several intracellular pathways for mechanotransduction signaling have been suggested, including focal adhesion kinase (FAK), paxillin, integrin-linked kinase (ILK-1), and mitogen-activated protein kinase (MAPK) (127, 206, 207, 241, 451, 618). MAPK is crucial for the conversion of mechanical load to tissue adaptation inducing signaling from the cytosol to the nucleus. It is well described that several cell types and subsets of MAPKs such as extracellular signal-regulated kinase 1 and 2 (MAPK-erk1+2, p44), stress-activated protein kinases p38 (MAPK-p38), c-jun NH2-terminal kinase (MAPK-jnk, p54), and extracellular signal-regulated kinase 5 (MAPK-erk5) can be activated by mechanical stress, as well as by lowered pH, growth factors, hormones, and reactive oxygen species (250, 414, 678, 693, 696). In regard to mechanical loading, it has been shown in muscle that MAPK can be activated both as a result of active muscle contraction (36, 37, 559) and after passive stretch (161, 439). The activation of MAPK results not only in a production of transcription factors, thus mediating gene expression, but also in an activation of the protein synthesis on the translational level through eukaryotic initiation and elongation factors (229). It has furthermore been suggested that the mode of mechanical load is coupled to a certain type of MAPK activation. In line with this, it has recently been shown in rat skeletal muscle cells that concentric activation of muscle associated with metabolic and ionic changes resulted in a preferential increase in MAPK-erk1+2, whereas intense eccentric tensile loading with barely any metabolic changes resulted in a marked increase in MAPK-p38 (as well as in MAPK-erk 1+2) (693). In another study that also used rat skeletal muscle, a strong relationship was found between peak tension (whether active and/or passive) and MAPK-jnk (439). This falls in line with the demonstration of MAPK-jnk activation and the induction of immediate early genes by mechanical stress in smooth muscle cells (253). Whether any marked increase in MAPK-p38 is found in skeletal muscle is still debatable. Whereas one study could not find any increase in MAPK-p38 in rat muscle during concentric, stretch, or eccentric muscle activity (439), another study found a marginal and late increase in MAPK-p38 (37, 241) while a third study found MAPK-p38 activation in exercised human skeletal muscle (674). It has been shown that activation in skeletal muscle of MAPK-p38 is fiber-type specific (243). Somewhat in contrast to muscle, it seems very clear that in connective tissue MAPK-p38 is mainly activated with mechanical stretching of the tissue (127). Findings on regulation of matrix metalloproteinase (MMP) activation in fibroblasts point toward a differentiated interplay between MAPKs, in which MAPK-p38 is important for induction of MMP, while MAPK-erk1+2 mediates the repression (534). Although not conclusive, these findings are compatible with stress pattern-dependent MAPK pathways in both the muscle cell and the fibroblast and suggest an intimate interplay between the muscle cell and intramuscular connective tissue in response to mechanical loading. Evidently, such pathways do not in any way rule out other mechanistic pathways (e.g., calcium-dependent pathways) to be involved in mechanotransduction also (36).
Taken together, it is likely that separate modes of tissue loading and thereby of physical training will differentially stimulate the subtypes of MAPK in both myocytes and fibroblasts. Most likely, endurance like oxidative loading of tissue stimulates MAPK-erk, whereas strength type of exercise is more likely to use the MAPK-jnk pathway (693). Furthermore, passive stretch both in muscle and connective tissue preferentially gives rise to stimulation of MAPK-p38 (89). In human models, stretch does not cause any increased muscle protein synthesis, and thus does not result in any hypertrophic effect on the muscle (211). The fact that the stretch of muscle cells and of fibroblast shows parallel MAPK activation suggests that adaptive processes in intramuscular connective tissue interact closely with those of skeletal muscle tissue when subjected to mechanical loading.
