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Physiological Reviews, Vol. 79, No. 3, July 1999, pp. 917-1017
Copyright ©1999 by the American Physiological Society
Centre for Experimental Surgery and Anesthesiology, University of Leuven, Leuven, Belgium
I. INTRODUCTION
A. Aim and General Outline
B. Ischemia Models
C. General Biochemical Changes During Ischemia and Reperfusion
II. ION CHANNELS AND TRANSPORTERS
A. Ion Channels and Transporters in the Plasma Membrane
B. Ion Channels in Intracellular Organelles
C. Gap Junction Channels
III. ISCHEMIA SYNDROMES
A. Changes in Ion Concentrations
B. Amphiphiles and Fatty Acids
C. Radicals
D. Catecholamines
E. Extracellular ATP, Adenosine, and ACh
F. Stretch and Volume Changes
IV. ELECTRICAL CHANGES AND ARRHYTHMIAS DURING ISCHEMIA AND UPON REPERFUSION
A. Electrophysiological Changes at the Cellular and Multicellular Levels
B. Arrhythmias
V. CONCLUDING REMARKS AND PERSPECTIVES
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ABSTRACT |
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Carmeliet, Edward
Cardiac Ionic Currents and Acute Ischemia: From Channels to
Arrhythmias. Physiol. Rev. 79: 917-1017, 1999.
The aim of this review is to provide basic information on the
electrophysiological changes during acute ischemia and reperfusion from
the level of ion channels up to the level of multicellular preparations. After an introduction, section II provides a
general description of the ion channels and electrogenic transporters present in the heart, more specifically in the plasma membrane, in
intracellular organelles of the sarcoplasmic reticulum and mitochondria, and in the gap junctions. The description is restricted to activation and permeation characterisitics, while modulation is
incorporated in section III. This section (ischemic
syndromes) describes the biochemical (lipids, radicals, hormones,
neurotransmitters, metabolites) and ion concentration changes, the
mechanisms involved, and the effect on channels and cells. Section
IV (electrical changes and arrhythmias) is subdivided in
two parts, with first a description of the electrical changes at the
cellular and multicellular level, followed by an analysis of
arrhythmias during ischemia and reperfusion. The last short section
suggests possible developments in the study of ischemia-related phenomena.
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I. INTRODUCTION |
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A. Aim and General Outline
The aim of the present review is to provide a description of ionic channels and electrogenic transporters present in the heart (sect. II), to describe the biochemical and ion concentration changes during acute ischemia and early reperfusion and the effects on channels (sect. III), to translate these findings in terms of modulation of electrical properties at the cellular and multicellular level and to analyze their role in the genesis of acute cardiac arrhythmias (sect. IV).
The reader mainly interested in the electrophysiological changes during ischemia and arrhythmias may directly start with section III and return later, when necessary for a better understanding of certain processes and notions, to section II. The reader with a main interest in the biophysics of ion channels is directed first to section II.
Section II provides a description of the activation-inactivation kinetics and the permeation processes of channels and carriers; their role in the genesis of electrical activity is briefly discussed in this section. Section II does not include information on modulation or pharmacology. It is subdivided in sections describing channels and transporters in the plasma membrane, in the gap junction, and in intracellular organelles (sarcoplasmic reticulum and mitochondrion). Although information about eventual changes during ischemia is lacking for a number of channels and transporters, it is hoped that a systematic description and analysis will be helpful to understand more fully the actual state of knowledge, to put this information into perspective, and eventually to help to plan adequate experiments.
Section III describes how channels and transporters are modulated. The modulatory processes are not described for each channel separately but are grouped into "syndromes" related to ischemia, such as changes in ion concentration ([K+]o, [Na+]i, [Ca2+]i, and [Mg2+]i, where the subscripts o and i refer to extracellular and intracellular, respectively); disturbance of lipids resulting into the accumulation of long-chain acylcarnitines, lysophosphoglycerides, fatty acids, and arachidonic acid; production of radicals; secretion of neurotransmitters, hormones, and metabolites, with concomitant stimulation of adrenergic, purinergic, and muscarinic receptors; and the genesis of stretch. The analysis of each syndrome includes a description of the changes, the mechanisms involved, the effect on channels and transporters, and the final outcome at the electrophysiological and arrhythmia levels.
Section IV contains two major subdivisions: the electrophysiological changes at the cellular and multicellular level and the genesis of arrhythmias. Section IVA contains a description of the changes in resting and action potential, in excitability, refractoriness, and conduction and is followed by a discussion of possible mechanisms. Section IVB starts with an analysis of the general processes involved in the genesis of arrhythmias, followed by a description of the type of arrhythmias encountered during ischemia and reperfusion, and finally a discussion of possible mechanisms.
B. Ischemia Models
Cardiac ischemia is characterized by a deficient energetic input as well as a deficient waste removal. The result is failure of contraction, deterioration of electrical behavior, and eventual death of the cell. At the organism level, the end point may be lethal arrhythmias or mechanical pump failure. To study ischemia, different experimental models have been used. Coronary artery ligation or obstruction by a local thrombus mimics closely the clinical settings of myocardial infarction but does not allow a direct analysis of the changes in the ionic currents involved. For this purpose, voltage-clamp measurements have been applied to multicellular preparations and single cells subjected to hypoxia, uncouplers of the mitochondrial oxidative chain, inhibitors of glycolysis, superfusion with a solution containing a high concentration of K+ and H+, and deficient in glucose. In other models, the complex nature of the ischemic process has been dissected in different facets of specific biochemical changes that occur during ischemia: amphiphiles, radicals, catecholamines, adenosine, ACh, and stretch.
C. General Biochemical Changes During Ischemia and Reperfusion
Under aerobic conditions, NADH and FADH2, formed during glycolysis and in the citric acid cycle, transfer their electrons to O2 through the electron transport chain. This provides the energy to build up the chemiosmotic gradient that drives the synthesis of ATP. Oxidative phosphorylation is coupled to the demands of the cell. A feedback is generated by the breakdown products of ATP (772) and the rise in mitochondrial Ca2+ that modulates the activity of the mitochondrial dehydrogenases (692) and of the ATP synthase (199).
The turnover rate of high-energy phosphates is 30-40
mol · g wet wt
1 · min1, while the
storage is only 15 mol/g wet wt. This means that the time limit for
exhaustion is short and only 15-30 s (339). When oxygen
falls below a critical level in the cytoplasm, the electron transport
and the process of H+ ejection in the mitochondrion stops.
The energy stored in the proton electrochemical gradient becomes
insufficient to synthesize ATP in appropriate quantity. Some of the
energy in ATP may be used to maintain the mitochodrion membrane
potential (Em) and to inhibit irreversible
reactions leading to cell death (772). As a consequence,
[ATP] may be expected to fall (Fig. 1)
and [ADP] to increase. The cell, however, has efficient means to
maintain the ATP level: 1) energy demand falls very rapidly
during the first 30 s of ischemia as a consequence of contraction
failure, 2) an important amount of phosphocreatine (PCr) is
continuously used to restore ATP from ADP with concomitant increase in
Pi, and 3) anaerobic glycolysis starts and
intensifies.
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The fall in the ATP/ADP stimulates the glycolytic pathway by activation of three important enzymes: 1) hexokinase responsible for the formation of glucose-6-phosphate; 2) phosphofructokinase, responsible for the transformation of fructose-6-phosphate to fructose-1,6-biphosphate; and 3) pyruvate kinase for the formation of pyruvate. The reactions leading to pyruvate are stimulated but glucose metabolism stops at the pyruvate-lactate stage, due to inhibition of pyruvate dehydrogenase. Pyruvate is transformed in L-lactate under simultaneous oxidation of NADH to NAD+. At the same time, phosphorylase a is transformed in its active form, facilitated by accompanying increases in AMP, Pi, Ca2+, and cAMP and increases the breakdown of glycogen. High glycogen content of cells postpones the development of contracture during ischemia (190). Finally, glucose transport through the cell membrane is stimulated, secondary to a translocation of glucose transporters from an intracellular pool to the plasma membrane (1155) and increases the flow down to pyruvate. Glycolytic enzymes are especially dense in the subsarcolemmal space; stimulation of anaerobic glycolysis at this level is important for the regulation of intracellular ion concentrations and channels (KATP) (1082).
Stimulation of the glycolytic pathway explains why [ATP] stays remarkably constant during the initial 10-15 min of ischemia. However, the free energy change upon hydrolysis of ATP falls immediately because of the rise in [ADP] (281). This fall has important consequences for a number of ATP-driven transporters such the Na+-K+ATPase and the Ca2+-ATPase. The anaerobic glycolytic process is self-inhibiting however. When acidosis becomes too pronounced, glycolysis in turn is inhibited, and ATP synthesis is further reduced.
Whenever the free energy of ATP hydrolysis exceeds the energy stored in
the proton electrochemical gradient, net ATP hydrolysis may occur; the
ATP synthase then acts as an ATP hydrolase, and the energy is used to
update the electrical gradient in the mitochondrion (224)
(Fig. 2). Although the arrest of electron
flow is expected to cause depolarization of the mitochondrial membrane
during anoxia, such depolarization only occurs after a delay
(244). The delay corresponds to the time needed to block
glycolysis to cause serious ATP deficiency and rise in
[Ca2+]i. Blocking ATP hydrolysis by
oligomycin slows the fall in [ATP] (808) and delays
activation of the mitochondrial mega-channel. When [ATP] drops to
levels <1 mM and at the same time the concentration of H+,
[Ca2+]i (>1 µM), PO43
(>10 mM), and long-chain acylcarnitines (LCAC) increase
(772), the mitochondrial membrane becomes abnormally leaky
through activation of the mega-channel or transition pore in the
inner membrane of the mitochondrion. Efflux of ATP through the plasma
membrane is an additional reason for a fall of intracellular ATP. In
the extracellular medium, this ATP is further broken down to adenosine
and may activate purinergic P1 and P2
receptors.
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Block of oxidative metabolism and fall in ATP/ADP will have consequences on lipid metabolism, the generation of oxidative stress, the release of catecholamines, and ion concentrations.
Upon reperfusion after 5-30 min of ischemia, oxygen consumption rapidly recovers. The NADH/NAD+ quickly decreases, although the level may remain higher than the control for some time. After brief (5-10 min) ischemic bouts, PCr concentration quickly returns to normal, but recovery of ATP is slow. After longer periods of ischemia, PCr concentration still recovers within 5 min, but ATP, which may have dropped to 50%, stays at this low level for 30 min or more (641).
The preischemic pattern of substrate utilization is restored, i.e., oxidation of fatty acids (FA), is the main contributor to ATP synthesis; the level of glycolysis, however, remains elevated in the early period of reperfusion and is important for ATP generation. This situation may explain why block of glycolysis at this time is disadvantageous and results in aggravation of Ca2+ overload and release of intracellular enzymes. Such a block occurs when FA are present in high concentration (621). A high rate of FA oxidation generates NADH which inhibits pyruvate dehydrogenase. Glycolysis which stops at the pyruvate level is accompanied by an overproduction of protons and may negatively affect the recovery of [Na+]i and [Ca2+]i (612). High levels of lactate are deleterious for recovery.
The high level of oxygen consumption early after reperfusion stays in contrast to the continuing deficiency of the contractile machinery or "stunning." This uncoupling between oxygen consumption and contractility is not due to impairment of the respiratory chain flux and insufficient ATP synthesis. Energy is sufficiently present but apparently not used. As a possible explanation for the high rate of oxygen consumption, the existence of futile cycles has been proposed, such as the cycling of Ca2+ between the cytoplasm and mitochondria (189). The absorption of Ca2+ by the mitochondria as well as its removal via the Na+/Ca2+ exchanger depends on the energy flux in the electron chain. The ejection of protons generates the negative matrix potential that drives the absorption of Ca2+ and creates the proton gradient that is required for removing Na+ from the mitochondrial matrix via the Na+/H+ exchanger and subsequently Ca2+ via the Na+/Ca2+ exchanger. Adenosine 5'-triphosphate, which for its synthesis is also dependent on the proton gradient, is used by the plasma membrane Na+-K+ pump to keep cytosolic [Na+] low (Fig. 2). Under normal conditions, this cycle only requires little energy, but in the case of Ca2+ overload, the expenditure may become excessive. The situation is aggravated by an eventual intermittent opening of the mega-channel activated by elevated matrix [Ca2+] (91). The leak created causes breakdown of the mitochondrial electrical gradient. Activation of the mega-channel may propagate from mitochondrion to mitochondrion, creating a Ca2+ wave in the cytosol (449).
Reperfusion after more than 30 min leads still to an important but only transient recovery of oxygen consumption, while contractile function remains severely and persistently depressed. Intracellular enzymes and other substances are lost. On the microscopic level, cells appear necrotic, and later important fibrosis develops.
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II. ION CHANNELS AND TRANSPORTERS |
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A. Ion Channels and Transporters in the Plasma Membrane
1. Na+ channels
In most excitable cells, the Na+ current is
responsible for the upstroke and the conduction of the action
potential. In heart, the density of the channel is low in sinoatrial
node (SAN) and atrioventricular node (AVN) cells and highest in
Purkinje cells. The Na+ channel is voltage operated and
shows activation and inactivation. It is specifically blocked by
tetrodotoxin (TTX) (for a general review, see Ref. 291); modulation of
channels is described in section III. A) ACTIVATION AND INACTIVATION. When a cardiac cell is
depolarized, an inward current is generated that rises rapidly and decreases afterward on a much slower time course. The rise is sigmoidal
(567) or exponential (685). The time to peak
shortens with depolarization (634). The decline in
macroscopic current or inactivation is best described by a sum of two
exponentials. The time constant of the first component becomes shorter
with depolarization. Peak current-voltage relation can be translated into a
conductance-voltage relation from which an activation curve can be constructed. Peak current is voltage dependent and increases for more
negative holding potentials. The relation has been explained as a
change in availability of the channel to become activated. Both
relations, activation and inactivation, can be described by a Boltzmann
distribution, with slopes of ~6 mV. Midpoints of voltages in
multicellular preparations are The time course and the steady-state properties of the current have
been explained in terms of two voltage-dependent processes, activation and inactivation independent from each other
(425). The opening and closing of the channel depends on
the movement of charged gates, and the hypothesis thus predicts the
existence of a gating current. In heart cells, such a gating current
has been measured for the activation process (50,
350, 372, 486) but not for the
inactivation process, as if inactivation is voltage independent. The
activation gating current, however, becomes smaller during the
development of inactivation. The gating charge becomes immobilized,
suggesting some coupling between the two processes (372,
486). Depending on the type of cell, coupling between activation and inactivation is variable. Coupling is strong in neuroblastoma cells (9) in which activation shows a
variable onset and inactivation follows rapidly. In cardiac cells, the coupling is weak; openings always occur at the beginning of the depolarizing pulse (activation is fast) and their duration is variable
(inactivation is slower and variable) (69,
90, 567, 587, 856). Recovery from inactivation is normally very fast (1-10 ms), with rates
increasing upon hyperpolarization. After long depolarizations, recovery
can become very slow (order of seconds). The process has been called
slow inactivation (125, 160,
825, 876). The existence of this phenomenon
may be important to understand changes occurring during and after
ischemia, where the cells are subjected to long depolarizations. Is there more than one type of Na+ current? In heart cells,
decay of the Na+ current is normally very rapid. In a
limited range of potentials, where activation and inactivation overlap,
a small noninactivating current or window current can be recorded,
which is due to the cycling of channels between the rested, activated,
and inactivated state (27, 325,
755). This is not due to any abnormal behavior of the
channel, but in pathological conditions, the overlapping may increase,
resulting in an enhancement of the current. A slowly inactivating Na+ current, which represents a small
percentage of the total Na+ current, can be recorded in
rabbit (125) and canine (325) Purkinje fibers, in rat ventricular cells (826), and in expressed
human Na+ channels (801) over a broad range of
potentials. In the rat, it becomes more pronounced during hypoxia
(489) (Fig. 3). Part of it
does not inactivate completely. Compared with the fast Na+
current component, activation is shifted in the hyperpolarized direction. This may cause more pronounced overlapping of activation and
inactivation, generating in this way a constant steady-state component. The current deactivates immediately upon hyperpolarization. At the single-channel level, it is characterized by a bursting behavior, i.e., clusters of repetitive short openings (5 ms) sometimes alternating with long openings (order of 200 ms) (1187).
An activity characterized by short openings (<0.5 ms), becoming
shorter with depolarization, has been described as "background"
current because it carries current at the resting potential
(1187). The current undergoes inactivation, however, and
is very selective for Na+. According to Böhle and
Benndorf (90), one and the same channel can show many
modes of gating behavior; this channel may thus not be different from
the slowly inactivating one. The slowly inactivating current has been
described as being more sensitive to block by TTX (174), a
finding which has been advanced in favor of the existence of a
different isoform; the result, however, can also be explained by the
slow kinetics of the TTX block (126).
30 mV for the activation process and
85 mV for the inactivation process.
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Fig. 3.
Hypoxia increases persistent Na+ channel activity.
Voltage steps from 120 to 50 mV were applied to a cell-attached
patch from a rat ventricular myocyte. A: control.
B: during hypoxia, different cells. Bottom trace
in each series is average current of 50 traces. [Adapted from Ju et
al. (489).]
After exposure of membrane patches to lysophosphatidylcholine
(108, 1002), a persistent current can be
recorded over a voltage range of 150 to 0 mV. It is probably due to a
modulation of the fast component. The current does not deactivate upon
hyperpolarization and is substantial at the resting potential. It may
play an important role as an inward leak current, causing
K+ loss during ischemia.
A Na+-dependent, TTX-insensitive background current is
induced by high concentrations of ACh in guinea pig ventricular
myocytes (659). The current reverses at 25 mV in normal
Tyrode solution, and its single-channel conductance has been
estimated by noise analysis to be ~2 pS (897). Because
of its different pharmacological and single-channel
characteristics, it can be regarded as a different isoform.
B) ION PERMEATION. In the presence of 150 mM
[Na+]o, single-channel conductance is
20-25 pS; it is dependent on the concentration of external
Na+ with a dissociation constant
(Kd) of 300-400 mM (885) and
shows different substates (554, 727,
856). The channel is permeable to Na+ and
Li+ and much less to K+ (9.5%) and
Cs+ (2%) or tetramethylammonium (<1%)
(885). A small Ca2+ permeability exists
(3) and may increase in the presence of ouabain and upon
-receptor activation (840). The current-voltage relation is linear in the absence of bivalent ions; deviation from this
linear behavior in normal bathing solutions is due to a
voltage-dependent block by [Ca2+]o and
[Mg2+]o; electrical distance is 0.3-0.4
(884). The block by
[Ca2+]o and [Mg2+]o
is characterized by a reduction in apparent single-channel conductance, suggesting a very fast block. Other bivalent ions efficiently but more slowly block the current: Cd2+ > Mn2+ > Co2+ > Ca2+ > Mg2+ > Ba2+. Of importance to
note is the highly sensitive block by Zn2+ and
Cd2+ (297, 1042); these
ions are much less effective in neuronal and skeletal muscle cells,
whereas the opposite sensitivity exists for TTX. Internal
[Mg2+] blocks outward current through the channel
(786). The block is slightly voltage dependent with an
electrical distance of 0.18 from inside.
At the molecular level, the channel consists of an 
-subunit (260 kDa) and two -subunits (36 and 33 kDa each) (291). The -subunit, which is sufficient for channel activity, is composed of
six transmembrane segments repeated four times in a tetrameric structure. The voltage sensor for activation has been related to the S4
segment. The short intracellular segment between domains III and IV has
been identified as the fast inactivation gate. Spontaneous deletion of
the KPQ segment in the junctional part gives rise to the LQT3 syndrome,
characterized by a slowing of the inactivation process and generation
of long action potentials. The absence or weak voltage dependency of
the inactivation process is consistent with the location of this
inactivation gate outside the electrical field of the membrane. Slow
inactivation involves conformational changes in the external pore
(C-type inactivation) (1058). The presence of sialic acid
at the external surface shifts the activation and inactivation in the
negative direction (64). Interaction with the cytoskeleton
modulates the inactivation and activation voltage dependence;
F-actin disruption and microtubule stabilization accelerate the
shift in the negative direction that occurs during whole cell recording
(636). The two -subunits exert a modulatory role, speed
up inactivation, and decrease block by local anesthetics
(633).
The permeation process is dependent on the hydrophilic part of the
-subunit between transmembrane segments 5 and 6. The selectivity for
Na+ is due to specific amino acids in this region, and
mutation of only two critical amino acid residues is sufficient to
confer permeability properties similar to the Ca2+ channel.
A cysteine in domain I is responsible for the high affinity of the
channel for Zn2+ and Cd2+ and low sensitivity
to TTX of the cardiac isoform.
2. Ca2+ channels
Different types of Ca2+-permeable channels have been described in the plasma membrane of heart cells: the L- and T-type channels, both voltage activated, and a background channel (see Ref. 673). They can be differentiated on the basis of their electrophysiological and pharmacological characteristics. The density of the T- and L-type channels differs in different sections of the heart. The ratio of T-type over L-type channel is highest in Purkinje and sinoatrial cells where it approaches the value of 0.2-0.6, and it is less in atrial and ventricular cells where the ratio only attains 0.015-0.025 (48).
A) BACKGROUND CA2+ CHANNELS.
Calcium-permeable channels are seen following incorporation of
plasma membrane protein fractions in bilayers (806). No
voltage steps are required for activation, and spontaneous
single-channel activity with long openings of >100 ms can be
recorded at negative Em values of 90 mV.
The conductance is 7 pS in isotonic Ba2+; the channels are
not blocked by dihydropyridines (DHP) and show no rundown. Their
selectivity is not pronounced with a permeability ratio
PBa/PCs of 10.
B) L-TYPE CA2+ CHANNEL. Calcium influx through the L-type Ca2+ channel is responsible for the upstroke of the action potential in the SAN and AVN and plays an important role in determining the plateau and eventual spike-dome appearance of the action potential in other cardiac cells. It is further responsible for the coupling between excitation and contraction, induces release of Ca2+ from the sarcoplasmic reticulum, and regulates intracellular Ca2+ load. In this way it determines activity of a number of mitochondrial and cytoplasmatic Ca2+-sensitive enzymes.
I) Kinetics. A) Activation and inactivation. Threshold for activation is around25 mV, and half-maximum activation is
attained at about 15 mV for most cells (see Ref. 673) and at more
positive potentials (3 mV) in the AV node (373). The
rise in current follows a sigmoidal time course, suggesting a multistep
process as the underlying mechanism. Activation is preceded by a gating current (50, 352, 486,
898). The density of the channels derived from the gating
current is much larger than the ionic current density, suggesting that
some of the channels although gating are not carrying current.
At the single-channel level, three modes of activity have been
distinguished (137, 403). In mode
1, the channel shows repetitive short (<1 ms) openings and
closures (0.2 and 2 ms), forming a burst of activity separated from
other bursts by longer closures. A number of consecutive bursts may be
grouped in a cluster. A variable waiting time precedes the openings; it
decreases at more depolarized levels and corresponds to the faster
activation and shorter time to peak values of the Ca2+
current. Mode 2 occurs in the presence of DHP agonists
(403) or after -receptor stimulation (137,
767) and is characterized by much longer open times.
Mode 3 is characterized by the complete absence of openings
or presence of only rare short openings. The frequency of this latter
mode increases with preceding depolarizations and corresponds to the
occurrence of steady-state inactivation.
The L-type Ca2+ current inactivates in two ways: a
voltage-dependent and a current-dependent way. The existence of
two types of inactivation explains the complex time course of current
decay and the presence of a dip in the inactivation curve.
Half-maximum steady-state inactivation occurs at 20 to 30 mV.
The curve shows a minimum at ~0 mV and increases again at more
positive potentials. When intracellular Ca2+ is well
buffered, this turning up is absent and the decay of the current during
a pulse is much slower. These observations have led to the conclusion
that inactivation is dependent on voltage as well as on
Ca2+ influx. The latter or Ca2+-induced
inactivation is the faster process, whereas the voltage-induced inactivation is rather slow. Activation and inactivation show a
remarkable overlapping (window current) (414,
900).
B) Intracellular Ca2+ or current-dependent
inactivation. Evidence for intracellular
Ca2+-dependent inactivation at the whole cell level is
based on the change in time course of current decay with changes in
[Ca2+]i (see Ref. 673) (Fig.
4, A and B). The
decay is faster the larger the Ca2+ current, and it is
slowed in the presence of intracellular Ca2+ buffers. At
the single-channel level (cell-attached patches), Ca2+
permeation through the channel reduces the open probability of subsequent reopenings of the channel and shifts the gating mode toward
a mode with long-lived closed states (454). In excised patches, with Ba2+ as the charge carrier, steady-state
elevation of Ca2+ in the range of micromolar concentration
or flash photolysis of Ca2+ reduces the open probability of
the Ca2+ channels. It is especially the Ca2+
originating from the sarcoplasmic reticulum (SR) that is responsible for the inactivation process during the first 50 ms of depolarization (949, 1154); at later times also
Ca2+ permeating through the Ca2+ channel
contributes to the inactivation (949). The increase in
cytosolic Ca2+ by the release from the SR is indeed 10-fold
greater than the Ca2+ entering the cell via the L-type
Ca2+ channel (915). In favor of this
explanation is the observation that inactivation is much slower after
depletion of the SR by caffeine or in the presence of ryanodine. It is
important to note that [Ca2+] seen by the channel may
importantly deviate from the cytosolic [Ca2+] as measured
by fluorescence techniques. Especially during the first 50 ms of a
depolarizing pulse the concentration seen by the channel may be much
higher. Such a difference may explain why the relative inhibition of
the current estimated from the Ca2+ transient is greater
during this initial period than later (611).
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1C-subunit; deletion eliminates
Ca2+-induced inactivation. The site is located near the
inner mouth but outside the electrical field (205,
719). The reduction of Ca2+ current by
[Mg2+]i also has been explained by a direct
binding to this site, reducing the number of functional channels
(1132). Because Ca2+ binding is a fast
process, it provides an explanation for the time course of inactivation
and the observation that Ca2+ oscillations are accompanied
by equivalent changes in current (911). Photolysis of
Ca2+ results in a rapid inactivation, within 20 ms
(351) to 75 ms (46); the process is not
accompanied by a change in gating current, confirming that
intracellular Ca2+-induced inactivation and
voltage-dependent inactivation are two distinct phenomena
(273).