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III. TENDON AND SKELETAL MUSCLE EXTRACELLULAR MATRIX CONTENT: ORGANIZATION AND PHYSIOLOGICAL FUNCTION |
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Tendons consist of a systematic and densely packed organization of connective tissue dominated by collagen organized into fibrils, fibers, fiber bundles, and fascicles, as well as by the presence of other ECM proteins. The nature of the individual components of the tendon is equipped to withstand high tensile forces (224, 627, 632). The division of tendons into fibrils ensures that minor damage does not necessarily spread to the entire tendon, and also provides a high total structural strength (Fig. 1). Tendon consists of 55–70% water, and a substantial part of this is associated with proteoglycans in the ECM (187, 307, 546, 659, 660). Of the tendon dry weight, 60–85% is collagen. This collagen is predominantly type I (
60%) arranged in tensile-resistant fibers, and composed of two 1- and one 2-chains. These are products of separate genes rather than a posttranslational modification of a single molecule. Also, collagen types III (reported between 0 and 10%), IV (2%) (12, 260), V, and VI are present (72, 74, 307, 324, 652, 653). In addition to this, a small amount of elastin fibers are present (2% of dry weight) (194, 195, 307). Apart from a very small amount of inorganic substance (<0.2%), the remaining substance consists of different proteins (accounting for 4.5%) (660), but very little information is present as to the relative contribution of these (10b). It has been shown that the inorganic substance is dominated by PGs, especially small leucine-rich proteins of which decorin (up to 1%) (164, 307, 660) and cartilage oligomeric matrix protein (COMP, up to 1%) (465, 601, 604) are probably the most abundant. In addition, other small leucine-rich PGs such as fibromodulin, biglycan (up to 0.5%), and lumican, together with osteoadherin, tenascin-C, proline argininerich end leucine-rich repeat protein, optican, keratocan, epiphycan, syndican, perlecan, agrin, fibronectin, laminin, vercican, and aggrecan are present in tendon tissue (300, 307, 537). The PGs and water are thought to have a spacing and lubricating role for the tendon, whereas the role for several of the small and nonaggregating leucinerich PGs is more unclear. The proteoglycans also seem to play an important role in fibril fusion, as do fibrillin molecules aligning along fibrils (50). Tendons vary markedly in design, most likely coupled to their function. In the quadriceps the tendon can be found to be short and thick, whereas several of the tendons to the fingers or toes are long and thin. Furthermore, tendons may vary in thickness along its length and are often surrounded by loose connective tissue lined with synovial cells, the paratenon, to allow for large movements of the tendon. The epitenon is the connective tissue sheet that immediately surrounds the tendon, and it consists of loose, fatty, areolar tissue that allows for the tendon together with the tendon sheet, the peritendon, to glide against adjacent tissue (574).
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B. Tendon Fibroblast Signaling
Tendons are dominated by fibroblasts. In addition, also other cell types like endothelial cells and mast cells as well as axons are, together with the ECM, also present in tendons. It has been demonstrated that tendon fibroblasts lie in longitudinal rows and have numerous sheet-like cell extensions that extend far into the ECM (447) (Fig. 1). Isolated tendon fibroblasts respond to mechanically induced loading with expression of several ECM components (53). In the intact tendon, cells are linked to each other via gap junctions as evidenced by immunolabeling for connexin32 and connexin43 (447, 528). Where the latter represents the meeting of cell processes as well as where cell bodies meet, the former only represents contact between cell bodies. In total, the architecture of the fibroblasts of the tendon and their interconnection provides a three-dimensional network that surrounds the collagen fibrils and provides a basis for cell-to-cell interaction. In vitro, tendon cells upregulate collagen and gap junction production under mechanical cyclic loading, and pharmacological inhibition of the gap junction leads to loss of this response (52, 662). Gap junctions must under loading be able to withstand high loads and have been shown to be coupled to the actin cytoskeleton (394, 395, 695).
In articular chondrocytes and compressed tendon regions, a compression-sensitive organization of intermediate filaments has been shown (179, 528, 529). As well actin filaments and fibers have been shown in developing intervertebral discs and in scar connective tissue (194). However, the demonstration of these has not been put into a functional perspective (331, 469). It has been demonstrated that in knockout mice for the intermediate filament vimentin, -smooth muscle actin organization is abnormal in dermal fibroblasts and that their contractile ability is impaired (180). Recently, it has been shown that tendons have actin-based cell-cell interaction (515) and that actin stress fibers run along the rows of fibroblasts (529). When mechanically loaded, junctional components n-cadherin and vinculin rose together with tropomyosin, without any change in actin levels. The rise in cadherin and vinculin suggests an increased cell-cell adhesion or cell-matrix adhesion. This suggests that mechanical load transforms fibers into partly contractile components that may contribute to an active mechanism in the recovery after stretch and that these structures can maintain the integrity of the longitudinal tendon rows and to monitor tensile load and contribute in the mechanotransduction during exercise (529).
C. Tendon Vasculature and Blood Flow Regulation
Compared with muscle, tendons have relatively limited vasculature, and the area occupied by vessels represents 1–2% of the entire ECM (373, 374). The vessels mainly emanate from the epitenon where longitudinal vessels run into the endotenon (10c, 341, 373, 374). Supplying arteries and arterioles may come from the perimysium at the musculotendinous junction and vessels from the tendon bone junction (113, 137, 574). Long tendons are supplied by several vessels along their length (271, 341). Due to the large excursion (up to 6 cm) that some tendons experience during movement, the vessels to such tendons need to be long and often winding in nature.
The ECM in relation to both muscle and tendon is extensively filled with blood vessels (508, 509), to provide the contracting muscle with oxygen and substrate for energy production, and to ensure an efflux from musculature of combustion products. It remains, however, unsolved to what extent the blood flow to connective tissue alters with mechanical loading of the tissue. In the resting state, rabbit tendons have been shown to have tendon flow of around one-third of that in muscle, and it is known that blood flow in both tendons and ligaments increase with exercise and during healing in animals (46). Both with the use of radiolabeled xenon washout technique from peritendon tissue as well as with application of near-infrared spectroscopy and simultaneous infusion of contrast substance (95), it has been possible to demonstrate in human models that blood flow within and around tendon connective tissue increases up to sevenfold during exercise, both in young, middle-aged, and elderly individuals (93, 94, 377, 378, 381). This increase is by far smaller that the 20-fold increase in adjacent skeletal muscle blood flow under similar exercise conditions (93, 94). However, compared with the metabolic activity of the tendon during exercise, it might be adequate. Furthermore, it can be shown that skeletal muscle blood flow during maximal exercise is close to what is possible to achieve with postocclusion reactive hyperemia, while the flow in tendon is still only 20% of that during maximal exercise (93, 94). This implies that tendon flow is not simply a function of skeletal muscle blood flow and that its regulation represents a separate regulatory system.
Vasodilatory agents have been measured simultaneously in skeletal muscle and its adjacent tendon during mechanical loading in vivo, and it has been found that adenosine concentrations rise in an intensity-dependent fashion in muscle, whereas the changes in tendon were less marked and unrelated to intensity (375). Furthermore, bradykinin concentrations rose in parallel in the two tissues during exercise, and already elicited its maximal response at low exercise loads (375). The changes in tissue bradykinin concentrations are in the range that has been found to cause a vasodilatory effect on endothelium (554). These findings indicate that these two substances are involved in blood flow regulation in skeletal muscle and tendon with exercise and that bradykinin is involved in the blood flow increase during lower work loads both in tendon and muscle. Whether bradykinin exerted its vasodilatory effect directly on the vasculature (109) or more indirectly via release of other substances as nitric oxide (NO) (490), prostaglandins (58), or endothelium-derived hyperpolarizing factor (EDHF) (279, 458) is yet to be established.
Interestingly, it has been shown that prostaglandin concentrations rise both in muscle (214, 317) and in connective peritendinous tissue (385) with exercise. Whereas inhibition of prostaglandin synthesis by itself did not inhibit total flow during exercise in skeletal muscle, but did so only if simultaneous blockade also of NO synthesis was performed (96), the peritendinous and tendinous blood flow during exercise was diminished by 40–50% compared with control exercise without blockade (376). This differentiated regulation of blood flow regulation in skeletal muscle and tendon tissue, respectively, can be hypothesized to imply also a differentiated regulation of blood flow within the skeletal muscle itself. This would be so if parts of the vasculature in muscle is located in regions with abundance of ECM, i.e., aponeurosis and perimysial tissue. The finding of flow heterogeneity within skeletal muscle as well as the demonstration of nutritive and nonnutritive vessels in skeletal muscle (94, 95, 137) are certainly supporting evidence for such an idea. It would also explain a separate role during exercise for vessels that were very responsive to vasodilation dependent on work load and thus providing maximal supplementation of substrate and oxygen to the muscle, and on the other hand vasculature located in connective tissue, both within the muscle and in relation to tendon tissue, where flow is coupled to inflammatory activity in repair processes for the ECM. The latter would also serve as a kind of shunt with the potential of partly limiting its vasodilation to share blood with the nutritive vessels during exercise.
With regard to the ECM, the main question remains whether the increase in flow is sufficient to meet the oxidative needs of the tendon and its cells during exercise. Determination of oxygen saturation and content of the Achilles tendon region in humans has been performed using near-infrared spectroscopy with the addition of a dye dilution method (94–96). When simultaneous recording of tissue oxygenation and blood flow of human tendon regions was performed both at rest and during muscular contractions, it can be demonstrated that a tight correlation exists between increasing blood flow and declining oxygen tissue saturation (334) (Fig. 2). This correlation could indicate a coupling and fits very well with what is found in skeletal muscle during exercise (96). This illustrates that during exercise the estimated oxygen uptake in humans tendon regions rises severalfold compared with the resting state and that even during intense mechanical loading of tendons, there is no indication of any tissue ischemia.
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D. ECM Components in Skeletal Muscle
Intramuscular connective tissue has multiple functions (301a, 407) (Fig. 3). First it provides a basic mechanical support for vessels and nerves. Second, the connective tissue ensures the passive elastic response of muscle. Third, it is now clear that force transmission from the muscle fibers not only is transformed to tendon and subsequent bone via the myotendinous junctions but also via lateral transmission between neighboring fibers and fascicles within a muscle (228, 338, 415, 626). It has been shown that tension developed in one muscle part can be transmitted via shear links to other parts of the muscle, and that even the cutting of an aponeurosis in a pennate muscle still maintains much of the force transmission (401). The perimysium is especially capable of transmitting tensile force (631). Although studies have also demonstrated a potential of the endomysium for force transmission, the orientation and curvilinearity of the collagen fibers provide high amounts of elasticity and thus not sufficient stiffness to function optimally as a force transmitter.