C) Voltage-dependent inactivation. The evidence for
voltage-dependent inactivation is based on the following
observations (see Ref. 673). 1) Under conditions where
intracellular Ca2+-dependent inactivation is excluded
(e.g., current carried by monovalent cations or current carried by
Ba2+ or Sr2+), the current decays with time,
and the rate of decay is faster the higher the depolarization.
2) Inactivation develops when prepulses are applied that do
not result in Ca2+ inward current. 3)
Inactivation is present in channels incorporated in lipid bilayers with
Ca2+ buffered (806). 4)
Inactivation is slowed by trypsin treatment, but the intracellular
Ca2+-dependent inactivation is not affected
(862). No charge movement occurs on inactivation, but as
inactivation of the current proceeds, the charge movement that
accompanies activation becomes smaller (351,
898).
D) Recovery from inactivation: Ca2+-induced
facilitation. Upon hyperpolarization, the Ca2+ current
recovers from inactivation induced by a previous depolarization. Because inactivation is voltage and intracellular Ca2+
dependent, it is logical to expect repriming also to depend on these
two parameters. For the voltage-induced inactivation, the rate and
the degree of recovery is greater the more hyperpolarized the membrane,
with time constants in the order of 300 ms at 50 mV and 100 ms or
shorter at 80 mV. A much slower component (seconds) is present after
long depolarizations, indicating the occurrence of slow inactivation
(94, 868). This slow inactivation may play a
role in overdrive suppression (1074). Recovery from
Ca2+-induced inactivation as such is voltage independent
(911, 912) but indirectly it is modulated by
voltage, since the fall in [Ca2+]i is
dependent in part on the Na+/Ca2+ exchange that
is faster the more negative the Em.
At negative holding potentials, recovery from inactivation may
show an overshoot, i.e., the Ca2+ current transiently
becomes larger than in steady state. The original finding of an
overshoot was made in Purkinje fibers treated with digitalis
(505) but can also be observed without Ca2+
overload (see references in Refs. 606, 770). The potentiated Ca2+ current is characterized by a larger peak and a slower
time course of decay (991, 1159,
1193) (Fig. 4).
At the single-channel level, facilitation is characterized by an
increase in open probability (Po) with
a larger proportion of long openings (413) (mode
2) and an increase in number of functional channels
(1132). In all these approaches, it is clear that a
moderate increase in Ca2+ is required (991,
1159, 1193); the overshoot in recovery is inhibited by rising intracellular Ca2+ buffering, or by
ryanodine or using Ba2+ as the current carrier. Excessive
rises in Ca2+ lead to inhibition by
Ca2+-induced inactivation (351).
How elevated Ca2+ causes facilitation remains a matter of
debate. Phosphorylation of the channel protein is a possibility. Flash photolysis induces facilitation with a delay, and the effect is counteracted by inhibitors of protein kinases (PK) (18,
1159). Other groups, however, did not find an effect of PK
inhibitors (46, 1132), and facilitation still
occurred with nonhydrolyzable ATP analogs (1132) or even
improved (46). The conclusion of these authors is that a
Ca2+-nucleotide complex directly potentiates
Ca2+ current (ICa) through
a phosphorylation-independent mechanism. The existence of two
phases in the recovery (decrease followed by an overshoot) has
practical consequences in determining down- or upregulation of
ICa as a function of frequency and
diastolic Em.
II) Permeation and selectivity. The channel is 500-1,000
times more permeable to bivalent ions such as Ca2+ and
Ba2+ than to monovalent ions. The exclusion of monovalent
ions depends on the presence of a minimum concentration of bivalent
ions. In the absence of bivalent ions, the channel becomes highly
permeable to monovalent ions (651). Although restricted,
the permeability for K+ is responsible for a substantial
current during the action potential, the reason being that the
concentration of intracellular K+ is quite high compared
with the nanomolar free Ca2+ concentration. The
K+ contribution also explains why the reversal potential
(Erev) of the Ca2+ current
is much less positive than expected for the equilibrium potential for
Ca2+. The bivalent ion current through the channel
increases with the concentration and shows saturation. Compared with
the single-channel conductance in isotonic Ba2+ (8-10
pS and 15-25 pS, see Ref. 673), the single-channel conductance at
the physiological concentration of 1 mMCa2+ was found to be
surprisingly high at 7 pS (1162). The presence of negative
charges at the pore mouth of the channel, which attract bivalent ions
and increase their local concentration (331), is probably
the reason for this behavior. In the presence of both Ca2+
and Ba2+, the current across the channel is smaller than in
the presence of either Ca2+ or Ba2+ alone
(997). This kind of behavior has been called anomalous mole fraction behavior and suggests a multi-ion channel. Different ions thus interact in such a way that the flux of one species is
hampered by the presence of the other species. Multi-ion occupancy also explains the high conductance and at the same time the high selectivity. High selectivity is conditioned by high affinity; high
conductance by the presence of more than one ion in the channel and
repulsion of one ion by the other. The multi-ion nature of the
channel also explains the flickery block behavior of the channel in the
presence of elevated proton concentration (784).
The channel can be blocked by a number of extracellular bi- and
trivalent ions such as Mg2+, Mn2+,
Co2+, Ni2+, Cd2+, Zn2+,
and La3+ (997). The ions Cd2+,
Zn2+, and La3+ block the channel in a
voltage-dependent way with an apparent electrical distance of 0.15 for Cd2+ and Zn2+ and 0.60 for
La3+. Intracellular Mg2+ is needed to activate
enzymes that phosphorylate the channel but may, on the other hand,
reduce the current that has been augmented by isoproterenol or BAY K
8644 by a direct blocking action and by activating phosphatases
(1090, 1132). The cAMP-dependent
phosphorylation reduces the sensitivity to
[Mg2+]i block (1131).
The cardiac Ca2+ channel molecular structure consists of
four subunits: two -subunits, 1 and
2, a -subunit, and a 
-subunit (a 
-subunit
is exclusively expressed in skeletal muscle). The -subunit is
sufficient to express channel activity. It resembles the
Na+ channel with four times six transmembrane segments, a
highly charged S4 segment that probably acts as the voltage sensor for activation, and an intracellular link between domain II and III responsible for inactivation. Trypsin treatment removes
voltage-dependent inactivation but not internal
Ca2+-dependent inactivation (862). Binding of
Ca2+ to the COOH terminal is a possible mechanism for
Ca2+-induced inactivation (205).
The highly conserved glutamate residues located in the pore region of
all four repeats are involved in high-affinity bivalent binding
(1146). A mutation from E to Q in domain III has shown that this group is the strongest determinant of Ca2+
binding (331).
The function of the -subunit is markedly modulated by the
-subunit. Coexpression of the two results in a fourfold increase of
peak current, which is not due to a change in single-channel conductance (331) but to a marked increase in density of
functional channels (488, 718,
759). The gating current remains unchanged, but coupling
between conductance and gating is improved (759).
The L-type Ca2+ channels is highly regulated; this
aspect is analyzed in section III.
C) T-TYPE CA2+ CHANNEL. A Ca2+ current of short duration is activated at potentials more negative than the threshold for the L-type Ca2+ current (48, 726; see review in Ref. 1026). The current is well represented in SAN cells, atrial cells, Purkinje cells, and nodal cells. In the embryonic chick ventricle, it is the major Ca2+ current. The current is not found in human atrium (258, 606) or human ventricle (76). The channel has been proposed to play a role in pacemaking. It may also interfere with steroidgenesis, cell proliferation, and cardiac growth (400).
I) Activation, inactivation, and repriming. Threshold for activation is around70 to 50 mV and maximum activation
is seen at 30 to 10 mV (see Ref. 1026). Inactivation is rapid and
complete, with time constants of 30 ms at 50 mV, becoming shorter at
more depolarized levels. This behavior is opposite to the L-type
channel, where inactivation is decelerated at positive potentials.
Steady-state inactivation extends from 85 to 40 mV with half
maximum around 60 mV and slope of 5.5 mV (14,
36, 412). An increase in [Ca2+]i does not induce inactivation but
rather facilitates the T-type current (13,
994). At the single-channel level, this increase in
current is characterized by a shift to long openings (mode 2 behavior).
Repriming is voltage dependent and becomes faster with
hyperpolarization: 250 ms at 70 mV and 100 ms at 90 mV
(145). It is slower the longer the preceding
depolarization, suggesting the existence of slow inactivation
(412).
II) Permeation. In 100 mM [Ca2+], the
single-channel conductance is 8 pS, compared with 20 pS for the L
type. Contrary to the behavior of the L-type channel, permeability
for Ca2+ and Ba2+ ion is the same
(48, 900). The channel is permeable to
Sr2+, blocked by Ni2+, but much less sensitive
to Cd2+. Extracellular protons inhibit the channel with
greater efficiency than the L-type current, whereas intracellular
protons have no effect (1000). Extracellular
Mg2+ reduce the current and shift the activation and
inactivation curves in the positive direction (1113).
Recently two isoforms, the G and H isoform of the -subunit, have
been cloned (760). Sialic acid probably forms an important component of the extracellular part of the channel (1150).
3. K+ channels
Because the equilibrium potential of K+ is rather negative, all cardiac K+ channels when activated will carry outward current, repolarize the membrane during the action potential, or stabilize the membrane at a hyperpolarized level. Among the many K+ currents, distinction can be made between voltage-activated currents (Ito, IKur, IKss, IKr, and IKs), ligand-activated currents (IKACh, IKATP, IKNa, and IKAA), and a current (the inward rectifier IK1) that apparently does not gate and can be called a background current. Under physiological circumstances the voltage-activated K+ currents, IKACh among the ligand-activated and IK1, play an important role in shaping the normal action potential. Under ischemic conditions, ligand-activated currents, especially IKATP and IKAA, become primordial, whereas some of the "physiological" currents are inhibited. Voltage-activated K+ currents show activation and inactivation upon depolarization; the rates of these two processes can vary from fast to ultra-slow. Ligands can bind to receptors, which then activate the channel via a G protein, or can interact directly with an intracellular site of the channel.
The amino acid composition of most K+ channels is known (201), and recently, the molecular structure of the pore has been elucidated from X-ray analysis of the crystallized molecule (234). The channels show a remarkable homology with the Na+ and Ca2+ channels. However, whereas Na+ and Ca2+ channels consist of tandemly linked four domains of six transmembrane segments that are connected in one long polypeptide, only one domain with six transmembrane segments is found for the K+ channel. Segment 4 shows a high density of positive charges and acts as potential sensor. Two types of inactivation have been described in expressed channels (794): N-type inactivation in which the negatively charged NH2 terminal acts as a ball and blocks the open channel and C-type inactivation (COOH terminal) in which conformational changes on the extracellular side close to the pore result in some kind of constriction. C-type inactivation is sensitive to drug binding and extracellular K+. The ligand-activated and background channels have a simpler structure and contain only two transmembrane segments. Recently, a new family of K+ channels with two pore segments in tandem and four transmembrane segments have been expressed. They act as background channels (530); some of them are activated by arachidonic acid and polyunsaturated fatty acids (280). A tetrameric structure for all K+ channels is highly likely. The pore in K+ channels is formed by a stretch of 19 amino acids in the link between S5 and S6 in the four repeats. The motif GYG (or of GFG) in the P-region is the signature of K+ selectivity, but other residues also participate in determining K+ selectivity.
A) K+ OUTWARD CHANNELS WITH FAST ACTIVATION. Upon depolarization, three different K+ currents are rapidly activated. They can be distinguished by their rate of inactivation, which is relatively fast for Ito, slow to ultraslow for IKur, and nonexistent for IKss, also called background current. On the latter current, no detailed information is available at the present time.
I) The fast transient outward current. The fast transient outward current (Ito) is a transient outward K+ current that is rapidly activated and inactivated and blocked by millimolar concentrations of 4-aminopyridine (4-AP). The criterion of 4-AP sensitivity is not exclusive. In some species, another K+ current, the IKur, is also blocked even by micromolar concentrations of the drug (see below), and in the dog ventricle part of the Ito is insensitive to millimolar concentrations (607). It should be distinguished from another transient outward current carried by Cl and
activated by [Ca2+]i; this current is
also called Ito2. In this review it will be indicated as IClCa.
Ito is partly responsible for the initial fast repolarization or phase 1 during the action potential. The density
of Ito varies among species and in a
particular species it varies in different parts of the heart (see Ref.
117). It is more expressed in the atrium and Purkinje fibers and in the ventricle more in the epicardial than endocardial fibers. The density
of Ito in the heart increases after birth
(259, 483, 639), although its
presence is variable (191, 342), an
observation which is possibly related to pathological downregulation.
A) Activation and inactivation. The current is activated upon
depolarization. Values for time course and steady-state vary among
species and experimental conditions. In the rabbit, time course is fast
and monoexponential (269); in the ferret it is sigmoidal
(118). Midpoint voltage values for steady-state
activation vary between 10 mV in the rabbit (269) and
+20 mV in the ferret (118).
|
50 and 15 mV (see Ref. 117) (Fig.
5A). Recovery from inactivation is very sensitive to
voltage, being faster the more hyperpolarized the membrane. It is also
facilitated by increasing [K+]o
(284); this supports the hypothesis that inactivation is
of the N type (750). For C-type inactivation, the
process is slowed by extracellular K+ acting as a "foot
in the door."
Actual time constants for recovery vary with species. In most species,
including humans (15, 284, 342),
recovery is fast with time constants in the order of 20-60 ms at 80
mV; frequency dependence is small (Fig. 5B). In rabbit
atrium and ventricle, in sheep and dog Purkinje fibers (see Ref. 117),
and in human subendocardial fibers (708), recovery is slow
to very slow (time constants of 1-6 s). In these latter preparations,
the current is markedly reduced (272) and shortening of
the action potential markedly less at elevated frequencies
(512).
B) Permeation. On the basis of measurements of
Erev (25, 118,
158, 416, 707, 708,
1088), the Ito current is
considered to be mainly carried by K+, although it seems
less selective than other K+ currents, such as
IK1. At the single-channel level, the
current-voltage relation is linear and single-channel
conductance in 145 mM [K+]o is on the
order of 10-30 pS (63, 158,
717) with the exception of a much lower value of 3-4 pS
for ferret ventricle (118). The conductance increases at
elevated [K+]o, with a
Kd of 200 µM (284).
As molecular substrates for Ito, Kv4.2
(922) and Kv4.3 (229) have been proposed. On
the basis of differences in voltage dependence, kinetics of
inactivation and recovery, and block by 4-AP, Kv4.2 is the better
candidate for Ito in the human, rat, and
ferret, and Kv4.3 in the canine and human subendocardium. Kv1.4 may be
the better choice for the sheep and the rabbit. In the rat, expression
of the protein shifts from Kv1.4 to Kv4.3 after birth and during
thyroid treatment (1094).
II) IKur. A rapidly activated K+
current, with no or very slow inactivation is present in different
heart preparations. The voltage dependency of the slow inactivation
process and the sensitivity to 4-AP is species variable, and on this
basis, it can be concluded that the current does not correspond to a
unique channel. On the basis of the sensitivity to 4-AP, the currents
can be subdivided into two groups. In the human atrium
(15, 192, 388,
1065), dog atrium (1164), cultured rat
neonatal ventricle (347), and mouse ventricle
(287, 1182), IKur is
exceptionally sensitive to 4-AP and completely blocked by
concentrations of 50 µM or less. In rat atrium (97) and
ventricle (25) and human ventricle (707), the
current is insensitive to 4-AP. In many publications it is described as
a noninactivating component of Ito.
A) Activation and inactivation. On depolarization to levels positive to
40 mV, an outward current remains after subtraction of a rapidly
inactivating Ito in many species: rat
atrium (97, 1021), rat ventricle
(25, 347, 1083), human atrium
(890, 1065) and human ventricle
(707, 1087), rabbit ventricle
(272), guinea pig ventricle (1161), and dog
atrium (1164). It is rapidly activated and shows no or
only very slow decay. The current inactivates however. Inactivation has
been determined using long (tens of seconds) conditioning pulses.
Midpoint inactivation potential is variable: 70 mV (25)
and 90 mV (1083) in the rat and much less negative
values of 9 and 20 mV in the human atrium (284, 890, 1065) (Fig. 5A). In accord
with the existence of slow recovery from inactivation, the current is
markedly reduced at elevated frequencies in the rat ventricle
(25), rabbit ventricle (272)), and human
atrium (272, 284, 890) (Fig.
5B). In these preparations, a rest current
(IKss) remains, which seems different from
IKur. It is reduced by -receptor
stimulation in rat atrium (1021) and by -receptor
stimulation in rat ventricle (849).
B) Permeation. IKur is assumed to be
carried by K+, but direct demonstration is mostly lacking.
In human atrium, tail currents reverse at negative potentials,
suggesting a predominant (388, 1065) but not
exclusive (191) permeability to K+. In favor
of the K+ nature is the observation of sensitivity to
tetraethylammonium (TEA) in rat ventricle (25) and dog
atrium (1164) and block by Ba2+ in guinea pig
ventricle (33, 1161) (in humans the current
is not sensitive to Ba2+). Single-channel conductance
in 5.4 mM [K+]o is in the order of 14 pS
for the guinea pig ventricle (1161) and 20 pS for the dog
atrium (1164) and is sensitive to
[K+]o. Fully activated
current-voltage relations show outward rectification (388, 1065).
The Kv.1.5 protein is a possible molecular candidate for the
IKur current in the human atrium
(271, 923, 1065). It shows a
high sensitivity to 4-AP (1067) and a limited, partial
inactivation at positive potentials but is insensitive to TEA. The
single-channel conductance is 17 pS. The protein is present in the
rat and the human atrium and ventricle, as determined by
immunolocalization; it is highly concentrated in the intercalated disks
(669). In the dog, Kv3.1 has been proposed as a molecular
candidate (1164).
B) K+ CURRENTS WITH DELAYED ACTIVATION: DELAYED K+ CURRENTS. On the basis of kinetics, rectification, sensitivity to blockers, and modulation by intracellular messengers, two delayed K+ currents, IKr and IKs, can be distinguished (153, 838); IKr shows activation and inactivation, IKs only activation. Both are present in the human atrium (1066), human ventricle (77, 556, 605, 1033), guinea pig ventricle (838), guinea pig atrium (433), dog ventricle (324, 613) and atrium (1165), rabbit atrium (700) and ventricle (832), mouse neonatal ventricle (1055), and rat ventricle (141, 1115). Only IKr has been clearly described for the cat ventricle (290), the ferret ventricle (617), and rabbit SAN (1032) (466). IKs seems the only delayed current in the guinea pig SAN (22). Density of the two currents varies in different layers of the myocardial wall. In midmyocardium of the dog ventricle, expression of IKs is small (613); this explains the longer action potential in these cells. In the ferret ventricle, the ERG protein (Kr) is most abundant in the epicardial layers (99).
I) Rapid delayed K+ current, IKr. A) Kinetics. IKr activates rapidly for depolarizations positive to40 mV, with a midpoint voltage between
20 and 5 mV; this value is [K+]o
independent (889). Time constants of activation vary among species; in the guinea pig, they are in the order of 175 ms at 30 mV
and shorten at more positive or more negative potentials to ~50 ms
(838). In the rabbit ventricular cell, time constants for
activation are longer, 500 ms at 40 mV to less than 100 ms at 0 mV
(128). Compared with IKs,
these time constants are shorter, a finding on which the distinction of
the two currents has been based. Deactivation does not follow the same
pattern. It is fast in the guinea pig but much slower in the rabbit
(128) and in the dog (324); in these
preparations, the time course is composed of at least two exponentials,
of which the first one is on the order of 0.5 s and the second on
the order of 5-10 s at 40 mV (129, 324).
At the single-channel level, the mean open time is on the order of
3 ms. Closed time distribution is biexponential with values of 0.6 and
22 ms (at 100 mV and 100 [K+]o)
(889).
In whole cell recordings, the time course of the macroscopic current
shows saturation with no indication of a secondary decrease. The tail
currents on hyperpolarization, however, are preceded by a "hook,"
and the current temporarily increases before it declines in an
exponential way (838, 889). The initial
increase in outward current has been interpreted (889) as
due to recovery from inactivation, a process supposed to be faster than
the deactivation process. The hypothesis implies that the current
during depolarization very rapidly undergoes inactivation, before there
is any substantial activation (Fig. 6).
It implies that steady-state inactivation extends over a voltage
range that is quite positive. The consequence is that the current
rectifies in the inward direction. Inactivation preceding activation
has also been demonstrated in the expressed HERG channel
(836, 932, 1141), which has been
shown to be responsible for the IKr
current. Fast recovery from inactivation is the reason for an increase
in the number of openings of the Kr channel upon repolarization during
early diastole in nodal cells (466, 1033).
|
13 mV (666) to 26 mV
(38). Kinetics are slow; the time course of the rise in
current is sigmoidal, whereas the decay of the tails is monoexponential
at voltages negative to 50 mV but biexponential at more positive
potentials (605, 666). Deactivation is slow in the guinea pig but relatively fast in the dog and the rabbit (128, 324, 613). At the
single-channel level, kinetics are complex with many open and
closed times (38, 242).
B) Permeation. The channel is less selective than
IKr. The Erev
is more positive than that for IK1 and
changes only by 49 mV for a 10-fold change in
[K+]o (666). The fully
activated current-voltage relation approaches linearity, except for
the current in frog atrial cells where inward rectification is present
(243).
Single-channel conductance is relatively low with estimations of 5.4 pS
in guinea pig ventricle (38), 3 pS in guinea pig atrium
(433), and a greater value of 20 pS in frog atrial cells (242).
Extracellular K+ has no direct effect on the conductance
but affects the current through changes in the chemical gradient; thus
in zero [K+]o, the current is greatly
increased, especially in Na+-free conditions
(850). Cobalt (1 mM) (265) and
La3+ (at 100 µM or higher) (38,
837) block the current. A rise in
[Na+]i or
[Ca2+]i enhances
IKs (978, 728).
C) Molecular structure. Coexpression of the minK and the Kv.LQT1
generates a current with the characteristics of the cardiac IKs (39, 835).
The KvLQT protein has the classical constitution of
voltage-activated K+ channels. The minK protein
consists of only 129 or 130 amino acids and a single putative
transmembrane domain, with the NH2 terminal turned to
the external side of the membrane (956,
1023). It plays an essential role in the function of
Kv.LQT1 and can be considered a regulator protein.
C) THE INWARD RECTIFIER. The inward rectifier current
(IK1) is the current responsible for
maintaining the negative resting potential in cardiac cells; it also
plays an important role during the final rapid repolarization during an
action potential (894). The density of the
IK1 is highest in the Purkinje and
ventricular system (445), less in atrium
(396); in the SAN, the IK1
current is absent (459). A substantial increase of the
current occurs during development from the neonatal to the adult stage
(487, 1049).
I) Activation-deactivation: inward rectification. Is
IK1 a background or voltage-activated current? The
IK1 current, the first K+
current to be characterized in cardiac cells, was considered initially
to be a time-independent background current. Its pronounced inward
rectification provided an explanation for the existence of a long
plateau in the cardiac action potential (see Ref. 133). With the
improvement of recording techniques, it became clear that
IK1 showed time-dependent changes,
which were analyzed as activation, deactivation, and inactivation
(569). Upon hyperpolarization from a holding potential of
50 mV to 100 mV, a quasi-instantaneous current jump is followed
by an exponential increase in inward current to a steady state
(385, 569, 982). On
depolarization, the reverse sequence is seen. This led to the
hypothesis that the IK1 channel opens and
closes by an intrinsic gating process not different from other
voltage-operated channels. More recently, the time-dependent
changes in the current or gating have been recognized to be generated
by a time-dependent block-unblock by Mg2+
(656, 1009) and polyamines (622,
1093). Magnesium and putrescine ions are responsible for
the very rapid phase, spermidine and spermine ions for the slower phase
(622, 723) (Fig.
7). The difference in rate corresponds to
the difference in positive charge (263,
1139). The block is voltage dependent, with an electrical distance of 0.3 for [Mg2+]i; polyamines
seem to penetrate deeper in the pore (622,
723). With time at the depolarized level, the block shifts
from fast Mg2+ to slow polyamine block. On
hyperpolarization, this is seen as an increase in the slower phase of
activation (463). This new concept of activation being an
unblocking is in accord with the information obtained on the molecular
structure of the inward rectifier family, in which a voltage sensor or
S4 segment is lacking. The IK1 molecule
consists of only two transmembrane segments with a H5 or pore sequence
in between (563, 954). The
IK1 channel can thus be considered a
background channel, and a distinction between gating and permeation
becomes less obvious. Whether on top of block-unblock there still
exists an intrinsic gating mechanism is not fully resolved, and recent
experiments on IRK1 channels expressed in oocytes have been explained
in this way (893).
|
D) LIGAND-ACTIVATED K+ CURRENTS. I) ACh-induced K+ current. Slowing of the heart beat is caused by activation of a specific K+ channel, the ACh-induced K+ current (IKACh), in the SAN (see review in Ref. 1095). In lower vertebrates and birds, the current is present in atrial as well as ventricular cells (381). In the mammalian species, the current is expressed in atrial cells, AV node cells, and Purkinje cells; in ventricular cells, the channel is not present in all species but has been described for the ferret (95), the rat (676, 1119), the dog (1142), and the human (558).
A) Activation. Activation of IKACh occurs upon binding of ACh to the M2 muscarinic receptor. The receptor is directly coupled to the K+ channel via a guanine nucleotide-binding protein or G protein, characteristically inhibited by pertussis toxin. Activation of the channel is due to binding of the-subunits to the channel as demonstrated in
isolated native (1126) and cloned channels (800). G binds to the COOH terminal of the channel
(456).
Activation of the G protein connected to the K+ channel is
also possible through stimulation of other receptors: adenosine (P1 receptors) (54, 926),
external ATP (P2 receptors) (665), somatostatin (602), calcitonin gene-related peptide
(522), endothelin (523), a serum
albumin-associated factor of phospholipid nature (105), and sphingosine-1-phosphate (1019).
The presence of an agonist, however, is not absolutely necessary, and
background openings of the channel have been observed in different
situations. Such activity has been related to the presence of ATP, of
increased [Na+]i, or of leukotrienes.
Leukotrienes act on the G protein and stimulate the exchange of GDP for
GTP (570). For activation by ATP, different mechanisms
have been proposed; local GTP formation from GDP through activation of
nucleotide diphosphate kinase (NDPK) (395,
492), an unknown effect of NDPK (1122),
phosphorylation (531, 930), which makes the
channel sensitive to [Na+]i
(943), or generation of phosphatiditylinositol
4,5-bisphosphate (PIP2) by ATP-dependent lipid
kinases (440).
B) Permeation and rectification. The channel is very selective for
K+. The conductance is highly sensitive to extracellular
K+ concentration. In symmetric conditions (150 mM
[K+]), the conductance is 40-44 pS. The open times are
very short (1-3 ms), occurring in bursts (829,
924). Open probability in human atria is very variable,
from 0.03 to 0.3 in the presence of 105 M ACh
(396).
At the whole cell level, the current is characterized by inward
rectification. The current activates on hyperpolarization and
deactivates incompletely on depolarization (731). The
phenomenon resembles the time-dependent changes in the
IK1 current, although they are of a slower
nature and smaller in amplitude. Similar to the
IK1 current, rectification is explained by
block of the open channel by intracellular Mg2+ and
polyamines (1127). The IKACh
is blocked by classical K+ channel blockers such as
Cs+ and Ba2+ (130,
698, 1170).
Fade or desensitization is the process by which the current through the
channel decreases, although the agonist concentration remains constant.
Upon washout of the agonist, the response to a second exposure remains
temporarily depressed (131). In the heart, fade is more
pronounced for the KACh channel and less for other channels coupled to
M2 receptors (ICa,
If, and IKs)
(95, 131, 430).
For relatively short exposures of ACh, fade occurs in two phases: a
rapid phase (up to 30 s) (524, 571)
occurs at the channel or G protein level followed by a slower one (up
to 3 min) in which the receptor is involved (1061,
1171). At the single-channel level (524),
the fast phase is characterized by a shortening of the open time from
4.3 to 1.1 ms; it is probably due to dephosphorylation of the channel
or of the G protein (903) or inhibition of the ATP effect
(531); the desensitization is heterologous and is not
restricted to a single type of receptor. The slower phase is
characterized by a reduction in the frequency of opening
(524) and is probably related to a phosphorylation of the
muscarinic receptor by a receptor kinase; this kind of desensitization
is thus homologous (1171). In accordance with this
explanation, the fast phase still occurs when the receptor is
short-circuited by acting directly on the G protein via guanosine
5'-O-(3-thiotriphosphate) (430) or arachidonic
acid metabolites (860), but the slow phase is absent.
A third phase of fade occurs when cells have been exposed to the
agonist for many hours. The underlying process in that case seems to be
internalization (106).
C) Molecular structure. From a structural point of view, a combination
of two inward rectifier channels is proposed to be responsible for
IKACh: the Kir3.1 and Kir3.4
(142, 563). Coexpression of the two proteins
in combination with the M2 receptor elicits an
activity that highly resembles KACh (562).
II) The ATP-inhibited K+ channel: KATP. The
KATP channel, first described in heart cells (729), is
present in many other cell types (465). In heart, it is
expressed in ventricular, atrial, as well as nodal cells from different
species, including the human heart (32). In heart cells it
seems to exert a protective role during an ischemic insult. By
shortening of the action potential, generation of inexcitability and
shifting the Em closer to the equilibrium
potential for K+, excessive loss of K+ is
avoided. Activation of the channel also seems responsible for
preconditioning or protection against a second insult.
A) Activation. In inside-out patches, a K+ channel with
a high conductance is activated when the cytoplasmic [ATP] is
decreased below a critical concentration. ATP normally decreases the
Po. The Kd
determined in inside-out patches is ~0.1 mM (540,
725, 729). This value should be considered a
mean value. For individual channels, the Kd
may differ by as much as three orders of magnitude and ranges from 9 to
580 µM (279). Hill coefficients vary between 1.0 and
5.0. Anionic ATP as well as MgATP are able to inhibit the cardiac
channel. The sensitivity to ATP is higher in the presence of free
Mg2+ (278). Kinetics of the single-channel
activity are complex with multiexponential distributions for open and
closed times and bursts (788). The burst duration is
inversely related to [ATP], and intraburst kinetics depend on
Mg2+ concentration (1186).
The Kd value is much lower than what is
considered the normal [ATP] of 5 mM or more. How is it then possible
to activate the channel? Two considerations should be made: the local
[ATP] seen by the channel can be lower than the bulk concentration,
and the sensitivity to block by ATP is variable.
1) The subsarcolemmal [ATP] that is seen by the channel is
much lower than the bulk value when the
Na+-K+-ATPase (781,
998) or the adenylate cyclase (435,
858) is activated, or when anaerobic glycolysis is blocked
(1081).
2) The sensitivity to block by ATP can be reduced by
acidosis, dinucleotides, an increase in lactate or decrease in taurine, congestive heart failure, an increase in
[Mg2+]i or
[Ca2+]i and polycations, receptor
activation, changes in the cytoskeleton, and, most important, changes
in PIP2 concentration at the intracellular side of the membrane.
Intracellular acidosis, although reducing single-channel
conductance (266), increases
Po as long as ATP is not totally depleted (193, 590). The concentration-response
curve is shifted to higher [ATP] (560). The presence of
Mg2+ is required and suggests a change in phosphorylation
of the channel (193). When acidosis is too pronounced and
the pH falls below 6.0, Po is markedly
reduced (560).
Lactate (366, 516) has an activating effect,
whereas taurine (364) and Cl
(215) reduce activity. The inhibitory effect of
Cl may be due to protein destabilization.
Dinucleotide and MgADP shift the inhibitory curve to higher [ATP];
the mechanism is not of a competitive nature, since both substances
bind at different sites (494, 729,
970). Adenosine 5'-diphosphate can also reactivate the
channel after rundown due to the absence of ATP or presence of high
[Ca2+]i. Dinucleotides thus exert
effects similar to K+ channel openers.
Higher concentrations of ATP are needed to block the channel when the
concentration of Mg2+ or Ca2+ and polycations
such as protamine, polylysine, and poly-arginine are small. These
cations are supposed to neutralize negative charges at the mouth of the
channel; in their absence, access of ATP to its binding site is
antagonized (212).
Activation is further facilitated by phosphorylation and G protein
interaction. Protein kinase C-induced phosphorylation
(439, 610) occurs after stimulation of
1, P1, P2,
M1, and M2 receptors (608, 619, 1062). A direct
coupling to the channel via a G protein is present for
P1 and M2 receptors
(469, 532, 540,
969).
The functional availability of the channel is dependent on the presence
of an intact cytoskeleton. Actin filament-depolymerizing agents
(cytochalasin and desoxyribonuclease I) accelerate rundown in excised
patches (968), whereas actin stabilizers (phalloidin) or
PIP2 (inhibitor of F-actin severing protein)
inhibits rundown (306). Disruption of actin filaments also
impairs the sulfonylurea inhibition of the channel (98,
1151).
Recently, a dramatic decrease in sensitivity to ATP of Kir6.2 expressed
channels has been described after addition of micromolar concentrations
of PIP2. The hypothesis is formulated that the COOH
terminal of the inward rectifier binds to PIP2 in the
membrane and keeps the channel activated unless [ATP] is raised to
the millimolar range (47, 904).
B) Permeation, conductance, and rectification. The ATP-dependent
channel shows a high conductance of 80 pS in symmetrical [K+] conditions (494,
729). It is very selective for K+, with a
Na+ permeability of only 0.01 relative to K+.
The conductance depends on [K+]o
(724). In physiological
[K+]o, the conductance falls to 25 pS
(277).
In the presence of high [K+]o, the
channel shows inward rectification. At normal physiological
[K+]o, however, the current shows no
rectification or even outward rectification up to 0 mV
(722, 934) (Fig.
8A). Outward rectification is
also characteristic for the current generated during metabolic inhibition (462), hypoxia (61), and in the
presence of potassium channel openers. Interpretation of rectification
in those cases should take into account the simultaneous inhibition of
the IK1 current. At positive voltages, a
weak inward rectification is seen, which is caused by Mg2+
block and not by spermidine or spermine ions (1127).
|
40
mV; the Hill coefficient is 1. The SUR molecules are characterized by multiple membrane-spanning domains and two
nucleotide-binding domains (NBD). They act as receptors for
sulfonylurea drugs and K+ channel openers. Activation by
MgADP also occurs at the SUR, probably at one of the NBD. Both ATP
(338) and GTP (989), on the other hand,
inhibit the channel by binding to the Kir6.2 subunit. The expression of
SUR-1 facilitates the incorporation of Kir6.2 in the plasma membrane of
HEK cells (484).
III) The Na+-activated K+
channel. The Na+-activated K+ channel
belongs to the ligand-activated channels and is activated by
[Na+]i. It was first described in
cardiac cells (497).
A) Activation by [Na+]i. The channel is
selectively activated by [Na+]i; other
substitutes such as Li+ are inefficient (497,
624). Relatively high concentrations of
[Na+]i, however, are required
(Kd 66 mM) (497). A similar
sensitivity has also been found by other authors (625,
804). The sensitivity to
[Na+]i is variable from one patch to
another and in the same patch for different channels. The
Po is independent of voltage between 100
and +60 mV and only declines for very negative potentials (834). The Po is greater in
cell-attached than in inside-out patches (804);
the response curve for [Na+]i is shifted
to lower concentrations, and maximum activity is increased
(804).
The channel's activity is characterized by two open states with time
constants in the order of 0.5 and 10 ms and three closed states with
time constants in the order of 0.5, 2, and 20 ms (497, 624). Sometimes the channel may enter a quiescent state
for many seconds.
B) Permeation. The KNa channel shows a very high conductance
(497, 625, 834,
1068) with values up to 200 pS. Its actual value depends
on the ratio of intracellular to extracellular [K+], on
the voltage, and on the presence of other ions, such as H+,
Na+, and Mg2+. Permeation shows
Goldman-type behavior with a current-voltage relation that is
linear, inward rectifying, or outward rectifying depending on the ratio
of intracellular to extracellular [K+] (625)
(Fig. 8B). Under physiological conditions, rectification is
outward and predicts the channel to carry large currents during the
action potential.
The channel shows many (up to 12) substates (834,
1068). An analysis of block by Mg2+ and
Na+ is not in favor of a multibarrel structure of the
channel (1068).
Selectivity of the KNa channel for K+ is high. The change
of the Erev with different K+
concentrations follows the predicted values of the Nernst equation (497, 625). Substitution of K+ by
Rb+ or Cs+ results in block of the channel
(625);
PNa/PK has been
estimated to be 0.02 (497). Increases in
[Mg2+]i and
[Na+]i (1068) cause the
channel to shift to a lower conductance state, especially at positive
potentials (625).
IV) K+ channel activated by fatty acids and
amphiphiles. Arachidonic acid, unsaturated fatty acids, and
phospholipids activate K+-selective channels in neonatal
rat atrial cells (527) and in adult rat atrial and
ventricular cells (528) (Fig.
9).
|
4. Cl channels
Four different types of Cl channels have been
decribed in cardiac cells (444): a channel activated by
PKA-dependent phosphorylation, which is probably identical to the
channel activated by PKC, a [Ca2+]i-activated channel, a
[ATP]o-activated channel, and a stretch- or
swelling-induced channel. All channels, except the PKA-induced Cl channel, are blocked by disulfonic stilbene compounds
(DIDS and SITS); the PKA-activated channel is blocked by
4,4'-dinitrostilbene-2,2'-disulfonic acid (DNDS). They show pronounced
outward rectification in symmetrical Cl conditions; this
rectification is much less present for the PKA-induced current. Two
channels have been cloned and expressed: the CFTR channel, which is
responsible for the PKA-stimulated channel (378, 596, 710), and ClC-3, which accounts for the
swelling-induced current (241).
Because the ECl is positive to the resting
potential, activation of Cl current causes depolarization
of the resting potential but accelerates repolarization early during
the action potential (237). The physiological role of
Cl currents, however, cannot strictly be delineated, the
reason being that the condition to activate the currents may only be present in physiopathological conditions: secretion of catecholamines for the IClPKA, cell swelling by osmotic
forces for IClswell, secretion of ATP in
the extracellular medium for IClATP. Even the IClCa that is activated by a
physiological processes, i.e., the release of Ca2+ from the
SR, does not seem to be expressed in all cells (absent in human atrial
cells, Ref. 604). The density and expression of the currents is very
variable; in general, IClPKA is better represented in ventricular cells and
IClswell in atrial cells.
A) PKA- AND PKC-DEPENDENT CL CHANNEL.
The PKA-activated Cl current (for review, see Ref.
308) is typically present in the ventricle, less in the atrium; it has been described for the ventricle of the guinea pig (34,
250, 660, 1012), rabbit
(especially in the subepicardium, Ref. 383), and cat
(1178) and in the human atrium and simian ventricle
(1071). It is not present in the rabbit SAN or atrium, the
dog atrium and ventricle, or the mouse and rat ventricle
(437).
-receptor stimulation (308). The
biochemical pathway involves phosphorylation of the regulatory domain
of the CFTR protein. This protein consists of two membrane domains each
of six transmembrane segments, two NBD and one regulatory domain, all
cytoplasmatic. -Receptors and histamine receptors (383, 447) are positively coupled to PKA; negative coupling
occurs via M2 (964), endothelin A
(475), and ANG II receptors (733). The nitric
oxide (NO) synthase pathway does not seem to be involved (1168). Activation via cAMP is facilitated by simultaneous
inhibition of tyrosine kinase (901) and previous exposure
to ACh (1167).
Phosphorylation results in a change from silent to active channels
(248) and modulates the kinetic behavior
(446, 448). The regulatory domain of the
channel can be partially or highly phosphorylated, each of these steps
corresponding to two types of activity. In a first phosphorylated
stage, the channel binds ATP at NBD1, followed by hydrolysis (this step
can be reversed by okadaic acid-sensitive phosphatase). With
Pi leaving the protein and ADP remaining on the NBD1
site, the channel is activated and shows brief openings; the channel
closes when ADP is released from the channel. A second phosphorylation
(reverse reaction is okadaic acid insensitive) makes it possible for
the molecule to bind a second ATP molecule at site NDB2. Binding of ATP
to this second site and its hydrolysis inhibits the release of ADP from site 1 and stabilizes the channel in the open state. The
activity is characterized by long openings (1 s) and closures of 0.2 and 10 s (250). As long as the hydrolysis products
stay on the protein, the channel is locked in the open state with a
Po close to 1.0. Egress of the hydrolysis
products suppresses the stabilization and causes channel closure.
Inhibition of phosphatase 1 and 2A by calyculin A increases markedly
activation of the channel by forskolin (1138).
II) Permeation and conductance. The single-channel
conctance is between 7 and 14 pS (710). In asymmetric
[Cl], the current-voltage relation is outwardly
rectifying for the whole cell as well as for the single channel.
Current can be carried by other anions, such as ATP, but the
conductance is small (871). In ischemia, it may be
responsible for the release of ATP from the cell. The permeability
sequence for small anions is Br > Cl > I (710, 744,
1012). The channel is slightly permeable to cations with a
PCl/PNa of
10-20 (149).
The channel is blocked by 9-aminoacridine (9-AC) and DNDS
(34, 382, 693) as well as
glibenclamide (981, 1133) but insensitive to
tamoxifen and DIDS and SITS (382).
III) Are PKA- and PKC-induced currents different
currents? Activation of PKC opens a Cl channel in
guinea pig (168) and feline ventricular myocytes (1178), which shows single-channel conductance (9 pS)
and kinetics identical to the PKA-activated channel
(168, 1178). The currents are not additive
and are blocked by the same substances, i.e., 9-AC and DNDS but not by
DIDS. The question remains whether the permeability sequence is
different (1052). The regulatory domain of the cloned
molecule contains consensus phosphorylation sites for both kinases. It
is interesting to note that in some preparations in which PKA is
ineffective in opening a Cl channel, activation occurs
via external ATP and PKC (500, 597, 664). In guinea pig ventricular myocytes, PKC activation
alone did not activate the channel, but it potentiated the channel
supramaximally stimulated by isoproterenol (683).
Activation of PKC may also be the mechanism underlying the occurrence
of a Cl current after stimulation of AT1
receptors by ANG II (693), since intracellular
Ca2+ is required to elicit the current.
B) SWELLING-INDUCED OR STRETCH-ACTIVATED CL
CHANNEL.
A volume-activated Cl channel is present in atrial
cells of the rabbit (357), guinea pig (902,
1012), dog (929, 992), and human
(745, 827) and in cultured cells of the chick
embryo (1176) and possibly in neonatal rat cells
(186).
> NO3 > Br > Cl > aspartate
(1012). Taurine and inositol efflux occurs upon cell swelling probably via this pathway (473). In the ClC-3
clone, one amino acid mutation (N579K) is sufficient to change the
permeability preference from I to Cl
(241). In neonatal rat, a channel has been described
(186) with a very large channel conductance of 400 pS and
a PCl/PNa of
25; this channel may be different from the other described above.
C) INTRACELLULAR CA2+-ACTIVATED CL
CURRENT.
A transient and intracellular Ca2+-dependent outward
current, carried by Cl, has been demonstrated in rabbit
myocytes (1192) and is called ito2. The current exists in rabbit Purkinje
cells (910), rabbit atrium (604), canine
ventricular (1191) and canine atrial cells (1165), ferret ventricular myocytes (985),
and cultured chick heart cells (618). It has not been
found in the human atrium (604).
current is activated by Ca2+ released from
the SR, subsequent to entry of Ca2+ via L-type
Ca2+ channel. Evidence supporting this hypothesis is based
on the observation that the current is prevented by block of the
L-type Ca2+ channel, block of the SR Ca2+
release channel, and intensive buffering of intracellular
Ca2+, whereas stimulation of Ca2+ influx by
isoproterenol has the opposite effect. Exposure to caffeine, which
releases Ca2+ from a preloaded SR store, elicits a current
even at negative Em values, suggesting that
the essential phenomenon leading to activation is the increase in local
[Ca2+] and not the depolarization (912).
Activation and inactivation curves have been described
(513); they should not be regarded as the expression of
voltage dependence of the Cl channel but generated mainly
by the voltage dependence of the Ca2+ current.
In Purkinje cells of the rabbit (910) and in ferret
ventricular myocytes (985), the Cl current
is much shorter than the intracellular Ca2+ transient and
already declines before the Ca2+ transient reaches its peak
value (Fig. 10). The rapid fall in current is not due to some type of accomodation or "inactivation." Steady activation with no time-dependent decline in activity is obtained in canine permeabilized ventricular myocytes exposed to
variable [Ca2+] (513); in inside-out
patches, single-channel currents did not show any decrease with
time (169). A possible explanation would be that the local
subsarcolemmal Ca2+ transient seen by the channel is much
shorter than the cytoplasmic Ca2+ transient. Subsarcolemmal
[Ca2+] as seen by the Na+/Ca2+
exchanger, however, does follow the time course of the intracellular Ca2+ transient (780). At the present time, the
simplest explanation is to assume that activation of the channel
requires an elevated [Ca2+] (high threshold), such that
the time course of the current is only determined by the short peak of
the transient. In Ca2+ overload, the Cl
current is prolonged and shows much slower kinetics, although the
duration remains shorter than the intracellular Ca2+
transient (747). It is important to note that the current
can be activated at negative potentials (912,
1191) and thus may play a role in the genesis of delayed
afterdepolarizations and arrhythmias.
|
> I > Br > Cl; the channel is,
however, impermeable to large organic anions such as aspartate (used as
substitutes to change the equilibrium potential). The
current-voltage relation has been described as outward rectifying
(1192) or linear in symmetric conditions
(910). In canine ventricular cells (169), the
single channel has a small conductance of 1.5-2 pS and shows long
openings. The apparent Kd is rather high,
150 µM; it should be mentioned, however, that [Ca2+]
seen by the channel can be very different from the bulk concentration. The current is blocked by SITS and DIDS (169,
910, 1192), by DIDS and niflumic acid
(594), and by glibenclamide (1133). No information is available on the structure.
D) EXTRACELLULAR ATP-ACTIVATED CL CURRENT.
I) Activation. Purinergic activation of a Cl
current has been demonstrated in atrial cells of the guinea pig heart
(664) and in ventricular cells of the mouse
(597) and the rat (500). Activation occurs
with ATP, adenosine 5'-O-(3-thiotriphosphate), nonhydrolyzable ATP analogs, ADP, and AMP. Adenosine activates the
channel in guinea pig but not in the mouse or rat. With respect to the
receptors, no clear distinction is possible between P1 and P2, since ATP, adenosine, and AMP were equally
active in the guinea pig (664). In rat ventricle, the
receptor seems to be P2 (500). The
channel is insensitive to internal cAMP or
[Ca2+]i. Activation is slow and takes
seconds, suggesting the intervention of an intracellular messenger.
Stimulation of phosphoinositol metabolism, with secondary activation of
PKC by diacylglycerol (1125) is a possibility.
5. Nonselective cation channels and the If channel
In the heart, a number of nonselective cation channels (NSC) are
activated by stimuli that also activate Cl currents, such
as a rise in [Ca2+]i, an increase in
[ATP]o, and stretch. Others are activated by oxygen
radicals and amphiphiles, by depolarization (sustained inward current
or Ist), and by hyperpolarization
(If current). Finally, a NSC current has
been described with no known activation mechanism; it is spontaneously
active and can thus be regarded as a background current. They all
differ by permeability and block characteristics.
Both Ist (only described in the sinus) and If (present in nodal, Purkinje, and plain atrial and ventricular cells) play an important role in pacemaking under physiological and pathological conditions. The other NSC currents seem only to be activated in pathological conditions (increase in [Ca2+]i, [ATP]o, stretch, radicals, and amphiphiles). Because these currents carry inward current at negative Em values, they cause the resting potential to be more positive than EK and thus favor K+ loss through K+ channels, of which the conductance is increased in ischemic conditions.
A) INTRACELLULAR CA2+-ACTIVATED CATION CHANNEL. Two types can be distinguished: one is activated by [Ca2+]i alone, and the other, in addition, requires depolarization.
I) First type. The first type has been described in inside-out patches (172), cell-attached patches (251), and whole cell recordings (650). A) Activation. Activation of the current occurs after exposure of the cell to Na+-free medium (251), high [Ca2+]o, low [K+]o solution (368), following intracellular injection of Ca2+ (650), application of caffeine, cell dialysis with solutions containing micromolar Ca2+ concentrations, and exposure to intracellular oxygen free radicals (472). The Kd for Ca2+ in inside-out patches is 1.2 µM with a Hill coefficient of 3. The channel activity is insensitive to large voltage changes (172, 251). B) Permeation. The conductance of the single channel is 30-40 pS in cultured neonatal rat cells (172) and 15 pS in the guinea pig (251). The current-voltage relation is linear and shows a reversal at zero or slightly negative Em values. The channel is equally permeable to Na+, K+, Li+, and Cs+ but excludes anions. Kinetics of the channel are voltage independent but largely determined by [Ca2+]i. At 10 µM, mean open times are 3.8 and 140 ms, and mean closed times are 1.8 and 15 ms (251). The long open time increases with [Ca2+]i while closed times decrease; sometimes openings of many seconds are seen. II) Second type. A second type of Ca2+-activated cation channel, characterized by completely different activation and conductance parameters, has been found by incorporation of sarcolemmal vesicles isolated from adult canine ventricle in lipid bilayers (410). The channel has not been found in cell-attached or inside-out patches, a result that could be due to the channel being situated in the clefts of the sarcolemma. The channel is activated by a combination of depolarization and rise in [Ca2+]i. In the presence of Ca2+ below 100 nM, the channel is activated for potentials at the cis-side (cytoplasmic side) positive to60 mV. The
Kd for Ca2+ is <1 µM, and
the Hill coefficient is 2. Kinetics can be described by two open times
(5.6 and 22 ms) and two closed times (9 and 29 ms). The
current-voltage relation is linear, and the channel is equally
permeable to Na+ and K+. The
PCa/PNa is 0.2.
B) EXTRACELLULAR ATP-ACTIVATED TRANSIENT CATION CHANNEL. I) Activation. A transient, rapidly desensitizing inward current is activated by [ATP]o in frog atrial myocytes (301), in rabbit SAN (899), in rabbit atrial cells and guinea pig atrial and ventricular cells (411, 752), and in rat ventricular myocytes (1180). The Kd for [ATP]o is 56 µM (301). Adenosine, AMP, and ADP are ineffective; channel activity is blocked by theophylline. On the basis of the action of agonists and antagonists, the receptor involved can be classified as P2x. The channel is not sensitive to [Ca2+]i (1180).
II) Permeation. The current-voltage relation is linear (49, 301) or slightly inwardly rectifying (411); the Erev is close to 0 mV, suggesting a nonselective permeability. Activation of the channel leads to elevation of [Ca2+]i (415). The current can be blocked by external Cd2+ (1180) but is insensitive to external Co2+ or Ni2+. A P2x receptor channel has been cloned from rat vas deferens (1008). Functionally, it resembles the cardiac channel activated by [ATP]o. The structure has similarities with the inward rectifier channel, and its single-channel conductance is 15 pS at100 mV. It is permeable to Na+, K+, and
Ca2+
(PCa/PNa is
4.8), Tris, and N-methyl-D-glucamine (NMDG)
(1008). Inward rectification of the expressed channel is
due to voltage-dependent gating and reduction in channel
conductance (1183).
C) STRETCH-SENSITIVE CATION CHANNEL. I) Activation. Stretch- or swelling-activated NSC currents have been described in rat atrial cells (526), neonatal rat cells (188), cultured chick hearts (815), guinea pig ventricular cells (841), and dog ventricular myocytes (162).