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Intramuscular connective tissue accounts for 1–10% of the skeletal muscle and varies quite substantially between muscles (208, 301, 391, 628) (Fig. 3). Whereas the endomysium encloses each individual muscle fiber with random arrangement of collagen fibrils to allow for movement during contraction, the multisheet-layered perimysium runs transversely to fibers and holds groups of fibers in place, while the epimysium is formed of two layers of wavy collagen fibrils to form a sheetlike structure at the surface of the tendon. It has been demonstrated in bovine muscles that the endomysial content can vary between 0.5 and 1.2% of the muscle dry weight, whereas the perimysium accounts for between 0.4 and 4.8% (520). This relatively small variation in the endomysial compared with perimysial connective tissue content between muscles could indicate that at least some functional differences between muscle groups related to connective tissue content are mainly defined by perimysial characteristics. The intramuscular connective tissue is dominated by collagen and ensures not only an organization into fasicles and fibers, but contributes importantly to the force transmission (39). Several collagen types have been identified in intramuscular connective tissue (up to 7) (174, 410, 411), and whereas type IV dominates the basement membrane adjacent to the plasma membrane of the sarcolemma (12, 342), the fibrillar collagen type I and III (and to some extent type V) dominates the epi-, peri-, and endomysium (the reticular layer). By far type I collagen dominates the intramuscular collagen content (reported from 30% and up to 97% of total collagen) (47, 48, 257, 407). At the other end of the scale, collagen types II, VI, IX, XI-XVI, and XVIII-XIX represent only very minor amounts (88a, 167, 252a, 410, 411, 440, 471, 486). It is likely that the difference in relative content of connective tissue in specific muscles is coupled to function and the role of connective tissue (247, 248). Differences between muscles with regard to their relative content and type of collagen is already present early in development (483, 485), and in cattle, the concentration of hydroxyproline as well as of collagen type I and III achieve their highest levels two-thirds through gestation (411). Interestingly, the highest collagen concentrations are achieved at the time when myotubes undergo their first phase of morphological and contractile differentiation (411). Furthermore, small leucine-rich proteoglycans of intramuscular connective tissue are expressed in parallel with development of skeletal muscle (506). Decorin and fibromodulin mRNA were markedly elevated for a few days, and biglycan and lumican for 1 wk postnatally (485). Interestingly, during this period the structure of the intramuscular connective tissue changes markedly, thereby the neonatal structure is less organized than that seen just 2–3 wk later (672). The increases in PG expression are paralleled by increases in myostatin expression and transforming growth factor- (TGF-) and could suggest an interplay between the development of skeletal muscle and intramuscular connective tissue (485, 672).
E. Functional Implications of ECM in Tendon and Muscle
It is important to accept that both tendon and intramuscular connective tissue interact closely with the contractile elements of the skeletal muscle to transmit force (521, 562, 566, 567, 573, 610, 675, 691). The dimensions of tendons will influence the ability to stretch, and the ability of the tendon and the intramuscular connective tissue to store and release elastic energy during movement reduces the overall energy need during walking or running (15, 71). Some of the evidence for the functional importance of ECM components stems from studies of mutant knockout models. Given its important role in basal membrane formation, it might be obvious that mice lacking laminin will result in growth retardation and muscle dystrophy. Furthermore, mutations of integrins will also lead to muscle dystrophy and in collagen type VI to myopathy (303, 442). In mice lacking collagen type IX or XI, abnormal collagen fibrils will be found especially in relation to joints (199, 397), while in animals lacking type X collagen chondrodysplasia will develop (666). Furthermore, a defect in types IV, IX, XIII, and type XV collagen will cause myopathy symptomatology (87, 88a, 185, 367). Knockout models for collagen type I, especially when accompanied by mechanical loading, have been difficult to study, in that these animals develop severe osteogenesis imperfecta (126). Finally, somewhat interestingly, in models for proteoglycan defects in the form of a fibromodulin-null mouse, irregular collagen fibrils in tendon structure was observed, whereas no changes were detected in bone or cartilage (615). Mice lacking biglycan and fibromodulin will experience ectopic tendon ossification (27). In line with this, in mice lacking COMP, no clear musculotendinous abnormalities could be found, whereas in humans without COMP, skeletal dysplasias are observed (267). The limitation of these models is the concept of redundance, a phenomenon that is likely to be present also in the ECM, as it can be demonstrated for regulation of circulation and release of hormones in relation to exercise (333, 334).
An important role in linking together the fibrous elements of the ECM whether in muscle or tendon are the proteoglycans (111, 572, 581, 583, 584, 673). Within muscle, it has been demonstrated that PGs in the perimysium are rich in chondroitin and dermatan sulfate. In contrast, those PGs that are present in the endomysium and the basal membrane are dominated by heparan sulfates (484). In addition, decorin has been demonstrated to be present in at least bovine muscle closely associated with chondroitin sulfate (183, 480), and this is dominant in muscles during the early embryonic and postnatal state (644, 645), whereas heparan sulfate is dominant in the late embryonic state. Although several of the ECM substances in addition to collagen have been located in tendon (and muscle), little information on its functional role has been provided. One of the leucine-rich small proteoglycans that envelopes the collagen fibrils is decorin (583). Knockout of decorin suggests the involvement of decorin in the formation of collagen fibrils and to some extent controls the diameter of the fibril and prevents any lateral fusion of collagen fibrils (111, 156). Furthermore, inhibition of decorin results in larger collagen fibrils and increased mechanical properties in healing ligaments (478, 479). The clear role of decorin, or any coordinated effect of either fibromodulin or lumican situated in the same region as decorin, but having different binding sites (268, 616), is not definitively clear (119, 196, 616). More recently, it has been shown in chick embryonic tendon that small leucine-rich PGs like decorin are bound to collagen even before collagen fibril assembly, and this suggests a much earlier involvement of decorin and other PGs than thought so far (245). Even though PGs and glycosaminoglycans are important for tendon function, it has been suggested that neither these nor the collagen fibril size in itself can explain the biomechanical capacities of tendon tissue, therefore suggesting a more complex interplay involving factors and component of tendon tissue yet to be described.