II) Permeation. The channel in rat atrial cells is permeable to monovalent cations and Ca2+ (PCa/PK estimated to be 0.9) (526) and potentially plays a role in generating Ca2+ overload and triggering atrial natriuretic factor secretion. The current-voltage relation is linear with a single-channel conductance of 21 pS (526). Gadolinium ions block the cardiac channel and the accompanying volume changes in dog ventricular myocytes (162) and inhibit swelling induced by osmotic gradients in rabbit ventricular cells (946). Negative results were obtained in the rat atrial channel (526). Stretch-induced increase in [Ca2+]i, probably due to activation of the stretch-induced channel, is blocked by streptomycin (313). A channel with a greater single-channel conductance of 120 pS, a preference of K+ over Na+ of 3.4, and impermeability to Ca2+ has been described in neonatal rats (188). The channel is insensitive to TEA (20 mM), 4-AP (1 mM), TTX (100 µM), or Cd2+ (1 mM).D) NSC ACTIVATED BY AMPHIPHILES.
In guinea pig ventricular cells (631) and in rabbit
ventricular cells (114), a NSC channel is activated by
amphiphiles. In the guinea pig, it shows a permeability sequence of
Cs+ > K+ > NMDG+ > Na+ > Ca2+. In the rabbit, the
current shows some inward rectification and reverses at 18.5 mV in a
solution containing a high Na+ concentration. It is
insensitive to TTX (10 µM) or Cd2+ (100 µM) but blocked
by La3+. Gadolinium blocks the channel in the rabbit but
not in the guinea pig.
E) NSC ACTIVATED BY OXIDATIVE STRESS. A NSC is activated by exposure of guinea pig ventricular myocytes to extracellular radicals; an increase in [Ca2+]i is not required (472). A similar current is activated by other oxidizing agents such as thimerosal and diamide (667, 881) and singlet oxygen (965). The channel activated by internal free radicals is blocked by Ni2+ and Gd3+ (472).
F) NSC BACKGROUND CHANNELS. An inward background current carried by cations has been described in SAN cells of the rabbit (356, 1038), in atrial and ventricular myocytes of the guinea pig heart (543), and in human atrial cells (191). In feline atrial myocytes, such a current is seen after addition of ACh (1062). In the SAN, the permeability sequence is K+ >> Rb+ > Cs+ > Na+ > Li+. The NMG ion is not permeant, and 10 µM Gd3+ partially blocks the channel (356). In atrial and ventricular cells, the presence of a background current causes the resting potential to be less negative than EK; in nodal cells it plays a role in pacemaker activity.
G) SUSTAINED INWARD CURRENT.
I) Activation, inactivation. In SAN cells of the rabbit
(346) and the guinea pig (345), an inward
current is activated on depolarizations from 80 to 60 mV and more
positive levels. In the potential range where spontaneous activity is
seen, the current appears as a sustained inward current. Inactivation
occurs slowly for relatively large depolarizations.
H) PACEMAKER CURRENT. The heartbeat in normal conditions finds its origin in the SAN. The electrical activity in the node is characterized by the existence of diastolic depolarization. Many currents participate in this process (730). One of the important currents, the pacemaker current If, is activated during the repolarization phase of the action potential and generates an inward current. The If current has been described primarily in SAN, AVN, and Purkinje cells (220) but is also present in atrial (124, 246, 779, 973) and ventricular cells (139, 1158). In the rabbit, the density decreases and the voltage dependence shifts in the negative direction from newborn to the adult stage (2, 803). The reverse shift occurs in primary cultures of rat ventricular cells and is accompanied by dedifferentiation (267). Recently, the channel protein has been cloned; the structure resembles that of other nucleotide-activated channels (318, 623).
I) Activation. Upon hyperpolarization of the SAN membrane from40 mV to more negative levels, an inward current is activated that slowly increases to a steady level (see Ref. 730) (Fig. 11). On return to depolarized levels,
the current is deactivated. Time constants are of the order of seconds
at depolarized levels but become shorter with hyperpolarization. In SAN
cells, the activation curve extends from 40 mV up to 100 mV, with a
half-maximum value at 60 mV and a slope of 10 mV. It is not
changed by [K+]o (293). In
Purkinje (115) and ventricular cells (1156),
the activation curve lies more negative than in the SAN. The difference in voltage dependence is translated in the diastolic depolarization occurring at more hyperpolarized levels. The difference in activation voltages may be due to differences in sympathetic tone and adenylate cyclase activity. In ventricular cells, the current may become important in pathological conditions as a consequence of the shift in
the activation curve, secondary to catecholamine secretion or to
dedifferentiation (267). Important modulatory processes by
neurotransmitters are described in section III,
A, D, and E.
|
10 to 20 mV, suggesting a
nonselective channel, which passes K+ and Na+
ions (420). Permeability to other cations such as
Li+, Rb+, and Cs+ is small or
nonexistent (217). In the range of diastolic potential, the current is inward and carried by Na+. At the
single-channel level, small currents have been recorded revealing a
conductance of only 1 pS (218) (Fig. 11). Because the
patches contained many small channels, information on the kinetics of
single-channel activity could not be determined.
The amplitude of the fully activated current is sensitive to
[K+]o (217,
646); an increase of [K+]o
enhances the inward current through the channel (293). In K+-free medium, the inward current is practically reduced
to zero. The channel still undergoes activation, and upon
depolarization a large outward current occurs. The voltage dependence
of the activation and the kinetics of the macroscopic current, however, are not changed with [K+]o, suggesting
no change in Po but a modulation of the
single-channel conductance. Selectivity for Na+ is
increased in low [K+]o (reversal shifts
in the depolarized direction) (420).
A rise of [Ca2+]i from
1010 to 107 M increases the amplitude of
the If current and shifts the activation
curve in the positive direction by more than 10 mV (354).
From a simple screening effect, the opposite result is expected; the
mechanism therefore is probably indirect (1172).
The permeation process is affected by the anion species in the
extracellular medium. Substitution of Cl by organic
anions results in a decrease of the macroscopic conductance without
change in reversal and without shift of the activation curve
(293). The channel is thus not permeable to
anions, but its conductance is modulated by the anion species.
The current is inhibited by Cs+ in a voltage-dependent
way. Block by 2-5 mM is efficient and complete at hyperpolarized
levels but incomplete close to the reversal (217). The
effect of Cs+ is asymmetric; inward current is specifically
blocked by external Cs+ but not by internal Cs+
(when [Na+]o is elevated )
(293, 420).
6. Electrogenic exchangers
A) NA+/CA2+ EXCHANGE.
The Na+/Ca2+ exchange (for review, see
Ref. 765) plays an important role in the regulation of
[Ca2+]i, in the
excitation-contraction coupling process (increase of tension as
well as relaxation), and in determining the time course of the action
potential (56, 247) and of the electrical
restitution following a stimulus (482).
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2ECa)/n 2, or in the
case of three Na+ for one Ca2+ as
ENa,Ca = 3ENa 2ECa.
Under normal resting conditions, the Erev
can be calculated to be approximately 30 mV (249).
Negative to this potential, Na+ is moving in and
Ca2+ out (forward mode) and inward current is generated;
positive to this potential, Ca2+ is moving into the cell
and Na+ is extruded (reverse mode), and outward current is
seen (409). The current-voltage relation shows outward
rectification. During the initial part of the action potential, the
carrier moves Ca2+ inward and favors repolarization. As
Ca2+ is released from the SR, the
Erev of the carrier changes (see Ref. 75)
with the result that the current reverses and slows repolarization. The
exchanger is blocked by Ni2+ and Cd2+
(423, 535).
III) Molecular structure. The amino acid sequence of the
transporter protein is known (see Ref. 765). The glycosylated
NH2 terminal is supposed to be in the extracellular
compartment. The large hydrophilic linker between transmembrane segment
5 and 6 represents the regulatory part; it contains an EF
Ca2+ binding site and the sites for Na+
binding. A section of this regulatory link acts as an inhibitor when
applied as a separate peptide (exchanger inhibitory peptide, XIP)
(152, 663).
B) NA+-K+ PUMP. The function of the pump is to transport Na+ to the outside and K+ to the inside of the cell, which are directions opposite to the passive movements of these ions. The pump, therefore, has to consume energy to make this movement possible. Experiments have shown that for each ATP molecule consumed three Na+ and two K+ are transported. This means that the mechanism generates an electrical potential but also that it is sensitive to the Em (for review, see Ref. 292).
Activation of the pump hyperpolarizes the resting Em or exerts a repolarizing effect during the action potential. These aspects are especially pronounced during high-frequency stimulation, because of the increase in [Na+]i that activates the pump. In the sinus node and Purkinje fibers, this hyperpolarizing effect counteracts the spontaneous diastolic depolarization and is known as overdrive suppression (see references in Ref. 1105). The density of the pump sites per square micrometer varies from 1,200 in the guinea pig ventricle (714) to 2,500 in the rat ventricle (941). The density is larger in the ventricle than in the atria (1054), and in the ventricle it is greater in the subepicardial than in subendocardial cells. The turnover rate is ~75-100/s (714, 941). I) Activation by extracellular K+ and intracellular Na+. The Kd value for [K+]o is ~1 mM and for [Na+]i is ~10 mM, with Hill coeficients of >1.0. In Na+-free medium, the Kd for [K+]o decreases to 0.2 mM, suggesting competition between [Na+]o and [K+]o. The low Kd value for K+ means that the pump rate is maximally stimulated at physiological [K+]o values. The sensitivity to [Na+]i decreases at higher [K+]i, although the maximum rate is not affected. At physiological concentrations, the current is directly proportional to [Na+]i, which explains the sensitivity of the pump rate to frequency of stimulation. In spontaneously active cells, an increase in frequency will result in an enhanced outward pump current acting as a negative feedback on the rate of firing, a phenomenon called overdrive suppression. The increased pump activity plays a role in the shortening of the action potential and suppression of spontaneous activity with frequency of stimulation (1025). II) Permeation, voltage dependence, and Erev. The transport through the pump is electrogenic and voltage dependent. The amount of energy consumed increases with hyperpolarization. When the energy required to move three Na+ out and two K+ in equals the energy obtained on hydrolysis of ATP, the pump will stop. At a given Em called the Erev of the pump, these two values are equal. The Erev is thus determined by the free energy of ATP hydrolysis and the equilibrium potentials for Na+ and K+. Under normal conditions of [Na+]i, [K+]o, [ATP], [ADP], and [Pi], Erev is about180 mV (Erev = 
GATP/F + 3ENa 2EK).
The Erev shifts to less negative potentials
when [ATP] decreases and [ADP] and [Pi]
increase, changes that occur during metabolic inhibition. When
Erev approaches the
Em, the pump rate will drastically reduce
and eventually completely stop. These predictions have been verified
experimentally (328). G is normally 61.5 kJ/mol, but during ischemia it can fall to values of 49 or less
(190). Under those conditions, the pump will stop at
moderate Em values (around 60 mV).
Pump rate as a function of Em shows a broad
maximum extending from 80 to ~0 mV; it declines for more negative
potentials with an apparent reversal at very negative potentials around
180 mV. The pump rate also decreases at potentials positive to 0 mV (85). The voltage dependence with its positive and
negative slope varies with [Na+]o and
[K+]o. When
[Na+]o is reduced, the positive slope of
the current-voltage relation becomes less pronounced, i.e., pump
rate increases at negative Em values.
Calculation of [Na+]o dependence on
Em shows that the apparent affinity of the
transport protein for [Na+]o increases
with hyperpolarization (Kd decreases). The
negative slope at positive potentials can be changed in a similar way
by increasing [K+]o; in other words, the
apparent affinity for [K+]o decreases
with depolarization.
The voltage dependence of the pump has been explained by assuming that
part of the molecule acts as a ion channel (310). In
support of this hypothesis, gating currents have been measured (405). The voltage dependence is supposed to be due to
cation binding sites of the pump buried in the membrane and only
accessible from the extracellular side through a narrow channel. The
actual concentration of Na+ and K+ at the
binding sites will depend on the voltage gradient in this access
channel. They are greater the more negative the
Em, and the reverse occurs at more positive
potentials. The higher [Na+], the more difficult is the
release of Na+ to the extracellular medium. Also,
[K+] will be greater at negative potentials, but this may
not be effective if the concentration is already close to the
saturating level in control conditions. At positive potentials, the
actual [K+] will decrease and may fall below the
saturating value and thus reduce the pump rate.
The pump is blocked by digitalis, but sensitivity is species dependent
(84, 399); the rat is very insensitive with a
Kd of 2.4 × 103 M for
dihydroouabain (DHO) compared with 1.4 × 105 M in the guinea pig. Of theoretical but also
practical importance is the observation that the sensitivity to
digitalis decreases with increase of
[K+]o, and the opposite effect is seen
when [Na+]i is elevated
(942). DHO and K+ however do not bind at the
same site. An increase in [K+]o
increases the cycling of the enzyme between different states and
decreases the time that the external site is available for DHO binding;
an increase in [Na+]i, on the other
hand, will force the enzyme to a state where Na+ is
delivered to the outside medium and thus a state where DHO can bind
(84, 942). The antagonism between
[K+]o and ouabain is voltage dependent;
the degree of inhibition by a given concentration decreases at more
hyperpolarized potentials. This observation is consistent with the
hypothesis of a binding site for ouabain within the membrane electrical
field where the actual [K+] changes with
Em (84).
III) Molecular structure. The pump protein consists of one
- and one -subunit and is probably present as a dimer. Both have been cloned. The -subunit is a large protein of 110 kDa spanning the
whole membrane; it carries the ATP binding and phosphorylation site at
the intracellular portion and the digitalis binding site at the
extracellular face. Three isoforms exist, of which the 1 shows low affinity and 2 and
3 high affinity for ouabain (1054).
B. Ion Channels in Intracellular Organelles
1. SR channels
The SR membrane is a leaky membrane with a high conductance for
K+ and Cl A) THE CA2+ RELEASE CHANNEL OR RYANODINE RECEPTOR
(RYR).
In mammalian cardiac cells, the SR represents the store from which
Ca2+ is released during the action potential. In this
process a Ca2+-permeable channel in the SR plays an
important role. The Ca2+ released also activates the
Na+/Ca2+ exchange current, a NSC current, and a
Cl (see review in Ref. 677). The only
important gradient is that of Ca2+, whereas K+
and Cl seem to be equally distributed across the
membrane. The concentration of free [Ca2+] has been
estimated to be 700 µM (877). With a cytoplasmic
concentration of ~0.1 µM, this means a gradient of 7.000. The
Ca2+ Erev is thus >200 mV,
whereas the SR potential is estimated to be 0 mV. This means that the
large chemical gradient is not antagonized by an electrical gradient.
During Ca2+ release, the SR interior becomes negative but
at most a few millivolts because of the counterion movement of
K+ and H+ inward and Cl outward
(315). Two release channels have been described: the ryanodine receptor channel and the inositol 1,4,5-trisphosphate (IP3) receptor channel. current, which may all participate in the genesis of
early and delayed afterdepolarizations. In conditions of
Ca2+ overload, the channel thus plays an indirect but
important role in generating triggered activity. The channel is
specifically blocked by ryanodine.
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B) IP3 RECEPTOR. Although the role of IP3 is well established in smooth muscle and nonexcitable cells (72), the evidence that IP3 might play a role in the heart is circumstantial. Three aspects should be mentioned: 1) IP3 is released in cardiac cells, 2) IP3 receptors are present, and 3) IP3 causes release of Ca2+ (see review in Ref. 643).
1) Inositol 1,4,5-trisphosphate is released from the plasma membrane after activation of different receptors (M2, M1, P2, endothelin) (394, 775, 936, 1041). Release of IP3 is especially pronounced early during reperfusion, an effect mediated via activation of-receptors (17, 1107).
2) Inositol 1,4,5-trisphosphate receptors can be localized
by immunolocalization to the region of the intercalated disk
(696). Expression is greater in Purkinje fibers than in
other cells (335). Binding studies also found
IP3 receptors enriched in fractions containing the
intercalated disks with little or no binding in fractions containing
the longitudinal SR (521). Compared with RyR, the density
is 50-fold less. Expression of IP3 receptors is
increased in failing hearts, whereas that of RyR is decreased (643).
From a structural point of view, the IP3 receptor
shares considerable homologies with the RyR, although the molecular
weight is much less. The large NH2 terminal lies in
the cytoplasmic side with the IP3 binding site at its
end. The COOH terminal probably plays a role in activation because
antibodies that bind to this region can induce or inhibit channel
opening. Three isoforms have been isolated (252).
3) Inositol 1,4,5-trisphosphate causes Ca2+
release. Cardiac microsomes fused to planar bilayers exhibit channel
activity modulated by IP3. Four substates have been
recognized. The conductance is smaller (85 pS) than that of the RyR,
but the permeability series of bivalent ions is the same with a
permeability ratio of bivalent over monovalent cations around 6 (78).
Tension measurements have shown that caffeine contracture is amplified,
and Ca2+-induced tension oscillations are enhanced in
magnitude and frequency by IP3 (321,
1043, 1185). The effects of
IP3 vary and depend on the state of Ca2+
loading of the SR and the cytoplasmic [Ca2+]. The
influence of cytoplasmic [Ca2+] is biphasic; the release
for a given concentration of IP3 increases with
cytoplasmic [Ca2+], reaches a maximum at ~300 nM, after
which it begins to be inhibitory (79). Like the RyR, the
IP3R may become regeneratively active in conditions of
Ca2+ overload (374, 515). The
IP3 receptor is modulated by PKC, PKA, and
Ca/calmodulin (CaM) kinase II, MgATP, and pH (see Ref. 451). The
positive inotropic effect (775) as well as the
proarrhythmic effect (887) of -receptor stimulation has
been related to IP3 production. Aminoglycoside
antibiotics block release (236) and suppress ventricular arrhythmias.
C) K+, CL, AND H+ CHANNELS IN
THE SR.
Outside the Ca2+-release channel, K+,
Cl, and possibly H+ channels constitute
high-conductance pathways in the SR membrane. Because of their
presence, the membrane of the SR is clamped at about zero potential. In
this way, a large gradient forcing Ca2+ flowing from lumen
to cytoplasm is guaranteed. The flux of K+, H+,
or Cl as counterions facilitates the passive release as
well as the active uptake of Ca2+ via the
Ca2+-ATPase.
channel. In cardiac SR, a
Cl channel stays open at 0 mV. Its voltage dependence is
small (809) or nil (514). Gating of the
single channel is characterized by two open times and two closed times
(514).
The current-voltage relation is linear with a high conductance of
50-120 pS in cardiac cells (514, 809). The
channel is permeable to Cl, less to
SO42, and impermeable to gluconate
(363). In skeletal muscle, the channel is also permeable
to cations (Ca2+), with a sequence of
PCl/PTris/PCa = 1.0/0.5/0.3 (944). The existence of channels that are
permeable to anions and cations is not exceptional (see mitochondrial
voltage-dependent anion channel, Ref. 171).
Phosphorylation of the channel prevents the rundown of channels
incorporated in lipid bilayers (514, 809).
Phospholamban acts as a modulator (204), and its
phosphorylation results in activation of the channnel.
2. Mitochondrial channels
In the mitochondria, energy is transferred from different substrates to ATP by way of oxidation. Because one of the key reactions exists in building up a proton gradient, the inner membrane of the mitochondrion is supposed to be relatively impermeable and to show a permeability that is highly regulated by electroneutral carriers and uniporters. The outer mitochondrion membrane, on the other hand, has long been considered to act as a sieve that allows fast exchange of ATP, ADP, acetyl CoA, and other metabolites. This classic view has changed dramatically during recent years.
A) THE OUTER MITOCHONDRIAL MEMBRANE. Permeability of the outer mitochondrial membrane has been studied after isolation and incorporation of protein fractions in artificial lipid bilayers. Two different types of channels have been found (637). One type is cation selective, of which the function is not clear. Because it is blocked by targeting peptides, it may play a role in protein transport (620). The second type is represented by a voltage-dependent channel permeable to anions and more specifically to ADP and ATP (Fig. 2). Together with the adenine nucleotide carrier in the inner membrane, it plays an important role in the traffic of ATP and ADP between the cytoplasm and the mitochondrial matrix.
I) Voltage-dependent anion channel. A) Activation. After incorporation in artificial lipid membranes the voltage-dependent anion channel (VDAC) shows a maximum Po at zero Em that falls off but not to zero on application of either a positive or negative voltage gradient. At the same time, the conductance shifts to a substate, and the channel changes selectivity, becoming more permeable to cations. The obvious question is whether this type of voltage sensitivity has functional implications. Although a direct measurement of a potential gradient over the outer membrane has not been made, it is generally assumed that a Donnan potential exists based on the presence of a difference in concentration of large, charged colloids between the cytoplasm and the intermembrane space. Phosphorylation and Ca2+ binding (during a contractile cycle) may modify this potential gradient (807). The intermembrane space contains metabolic enzymes such as adenylate cyclase, creatine kinase, nucleoside diphosphate kinase, and deoxyribonuclease. Voltage gating is modulated by a modulatory protein and the NADH concentration. Sensitivity to voltage is increased in the presence of a modulatory protein (427) situated in the space between the inner and outer membrane. The increase in voltage dependence means that the channel closes for smaller changes in potential (0-5 mV) and becomes less permeable to anions (ATP, ADP). Voltage gating of the channel is further regulated by micromolar cytoplasmic concentrations of NADH that act as a ligand for the channel (1188). It is estimated that fluctuations of NADH could result in a sixfold change of the permeability to adenine nucleotides with consequent changes in the intensity of respiration (615). The VDAC should thus be considered a sensor of glycolysis, since cytoplasmic NADH is mainly determined by glycolysis. The closure of VDAC may be the mechanism by which glycolysis inhibits oxidative phosphorylation (Crabtree effect). In this connection it is of interest to note that the VDAC structure binds hexokinase and glycerolkinase. B) Permeation of the VDAC. The VDAC is permeable for molecules up to 1 kDa. It stays open with a very large single-channel conductance of 650 pS at zero Em and is slightly more permeable to anions than to cations (2/1). It allows passage of ATP and ADP. Permeability to anions ranges from 100/10/1 for Cl/succinate/ATP. The flux of ATP is high
(106 molecules ATP/s, Ref. 807). The outer membrane behaves
thus quite differently from the classic sieve but should be considered a highly regulated structure.
C) Structure of VDAC. The channel has been crystallized
(972) and forms groups of six transmembrane pores, each
pore consisting of a single 30- to 35-kDa VDAC polypeptide
(87). The polypeptide contains numerous stretches of
10-14 amino acids that show an alternating polar and nonpolar
character. Partial closure is supposed to be due to a positively
charged stretch of the molecule moving out of the membrane (gating
current, Ref. 925) and leaving the pore negatively charged and
permeable to cations.
B) THE INNER MEMBRANE. The study of the permeability of the inner membrane (see Ref. 538) has been possible by application of the patch-clamp method to mitoplasts (928). Mitoplasts are mitochondria stripped of their outer membrane. Under physiological conditions, the permeability of the inner membrane is low. In ischemic conditions, however, this permeability barrier can be weakened or completely vanish by opening of large channels. Five different channels have been described (Fig. 2): 1) a multiple conductance channel (MCC) or mega-channel permeable to large cations and anions, 2) an ATP-dependent K+ channel, 3) a 107-pS slightly anion-selective channel, known as the mCS channel, acronym for mitochondrial centopicosiemens channel, and two channels activated when the matrix side is exposed to very alkaline conditions: 4) a cation-selective 15-pS channel, and 5) an anion-selective 45-pS channel.
I) The MCC or mitochondrial permeability transition pore. A) Activation. Under normal physiological conditions, the channel is closed mainly because of the high [ATP] and low [Ca2+]. ATP is normally needed to maintain a low cellular [Ca2+] and a negative mitochondrial Em (908). The channel is activated by elevation of intramitochondrial [Ca2+] in the micromolar range, in combination with low [ATP] (189, 244, 952). Activation is facilitated by long-chain acyl CoA (771), pro-oxidants, and dithiol oxidatives (reversed by disulfide reduction), high O2 tension in combination with low [ATP], and high [Ca2+]; it is inhibited by H+, Mg2+, and the immune suppressive drug cyclosporin (953). It is also sensitive to drugs that bind to the benzodiazepine receptor. Typical during activation is the increase of conductance in steps. A possible underlying mechanism is the assembly of subunits into a large channel by the action of Ca2+ (953). The presence of a high conductance in the inner membrane poses a problem. Open pores spanning the inner membrane would dissipate the energy-transducing gradients and so uncouple oxidative phosphorylation (comparable to the effect of the uncoupling protein in brown fat cells). As intramitrochondrial [Ca2+] is normally low and matrix [ATP] high, the MCC is not expected to be activated under normal conditions. In ischemia or Ca2+-overload conditions, and especially upon reperfusion with high O2 tension restored, the channel may be activated and is probably responsible for the permeability transition. Activation is accompanied by depolarization (see Fig. 19) and release of the mitochondrial content, especially Ca2+ itself. This release of Ca2+ may trigger a Ca2+ wave from one mitochondrion to the other. The mitochondrion may still be operating in a low-conductance mode, and the activation of the MCC remains reversible; the mitochondrion acts as an excitable organelle (449). If activation is steady, however, the cell will die (244, 953). Pore opening disrupts mitochondrial energy transduction by providing a short circuit for protons, bypassing the chemiosmotic proton circuit that normally links the oxidation of substrates with phosporylation of ADP. Once uncoupled, mitochondria act as a drain for glycolytic ATP. The molecular nature of the mega-channel as well as all other inner membrane channels is not known. B) Permeation. The mega-channel has a variable conductance between 40 and 1,000 pS and is unselective to cations or anions. Large anions such as ADP and ATP can permeate. II) The ATP-dependent K+ channel. The ATP-dependent K+ channel is similar to the channel in the plasma membranes of many cells (458). It has the same conductance and the same activation pattern. It opens by reduction of [ATP] below a critical concentration. It is also blocked by glibenclamide and sensitive to K+ channel openers. The opening is facilitated by PKC-dependent phosphorylation (846). In contrast to the cardiac plasma membrane channel, it is sensitive to diazoxide and resembles more the pancreatic channel (316). Its role in mitochondria is not obvious; its activation may stabilize the mitochondrial Em and avoid activation of the mega-channel. A role in preconditioning (316, 619, 846) and in volume regulation has been proposed; during ischemia, mitochondria swell, and opening of large-conductance channels may induce efflux of solutes and water and reduce volume. III) The 107-pS anion channel or mCS channel. The 107-pS anion channel of mCS channel (928) is slightly anion selective. It is activated at positive matrix potentials, exponentially increasing with positivity. Its anion selectivity is poor (PCl/PK of 4.5). It is not pH sensitive but inhibited by oxidative uncouplers. Under normal conditions, the channel is thus closed. For its function, different hypotheses have been offered: 1) a function as uncoupling protein analogous to that in brown fat mitochondria, 2) a role in volume homeostasis, and 3) a function as import channel for proteins and mitobiogenesis. The majority of mitochondrial proteins are synthesized in the cytoplasm and hence have to be imported into the organelle; this requires a two channel assembly in tandem in the inner and outer membrane. 4) Recently, it has been proposed (37) to function as a protective mechanism against a fall in mitochondrial potential. Permeability under physiological conditions is supposed to be small but large enough to allow passive equilibrium for Cl. In a situation of metabolic inhibition when the
matrix negativity would normally drop, this process might be inhibited
by opening of the mCS channel clamping the mitochondrial membrane at a
very negative matrix potential.