One of the large chondroitin sulfate PGs, aggrecan, is largely upregulated upon compressive loading of the tendon tissue, whereas decorin only responds to tensile loading (548, 549), and is likely to be involved in the preference to synthesize type II collagen in regions of tendons that are subjected to compressive forces (61, 62, 528). Compressed areas of tendon are found to have increased amounts of larger weight PGs (200, 557). This illustrates the differentiated response to tensile and compressive loading, respectively, on collagen and ECM proteoglycans (663, 664).
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IV. REGULATION OF COLLAGEN AND OTHER EXTRACELLULAR MATRIX PROTEIN SYNTHESIS: INFLUENCE OF CHANGES IN MECHANICAL LOADING |
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The major component of the ECM, collagen, is produced in principal by fibroblasts either on the membrane-bound ribosomes of the rough endoplasmic reticulum (ER) or placed within the ECM, respectively. Collagen biosynthesis is characterized by the presence of an extensive number of coand posttranslational modifications of the polypeptide chains, which contribute to the quality and stability of the collagen molecule (Fig. 4). Intracellularly, translation of preprocollagen mRNA occurs in ribosomes and procollagen assembly in the endoplasmatic reticulum (437, 443, 661). C-propeptide domains of polypeptide -chains fold, and trimerization initiates the triple helix formation in fibrillar collagen types. These events depend on a well-matched interaction of ER enzymes like prolyl-4-hydroxylase (P-4-H), galactosylhydroxy-lysyl-glucosyltransferase (GGT), lysyl hydroxylase, prolyl-3-hydroxylase, hydroxylysylgalactosyltransferase as well as on heat shock protein 47, glucose regulating protein 94, and protein disulfide isomerase (201, 202, 368, 473, 539, 561, 568, 604). Genetic information for procollagen chain formation is divided into several exons in the DNA separated by relatively large intron areas and thus demands extensive processing of RNA prior to that mature mRNA being available for protein synthesis (661). Procollagens are transferred from ER to the extracellular space through the Golgi apparatus and contain NH2-terminal and COOH-terminal extension peptides at the respective ends of the collagen molecule (264). The fact that procollagen is larger that the conventional transport vesicles necessitates transport within the Golgi apparatus (88).
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After secretion into the extracellular space, the amino-propeptides are cleaved by specific proteinases and the collagens self-assemble into fibrils or other supramolecular structures (516) (Fig. 3). The synthesis of collagen fibrils occurs first as an intracellular step with assembling and secretion of procollagen, followed by an extracellular step converting the procollagen into collagen and subsequent incorporation into stable cross-linked collagen fibrils (Fig. 3). The synthesis of type I collagen is used here to illustrate the fibrillar collagen formation, due to its dominance in the connective tissue of tendon and muscle, but synthesis of other collagen types shares many similarities with that of collagen type I, but is described further in this review. Following the transcription of genes coding for the formation of collagen type I, the pro--chains initially synthesized undergo marked posttranslational reactions. First, hydroxylation converts residues to 4-hydroxyproline or 3-hydroxyproline by three different hydroxylases (473). Interestingly, the hydroxylases only act on nonhelical substrates and do not act on collagen or collagen-like peptides that are triple helical. Newly synthesized collagen polypeptides are glycosylated, and this process ends before the folding of collagen into a triple helix structure. Finally, the intracellular processing completes with the synthesis of both intrachain and interchain disulfide bonds (170). This last process is not started before the translation is completed and probably not before the chains are released from ribosome. The procollagen is then secreted from the cell, and it is well described that the rate of secretion depends on the intracellular processing of the protein. If folding of the pro--chains into the triple helical confirmation is prevented, secretion of the protein is delayed. The three polypeptide chains form a triple-helical structure. The -chains forming the structure are composed of repeating amino acid sequences Gly-X-Y, where the glycine residue enables the three -chains to coil around one another. Proline and 4-hydroxyproline residues appear frequently at the X- and Y-positions, respectively, and promote the formation of intermolecular cross-links. The stability and quality of the collagen molecule is largely based on the intra- and intermolecular cross-links. The 4-hydroxyproline formation is catalyzed by P-4-H and is a unique feature of collagen. Thus its assay is suitable for evaluating collagen content. Levels of P-4-H activity generally increase and decrease with the rates of collagen biosynthesis, and assays of the enzyme activity have been used for estimating changes in the rate of collagen biosynthesis in various experimental and physiological conditions (256–258, 318, 474, 569–571, 619, 620).