IV) Alkaline-activated channels. When the matrix side of
a mitoplast preparation is exposed to very alkaline solutions, two channels can be observed: one is cation selective and shows a conductance of 15 pS and a second is anion selective and shows a
conductance of 45 pS. The role of these channels is unknown.
C. Gap Junction Channels
1. Introduction
Gap junctions are responsible for the syncitial nature of cardiac
tissue. Their high conductance and permeability allow for fast
conduction of the action potential (electrical coupling) and for an
efficient flow from cell to cell of molecules or metabolites with a
molecular mass up to 1.2 kDa (K+, Na+,
Ca2+, cAMP, cGMP, IP3) (metabolic
coupling). In the canine heart, each ventricular cell is connected to
11 other cells by way of gap junctions (824). Different
values for the conductance between cells have been published from 250 to 2,500 nS/cell. For a normal conduction of the action potential
between a pair of cells, ~35 gap channels seem to be sufficient
(1078). Redundancy is thus great. This contrasts with the
recent finding that mice heterozygous for connexin (Cx)43 null mutation
show a 45% reduction in conduction velocity of the action potential
(344). The distribution of gap junctions in the normal heart is nonuniform or
anisotropic (761). Gap channels are found almost
exclusively in the intercalated disks. Large intercalated disks exist
at the end of the cells, smaller along the length. A small number is present at the SAN-atrium junction and the Purkinje-muscle
junction. These observations explain why conduction in the transverse
direction or between the SAN and the atrium or between the Purkinje
system and the ventricular muscle can be critically reduced. In
culture, the number of junctions is smaller, which translates in
slower conduction of the action potential and absence of
propagated Ca2+ waves. 2. Formation of gap junctions and structural considerations
Gap junctions can be induced by forcing two single cells into
physical contact (1030). Precursors are present in the
plasma membrane from which cell-to-cell channels can form rapidly upon pairing. Analysis with different biophysical techniques has shown that
the channels responsible for the large conductance of the gap junction
membrane consist of two hemichannels or connexons in two apposing cells
(933). Each hemichannel is composed of six polypeptide
subunits or connexins. Subunits derived from different genes differ in
molecular mass varying between 26 and 50 kDa and in conductance between
40 and 160 pS. In the heart, three connexins are expressed: Cx40, Cx43,
and Cx45 (197). They show a different distribution and
differ in functional characteristics. Connexin43 is the most
abundant. Connexin40 is preferentially present in atria, nodal tissue,
and Purkinje system (501, 574). Connexin43 and Cx45 turnover quite rapidly (2-3 h) in cultured neonatal rat heart
cells (197). In the formation of gap junctions, hemichannels act as ligands for each
other. Of importance for this activation is the presence of six
cysteines in the extracellular loop of the connexin molecule. Docking
and/or opening of channels involves disulfide exchange. Each subunit or connexin is supposed to consist of four transmembrane
segments, M1 to M4. M3 is amphiphatic and probably part of the pore
structure. Amino acids at or near the NH2
terminal and the M1/E1 border form part of a charged complex that may
act as a voltage sensor. The COOH terminal together with the
intracellular loop between M2 and M3 form the so-called proton gate
(195). The gap junction channel is gated via two important mechanisms called
chemical and voltage gating. 3. Chemical and voltage gating
A) CHEMICAL GATING BY PROTONS AND CALCIUM IONS: A SLOW PROCESS.
Protons cause the gap channel to close (see Ref. 104) (Fig.
14). The mechanism is proposed to be a
ball-receptor inactivation process; protons increase the positive
charge of the receptor site and allow the negatively charged ball of
the COOH terminal to bind and to occlude the channel pore. Deletion of
part of the COOH terminal causes a significant fall in pH sensitivity
(695). In the cytoplasmic loop between M2 and M3, which
probably acts as the receptor site of the Cx43 gap junction, histidine
residues are present that can be titrated by protons
(933). The presence of positive charges at critical
positions in this loop leads to channel closure.
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Fig. 14.
Relationship between pH and junctional conductance
(Gj) measured in cardiac paired cells of
guinea pig. Most data were obtained in absence of free Ca2+
( 
). In 4 experiments, results were obtained in
presence of [Ca2+] (pCa 6.8 and 7.0). At these
concentrations, effect of pH was not different. [From Noma and Tsuboi
(732).]
B) VOLTAGE GATING: A FAST PROCESS. Gap junction channels in cardiac cells are sensitive to transjunctional voltage (691, 1053). Upon application of a voltage gradient, the instantaneous current-voltage relation is linear. With time, channels close however and the relation becomes S type, a process that can be compared with inactivation (1053) (Fig. 15). Such a process accelerates uncoupling during ischemia.
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4. Permeation
The gap junction shows a high permeability and passes substances with a molecular mass up to 1 kDa, including IP3 and cAMP (1030). The single-channel conductance decreases markedly for TEA and tetrabutylammonium. The ionic radius of these molecules is ~12 Å. This dimension may thus be close to the limiting pore diameter. Calcium ions easily permeate, but propagation of spontaneous Ca2+ waves, occurring under conditions of Ca2+ overload, is rather rare, with a probability of 0.15 (584).
Values for the single-channel conductance vary, and this variability may be related to the relative expression of the three connexins, the state of phosphorylation, and the voltage (111, 690, 955, 1031). Connexin43 has a conductance of 45-100 pS, depending on the phosphorylation state, is equally permeable to anions and cations, is highly permeable to dyes, but is insensitive to transjunctional voltage. Connexin40 shows a conductance of 120-160 pS, and its permeability to cations is about fivefold greater than to anions. Connexin45 has a smaller conductance of 30 pS, and its permeability to anions and dyes is limited; it is very sensitive to voltage (501). The current-voltage relation is linear, but rectification can be generated by expression of chimeras consisting of proteins with different voltage dependency (1040). If such asymmetry exists in native cells, it could play a role in unidirectional conductance.
For modulation by phosphorylation, see section IIID.
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III. ISCHEMIA SYNDROMES |
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Block of oxidative metabolism and fall in [ATP]/[ADP] causes important changes in ion concentrations ([K+]o, [H+], [Na+]i, [Ca2+]i, [Mg2+]i); disturbs lipid metabolism with accumulation of long-chain acylcarnitines, lysophosphoglycerides, fatty acids, and arachidonic acid; leads to the production of radicals, secretion of neurotransmitters, hormones, and metabolites, with concomitant stimulation of adrenergic, purinergic, and muscarinic receptors; and causes mechanical changes and stretch in the ischemic regions of the tissue. These changes have important modulatory effect on channels and carriers. In this section, this type of information is grouped in physiopathological topics, characteristic for acute ischemia. The analysis of each syndrome includes 1) a description of the changes, 2) the mechanisms involved, 3) the effect on channels and transporters, and 4) the final outcome at the electrophysiological and arrhythmia level.
A. Changes in Ion Concentrations
In this section we distinguish successively accumulation of [K+]o, intra- and extracellular acidosis, accumulation of [Na+]i, accumulation of [Ca2+]i, and depletion of [Mg2+]i.
1. Increase of [K+]o
A) DESCRIPTION OF EXTRACELLULAR K+ ACCUMULATION.
Under aerobic conditions, [K+]i is high
and [K+]o is low. Passive K+
efflux is compensated by active K+ influx via the
Na+-K+ pump. During ischemia, this dynamic
equilibrium is broken, and external K+ accumulates.
Potassium loss during ischemia typically occurs in three phases (Fig.
16A). Within 20 s after
the occlusion of a coronary artery there is a fast accumulation of
K+ in the extracellular space that reaches a plateau after
3-10 min and is followed by a third slower increase starting between 15 and 30 min (see Ref. 1099).
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Fig. 16.
A: increase of [K+]o
during ischemia in Langendorff-perfused quiescent and stimulated (4 Hz) rat hearts, measured with K+-sensitive electrodes
inserted in mid left myocardium. Note faster rise but lower plateau
during stimulation; effect on plateau has been interpreted as due to
activation of Na+-K+ pump. [Adapted from Wilde
and Aksnes (1099).] B: increase of
[Na+]i, measured by a floating
Na+-sensitive microelectrode, in a control group
( 
) and a group treated with a thrombin receptor
activator (SFLL 100 µM; ) after onset of ischemia in
isolated, blood-perfused rabbit papillary muscles. Stimulation of
thrombin receptors causes a larger and faster increase in
[Na+]i. [Adapted from Yan et al.
(1136).] C: fall in intracellular pH
(31P-NMR) during ischemia and fast recovery upon
reperfusion in perfused ferret hearts. [Adapted from Marban et al.
(640).] D: changes in free
[Ca2+]i (NMR with 5F-BAPTA) in perfused
ferret hearts during 20 min of ischemia followed by reperfusion.
Increase in [Ca2+]i is delayed compared
with changes in [K+]o,
[Na+]i, or pH. [Adapted from Marban et
al. (640).]
1, but not above this value (377,
418, 544, 1080). In the absence of stimulation, [K+]o accumulation
starts with a delay and its rate of accumulation is clearly less rapid;
an outspoken plateau is not present.
A delay is also obtained in the presence of -receptor blockade
(418, 1097); the plateau level, however, was
not different. Stimulation of the sympathetic nervous system on the
other hand causes a faster rise in [K+]o
(1101).
During the plateau, [K+]o is constant or
slowly changing in the positive or negative direction
(288, 418, 1101). The
preparation remains excitable or regains its excitability. When
stimulation is stopped, [K+]o falls,
suggesting an active Na+-K+ pump
(1080). During the plateau, catecholamines are massively released from nerve endings and cells (865), glycolysis is
stimulated, and lactate production is increased. The plateau level of
[K+]o is dependent on glycolytic
activity (1101). When the heart is depleted of glycogen
during successive ischemia periods, lactate production
(348) and acidification (288) are less, and
[K+]o rises faster.
The changes in [K+]o are far from
homogeneous (857). They are most pronounced in the center
of the infarct. Higher levels are present in subepicardium compared
with subendocardium.
When ischemia is continued over 20-30 min,
[K+]o starts to rise again. This phase
is accompanied by inexcitability, an increase of longitudinal
resistance, elevation of the resting tension, and development of rigor
contracture (135, 1079). Lactate production stops (281), and extracellular pH (pHo)
remains at a low level. The metabolic changes suggest that anaerobic
glycolysis is blocked.
Upon reperfusion, [K+]o recovers
completely and rapidly during the first rising phase or the plateau
(8). The [K+]o may even
fall transiently below the control level. Recovery is incomplete, and
[K+]o remains elevated when reperfusion
is started after 30 min of ischemia when the secondary rise of
[K+]o is already pronounced. Under those
conditions, the resting tension of the muscle may increase and often
shift into hypercontracture. At the same time, intracellular enzymes
are released, evidencing an irreversible increase in membrane permeability.
B) MECHANISMS RESPONSIBLE FOR THE CHANGE IN [K+]O. Three main factors contribute to accumulation of [K+] in the extracellular space (scheme 1): shrinkage of the extracellular space, decrease of active K+ influx, and increase of passive K+ efflux.
|
15
mol/s) would totally deplete the extracellular space of K+
(5 mM) in a very short time of 17 s (change in concentration is 18 mmol · l1 · min1; extracellular
space assumed to be 50% of the intracellular space or 10 pl/cell; [K+]o is 5 mM; 1 Coul = 105 mol). Even if the pump is only working at half the
maximal value and inhibition in early ischemia is only 50%, the change
in [K+]o will still be 4.5 mmol · l1 · min1, which is more
than needed.
Ischemic conditions make a moderate inhibition of the pump plausible:
LCAC (886) and oxygen free radicals (880)
have been found to reduce pump activity. Metabolic energy needed to
fuel the pump becomes deficient. It is not so much the [ATP] alone, however, that should be considered (a fall to 150 µM which is the
Kd value of the pump is probably an
unrealistic low value during early ischemia) but much more the increase
in [ADP] and [Pi] which cause a substantial fall
in the energy delivered from ATP hydrolysis or the phosphate potential.
Although the normal value is ~61 kJ/mol and the
Erev of the pump is 180 mV, the phosphate potential may drop below 50 kJ/mol, and the
Erev of the pump to 60 mV, which is about
the Em of the cells during the plateau of
[K+]o accumulation. The pump, in other
words, will stop functioning at the Em
expected under ischemic conditions (328). In accord with
the assumption of a partial block of the pump activity, recent measurements reveal substantial increases in
[Na+]i (see sect.
IIIA3) and internalization of pump
molecules by endocytosis in [ATP] depletion (M. J. Shattock,
personal communication).
3) There is an increase in K+ outward movement.
The first question is whether K+ loss, in the form of
potassium lactate or KCl cotransport, exists? On a molar basis, the
amount of lactate production is four times larger than the net loss of
K+ (502). If only part of the lactate movement
were linked to K+ efflux, this process could make an
important contribution to [K+]o
accumulation. However, the following observations are against a tight
coupling between K+ and lactate: 1)
K+ loss still occurs after blockade of anaerobic
metabolism, when lactate production is strongly reduced; 2)
K+ loss can be reduced by drugs, e.g., glibenclamide,
whereas lactate production is not changed (502); and
3) there is no evidence for an electrogenic transport of
lactate (892). When cells are exposed to lactate, the
intracellular medium becomes acidic, a process which is not accompanied
by any current under voltage-clamp conditions or is not affected by
a change in Em. Acidification was
saturable, partially stereospecific for L-lactate over
D-lactate and inhibited by monocarboxylate carrier
inhibitors, consistent with a carrier-mediated transport mechanism.
Part of K+ moves out of the cell via a KCl cotransport,
which is stimulated by an increase in cell volume as occurs in
ischemia. Inhibition of the KCl cotransporter by bumetanide causes a
decrease, and stimulation by ethylmaleimide an increase in
[K+]o accumulation (1136).
The quantitative contribution of this mechanism is unknown.
The next question is whether evidence exists for electrogenic
K+ outward movement.
Quantitatively electrogenic K+ movement can be expressed by
the product of the conductance to K+ and the
electrochemical gradient, according to the equation: IK = gK(Em EK). It tells us that net outward movement
will take place and increase when K+ conductance
(gK) is elevated, but only when the
Em is positive to the equilibrium potential
for K+ (EK). Evidence for the
existence of increased gK is given below. It should be stressed that an increase in
gK alone may be self-limiting because
it causes hyperpolarization and moves the
Em closer to EK. This is the reason why K+
loss during ischemia is less when the preparation has been pretreated with a K+ channel opener (499). As long as the
preparation is excitable, K+ loss occurs during the action
potential, and loss will be greater when gK
is increased. Computations have shown that, although the action
potential is shortened, net K+ efflux is enhanced (134, and
see Ref. 1099). This process is responsible for the initial phase of
K+ loss when the cells are still excitable. Later, when the
cell becomes inexcitable, the rate of K+ loss will be
dependent on the existence of an inward leak current.
Evidence for an increased inward leak current as well as an increased
K+ conductance during ischemia has been obtained on models
simulating the ischemic condition, because measurements of ionic
currents using the voltage-clamp technique cannot be done in real
ischemia. These models include multicellular and single cells exposed
to hypoxia, metabolic inhibition, elevated
[K+]o, and increased [H+].
In preparations exposed to hypoxia or metabolic inhibitors (see Ref.
134), an increase in outward K+ current has been measured.
The current reverses at the expected EK,
and its current-voltage relation is linear or even outward rectifying, over a broad range of negative and positive potentials. The
IK1 channel is an unlikely candidate, since
the IK1 current is strongly inward
rectifying, and inward rectification should even become more pronounced
in the presence of an increased [Mg2+]i
(652), [Na+]i, and
[Ca2+]i (654,
671). The IK1 current
furthermore was found to be decreased in the presence of
lysophosphatidylcholine (LPC) (161, 542), free oxygen radicals (542), and intracellular acidosis
(468).
A better candidate is the ATP-dependent K+ channel, and
a number of observations are consistent with this hypothesis.
1) Single-channel activity typical for the ATP channel
is seen in cell-attached patches during exposure of the cell to
dinitrophenol (DNP) (990) or cyanide (CN)
(729). A correlation in time is found between the
shortening of the action potential and single-channel activity (694, 725); shortening of the action
potential was reversed by intracellular injection of ATP
(963). 2) Glibenclamide, a blocker of the KATP,
reverses the outward current induced by DNP or hypoxia
(300) and the shortening of the action potential duration (317). Results on K+ efflux and
[K+]o accumulation, however, are less
convincing (1137).
A serious objection against the KATP channel being responsible for the
increased efflux is its high sensitivity to ATP block (Kd 100 µM), whereas the [ATP] during
the early minutes of ischemia remains at a sensibly higher level. The
following remarks should be taken into consideration. 1)
During ischemia, the sensitivity of the channel to ATP block is
decreased by the simultaneous rise in [ADP]i
(970), Pi, and lactate (366,
516); acidosis (193, 266); the
presence of free oxygen radicals (471) and extracellular adenosine (469, 540, 969); a
decrease in taurine concentration (364); and the existence
of stretch (1020). 2) The [ATP] in the subsarcolemmal space or the concentration seen by the channel may be
different from the bulk concentration. Block of anaerobic glycolysis, a
pathway important for local subsarcolemmal [ATP] together with
continued consumption of ATP by the
Na+-K+-ATPase and adenylate cyclase may
decrease the local [ATP] far below the bulk concentration
(435, 781, 858,
998). 3) Because of the large
single-channel conductance and the high density of the channels,
only a small proportion of the channels need to be activated to
generate a relative large increase in conductance or current
(725).
Two other candidate channels with outspoken outward rectifying
properties are KNa and KAA. The KAA is a channel with a conductance slightly larger than the KATP; its Po is
enhanced in acidosis (527, 528) and by
stretch (525), two conditions present during ischemia.
With respect to the possible role of the KNa channel, the problem is
similar to that of the ATP-dependent channel, the question being
whether the [Na+]i concentration can
reach the high values needed to activate the channel (497,
625). Recent measurements usuing the NMR technique show
substantial increases in [Na+]i. It is
furthermore important to realize that
[Na+]i close to the membrane can be much
higher than in the bulk; gradients and local accumulation exist (642, 1085; and see Refs. 127, 591). For these reasons, activation of
IKNa during ischemia and especially during
reperfusion (enhanced Na+/H+ exchange) remains
a real possibility.
Three pathways for an increased inward leak probably participate in
keeping Em away from
EK: modified TTX-sensitive
Na+ channels, NSC channels, and Cl channels.
Sodium channels are modified by LPC in such a way that they open and
show continuous activity at the resting potential level (107, 108, 1002). Inward current
via NSC channels can be induced by stretch (188,
529, 815), [ATP]o
(301, 411), free radicals (472),
long-chain fatty acids (441), and a rise in
[Ca2+]i (172,
251, 410). Some of the NSC channels activated
by stretch are equally permeable to Ca2+
(529). Calcium leak channels as such are induced by
radicals (1056). An important Cl current is
activated via -receptor stimulation (443), a rise in
[Ca2+]i (910,
1192), hypotonic distension (357,
929, 992), and [ATP]o
(500, 597, 664). Because
ECl in cardiac cells is positive to the
resting potential, an increase of Cl conductance
generates an inward current at negative potentials. Outward movement of
lactate through channels would generate an important inward current. At
present, no evidence for such a mechanism exists, and the major efflux
seems to occur through a proton-coupled carrier mechanism.
Finally, outward current is reduced through a partial block of the
Na+-K+-ATPase by the fall in free enegy of
hydrolysis, LCAC (959), and oxidative stress
(880), which thus favors depolarization. In conclusion, it
may be said that three K+ channels with large conductance
can be activated during metabolic inhibition, whereas a plethora of
inward currents carried by Na+, Ca2+, and
Cl are available to generate an electrical gradient for
K+ loss.
C) EFFECTS OF INCREASED [K+]O ON CHANNELS AND CARRIERS. The effect of the increase in [K+]o on channels, other than K+ channels, is mainly indirect via the depolarization it causes (for references, see sect. IIA). The extent of depolarization during acute ischemia is responsible for a partial or complete inactivation of the fast Na+ channel, the T-type Ca2+ channel, the Ito, and the IKur. Recovery from inactivation of these currents is slowed and is accompanied by a decrease in excitability, prolongation of refractoriness, and conduction slowing of the action potential.
For a number of K+ channels, but not for all, the conductance is increased when [K+]o rises; the IK1 current (123) is the most sensitive, but voltage-activated currents such as IKr (851) and Ito (284) and ligand-gated K+ channels such as KACh, KATP, KAA, and KNa all increase their conductance and carry more current at elevated [K+]o. In the case of IK1 and IKr, the mechanism is less pronounced inward rectification, consequent to a smaller block by intracellular cations (IK1) or smaller inactivation (IKr). These changes again will stabilize the Em and reduce excitability. Worthwhile to mention is the enhanced conductance of the If current of which the Na+-carrying capability is increased (293, 419). Because this current is only activated at hyperpolarized levels, the functional implications of this change remain limited, except transiently during reperfusion. Extracellular K+ stimulates the Na+-K+ pump, but because the normal K+ concentration is already sensibly greater than the Kd of 1 mM (715), the increase in pump current via this mechanism is of no functional importance.D) EFFECTS OF [K+]O ON ELECTROPHYSIOLOGICAL CHARACTERISTICS. At the multicellular level, the changes by elevated [K+]o will cause the cell to depolarize and the action potential to be reduced in amplitude, rate of rise, and duration. Inactivation of the Na+ conductance, concomitant to the depolarization, is responsible for the fall in action potential amplitude and rate of rise and the decrease in excitability. Transiently, however, excitability may be increased, the reason being that the depolarization, although causing inactivation of the Na+ current, at the same time moves the Em closer to threshold with as result a reduction in the current required to reach threshold (231). At depolarized levels, the recovery from inactivation is slower; in the presence of increased [K+]o, the result is a prolongation of the postrepolarization refractoriness (see Ref. 480). Conduction velocity is depressed and slowed, especially during the relative refractory period (545). Shortening of the action potential is mainly due to an increase of the IK1 and IKr conductance and to a lesser extent to a decrease of Na+ conductance. The changes in excitability, refractoriness, and conduction, together with the shortening of the action potential, favor the occurrence of reentry arrhythmias.
E) SYNOPSIS. Potassium loss during early ischemia occurs in three phases: a fast increase results in a plateau (10-20 mM) after 3-10 min and is followed (after 15-30 min) by a secondary and irreversible increase. Upon reperfusion before the irreversible phase, [K+]o recovers rapidly. Three main factors contribute to the rise: shrinkage of the extracellular space, inhibition of active K+ influx, and increase of passive K+ efflux. The increase in K+ efflux is due to the existence of an inward leak current (INa, INSC and ICl) which keeps the Em positive to the EK, concomitant with an increase in K+ conductance (activation of IKATP, IKAA and IKNa). The effect of elevated [K+]o, apart from causing an increase in conductance of K+ channels, is indirect via the depolarization. The final result is inexcitability and block of conduction.
2. Intracellular and extracellular acidosis
A) PROTON DISTRIBUTION UNDER NORMAL AND ISCHEMIC
CONDITIONS.
Under normal perfusion and oxygenation, pHi is
slightly more acidic than pHo. During ischemia,
CO2 retention and the net production of protons shift
pHi as well as pHo in the acidic
direction. External pH has been described to change rapidly and
monotonically from 7.4 to values as low as 6.0 (159).
Diverging results have been obtained on pHi, depending
mostly on the experimental model and the technique used [ion-sensitive
electrodes (ISE), distribution of weak acids, 31P-NMR
spectroscopy, and fluorescence indicators]. With the use of NMR and
fluorometric measurements, a pronounced fall in pHi and pHo has been found in preparations subjected to
total ischemia. From a control value of 7.15-7.2, pHi
fell to 6.5 after ~4 min and to 6.2-6.0 after 10-20 min (rat, Refs.
116, 159, 962, 1047; ferret, Ref. 640; rabbit, Ref. 689) (Fig.
16C). A short delay of 1 min before the decline in
pHi has been observed by Vandenberg et al.
(1011). Smaller changes in pHi have been
found using the ISE. This in part is due to a selection of
superficially located cells for measurement that is an unavoidable bias
with this technique. In a study on gas-superfused but
blood-perfused papillary muscles of the rabbit heart subjected to
ischemia (1135), internal pH was found to be rather
resistant to any change when PCO2 was held at
the initial value; however, if the CO2 tension in the
experimental chamber surrounding the preparation was gradually
increased to values that are probably present in vivo during ischemia,
pHi underwent a fall from 7.0 under control conditions
to 6.55 after 18 min of ischemia. At the same time, changes in
pHo were more pronounced and decreased from 7.39 to
6.13. In other words, the pH ratio reversed, an effect that has been
correlated to the higher buffering capacity of the intracellular
medium. The finding that pHi depends largely on
PCO2 predicts heterogeneity in
pHi changes, depending on the depth of the myocardial
layer. The resistance of pHi to undergo dramatic
changes further demonstrates that protons continue to be transported
against their electrochemical gradient even during ischemia. A more
pronounced fall in pHo than pHi has been
described in another study in which ischemia was simulated by bathing a
papillary muscle in oil saturated with nitrogen (1018).
B) REGULATION OF PROTON CONCENTRATION IN NORMAL, ISCHEMIC, AND
REPERFUSION CONDITIONS.
Intracellular proton concentration is kept at a much lower level than
predicted for a passive distribution. For a 80 mV resting potential,
the pHi at equilibrium should approach 6.0. The fact that the pHi is around 7.2 means that protons are
actively transported outward. In aerobic conditions, the continuous
production of protons is compensated by CO2
elimination, Na+/H+ exchange, and
Na+-HCO3 cotransport, the latter two
processes being ultimately coupled to the energy-consuming
Na+-K+ pump (493, 595a, 616).
1-receptor stimulation and activation of PKC
(298). -Receptor activation however inhibits
(580). The exchanger is also inhibited by extracellular
acidosis (1114).
The second important mechanism to protect the cell against acidosis is
the Na+-HCO3 cotransporter. According to
a recent publication (7), it is electrogenic (2 HCO3 for 1 Na+ ). In the guinea pig
heart, the cotransporter is responsible under physiological conditions
for about the same amount of acid extrusion as the
Na+/H+ exchanger (198,
580). The cotransporter is also coupled to - and
-receptors but in an opposite way to that of the
Na+/H+ exchanger. -Receptor stimulation in
other words depresses whereas -receptor stimulation enhances the
activity of the cotransporter. The final effect will thus depend on the
relative importance of the versus and cotransporter versus
exchanger pathways. In the guinea pig, the -effect is predominant
over the -effect, and the effect on the symporter is stronger than
on the exchanger (580). Sympathetic stimulation therefore
will decrease the rate of acid extrusion in this species. These effects
are mimicked by application of extracellular ATP, whereas adenosine or
ADP is without effect. In the rat, -receptor stimulation has been reported to result in alkalinization, implying a larger effect on the
exchanger (470). Lysophosphatidylcholine impairs the
symporter and slows the recovery from an acid load (1129).
The fact that two different mechanisms reduce the proton load explains
why block of one does not necessarily result in acidosis.
In heart cells, two other processes, the
Cl/HCO3 and
Cl/OH exchange, act in the opposite way and
may cause acid load (592, 948). They are
stimulated by activation of -receptors and
[ATP]o. Whether the stimulation involves a direct
modification of the exchanger or is due to an indirect effect is not
known. -Receptor stimulation in addition stimulates glycolysis,
which indirectly contributes to the acidification.
During ischemia, the fall in pHi and
pHo is caused by an increased production of protons
and insufficient removal (210).
1) Glycolytic ATP turnover in contrast to oxidative ATP
turnover is accompanied by a net H+ production. During
ischemia, ATP production shifts from the mitochondrial system to
glycolysis, and this shift is accompanied by an obligatory increase in
protons. The finding that inhibition of glycolysis abolishes the
acidosis during CN exposure is consistent with this explanation
(10).
2) Net ATP hydrolysis is accompanied by net production of
protons. One mole of ATP breakdown produces 0.8 mol H+.
This contrasts with the absorption of 0.35 mol H+ upon
breakdown of PCr which thus may shift the pH early in ischemia in the
basic direction. The ratio of [PCr] to [ATP] is normally around
two. Metabolic inhibition, at least in the beginning, can result in
alcalosis or acidosis depending on the relative amounts of PCr or ATP
that disappear (253). A delay in pHi
decline has been observed for global ischemia of the ferret heart
(1011).
3) Removal of protons is insufficient. Under normal
conditions, a large amount of the acidic load is removed by simple
CO2 diffusion. Although the production of
CO2 by aerobic metabolism ceases after coronary
occlusion, it is still formed from HCO3 as a
consequence of metabolic acidosis. The acidosis will thus be different
across the ventricular wall with much more acidic conditions in the
subendocardial regions (1077). Differences also exist
between the central and border zones of the infarct. Acidosis will also
greatly depend on the presence of buffer systems, e.g., blood or saline
solutions (253, 390). These considerations
should be taken into account when interpreting the data obtained in
experimental models where the oxidative as well as the glycolytic
pathways are inhibited by metabolic blockers but superfusion and
elimination of CO2 persist. The two other mechanisms
for acid removal, i.e., the Na+/H+ exchanger
and the Na+-HCO3 cotransporter, are
infavorably affected by the increase in
[Na+]i. Upon reperfusion, pH in general
rapidly recovers (398, 1011). The underlying
mechanisms are washout of acid metabolites (H+,
lactate, and CO2) and
Na+-coupled acid extrusion
(Na+-HCO3 cotransport and
Na+/H+ exchange). The pH does not recover when
hearts are perfused with HEPES buffer (no
Na+/HCO3 symport), and
Na+/H+ exchanger and lactate transport are
simultaneously inhibited. The most important of the three mechanisms is
washout of CO2 and lactate, whereas
Na+-dependent extrusion (Na+/H+ and
Na+/HCO3) accounts for 20-35% of the
recovery (1011). Among these two mechanisms, the relative
contribution of Na+-HCO3 cotransport is
larger. The minor contribution of the Na+/H+
exchanger is evident from the small effect of blockers on the recovery
of pH (398, 769, 864). Both
extrusion mechanisms and the accompanying Na+ influx,
however, should be taken into account to understand the possible
increase in Ca2+ load of the cell upon reperfusion. This
explains why improved recovery of contractile behavior occurs
(398), and arrhythmias are less frequent (29,
1123) when the exchanger is blocked.
C) EFFECTS OF THE PH CHANGES ON CHANNELS AND CARRIERS. Because channel proteins act like enzymes, large changes by protons may be expected. Most plasma membrane currents, with the exception of IKATP and IKAA, are inhibited, some by extracellular (INa, ICa, Ito, IKr, IKs), others by intracellular acidosis (IKNa, IK1) (for references, see Table 1) (Fig. 17). The mechanism is a decrease in single-channel conductance, Po, or both and eventual voltage shifts in the activation-inactivation kinetics. The change in conductance in general is not voltage dependent; binding of protons outside the electrical field probably reduce the local concentration of permeating cations or shift the conductance to a lower substate by an allosteric effect. The Po is diminished by shortening of the open time (INa, ICaL), prolongation of the closed time (IK1), and increase in nulls (ICaL). External acidosis normally causes also positive shifts in the activation-inactivation curves (INa, ICaT, Ito, IKr). In case of shift, the effect on macroscopic current for a channel with activation-inactivation kinetics will thus depend on the resting (holding) Em (938). In normal polarized cells, the current is not changed at positive test potentials but reduced for smaller depolarizations; the difference is due to the shift of the activation curve. When the resting potential is depolarized, however, the current may paradoxically be increased, an effect due the positive shift of the inactivation curve (442, 938). An exception to the rule of voltage independence can be found in the change of macroscopic conductance of the IKr current by extracellular protons (Fig. 17), which cause a larger reduction of the current at hyperpolarized potentials (129).
|
|
D) ELECTROPHYSIOLOGY AND PH CHANGES. The effects of acidosis at the channel level are translated at the multicellular level into a fall in resting potential, a reduction of upstroke velocity, a prolongation of the action potential duration, with eventual oscillations at the plateau level and occurrence of early afterdepolarizations (EAD) (175). Refractoriness is prolonged, due to a slowing of the Na+ current recovery. Conduction of the action potential is retarded (319) as a consequence of the decrease in excitatory current and a fall in gap conductance. Of these changes, the prolongation of the action potential and the occurrence of EAD are not seen during ischemia but may be present upon reperfusion. During ischemia, the action potential is shortened as a consequence of extracellular K+ accumulation and activation of outward currents, mainly carried by KATP and KAA (see sects. IIIA1 and IVA). All the acidosis-induced changes are proarrhythmic and may explain why the threshold for ventricular fibrillation is reduced in acidosis (950).
E) SYNOPSIS.
Under aerobic conditions, the continuous production of protons is
compensated by CO2 elimination,
Na+/H+ exchange, and
Na+-HCO3 cotransport. During ischemia,
pHi and pHo fall by one pH unit or
more. The fall is caused by an increased production and reduced removal
of protons. Most plasma membrane channels with the exception of KATP
and KAA, as well as gap junction channels, the SR Ca2+
release channel, and Na+/Ca2+ exchange current
are inhibited, some by extracellular and others by intracellular
acidosis. The mechanism for the channels is a decrease in
single-channel conductance and/or a reduction in
Po. In most instances, except for
IKr, the effect is voltage independent. These changes result in depolarization, prolongation of the action potential, and occurrence of EAD.
3. Accumulation of [Na+]i
A) DESCRIPTION OF NA+ DISTRIBUTION.
Cytoplasmic free [Na+] concentration measured with ISE,
NMR, and fluorescent dyes varies between 5 and 10 mM intracellular water for different animal species (see Refs. 30, 1037). Species differences exist, and [Na+]i is higher
in the rat and mouse ventricle cells (15-16 mM) (1143). In single cells, actual values depend on the cell isolation procedure; the presence of taurine, for instance, improves the quality of the
cells and reduces [Na+]i in rat cells
from 20 to 10 mM (232, 945). The
[Na+]i is significantly higher in
subepicardial than in subendocardial cells (173).
B) MECHANISMS INVOLVED IN THE CHANGES OF [NA+]I DURING ISCHEMIA. During ischemia, an increase in [Na+]i is caused by a reduced active outward movement and an increased passive inward leak.
A partial block of the pump during early ischemia is highly probable and may explain the observed changes in [Na+]i (232). Inhibition of the pump is due to the fall in free energy of ATP hydrolysis as a result of changes in [ATP], [ADP], and [Pi]; a reduction in enzymatic activity under the influence of LCAC (959) and oxidative stress (880); and a reduction in the number of Na+ pump molecules in the plasma membrane consequent to endocytosis (Shattock, personal communication). Theoretical calculations show that a reduction of the pump activity by one-fourth of its maximal activity will result in a change of [Na+]i amounting to 3.4 mmol · l1 · min1 (Na+
influx is assumed to remain constant). Under normal conditions, the
energy available from ATP hydrolysis amounts to 61.5 kJ/mol, but during
ischemia, it can fall to 49 kJ/mol or less. Concomitant with this fall,
the Erev of the pump shifts in the
depolarized direction and approaches the resting potential
(190).
Inward leak of Na+ occurs via carriers and channels. An
important carrier is the Na+/H+ exchanger;
extrusion of protons via the Na+/H+ exchanger
causes Na+ to move inward. Block of the
Na+/H+ exchanger has been found to reduce
[Na+]i increase during ischemia
(19, 769), although it had no effect on
pHi (398). In other experiments, the
beneficial effect of Na+/H+ exchanger block was
restricted to reperfusion (962).
Sodium ions move inward through TTX-sensitive Na+
channels and NSC channels. The fast Na+ channel is
modulated by the rise in LPC to open repetitively and show continuous
activity at the resting potential level (107, 1002). Block of this pathway by lidocaine reduces this
leak inward current (112, 1016).
Activity in NSC channels is induced by stretch (188,
529, 815), [ATP]o
(301), free radicals (472), and a rise in
[Ca2+]i (172,
251, 410), all changes which occur very early
during ischemia. Some of these NSC channels activated by stretch are equally permeable to Ca2+ (526). In pacemaker
cells (Purkinje fibers), an important influx of Na+ occurs
via the If channel (327).
The fast recovery of [Na+]i upon
reperfusion is somewhat unexpected, since washout of protons from the
extracellular space should stimulate Na+/H+
exchange and lead to a net Na+ influx. Activation of the
Na+-K+ pump however seems to prevail in most
cases. Results may vary depending on the relative activity of the
Na+/H+ exchanger and the pump. When glycogen is
still available, glycolysis causes acidification and an increase in
Na+/H+ exchange. In those instances, inhibition
of the exchanger improves recovery of contractile activity and prevents
arrhythmias (28, 29, 1123). When
cells are depleted of glycogen, acidification is less, and activity of
the exchanger remains limited. Under those conditions,
[Na+]i does not increase upon
reperfusion, and only a rapid fall is observed.
C) EFFECT OF [NA+]I INCREASE ON CHANNELS AND CARRIERS. Intracellular [Na+] if sufficiently elevated, activates specifically the IKNa (497, 625), and enhances IKs (978) and IKACh (943). In the case of IKNa, an increase in [Na+]i also reduces the single-channel conductance (1068), but this effect is largely compensated by the increase in Po. Outward current through other K+ channels, such as IK1 (654, 848) and IKATP (494, 434), is blocked by intracellular Na+; the block is voltage dependent, rapid, and seen as a decrease in overall conductance.
Intracellular Na+ is the main substrate that activates the Na+-K+ pump current. The process becomes important during reperfusion when oxygen tension is restored. On the Na+/Ca2+ exchanger, a rise in [Na+]i favors reverse-mode activity, causing extra Ca2+ load. The relation between Na+ and Ca2+ load explains the close relationship between [Na+]i and the frequency of spontaneous Ca2+ release (216). Via interaction with the endogenous XIP region, a rise in [Na+]i causes a state of inactivation of the exchanger (662, 663). The simultaneous rise in [Ca2+]i, however, will partly compensate for this inhibitory mechanism.D) EFFECT OF [NA+]I INCREASE ON ELECTROPHYSIOLOGY. All changes in channels and carriers, except for the reduction in IK1 and IKATP, tend to hyperpolarize the membrane, but the effect will only be visible during reperfusion. The marked hyperpolarization of the diastolic Em and the excessive shortening of the action potential, despite the low [K+]o, can be ascribed to stimulation of the pump and eventual activation of outward K+ currents. Insufficient recovery of the Na+ pump or excessive Na+ influx via the Na+/H+ exchanger may be responsible for increased Ca2+ influx via the reversed Na+/Ca2+ exchanger, causing Ca2+ overload, absence of recovery, and eventual arrhythmias. Prevention of Na+ and Ca2+ overload by blocking the Na+/H+ exchanger (19, 548, 769, 863) explains the inhibitory effect of such intervention on reperfusion arrhythmias.
E) SYNOPSIS. During ischemia, [Na+]i increases from 10 to 20 mM after 20-30 min. The mechanism is reduced active outward pumping (inhibited by the fall in free energy of ATP hydrolysis by LCAC and radicals) and an increased inward leak (through the Na+/H+ exchanger, INa, INSC, and ICl). The effect of increased [Na+]i is activation of the pump and eventually of IKNa. During reperfusion, these effects result in hyperpolarzation and short action potentials combined with low [K+]o. Dispersion is pronounced and is favorable to arrhythmias.
4. Changes in the distribution of
[Ca2+]i
A) CA2+ DISTRIBUTION UNDER NORMAL CONDITIONS AND
CHANGES DURING ISCHEMIA AND REPERFUSION.
I) Ca2+ distribution under normal conditions.
Under normal conditions, concentrations of Ca2+ differ in
the cytosol, the SR, the mitochondria, and the nucleus (for review, see
Ref. 773). In each compartment, it plays a different role. It modulates
the activity of myofilaments in the cytosol and of ionic channels in
the plasma membrane. The SR acts as a store for Ca2+ and
modulates contraction and relaxation. In the mitochondrion, the free
[Ca2+] regulates the activity of three dehydrogenases
(pyruvate dehydrogenase, -oxoglutarate dehydrogenase, and
NAD-dependent isocitrate dehydrogenase) and adapts in that way
energy production to energy utilization. In the nucleus,
Ca2+ modulates gene expression.
150 and 180 mV), this means that mitochondrial [Ca2+] is far from equilibrium.
The [Ca2+] in the mitochondrion is variable depending on
the cytoplasmic load. When cytoplasmic [Ca2+] is <500
nM, the gradient of mitochondrial [Ca2+] to cytoplasmic
[Ca2+] is less than unity but increases in an exponential
way at higher values (687). Mitochondrial
[Ca2+]-to-cytoplasmic [Ca2+] ratios larger
than unity have been measured during stimulation of isolated cells at 2 Hz (225). Total mitochondrial [Ca2+]
measured by the electron probe transiently increases during a
depolarization (461, 1084); the increase in
free Ca2+ transient during metabolic inhibition has been
interpreted as due to a fall in Ca2+ uptake by the
mitochondria during the cardiac systole. The rise in free
[Ca2+] has been confirmed using a combination of a
fluorescence and confocal microscopy technique (140). In
previous fluorescence measurements, such fast changes were not
detected. The reason for this divergence is not clear but may be
related to the buffer characteristics of the fluorescent probe
(461). Mitochondria are thus no source of Ca2+
for the cytoplasm during stimulation but rather act as a buffer. Because mitochondria represent a large fraction of the cell volume, they can accumulate massive amounts of Ca2+
(43), a situation which occurs during pronounced and
prolonged ischemia. The amount of Ca2+ stored in the
mitochondrion is sufficient to lead to a contracture when the
mitochondrial Em breaks down after
application of an uncoupler (1106).
In the cardiac SR, total [Ca2+] expressed per liter
cytosolic volume has been measured to be between 100 and 200 µM (see
Refs. 73, 323, 1024), with estimations of free [Ca2+] per
liter SR volume at 700 µM (877). An enormous gradient of 7.000 thus exists between the SR and cytoplasm.
II) Changes of [Ca2+]i during ischemia
and reperfusion. During anoxia, metabolic inhibition or ischemia,
diastolic free [Ca2+]i rises with some
delay. The delay is variable, and quite important differences in time
course have been reported, depending on the model. In studies of
ischemia (coronary ligation), the delay attained 10-20 min and the
rise in [Ca2+]i slightly preceded the
development of contracture and uncoupling of the gap junctions
(640, 1047) (Fig. 16D). A much
shorter delay between 2 and 5 min has been described in other studies
(519, 689). A large variation in time course
also exists in experiments on single cells subjected to hypoxia or
metabolic inhibition (see references in Ref. 817). The reasons for
these divergent results are not known but are probably related to the
intensity of the metabolic blockade.
Results on systolic [Ca2+] are variable. A transient rise
may occur (Fig. 18A)
(461) but is not always present [total heart (689, 1047), ventricular muscle
(920), and singe cells (253, 461)] and is followed by a decrease when diastolic
[Ca2+]i rises (519,
640). The initial rise is not accompanied by an
enhancement of the contraction and is probably due to a shift of
Ca2+ from the mitochondrion to the SR (461)
(Fig. 18A) and/or a release from binding sites by the
increase in intracellular acidosis (312). The fall in
systolic [Ca2+]i may be accompanied by
oscillations during diastole, caused by release from the SR
(906).
|
B) MECHANISMS FOR [CA2+]I CHANGES. The increase in cytoplasmic [Ca2+] in the cells submitted to ischemia is due to a less efficient removal from the cell via the Na+/Ca2+ exchanger and a reduced Ca2+ uptake in the SR, an increased inward leak through the plasma membrane, and displacement of Ca2+ from binding sites in the cytoplasm and in mitochondria by protons. Mitochondria are probably not responsible for an increase in cytoplasmic [Ca2+] except in excessive overload; in most instances, the mitochondria absorb Ca2+ during ischemia.
In the early stage of ischemia, free [Ca2+] in the cytoplasm increases due to a displacement of Ca2+ by H+ from binding sites (312). The experimental basis for this mechanism was provided by the observation that an increase of CO2 tension from 5 to 20% caused [Ca2+] in the cytoplasm to rise from 130 to 221 nM. The effect was not changed by application of ryanodine (block of the SR Ca2+ channel) or ruthenium red (block of mitochondrion uniporter). Calcium influx and efflux through the plasma membrane that are normally in equilibrium (986, 1057) go out of balance during ischemia with Ca2+ influx exceeding Ca2+ efflux. The most important mechanism for removal of Ca2+ from the cell is the Na+/Ca2+ exchanger, and it is responsible for 77% of the Ca2+ extrusion (583, 986). During ischemia, the Na+/Ca2+ exchanger is less efficient because of an increase in [Na+]i and [H+]i. An increase in [Na+]i promotes the reversed mode of the Na+/Ca2+ exchanger whereby Ca2+ is entering the cell and Na+ is removed (358, 920, 962). All mechanisms responsible for an increase in [Na+]i thus also contribute to the increase in [Ca2+]i via the exchanger. The efficiency of the exchanger is furthermore reduced by the acidosis (230, 764) and the influence of radicals (163). Whereas Ca2+ efflux is reduced, Ca2+ influx is increased during metabolic ihibition. The role of T- and L-type Ca2+ channels in this respect is negligible. Background channels carrying Ca2+ inward become more important during ischemia as they are activated by radicals (472, 1056). Inward leak of Ca2+ also occurs via NSC channels (see sect. IIIA5) that are activated by [ATP]o (301, 411), mechanical stretch (188, 526, 841), and a rise in [Ca2+]i itself (172, 251, 650). During ischemia, less Ca2+ is stored in the SR, an effect mainly due to block of Ca2+-ATPase activity, whereas changes in the release mechanism are less important. The decrease in Ca2+ reuptake is due to a reduction of Vmax, not to a change in Kd (0.5-0.7 µM) or the Hill coefficient (503). The effect is related to the reduction in free energy change of ATP hydrolysis (339, 644) and modification of the carrier by oxygen radicals (disulfide bridge formation) (565). Although the SR Ca2+ channel can become activated by exposure to oxygen radicals (429) or to arachidonic acid and its metabolites (196), the increased levels of [H+], [Mg2+], and [PO43] exert a blocking
effect. The overall result is probably of less importance.
Influx as well as efflux of Ca2+ in the mitochondria depend
on the electrical and proton gradient (Fig. 2). Influx through the uniporter is driven by the electrical potential difference that is
estimated to be 150 to 180 mV. Efflux from the mitochondria depends
on the proton gradient and is continuously guaranteed by the
Na+/Ca2+ exchanger and the
Na+/H+ exchanger which act in concert to keep
intramitochondrial [Na+] and [Ca2+] at low
levels. The immediate source of energy lies in the Na+
gradient, but the final energy source is respiration and the proton
gradient. Block of the oxidative metabolism, resulting in the
disappearance of the electrical and proton gradients, is thus expected
to result in equilibration of the mitochondrial [Ca2+]
and the cytoplasmic [Ca2+] with a ratio of
mitochondrial-to-cytoplasmic [Ca2+] approaching unity.
Because the cell volume fraction of mitochondria is 30-36%, they may
absorb a large amount of Ca2+ and act as a buffer.
There are mechanisms responsible for
[Ca2+]i changes during reperfusion.
Depending on the severity and duration of the ischemic period, the
cytosolic [Ca2+] concentration may normalize or on the
contrary increase to dramatic levels leading to cell death.
The rapid initial recovery phase seems to depend on the activity of the
SR Ca2+-ATPase (see Refs. 503, 697). Although the
Ca2+-ATPase activity and the number of ryanodine receptors
(1005) are reduced, uptake in the SR and increase of the
luminal [Ca2+] may be so large that threshold for release
is attained resulting in oscillations (987). If, in the
mean time, removal of Ca2+ from the cell through the
Na+/Ca2+ exchanger is restored, the
Ca2+ level may return to normal values (773,
906). In some cases, however, the oscillations are
followed by a secondary increase. Recovery depends much on the
restoration of the Na+ gradient and is facilitated by
blockade of the Na+/H+ exchanger
(578, 864) or acidosis (see Ref. 91).
Protection against Ca2+ overload contracture is also
provided by the natriuretic peptide that stimulates guanylate cyclase
(397). On the other hand, production of
IP3 secondary to 1-receptor
stimulation may inhibit recovery (17, 236).
A critical situation may occur when the ischemic period has been too
long and restoration of the Na+-pump activity has been
retarded. In this case, the [Ca2+] remains elevated. On
restoration of the oxygen supply, the electron flow through the
oxidative chain generates an electrical gradient in the mitochondrion
causing a massive absorption of Ca2+ from the cytoplasm.
The presence of such an abnormally high [Ca2+] together
with low [ATP] and oxidative stress may cause the opening of the
mitochondrial mega-channel (Figs. 2 and
19). The ATP, instead of being exported
to the cytoplasm, may even be hydrolyzed to generate a proton gradient
and limit the absorption of Ca2+ in the mitochondrion. The
final result is disappearance of all mitochondrial gradients,
hypercontracture, and cell death (244, 340).
It is important to note that high [Ca2+] alone does not
cause the permeability transition as shown by experiments with toxic
concentrations of digitalis in which [Ca2+] increases to
much higher levels than in ischemia and cells remain viable
(595).
|
C) EFFECTS OF [CA2+]I ON CURRENTS AND CARRIERS. An increase in [Ca2+]i modulates multiple currents in cardiac cells. The L-type Ca2+ current itself is very sensitive to increases in [Ca2+]i and shows an increased rate of inactivation, limiting in this way the Ca2+ load of the cell (see Ref. 673). In contrast, the current may also undergo facilitation for moderate increases of the Ca2+ level (505) (see sect. IIA2).
Some currents are dependent on [Ca2+]i for their activation: ICl(Ca) (910, 1192), INSC (172, 251), the Ca2+ release channel (120, 261), and the Na+/Ca2+ exchanger (686). An increase in [Ca2+]i stimulates the Na+/Ca2+ exchanger and counteracts in this way the Na+-induced inactivation (663). Other currents are modulated; an increase is seen for the T-type Ca2+ current (994), IKs (728, 978), IKr (851), If (354), the Na+-K+ pump (314), and the mitochondrial MCC (189, 952). Two currents are inhibited: the IK1 (654, 670, 671), a phenomenon comparable to the block by [Mg2+]i, and the gap junction channel (206, 732). The [Ca2+]i influences furthermore a number of enzymes. Adenylyl cyclase is inhibited (1154), and others are stimulated, e.g., mitochondrial dehydrogenases (692), the ATP synthase (199), PKC, phosphatases, and proteases (causing rundown of channels) (see Ref. 496).D) ELECTROPHYSIOLOGICAL EFFECTS OF
[CA2+]I.
Enhancement of Ca2+-induced inactivation of the
Ca2+ current, increase of the transient
Ca2+-activated Cl current, and of the two
delayed K+ currents will lead to shortening of the action potential.
channel
(747, 912), the NSC channels
(121, 368), and the Na+/Ca2+ exchanger (369,
579). Although the primary activator for Ca2+
release is the cytoplasmic Ca2+, other factors such as ATP
(1120), the phosphorylation state of the channel
(754, 1006), and luminal [Ca2+]
(917) determine the efficiency of the release mechanism
or, what has been called, the gain of the system (120).