The exact location of the processing of procollagen to collagen might however be more complex than that (533). Procollagen N-proteinase (called ADAMTS-2) has recently been cloned (142, 143), and procollagen C-proteinase has been documented to be identical to bone morphometric protein (BMP-1) (327, 398) in which, at least in mouse embryonic tendon, all three protein variants of BMP-1 are expressed (541, 580). Recent experiments in embryonic chicken tendon using pulse-chase followed by sequential extraction have revealed that both intra- and extracellular pools of active procollagen C-proteinase (BMP-1) are present in fibroblasts, whereas the N-proteinase is located in close proximity or within the plasma membrane (91, 111). Conversion of extracellular procollagen to collagen was prevented when procollagen C-proteinase was blocked, whereas on the other hand, collagen fibrils were seen in post-Golgi vesicles and tubules (111). Therefore, despite demonstrated procollagen to collagen conversion being completed extracellularly, some collagen formation may be completed intracellularly and the sequence of C- and N-proteinase in converting procollagen into collagen appears to be more random than so far thought (111).
Fibril segments are prerequisites for fibril formation (496, 497) and have been shown to increase in length from a few microns to 100 µm (76–78), and gradually develop increasing diameter (203, 204). It is likely that fibrils develop into different mass profiles, where some are regular linear ones, some are very short and spindle-shaped, and some are intermediary fusing fibers (245, 492). The collagen molecules are arranged either unipolar or bipolar (245, 312, 313) and fuse end to end (77, 78). Most likely as a result of MMP activity from the fibroblasts, this end-to-end fusion is followed by increased decorin formation (77, 78) and removal of collagen type XIV from fibril surfaces (703). The fibrillar structure and the tissue's resistance toward loading is yet to be clarified (135, 231), but it is known that the cross-link-deficient tendons are less resistant to loading (680) and that the elongation of the tendon depends on molecular gliding within the collagen fibrils (215, 519). Proteoglycans are important for fibrillogenesis (156, 164, 702), and when decorin/fibronectrin binding is inhibited, tendon length is increased (112). Determination of pyridinoline (Pyr), which represents important components of cross-links of mature collagen fibers within the ECM, has shown that the content of Pyr is especially high in tendon and ligaments, and interestingly also that the Pyr-to-collagen ratio appears to be high in tendon and ligament compared with, e.g., bone (237). This underlines the likely importance of cross-links in these structures (339) and stresses the complexity in studying structure-function relationship with regard to connective tissue as tendon, ligaments, and to some extent skeletal muscle (194).
B. Determination of Collagen Turnover in Humans
To determine turnover rates for collagen, radiolabeled amino acids have been introduced (387–389). These allow for labeling of substances such as proline, which are incorporated into the collagen. If infused they will result in an increase in the specific activity of a certain tissue removed from the experimental species at a selected time point. The calculation of turnover rates initially however was based on first-order decay curves that rely on the prerequisite that all collagen molecules are equally likely to be degraded. If, which has been shown, the collagen pool is subdivided into a more fast and a more slow exchanging pool, this method likely gave a pessimistic picture of the capability to turnover collagen (387). More recently, the use of techniques "flooding" the precursor pool to reduce or eliminate reutilization and using infusion over a relatively short time has been performed successfully (44).
Microdialysis of both muscle and connective tissue has been performed with the intention to mimic the function of a capillary blood vessel by perfusing a thin dialysis tube with a physiological fluid implanted into the tissue (384–386). This allows for collection of extracellular fluid both in animals and in humans in vivo, both with the organism in a basal state as well as during conditions where physiological perturbations are performed, whether these are chemical or mechanical. The collection of dialysate allows for calculation of interstitial concentration of unbound substances that are able to cross the membrane of the catheter, provided the technique is supplemented by calibration methods that allow for quantitation of this. Using microdialysis fibers along the peritendon also provides the possibility to study during exercise. For several metabolic parameters it has been shown that determinations peritendinously reflect the changes that occur intratendinously (379).
C. Responses to Increased Loading: Acute and Chronic Exercise
Muscular and tendinous collagen and the connective tissue network are known to respond to altered levels of physical activity (347–350, 507, 612, 613, 623, 658, 682, 688, 706). The specific activities and content of collagen components are known to be greater in the antigravity soleus muscle than in the dorsiflexor tibialis anterior, which is not tonically active (349, 569). Furthermore, ECM in skeletal muscle is known to respond to increased loading caused by endurance training (348, 349, 619, 711), acute exercise (474), or experimental compensatory hypertrophy (676) by increased collagen expression, synthesis, and collagen accumulation in the muscle. Strenuous exercise, especially acute weight-bearing exercise that contains eccentric components, is known to cause muscle damage (33). Upregulation of collagen synthesis may be a part of the repair process but may also occur without any evidence of muscle damage (256). Acceleration of collagen biosynthesis after exercise may thus reflect both physiological adaptation and repair of damage.