Under conditions of moderate Ca2+ load, the release is
linearly related to the luminal [Ca2+] concentration and
not propagated; the gain is constant. With increase of Ca2+
load however, gain increases nonproportionally, and release becomes regenerative and propagated (44, 367,
476, 847, 940). Under these
conditions, release may be spontaneous and occur in the absence of
trigger Ca2+. The result is EAD during the action potential
or DAD, following repolarization. Important to note is that
Ca2+ overload also increases the tendency in Purkinje
fibers to become spontaneously active; the activation curve of the
If current is shifted in the positive
direction (354, 1172), which favors
activation of the current and increases the rate of diastolic
depolarization. The fall in gap junction conductance by
[Ca2+]i further enhances the probability
for arrhythmias by reducing conduction (206,
732). In a final stage, activation of the mitochondrial mega-channel uncouples the ATP synthase, causes hydrolysis of ATP,
and leads to rigor and contracture (189,
244). High [Ca2+] alone, however, is not
sufficient (595); a simultaneous fall in [ATP] and the
presence of oxidative stress that may occur during reperfusion creates
a favorable condition for activation of the channel (189,
244) (Fig. 19). Once activated, the opening of the MCC
channel leads to irreversible processes and cell death.
E) SYNOPSIS. The [Ca2+] differs in the cytoplasm, the SR, and the mitochondria. Whereas systolic [Ca2+] may transiently increase during the first minutes of ischemia, diastolic [Ca2+] only increases after a delay of 10-20 min. In the mitochondria, [Ca2+] follows the changes in cytosolic level, although there may be an initial release if the [Ca2+] in the mitochondria is higher than in the cytoplasm at the start of ischemia. The increase in cytosolic [Ca2+] is due to a less efficient removal to the extracellular space via the Na+/Ca2+ exchanger, a reduced uptake in the SR, an increased inward leak, and a displacement by protons from binding sites. An increase of cytosolic [Ca2+] activates a number of channels, carriers, and enzymes (INSC, ICl, and INa,Ca) and modulates others (ICaL, ICaT, IKs,IKr, If, INa,K, and gap junction channels). The result is the occurrence of EAD, DAD, and arrhythmias. If on reperfusion, removal of Ca2+ to the extracellular space and into the SR is insufficient, while O2 tension is restored and the mitochondrion Em energized, a massive absorption of Ca2+ may occur in the mitochondrion and result in activation of the mitochondrial mega-channel, which signals irreversibility.
5. Changes in [Mg2+]i
A) DISTRIBUTION AND REGULATION OF [MG2+] UNDER
AEROBIC CONDITIONS AND ISCHEMIC CONDITIONS.
Estimations of total Mg2+ in heart cells using EPMA (see
Ref. 701) vary between 50 and 80 mmol/kg dry wt for the cytoplasm (A
band), 20-80 mmol/kg dry wt for mitochondria, and 20-75 mmol/kg dry
wt for junctional SR. From the estimated dry weight-to-wet weight ratio
of 10% for the cytoplasm and 25% for mitochondria, these values are
translated in 5-8 mM for the cytoplasm and 6-20 mM for mitochondria.
B) MECHANISMS OF [MG2+] REGULATION.
Free [Mg2+] in the cytoplasm is of the same order of
magnitude as the extracellular concentration. With the fact that the
intracellular medium is negative taken into account, the distribution
of free [Mg2+] is thus far from equilibrium.
Thermodynamically active transport is required. Two mechanisms are
involved: a Mg2+-ATPase and a
Na+/Mg2+ exchange process (361,
907). In favor of a Na+/Mg2+
exchange mechanism are the observations that
[Mg2+]i increases upon application of
ouabain. Reduction of [Na+]o also
results in an increase of [Mg2+]i
(214). The [Mg2+] content can be modulated
by -receptor stimulation, causing a release of intracellular
Mg2+, and by M2 receptor stimulation,
leading to the opposite effect. These changes may be secondary to
mobilization of the mitochondrial [Mg2+] pool by an
effect on the adenine nucleotide metabolism.
C) ELECTROPHYSIOLOGICAL EFFECTS OF INTRACELLULAR MG2+.
Intracellular [Mg2+] is needed to activate enzymes that
phosphorylate or dephosphorylate channels (Na+,
Ca2+, K+, Cl, and f channels). On
the other hand, as a cation interacting with the pore structure, it
blocks a number of channels, and in the case of K+ channels
generates inward rectification.
D) SYNOPSIS. Free [Mg2+] in the cytoplasm and the mitochondrion has been estimated to be 0.5-0.8 mM or 10% of the total amount present. During ischemia, the free concentration may increase to 2-6 mM. This change is due to hydrolysis of ATP to which Mg2+ are bound, and partly to a deficient removal via a Mg2+-ATPase and the Na+/Mg2+ exchanger. The behavior of channels and carriers is changed by Mg2+ via effects on phosphorylation, by blocking the pore (Na+ and Ca2+ channels), or by causing inward rectification in the case of K+ channels. At the multicellular level, Mg2+ may be considered to exert a stabilizing effect.
B. Amphiphiles and Fatty Acids
1. Accumulation of LCAC, lysophosphoglycerides, fatty acids,
and arachidonic acid
Under aerobic conditions, fatty acids (FA) are taken up from the
extracellular medium or derived from hydrolysis of triacylglycerols and
phospholipids and transformed to FA-CoA (acyl CoA). At the outer
leaflet of the inner mitochondrion membrane, FA-CoA is transformed to FA-carnitine under the influence of the enzyme carnitine
acyltranferase I (CAT-I) and then transferred to the matrix via the
translocase. At the inner site of the membrane, the reverse reaction
(CAT-II) results again in the formation of FA-CoA. Degradation
occurs by sequential removal of two carbon units ( The concentration of FA rises because of enhanced breakdown of membrane
phospholipids, while at the same time import in the cell continues. The
result is a 4-7 times increase in FA during global ischemia in rats
and even more up to 20 times during reperfusion (1014). Metabolism of phospholipids is drastically disturbed by activation of a
number of lipases. Arachidonic acid is released from phosphatidylcholine (PC) and PIP2 secondarily to
activation of a number of phospholipases (PL), such as
PLA2, PLC, PLD, and diacylglycerollipase. At what time this
activation occurs during ischemia is still a matter of discussion
(1013). Under the influence of lipoxygenase, AA is
metabolized in stimulatory metabolites, such as leukotrienes (LT) while
the cyclooxygenase pathway results in inhibitory metabolites, such as
prostaglandins and thromboxanes (see Ref. 1014). Concomitantly with AA, formed by PLA2 activation,
lysophosphoglyceride (LPG) and more specifically
lysophosphatidylcholine (LPC) concentrations increase (see Refs. 674,
675). Under aerobic conditions, catabolism of LPC is so efficient that
the concentration never rises substantially. In ischemia, catabolism is
inhibited and production is stimulated. Enzymes responsible for the
formation of LPC are activated through the rise in
[Ca2+]i, acidosis,
In the further analysis and description, distinction is made between
amphiphiles (LCAC and LPG) and fatty acids (including AA). They exert
quite different effects on channels and carriers, effects which may be
related to their different electrical charge: amphiphiles act as
zwitterions or cations at physiological pH, whereas fatty acids are
negatively charged. 2. Mechanisms of action and specific effects on channels and
carriers
A) AMPHIPHILES (LCAC AND LPG).
I) Mechanisms of action. Amphiphiles are molecules with an
hydrophilic, electrically charged part and an aliphatic, hydrophobic part. The most important amphiphiles in heart are LCAC and LPG, of
which LPC is an important representative. At normal pH, LCAC act as
cations, and LPG act as zwitterions.-oxidation)
(1014). Under anaerobic conditions, the arrest of
-oxidation results in accumulation of FA-carnitine or LCAC and
with some delay in the increase of fatty acids.1-receptor stimulation, LT, radicals, and thrombin, whereas acidosis and LCAC inhibit the enzymes active in the catabolic pathway.
-receptors. The increased number
of receptors is caused by an uncovering of receptors already present in
the membrane (185) (see Ref. 268).
Mechanical destabilization and dysfunction of membrane proteins can
also be the result of disruption of the cytoskeleton. Such
destabilization has been shown to occur with lyso-compounds (1003).
II) Specific effects on channel and transporter function.
The current through the fast Na+ channels is sensibly
affected in such a way that peak current is inhibited
(845), but inactivation slowed (1111) and
repetitive channel activity generated at rather negative
Em values, causing an important inward
leak. This persistent Na+ current does not deactivate on
hyperpolarization and is characterized at the single-channel level
by a bursting mode of openings (107, 108,
1002, 1003). It plays an important role as
leak current. In this context, it is important to note that a
nonselective cation current (INSC) is
activated by lyso-compounds (113, 631).
The L-type Ca2+ current has been reported to be reduced
(161, 1110), or not changed
(614).
Potassium currents are inhibited. Lysophophatidylcholine reduces the
single-channel conductance of IK1 with
no change in Po, whereas
L-palmitoylcarnitine has no effect on the
single-channel conductance but reduces the
Po (161, 541,
542, 844). In both cases, current is markedly
reduced. Also, IKATP is inhibited by LCAC
(528).
Lysophosphatidylcholine inhibits the Na+/Ca2+
exchanger (74) and the
Na+-HCO3 symporter (1129),
and LCAC block outward current through the Na+ pump
(886, 959).
Long-chain acylcarnitines specifically are concentrated in gap
junctions during ischemia even during short intervals and cause a
marked decrease in the single-channel
Po (1112, 1124).
Long-chain acylcarnitines may thus be responsible for the change in
conduction that occurs after 10 min of ischemia, concomitantly with the
secondary rise in [K+]o. The rise in
LCAC and the increase in longitudinal resistance are delayed by
blockers of the CAT enzyme. Simultaneous increases in
[Ca2+]i, which is also favored by LCAC,
may exert an additive effect.
In the mitochondria, LCAC, in conjunction with Ca2+,
phosphate and oxidative stress, facilitate the activation of the
mega-channel (771). Activation of the channel causes
complete uncoupling of oxidation from ATP synthesis and signals
irreversible changes in ischemia.
B) FA AND AA: MECHANISM OF ACTION AND SPECIFIC EFFECTS.
I) Mechanism of action. To a certain extent FA affect
channel behavior via the same mechanisms as amphiphiles; more specific effects are related to AA metabolites. Fatty acids interact with channels in different ways. 1) Fatty acids affect the
concentration of permeating ions by adding negative charge to the
channel protein, and more specifically cause an increase of cations
(stimulation of cation channels) but a decrease in anions (inhibition
of Cl channels). This hypothesis has received support by
experiments on smooth muscle K+ channel activation in which
incorporation of lipophilic substances with negative or positive charge
(763), respectively, caused activation or inhibition of
K+ channels. 2) At high concentrations, FA exert
a detergent action and increase membrane fluidity with consequent
destabilization (1112). 3) A third type of
interaction is due to the metabolites of AA, such as LT, which interact
with and activate G proteins and PKC (859,
1128).
-subunit of the human
channel (1118), and reduce the gating current
(57).
L-type Ca2+ current is variably affected: an increase by FA
and AA has been observed in guinea pig ventricular cells
(441); on the contrary, inhibition occurs in the frog
(762) by AA and in rat ventricular myocytes by
polyunsaturated FA (1116).
Among the K+ currents, IKACh
and IKur are stimulated, whereas
Ito and IKATP
are inhibited. Activation of IKACh in the
absence of ACh or other agonists occurs upon application of AA. The
effect is indirect after metabolism to leukotrienes. The
LT4 activate the G protein by stimulating GDP/GTP
exchange (570, 859, 1128). Also,
the stimulatory effect of AA on IKur is due
to its metabolites (89).
An outwardly rectifying K+ current
(IKAA) with large single-channel
conductance (60-90 pS) is activated in rat ventricular myocytes by AA
and other unsaturated FA (527, 528). The same channel is stimulated by stretch and acidosis and its
Po is slightly increased with
depolarization (525, 1050) (Fig. 9). The
channel is different from the KATP, which is inhibited by AA
(527, 1050). The stimulation by stretch does
not occur through activation of phospholipases and generation of FA
(742), because addition of albumin which binds to FA does
not eliminate the effect (525). Polyunsaturated FA and AA
cause inhibition of Ito while increasing IKur (89).
The Na+/Ca2+ exchanger is stimulated by
negatively charged FA and especially unsaturated FA (766).
This effect is secondary to an increase of the local
[Ca2+] concentration at the intracellular side secondary
to a translocation of phosphatidylserine (408,
585).
Gap junction conductance is inhibited by concentrations of AA and FA in
the micromolar concentration (110, 289,
647). Fatty acids are highly concentrated in gap junctions
during ischemia and probably destabilize the channels
(1112). Among the FA the long-chain unsaturated and
short-chain saturated FA are the most effective (110).
3. Electrophysiological effects of amphiphiles and fatty acids
On the basis of the effects on individual channels and carriers, predicted electrophysiological effects are different for amphiphiles and FA. Long-chain acylcarnitines and LPC generate preferentially depolarizing currents, whereas FA and AA and metabolites increase outward currents. The simultaneous occurrence of these apparently opposing effects is important, however, in the genesis of K+ loss: opening of K+ channels will facilitate K+ loss but only when the Em is kept away from the Erev of K+ by an inward leak.
Both LCAC and LPC, by favoring inward over outward current and inhibiting transporters, cause a fall in resting potential, reduction of upstroke velocity, initial prolongation of the action potential, appearance of DAD, and rhythmic activity (see Ref. 675). On the contrary, FA and AA do not cause prolongation but rather shortening of the action potential mainly by activation of K+ outward current (528, 1050).
Amphiphiles as well as FA (1112) destabilize the gap junction, reduce its conductance, uncouple cells, and decrease conduction of the action potential. The combination of all these effects provides the conditions for the initiation and the maintenance of arrhythmias. Because of the role of LCAC and AA in the generation of arrhythmias, possible therapies are based on inhibition of their production by acting on the CAT-I and PLA2 enzymes (183, 675).
4. Synopsis
During ischemia, amphiphiles (LCAC, LPG) and FA including AA and its metabolites accumulate in the plasma membrane, the gap junction, and intracellular membranes of the SR and the mitochondrion. Amphiphiles and FA may interact directly with channel proteins, with the phospholipids surrounding the channel proteins, or change the membrane fluidity. Amphiphiles increase inward current at the resting potential (fast Na+ channel and INSC) with simultaneous reduction of outward current through K+ channels (IK1 and IKATP). Carriers such as the Na+/Ca2+ exchanger and Na+-K+ pump are inhibited. On the contrary, FA activate outward currents, such as IKACh, IKur, and IKAA and stimulate the Na+/Ca2+ exchanger. The simultaneous activation of inward and outward currents favors K+ loss and Ca2+ overload. Together with the induced cell uncoupling, conditions are created to generate arrhythmias.
C. Radicals
1. Generation of oxidative stress and basic mechanism of
interaction
Oxidative stress results from the excessive generation of
radicals, peroxides, and singlet oxygen on one hand, and from the deficiency of protective mechanisms (enzymes and scavengers) that normally eliminate these radicals on the other hand. Free radicals are
molecules with one or more unpaired electrons; singlet oxygen is an
oxygen molecule with an unpaired electron moved to a higher orbital.
The unpaired electron alters the chemical reactivity of an atom or
molecule, making it usually more reactive. The lifetime of radicals is
in the order of 10 Free radicals are a normal product of the metabolic chain and, in some
instances, functionally very useful (e.g., NO). During aerobic
metabolism, between 1 and 5% of total oxygen is reduced to superoxide
(52). During aberrant metabolism (ischemia or reperfusion)
however, when high-energy electrons leak out of the metabolic
chain, radicals are formed to a higher degree (564, 736). Superoxide radical is normally generated from O2
during the oxidation of NADPH to NADP+. In the presence of
sufficient superoxide dismutase (SOD), the superoxide anion is directly
transformed with H+ to
H2O2. Superoxide anion and
H2O2 are also formed during the oxidation of xanthine by xanthine oxidase, auto-oxidation of
catecholamines, and during arachidonic acid metabolism and other
reactions such as those catalyzed by monoaminooxidase, tyrosine
hydrolase, and L-aminooxidase. The danger arises when
superoxide radical, NO radical, and
H2O2 lead to the formation of the
very reactive hydroxyl radical. During ischemia, the formation of radicals is amplified. The excessive
amount of radicals leads to changes in proteins (oxidation of sulhydryl
groups), DNA and RNA molecules, but especially in membrane lipids.
Lipids undergo peroxidation; polyunsaturated fatty acids more
specifically are an easy target because of the easier removal of
protons from the double bounds. The result is a lipid peroxyradical.
These peroxyradicals oxidize proteins, cholesterol, and other FA
especially polyunsaturated FA, thus propagating the reaction in a
chain-type way (561). Two peroxyl radicals can collide
and result in the generation of singlet oxygen, which can then directly
oxidize polyunsaturated FA to hydroxyperoxides. Decomposition of lipid
peroxides generates new radicals and noxious products, such as
malondialdehyde; these latter reactions occur only late during ischemia
(90 min of anoxia). In many cases, evidence for the generation of radicals during ischemia
or during reperfusion is indirect and based on findings in which agents
that inhibit the production of radicals or speed up their elimination
also reduce the expected electrophysiological changes. More recent
experiments using electron-spin resonance, chemiluminescence, or
HPLC with salicylate as the trapping agent have added direct evidence
for the production of radicals at least during reperfusion. The
intensity of radical production during the first minutes (2-5 min) of
reperfusion depends on the duration of the preceding ischemia and is
already evident after 15 min of ischemia (see Refs. 561, 564, 736). A second important factor in the genesis of oxidative stress is
deficiency in the protection. The cell is protected against abnormal
generation or too long persistence of radicals by a number of enzymatic
reactions and the presence of antioxidants or scavengers, but in
ischemia and reperfusion, these substances are less available. 1) Among the enzymatic reactions that eliminate free
radicals, SOD, catalase, glutathione oxidase, and peroxidase play an important role. Superoxide dismutase transforms the superoxide anion
with protons to H2O2 and
O2, and catalase or glutathione peroxidase finishes
the reaction with the formation of water from H2O2. Glutathione oxidase also
eliminates lipid peroxides under simultaneous oxidation of two
molecules of reduced glutathione. Mice lacking glutathione peroxidase
are susceptible to reperfusion injury (1153), whereas mice
with overexpression of catalase are more resistant to
ischemia-reperfusion injury (603). 2) A number of scavengers or substances normally present in
the cell neutralize radicals. Examples are vitamin E or 2. Effects of oxygen radicals on ion channels and
transporters
Radicals attack proteins and lipids (80). Sulfhydryl
groups of proteins are oxidized and disulfide bridges are formed,
resulting in disturbances of the ion permeation or gating of ionic
channels, decrease of transport capacity of carrier molecules, and
activation of enzymes (957, 1070). Membrane
lipids undergo peroxidation and change indirectly the behavior of channels. Although Na+ and Ca2+ currents are
inhibited, the cells are sensitive to Ca2+ overload; this
is the consequence of induction of leak channels, inhibition of the
Na+-K+ pump and of the
Na+/Ca2+ exchanger, and block of K+
current causing depolarization. In a later stage,
IKATP is activated either by depletion of
[ATP] or a direct effect of free radicals on the channel. Information on the effect of free radicals on the Na+
channel is scarce. In the frog ventricle, application of
tert-butylhydroxyperoxide causes a gradual reduction in fast
Na+ current with a shift of the
Erev in the negative direction.
Transiently, window current shows a dramatic increase (12-fold) due to
an increased overlap of activation and inactivation curves
(81). Such an increase in window current was not seen in
feline ventricular myocytes (40). In rat ventricular
cells, the most prominent effect of
H2O2 is a slowing of inactivation of
the TTX-sensitive current, an effect dependent on activation of PKC
(1070). Oxidation of sulfhydryl groups in the
pore-forming structures by agents like NO radicals inhibit the
Na+ current (151). Inhibition of the ICaL occurs in the guinea
pig (138) and is accompanied by a fall in Ca2+
transient (330). In the ferret, the effect depends on the
type of radicals; exposure to oxygen free radicals and
S-nitrosothiols increases the current, whereas the opposite
effect is obtained with the NO radical (119). The latter
effect is probably due to activation of guanylate cyclase. Under the influence of H2O2, a
Ca2+-permeable leak channel is induced in cultured rat
ventricular myocytes (1056). The channel resembles the
Ca2+-permeable channel described by Coulombe et al.
(187); it has a larger single-channel conductance than
the channel described by Rosenberg et al. (806) but is
also insensitive to DHP. A nonselective cation channel is activated in guinea pig ventricular
myocytes after extracellular or intracellular exposure of radicals
(472). It has the characteristics of the
[Ca2+]i-induced
INSC with a reversal at Most of K+ currents with the exception of
IKATP are inhibited by exposure to oxygen radicals. The Ito current in rat atrial myocytes is
decreased secondary to a shift in the inactivation curve to the left by
exposure to 1 mM tert-butylhydroperoxide or after
photoactivation of 100 nM Rose Bengal (768). A decrease
also occurs by exposure to oxygen radicals for the delayed
K+ current (138, 966,
1007) and the IK1 current
(471, 881). In the frog, the interesting
observation was made that block of the delayed K+ current
is dependent on the state of the channel (966). The largest block was obtained when singlet oxygen was exposed to the
channel in the rested state (activation gate closed); on the other
hand, if the channel was activated before application of singlet
oxygen, the blocking effect was less rapid and less pronounced (1007). In contrast to the well-documented decrease in
delayed K+ current by exposure to oxygen radicals, exposure
to thimerosal, known to oxidize sulfhydryl groups into disulfide
bridges (1144), increases IKs
in canine ventricular myocytes. Bisulfide group formation between the
NH2-terminal ball and part of the channel protein,
thus immobilizing the inactivation gate (816), is the mechanism proposed for the marked slowing of inactivation in expressed Kv.1.4 channels. The effect was reversible upon addition of reducing agents such as glutathione or dithiothreitol. An outward K+ current with the characterisitcs of the
IKATP appears after some delay upon
exposure to oxidative stress (471). The delay may
correspond to the time required to deplete the cell of [ATP]
(980), but a direct effect on the channel with changes in
the affinity for ATP is another possible explanation
(450). Oxidation of sulfhydryl groups by thimerosal
treatment indeed changes the ATP sensitivity of the channel and causes
faster activation during ischemia (164). A decrease in
sensitivity to ATP has been described after a period of ischemia, but
the type of covalent chemical change is not known (213).
Similar effects can be obtained by partial proteolysis in the presence
of trypsin (266). Direct measurement of the of the Na+/Ca2+
exchange current in patch-clamp experiments with controlled
Ca2+ and Na+ concentrations showed a decrease
under oxidative stress (163). In contrast, stimulatory
effects have been described in flux studies in cardiac vesicles
(799), in hearts subjected to hypoxia-reoxygenation (228), and in voltage-clamped guinea pig ventricular
myocytes treated with H2O2
(329). In those cases, Ca2+ and
Na+ concentrations were not "clamped," and stimulation
may be due to indirect concentration changes. The Na+-K+ pump current is reduced
(880). The inhibition correlates with a decrease in
specific ouabain binding and enzyme activity (495,
533). Oxidative stress activates the SR Ca2+ channel probably by
disulfide bound formation (511, 879). Binding
of ryanodine is decreased (428). The
Ca2+-ATPase of the SR is blocked in a sensitive way by
oxidative stress, and the inhibition can be neutralized by
dithiothreitol (254, 565). The mitochondrial mega-channel is activated in severe metabolic
challenge by oxygen radicals in combination with Ca2+,
phosphate overload, and ATP shortage (189,
244). As a consequence, the mitochondrial
Em breaks down, which is the sign of
irreversible damage (Fig. 19). 3. Electrophysiological changes caused by radicals
Two stages can be distinguished in the development of
symptoms in cells exposed to oxygen radical-generating systems
(Fig. 20). In a first stage, upstroke
velocity and conduction of the action potential are reduced; the
plateau is prolonged, and EAD may appear on the slow phase of
repolarization (283, 391, 716, 746, 1070). Repolarization is followed by DAD
(283, 391, 565). Eventually, the
cell may depolarize and show continuous oscillations at the plateau
level. In a second stage, extra systoles or spontaneous activity is
still present, but the action potential gradually decreases in duration
(68). When the action potential becomes very short
(471), the diastolic potential shifts in the
hyperpolarized direction (41), and the cell becomes
inexcitable and goes into irreversible contracture.
6 to 109 s.-tocopherol, vitamin C, glutathione, histidine and other amino acids, carotenoids, and flavonoids. These substances react with radicals and in the process
become themselves radicals; the reactivity of these latter however is
much less pronounced.20 mV. Activation
by oxygen radicals, however, is independent of changes in
[Ca2+]i, since it persists in the
presence of EGTA or ryanodine. Activation can be prevented by
dithiothreitol and stimulated by oxidizing agents such as diamide and
thimerosal. The data suggest activation by extracellular attack of the
channel protein and oxidation of sulfhydryl groups (472).
A similar conclusion was reached for the effect of singlet oxygen on
the frog ventricle (965). Because radicals cause
Ca2+ overload, activation of a
INSC via
[Ca2+]i is also possible; in this case,
the induction of a INSC by extracellular
oxygen radicals is blocked by buffering intracellular Ca2+
(881). Because in ischemic conditions a rise in
[Ca2+]i occurs, the two inducing
processes will cooperate.
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Fig. 20.
A: early changes in electrical activity and ionic
currents during exposure to free radical stress in a guinea pig
ventricular myocyte. Trace a was obtained upon gaining cell
access, and traces b d were obtained after 9, 11, and 13 min of free radical exposure, respectively. Depolarization of diastolic
membrane potential was accompanied by a loss of plateau, and membrane
potential finally stabilized at 30 mV (left). Current
tracings (right) were obtained from ramps that followed
voltage recordings. Note positive shift in reversal potential, increase
in outward current positive to 20 mV, negative shift in holding
current, and transient oscillations after stepping back to holding
potential. Changes can be explained by genesis of a nonselective cation
current and a decrease in IK1. [From Jabr
and Cole (472). Copyright 1995 American Heart
Association.] B: late changes in action potential duration
(left) and current-voltage relation (right)
during oxygen free radicals exposure in a guinea pig ventricular
myocyte. Action potential shortened while an important outward current
with outward rectification (IKATP)
developed. [From Jabr and Cole (471). Copyright 1993 American Heart Association.]