It has been found in mice that acute exercise increases activities of enzymes of collagen turnover 48 h after exercise. Enzymes responsible for collagen synthesis were increased, and the most in muscles dominated by red rather than white muscle fibers (474, 650, 651). This fits with the demonstration of higher content of collagen, as measured by amount of hydroxyproline, in red versus white muscle fiber dominated muscles, and with a high recruitment of red fiber muscles during the specific exercise protocol (349). Interestingly, the changes in collagen enzyme activities were accompanied by a rise in both hydroxyproline and collagen content of the exercised muscle, which persisted up to 3 wk after exercise (474, 650, 651). Later studies have demonstrated that expression of types I and III collagen was increased at the mRNA level within 1 day (257, 345) and that of type IV collagen as early as 6 h after acute exercise (344).
Extracellular conversion of procollagen to collagen requires at least two enzymes: a procollagen aminoprotease that removes the aminopeptides and a procollagen carboxyprotease that removes the carboxy propeptides. The cleavage of the carboxypeptide allows for indirect determination of collagen type I formation. Development of assays for such markers of type I collagen synthesis [the COOH-terminal propeptide of type I collagen (PICP)] and degradation [the COOH-terminal telopeptide region of type I collagen (ICTP)] has made it possible to study the effect of exercise on collagen type I turnover (99). When determined in circulating blood, these markers have been shown to be relatively insensitive to a single bout of exercise, whereas prolonged exercise or weeks of training were shown to result in increased type I collagen turnover and net formation (383). However, as bone is the main overall contributor of procollagen markers for collagen type I turnover in the blood, and as serum levels of PICP and ICTP do not allow for detection of the location of the specific type of region or tissue in which changes in turnover are taking place, from these studies it cannot be concluded whether changes in collagen turnover of tendon-related tissue or intramuscular connective tissue occur. The use of the microdialysis technique has recently been applied to the peritendinous space of the Achilles tendon in runners before, immediately after, and 72 h after 36 km of running (386) (Fig. 5). With this technique it was demonstrated that acute exercise induces changes in metabolic and inflammatory activity of the peritendinous region (386). In addition, acute exercise caused increased formation of type I collagen in the recovery process, suggesting that acute physical loading leads to adaptations in non-bone-related collagen in humans.
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Furthermore, when type I collagen synthesis and degradation in connective tissue of the Achilles peritendinous space was studied before and after 4 and 11 wk of intense physical training, an adaptive response of the collagen type I metabolism of the peritendinous tissue around human Achilles tendon was found in response to physical training (382) (Fig. 5). The increase in interstitial concentrations of PICP rose within 4 wk of training and remained thereafter elevated for the entire training period, indicating that collagen type I synthesis was chronically elevated in response to training. As blood values for PICP did not change significantly over the training period, it is reasonable to suspect that the increased collagen type I formation occurs locally in non-bone tendon connective tissue rather than reflecting a general rise in formation of collagen type I throughout the body (382). Also, tissue ICTP concentrations rose in response to training, but this rise was transient, and interstitial levels of ICTP returned to basal levels with more prolonged training. Taken together, the findings indicate that the initial response to training is an increase in turnover of collagen I, and that this is followed by a predomination of anabolic processes resulting in an increased net synthesis of collagen type I in non-bone connective tissue such as tendons (382). The pattern of stimulation of both synthesis and degradation with the anabolic process dominating in response to exercise in tendon-related connective tissue is a pattern that is in accordance with events occurring with muscle proteins in response to loading (540). As individuals in the training study (382) were training on a daily basis, it can be difficult to differentiate effects of each bout of acute exercise from the chronic training adaptation. Previous acute bouts of exercise will influence the outcome of each subsequent one. This probably explains why highly trained runners (training up to 12 h/wk) in one study had high basal levels of interstitial levels of PICP (386). Thus it cannot be excluded that the effect on collagen metabolism found during a program with daily training simply reflects an effect on collagen formation from the last training bout, rather than chronic effect of training.
On the basis of these findings it can be concluded that both an increased collagen turnover is observed in response to training and that with prolonged training a net synthesis of collagen type I is to be expected. Whether a net synthesis of collagen type I is transformed into morphologically detectable increases in tendon size is far from clear. However, in accordance with this view, it has been demonstated in animal models that training results in enlargement of tendon diameter (74, 605, 689). Furthermore, recent cross-sectional observations in trained runners versus sedentary humans have shown that magnetic resonance imaging (MRI)-determined Achilles tendon cross-sectional area was enlarged in trained individuals compared with untrained controls (553). It can also be speculated that training initially results in an increased turnover of collagen type I to allow for reorganization of the tissue and that more prolonged training results in a net increase in tendon tissue and probably alterations in tissue strength.
No clear dose-response relationship between type and amount of training and adaptive responses of collagen formation exists, but in equine tendon it has interestingly been found that in tendon subjected to low-level repetitive stress (extensor tendon) the collagen level is higher that in flexor tendons subjected to high stress (497). This indicates that intensity and loading pattern including recovery periods between training bouts likely play an important role in adaptation of ECM. Stretch-induced hypertrophy of chicken skeletal muscle has been shown to increase muscle collagen turnover using tracer methods (389), which is in accordance with the present findings on humans in which collagen synthesis increased markedly at the beginning of training. From studies by Laurent and co-workers (387–389), it was concluded that a large amount of newly synthesized collagen was wasted, resulting in disproportionate high collagen turnover rate compared with the magnitude of net synthesis of collagen. Likewise, in human muscle, type IV collagen degradation, as indicated by an increased MMP-2, increased over a period of 1 year with electrical stimulation of spinal cord injured individuals, without any detectable change in type IV content, indicating an increased collagen turnover rate with no or very little net synthesis (342). Measuring the racemization and isomerization of its C-telopeptide (CTx) has been used to determine collagen type I turnover. As these processes are coupled to protein turnover it is likely that they reflect an index of collagen turnover (237). With this method it has been shown in human cadaver tissue that tissues like tendon and ligament have a turnover rate comparable to that in bone, and that the turnover of collagen type I is in fact quite pronounced in skeletal muscle (237). These data agree with studies using radiolabeled proline/hydroxyproline in animals showing a relative turnover (3%) of collagen per day in skeletal muscle (389).
The present studies in humans and animals support the idea of a simultaneous activation of both formation and degradation in collagen of both muscle and tendon tissue in response to loading. Interestingly, for type I collagen in tendon, timewise this is followed by a more pronounced imbalance in favor of formation and resulting in a net collagen synthesis, whereas in muscle, type IV collagen does not seem to reveal any net synthesis. In addition to an increased turnover of collagen, indirect evidence for loading increased both synthesis and degradation rates has been demonstrated also for non-collagen components of the ECM (53, 537, 549).
An important step in collagen type I formation is the enzymatic regulation by procollagen C-proteinase (PCP) of the cleavage of PICP and PINP of procollagen to form insoluble collagen (311). It has been demonstrated that mechanical load can enhance the expression of the PCP gene, but not of the procollagen C-proteinase enhancer protein (PCPE) in dermal fibroblasts (498). In addition to this, it was shown that both the synthesis and the processing of procollagen were enhanced by loading in vitro. This effect was demonstrated to be specific as non-collagen protein synthesis in that study was not elevated (498). Furthermore, an enhanced processing of procollagen to insoluble collagen was found, as evident by a larger increase in the actual amount of insoluble collagen produced compared with the increase in procollagen synthesis. Interestingly, in that study it was demonstrated that the above-described changes only occurred if cells were in tissue cultures containing TGF- or serum (498), indicating that mechanical loading by itself is not able to cause changes unless certain growth factors were present.
D. Immobilization and Collagen Turnover
In contrast to physical loading, immobilization of rat hindlimb leads to a decrease in the enzyme activities of collagen biosynthesis in both skeletal muscle and tendon (569, 570), which suggests that the biosynthesis of the collagen network decreases as a result of reduced muscular and tendinous activity (181). The rate of the total collagen synthesis depends mostly on the overall protein balance of the tissue, but it seems to be positively affected by stretch in both muscle and tendon (569, 570). Changes in the total collagen content of muscle, measured as hydroxyproline content, are usually small or absent during immobilization lasting for a few weeks, which is probably due to the turnover rate of collagen (257). Collagen expression during immobilization has been shown to be at least partially downregulated at the pretranslational level (11). The mRNAs for type I and III collagens were already decreased after 3 days of immobilization, whereas stretch seemed to counteract this decrease (11). The content of type IV collagen was also reduced as a result of immobilization (12).
In other studies of rats, amounts of collagen in skeletal muscle were studied in response to immobilization at increasing muscle length to cause either atrophy or hypertrophy (569). Whereas P-4-H decreased and proteolytic enzyme activity increased in shortened muscle, no increase in P-4-H or GGT could be detected in lengthened muscle (569). Electrical stimulation could, at least in some muscles, counteract the immobilization-induced drop in muscle mass together with the decrease in content of hydroxyproline and collagen-related enzyme activities (571). To some surprise, it was found that denervation of muscles in rats resulted in an elevation of hydroxyproline content and in P-4-H and GGT activities of muscle (571). Although non-collagen proteins of the atrophying muscle were degraded at a high rate during denervation, this could not solely explain the rise in collagen content and enzyme activity with denervation. In tendons of immobilized and denervated muscle, activities of collagen-synthesizing enzymes fell during immobilization in the shortened position but were unaffected when immobilized in the lengthened position (571). This indicated that the regulation of collagen synthesis to varying mechanical loading possessed similarities in skeletal muscle and tendon tissue. Further studies of remobilization of skeletal muscle after 1 wk of immobilization in rats showed that activities of P-4-H and GGT as well as concentrations of hydroxyproline (HP) rose within days, whereas remobilization exercise did not cause any rise in tendon P-4-H or GGT (318). This could imply that although activity can activate collagen synthesis in both tendon and skeletal muscle, a higher activity is needed to stimulate tendon than muscle.