An explanation for the electrophysiological changes can be given in terms of the changes in ionic currents described above. In the early stage, outward currents (Ito, IKs, IK1, and the Na+-K+ pump current) are reduced, whereas inward currents (NSC, Ca2+leak, and Na+ current) are increased. These changes explain the depolarization at rest and the prolongation of the action potential with eventual EAD and oscillatory behavior during the plateau. The reduction of action potential amplitude and upstroke velocity are due to inhibition of the fast Na+ current, partly as a direct effect, partly secundary to the depolarization. These changes result in a marked depression of the conduction of the action potential, an effect potentiated by a fall in gap junction conductance when the cell becomes Ca2+ overloaded. Calcium overload is due to facilitated Ca2+ influx, in addition to a reduced removal from the cell via the Na+/Ca2+ exchanger and the Ca2+-ATPase and reduced active uptake in the SR. Calcium overload also provides an explanation for the occurrence of DAD.
The shortening of the action potential in the second stage accompanied by hyperpolarization is probably due to activation of IKATP, by a direct effect of oxygen radicals on the channel, and a decrease in ATP sensitivity or secondary to ATP depletion. Such depletion is accentuated when the mitochondrial mega-channel is activated.
Although proarrhythmic effects of oxygen radicals can be expected (881) from the occurrence of EAD and DAD and from the fall in upstroke velocity and gap junction conductance, their role in (reperfusion) arrhythmias has been answered with pros and cons. Arguments have been based on the use of antioxidants, but their effect seems to be species dependent. In the rat, an antioxygen radical treatment mostly provides protection against arrhythmias (71, 1108; but see Ref. 353), whereas such protection is absent in the dog (260). Antioxygen radical treatment may provide a better protection against delayed rather than early ventricular arrhythmias (166). Effects furthermore may be due to changes at the level of the coronary vasculature, radicals being responsible for the no-reflow phenomenon upon reperfusion (6).
4. Synopsis
Oxidative stress during ischemia and especially upon reperfusion results from the excessive generation of radicals and the deficiency of protection by enzymes (SOD, catalase, and glutathione oxidase) and scavengers. Radicals attack proteins (sulfhydryl oxidation) and cause lipid peroxidation, resulting in increased leak current (INSC), block of most K+ currents, activation of the SR Ca2+ release channel, and eventually the mitochondrial mega-channel. Electrophysiologically, upstroke and conduction velocity of the action potential are reduced, and the plateau is prolonged with the appearance of EAD and eventual depolarization to the plateau level. In a second stage, the cell may repolarize, again showing very short action potentials (activation of IKATP). In a final stage, the cell becomes inexcitable and goes into contracture.
D. Catecholamines
1. Release of catecholamines during ischemia
During ischemia, two distinct periods of catecholamine release
occur. An immediate release in the systemic circulation occurs after
stimulation of pain receptors and afferent nerve fibers in the ischemic
zone (depolarization due to increased
[K+]o and shortage of
O2). At that time, the local release is negligible; during a 5-min ischemia (angioplasty) in humans, no local
norepinephrine spillover occurs, despite evidence for a generalized
sympathicoadrenal activation (721). This early stimulation
is of short duration, and the local release is rapidly inhibited by
The effect of this excessive release of catecholamines is amplified by
increases in Although 2. Receptors and coupling to effectors
Catecholamines can bind to different types of receptors. In the
heart, one distinguishes The biochemical pathways involved in the modulation of ionic channels
and transporters by 2-receptor activation at the presynaptic level,
inhibition of the exocytotic process by acidosis, and inexcitability of
the nerve fibers due to the rise in
[K+]o (866). At ~10-15
min, a second "metabolic" release phase starts that is
quantitatively much more important (100-1,000 times larger). The
underlying mechanism of this phase is different from the first "exocytotic" phase and is accompanied by a reversal of the
Na+-dependent carrier that normally is responsible for the
transmitter reuptake. Because of shortage of metabolic energy, the
Na+-K+-ATPase is blocked, and
[Na+]i rises and causes the
Na+-dependent carrier to reverse. The increase in
Na+ is amplified by an acidosis-enhanced
Na+/H+ exchange. Storage of the
neurotransmitter in the vesicles furthermore falls
because of inhibition of the H+-ATPase. Evidence for
reversal of the transporter has been provided by the use of
desipramine, which blocks the carrier and markedly reduces the second
release (865).- and -receptor densities during ischemia. The
underlying mechanism for -receptor upregulation is a translocation from an intracellular pool to the plasma membrane (632).
For -receptors (185), it is not a translocation but an
uncovering process by the increased level of LCAC and consequent
increase of membrane fluidity.-receptor density is upregulated during ischemia
(632), -receptor kinase, which inactivates the
receptor, is stimulated at the same time (1004) with less
cAMP as a result (1028). Initially during ischemia, the
response may be increased, but later desensitization takes over.- and -receptors, which are further subdivided on a pharmacological basis in 1-
[1A (or A/C),
1B and others], 2-,
1-, 2-, and
3-receptors. About 80% of the -receptors are of
the 1B-type (268). The density of
-receptors is variable with species. In the rat, the receptors are
numerous, and the ratio of - to -receptors is 1.0. In primates
and humans (937), 1-receptors are less
expressed, and the ratio of - to -receptors is 2.5. Similarly to
-receptors, the ratio of 1- to
2-receptors is species dependent. In the adult
mammalian heart, this ratio is 0.8, whereas the reverse situation
occurs in the frog (919).-receptor stimulation in heart tissue are
multiple (268) (scheme
2).
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Scheme 2.
Coupling of - and -receptors to channels.
This complexity may explain the various effects described in the literature.
-Receptors are connected to effectors via G proteins. In most cases,
the nature of the G protein is of the pertussis toxin-insensitive type, belonging to Gq and Gh type
(336). In canine Purkinje fibers (23) and in
hamster cardiac myocytes (873), pertussis
toxin-sensitive pathways have also been described. Different
phospholipases (PLC, PLD, and PLA2) are activated via
-receptors (336). Phospholipase C activation, which is
the most important mechanism, increases turnover of inositol
phospholipids and leads to the formation of IP3
(775) (or inositol 1,4-bisphosphate, Ref. 16) and DAG. Diacylglycerol in turn activates PKC, and more specifically the classical or Ca2+-activated PKC and the novel or
Ca2+-independent PKC. Phospholipase A2
stimulation causes an increase in lysophospholipids and in AA with
subsequent formation of leukotrienes; leukotrienes in turn may again
activate PKC. 2-Receptors are negatively coupled to
adenylate cyclase, and stimulation leads to a decrease of cAMP. In
other instances, they are positively coupled to a
Ca2+/CaM-activated phosphodiesterase (PDE) with
the same result, i.e., a decrease in cAMP (833).
For 1-receptors, three types of coupling with their
effectors have been described (scheme 2): a direct coupling via a G
protein, a second type of coupling via cAMP without phosphorylation,
and a third way via phosphorylation by PKA, and probably other kinases. For 2-receptors, a coupling to adenylate cyclase
has been demonstrated (919).
Coupling between 1-receptor and effector has been
best studied in the case of the ICa, the
If, and the
ICl.
Direct coupling of the -receptor to the L-type Ca2+
channel via a stimulatory G protein has been demonstrated in excised
patches and in channels incorporated in lipid bilayers
(453) and is further suggested by the development of the
effect in two phases with time constants of 150 ms and 35 s (see
Ref. 1147). A direct coupling may not be universal, however, since a
fast phase is not present in all species. In preparations of the frog,
rat, and guinea pig, with only a slow phase, the whole effect can be
blocked by PKA inhibitors (380). A direct coupling has
also been proposed for stimulation of the
If current in the SAN (1148),
but its functional importance is not clear.
A second type of coupling between -receptor and effector occurs via
a direct effect of cAMP on the effector and has been demonstrated for
the If current in the SAN
(223).
The third type of coupling consists of PKA-dependent phosphorylation of the channel and has been clearly demonstrated for the ICa, If, and ICl. Other currents also are probably stimulated via this pathway: Ist, IKs, IKur, IKATP, Igap, and ICaSR. The pathway involved in the inhibition of IK1, IKss, and Ito is not known.
In the Ca2+ channel, two sites probably undergo
phosphorylation: phosphorylation of site 1 makes the
Ca2+ channel available and phosphorylation of site
2 causes the shift in the modal gating (720,
737). The existence of two phosphorylation sites is
consistent with the observation that stimulatory but differential
effects are obtained using specific phosphatase inhibitors (720, 1096): okadaic acid (acting on
phosphatase 2A) promotes mode 2 behavior, while calyculin A
(acting on phosphatase 1) increases availability. The -subunit may
undergo phosphorylation (1152), but expression of the
-subunit seems required to observe the increase in current
(549).
Phosphorylation of the If channel is of prime importance for modulation of the pacemaker current in ventricular and Purkinje cells. Experimental manipulations, using forskolin, PDE inhibitors, and permeable cAMP analogs (143), shift the activation curve in the positive direction while the opposite effect is seen when phosphorylation is inhibited using different PKA inhibitors. Inhibition of phosphatase by calyculin A shifts the activation curve in the positive direction with no change in the maximal conductance (1157, 1158). Whether PKA-dependent phosphorylation of the If is also occurring in the SAN remains controversial.
Activation of a Cl current by -receptor stimulation
occurs via a two-step phoshorylation of the regulatory domain of
the CFTR protein. Phosphorylation allows binding and hydrolysis of ATP.
The two steps correspond to two levels of activity, one characterized by short openings, the second by long-duration openings
(34, 446, 448).
3. Effect of A) EFFECT OF -receptor stimulation
-RECEPTOR STIMULATION ON CHANNELS AND
CARRIERS.
Major effects of -receptor stimulation occur on the K+
currents (decrease and increase), gap junction (decrease), and
exchangers (stimulation).1B-receptors coexpressed with the
human cardiac Na+ channel in oocytes (704)
causes a 40% reduction in current without changes in kinetics. The
same effect is obtained with phorbol esters, suggesting the involvement
of PKC stimulation.
-receptor
stimulation was characterized at the single-channel level by a
shortening of the open time and an increase in blank sweeps but no
change in the single-channel conductance (147). The
T-type channel activity is mostly reported to be increased (14, 993); such an effect may be important
for Ca2+ loading of the cell, since the T-type channel
is activated at rather negative Em values.
Stimulation of PKC leads to results similar to -receptor
stimulation, i.e., variable effects on the L-type
(233, 577) and stimulation of the T-type
current (993). It is not known whether the T-type
channel protein is phosphorylated. An indirect effect via
alkalinization secondary to activation of the
Na+/H+ exchanger by PKC can be excluded, since
pHi changes between 6.5 and 8.0 do not induce changes
in the T-type current (1000). Facilitation by
increased [Ca2+]i, secondary to
Ca2+ release by IP3, is a possibility that
remains to be investigated. Arachidonic acid and leukotrienes that are
produced during -receptor stimulation enhance the Ca2+
current (441).
Among the K+ currents, some
(Ito, IKur, and
IK1) are decreased, and others
(IKs, IKACh,
IKATP, and
IKAA) are enhanced. -Receptor stimulation substantially decreases Ito in
rat (24, 256, 796, 977) and rabbit ventricular myocytes (270). A
pertussis toxin-insensitive G protein plays the role of
transduction factor, but the subsequent biochemical pathway is not
known. Phorbol ester application and PKC activation yielded divergent
results, and an increase as well as decrease have been described. In
some cases, -receptor stimulation decreased the current, whereas PKC
activation had the opposite effect (270). In Kv.4.2 and
Kv.4.3 expressed channels, PKC stimulation inhibited the current
(713).
-Receptor activation reduces IKur (human
atrial cells, Ref. 605; rat ventricular myocytes, Ref. 796) and the
inward rectifier IK1 (24,
256, 796, 843,
874). The addition of purified PKC to inside-out
patches, containing IK1 channels, has the
same effect (843).
The effect on IKs is species dependent. In
the guinea pig, -receptor stimulation activates PKC and causes
enhancement of the current (979, 1051); the
effect is additive to PKA activation (978). In the mouse
(431) and rat (24), activation of PKC reduces
IKs. It is interesting to note that the
cloned minK protein, which can be regarded as a modulator or an
essential part of the KvLQT1 channel, differs by only one amino acid in
these species (404).
In oocytes, 1C
(1A/C)-expression causes a rapid upregulation of
the Kmin current by elevating
[Ca2+]i (996). The
IKACh current is enhanced by -receptor
stimulation via activation of PLA2, generation of AA
and subsequently of LT. Leukotrienes activate directly the pertussis
toxin-sensitive G protein connected to the KACh channel
(570).
The IKATP in human and rabbit ventricular
myocytes is activated by norepinephrine via stimulation of PKC
(439); the simultaneous increase of
[Ca2+]i facilitates the activation of
PKC (1063). Excessive turnover of cAMP, a result of
massive -receptor stimulation, may furthermore deplete
subsarcolemmal [ATP], which again enhances
IKATP (435, 858).
Currents with characteristics similar to, but not identical to,
IKATP are activated by AA
(527) or by LPC (1050).
Information on Cl channels is scanty. Although a
Cl channel is activated by PKC (168,
1052, 1178), the role of -receptors has
not been verified. The swelling-induced Cl current is
reduced by -receptor stimulation (238,
241).
Phosphorylation by PKC of the gap junction causes a fall in
single-channel conductance but an increase in
Po (690, 699); the overall effect of phenylephrine is a reduction of the gap junction
conductance by 45% (209). Such a reduction may play a
role in the genesis of reentry arrhythmias.
-Receptor stimulation activates the Na+-K+
pump current (874, 1103). The -receptor
involved is 1B, which is connected via a pertussis
toxin-sensitive G protein (874) to PKC; sensitivity of
the coupling is increased at elevated
[Ca2+]i (1059). In part,
the stimulation of the pump may be secondary to an increase in
[Na+]i as a consequence of an enhanced
Na+/H+ exchange.
Activity of the Na+/Ca2+ exchanger is enhanced
(939) probably after PKC stimulation. The increase in
activity can occur in the absence of pHi change
(939), but the simultaneous activation of
Na+/H+ exchange (470) will add to
the final effect, since alkalinization as such is a strong stimulator
of Na+/Ca2+ exchange (230,
245).
B) ELECTROPHYSIOLOGICAL ASPECTS OF -RECEPTOR
STIMULATION.
Because pump current is enhanced but IK1 is
inhibited, the outcome of -receptor stimulation on the resting
potential is difficult to predict and will depend on the relative
contribution of the two currents to the resting potential. A
depolarization of the resting potential is seen in the rat atrium,
whereas hyperpolarization occurs in the rat ventricle
(256, 977). Inhibition of the
IK1 has been invoked to explain the
positive chronotropy in canine Purkinje fibers (874) and
Langendorff-perfused guinea pig hearts (150).
1-Receptor stimulation prolongs the action
potential in most preparations (see Ref. 268), the underlying
mechanisms being block of Ito,
IKur, and IK1.
Exception to this rule is the guinea pig, where no effect or even
shortening of the action potential is observed (227). This
effect is related to the fact that in this species
Ito is not pronounced; in contrast,
IKs current is well represented, and this
current is increased after PKC activation.
Delayed afterdepolarizations can be enhanced or inhibited by
-receptor stimulation, depending on the mechanism by which they are
generated. When elicited after exposure to high
[Ca2+]o, -receptor stimulation
potentiates DAD (274). The potentiating effect could be
due to reduction of the resting K+ conductance
(IK1), to enhanced IP3
production with release of intracellular Ca2+, and to
increase of ICl and
INa,Ca. In this context, it can be mentioned that -receptor stimulation favors reperfusion arrhythmias (757), an effect that has been related to Ca2+
overload. Once started by abnormal automaticity, the arrhythmia may be
stabilized as a reentry arrhythmia by cell uncoupling as a consequence
of a fall in gap junction conductance (209). When DAD are
caused by strophanthidin-induced block of the
Na+-K+ pump (274), -receptor
stimulation inhibits DAD, an effect which can be explained by the
stimulatory effect on the pump, reducing [Ca2+]i overload. An inhibitory effect
was also seen when DAD were elicited by -receptor activation
(833); in this case, a decrease in cAMP by activation of
2-receptors is a possible explanation.
4. Effects of A) EFFECTS OF -receptor stimulation
-RECEPTOR STIMULATION ON CHANNELS AND
CARRIERS.
-Receptor activation results in stimulation of a number of currents
[ICa, If,
Ist, IKs,
IKur, IKATP,
ICl, Igap,
ICa(SR)], whereas only a few are reduced
(Ito, IKss, and
IK1).-receptor stimulation on the Na+ channel
has been a controversial issue in cardiac electrophysiology. All
published effects can be explained, however, when it is assumed that
the basic mechanism consists of a shift of both activation and
inactivation curves to the left, as shown experimentally
(738). The shift explains why an increase in current is
seen when both the holding potential and the test potential are rather
negative, why no change is seen when the test potential is positive and
holding potential negative, and why a decrease is obtained when the
holding potential is less negative (see references in Ref. 738). In
depolarized, but still excitable cells (early ischemia, border zone),
the negative shift of the inactivation curve will cause further
inhibition of the Na+ current, with reduction in upstroke
velocity and in conduction of the action potential. This inhibitory
effect, however, may be counteracted by some hyperpolarization of the
resting potential.
-Receptor stimulation by epinephrine and norepinephrine increases
peak ICa and slows inactivation (see Ref.
673); the slowing is clearly seen when Ba2+ is the charge
carrier but is very variable with Ca2+ because of
Ca2+-induced inactivation. Activation and inactivation
curves are shifted to the left. At the single-channel level, the
conductance is not changed, but the Po
during a pulse as well as the number of functional channels are
increased (decrease of blank sweeps). A shift from short to long
openings is manifest and explains the slowing of inactivation
(1160). Gating current is not changed, suggesting that
coupling between gating and channel opening is improved.
-Receptor stimulation has been reported to be ineffective on the
T-type Ca2+ current (48, 355,
1001), but recently a facilitatory effect has been
described in bullfrog atria (13). -Receptor stimulation furthermore may indirectly increase the currrent via an increase in
[Ca2+]i, which is known to facilitate
the current (994). The increase in current by ouabain can
be explained in a similar way (13).
In canine Purkinje fibers, the first component (time constant of 50 ms)
of the transient outward current Ito is
decreased by -receptor stimulation, whereas the slowly inactivating
component (partly IKur?) is substantially
increased, such that the ratio of fast to slow component changes from
0.65 to 0.15 (717). The effect is duplicated by forskolin
and cAMP. In contrast, in feline ventricular cells, forskolin or
isoproterenol has no effect on Ito
(268).
Analysis of the effect of -receptor stimulation on
IKur and IKss
currents is incomplete. In canine Purkinje fibers, the steady-state current that follows the two phases of the
Ito is increased (717); it is
possible, however, that the records included
ICl. An increase in
IKur was observed in the human atrium
(605) and in cultured neonatal rat ventricular myocytes
(345). The IKss in rat
ventricular myocytes on the other hand was very sensitively reduced
(nanomolar concentrations) (849).
-Receptor stimulation does not change
IKr but upgrades the
IKs current after phosphorylation of the
channel by PKA (65, 384, 1051,
1149). Enhancement of the current may also occur in a
phosphorylation-independent way (296). In the frog,
the total conductance is dramatically increased, and the activation
curve is shifted in the negative direction (243). The
single-channel conductance is not changed, but
Po is increased (242).
The IK1 current is reduced, an effect
opposite to M2 stimulation (559).
Activation of IKATP on the other hand is
facilitated by -receptor stimulation (435,
858). Subsarcolemmal depletion of [ATP] due to ATP
consumption by a stimulated Na+-K+ pump and by
enhanced cAMP synthesis (435, 858) has been
proposed to be the underlying mechanism. The same mechanism may be
responsible for activation of IKATP upon
simultaneous application of isoproterenol and a subthreshold
concentration of a potassium channel opener; the effect does not
require phosphorylation (995).
-Receptor stimulation activates a Cl current in a
number of ventricular preparations (see Ref. 308). The current is
activated by a two-step phoshorylation of the regulatory domain of
the CFTR protein (34, 446, 448).
The results on the swelling-induced Cl current are
divergent; forskolin increases (745), whereas cAMP inhibits the current (362).
-Adrenergic stimulation increases the pacemaker current
If by inducing a shift in the positive
direction of the activation curve without changing the fully activated
current (779) (Fig. 21) or
the single-channel conductance (220,
222). Such an effect implies that the current activates at
an earlier stage during the final repolarization of the action
potential. Diastolic depolarization is faster, and frequency of
spontaneous activity is increased. The effect on
If is obtained at concentrations that also
enhance ICa
[Kd = 7 nM (1173) for
ICa and 14 nM for
If].
|
-Receptor stimulation strongly enhances a sustained inward current
in the SAN of the rabbit (346). The current carried by Na+ is activated by depolarizations to 70 mV and more
positive and shows minimal inactivation in the range of potentials
corresponding to the diastolic depolarization. Its enhancement by
-receptor stimulation favors spontaneous activity in the sinus node.
Earlier experiments on multicellular preparations using radioactive
K+ uptake (706) are consistent with
stimulation of the Na+-K+ pump after
-receptor activation. Direct measurements of the pump current have
revealed variable effects. A stimulatory effect is present in sheep
multicellular Purkinje preparations but not in the single cell,
suggesting that the increase might have been indirect and due to
extracellular [K+]o accumulation
(326). However, in single cells from the rabbit, [Na+]i is reduced (211).
Little or no response was seen in rat myocytes (464). In
guinea pig ventricular myocytes, the effect requires PKA-dependent
phosphorylation, does not depend on changes in
[Na+]i or
[K+]o, but is highly dependent on
[Ca2+]i (314). Inhibition
of the pump by isoproterenol occurs at low [Ca2+]i and stimulation at high
[Ca2+]i. Gao et al. (314)
conclude that the direct effect of -receptor stimulation by
isoproterenol is inhibition (similarly to results of biochemical
studies), but stimulation of the pump may occur as a result of the
simultaneous increase in [Ca2+]i to the
micromolar range and the concomitant shift of the current-voltage relation of the pump in the negative direction. The stimulatory effect
is especially pronounced at hyperpolarized potentials.
Phosphorylation of the SR Ca2+ channel by PKA shifts the
Po curve as a function of pCa or of [ATP]
upward and to the left (see Ref. 359). Open times are increased and
sensitivity to Mg2+ block is decreased (1006).
Coupling between cells is improved by cAMP (207,
823). Phosphorylation of the gap channel protein by PKA
causes a fall in single-channel conductance (690), but
gap junction conductance increases (208,
823), suggesting a rise in Po
at the channel level.
B) ELECTROPHYSIOLOGICAL EFFECTS OF -RECEPTOR
STIMULATION.
-Receptor stimulation increases spontaneous pacemaker frequency in
the SAN secondary to an enhanced rate of diastolic depolarization and a
shortening of the systolic period (101), improves
conduction in the AVN, elevates the plateau level, and shortens the
action potential duration in atrial and ventricular cells
(506). Excessive stimulation may result in
Ca2+ overload and genesis of triggered spontaneous activity.
-receptor stimulation. Rate of repolarization may further be
enhanced by stimulation of the Na+ pump and by
activation of Cl channels, but the presence of the latter
current in pacemaker cells has not been documented.
Similar changes in action potential occur in atrial and ventricular
cells: the plateau is shifted in the positive direction and its
duration is shortened (506). Shortening of the action potential is amplified when the cell is partially depleted of [ATP]
during ischemia; -receptor stimulation enhances local [ATP] depletion, via consumption by the Na+-K+ pump,
the contractile system, and cAMP synthesis, resulting in activation of KATP.
-Receptor stimulation enhances the tendency to spontaneous activity
in atrial and ventricular cells. The mechanism is twofold: possible
activation of If current and facilitation
of triggered activity. The If current is
present in Purkinje cells but also in plain atrial and ventricular
cells. Because -receptor stimulation shifts the activation curve in
the positive direction, threshold may be reached at the resting
potential. Triggered activity is favored by the Ca2+
overload that accompanies excessive -receptor stimulation
(782). Calcium influx is increased via the L-type
Ca2+ channel and the amount of Ca2+ stored in
the SR is enhanced, by stimulation of the Ca2+-ATPase,
secondary to phospholamban phosphorylation (see Ref. 503). The high
level of luminal Ca2+ in the SR may result in spontaneous
release and genesis of DAD or EAD. The occurrence of EAD is especially
favored by the increased ICa. In
depolarized cells, e.g., in the presence of increased [K+]o concentration, enhancement of the
Ca2+ current may generate Ca2+-dependent action
potentials (132). These action potentials are slowly
conducted and may play a role in reentry arrhythmias.
5. Synopsis
Obstruction of the coronary circulation is immediately accompanied
by a release of catecholamines in the systemic circulation, followed
later after 10 min by a local release caused by block of the reuptake
mechanism in nerve endings. At the same time, the number of - and
-receptors in the plasma membrane are increased. Both - and
-receptors are coupled to their effector channels via complex
pathways: the coupling may be direct via a G protein or indirect via a
number of phospholipases (-receptors) or adenylate cyclase
(-receptors). -Receptor stimulation causes increase of some but
decrease of other K+ curents, decrease in gap junction
conductance, and stimulation of the Na+/Ca2+,
Na+/K+, and Na+/H+
exchangers. At the cellular level, the resting potential may depolarize
or hyperpolarize; the action potential in most preparations is
prolonged. Delayed afterdepolarizations are enhanced when they have
been caused by increased inward Ca2+ leak; they may be
inhibited when they have been induced by block of
Na+-K+ pump inhibition. -Receptor activation
results in stimulation of most plasma membrane currents (inward and
outward currents), gap junction channel, and SR Ca2+
release channel. Electrophysiologically, -receptor activation increases pacemaker activity, improves nodal conduction, and elevates and shortens the plateau of the action potential. Excessive stimulation may result in Ca2+ overload and triggered activity.
E. Extracellular ATP, Adenosine, and ACh
1. Extracellular ATP
A) RELEASE OF ATP.
Extracellular ATP is generated from two sources: 1)
sympathetic nerve endings secrete ATP upon electrical stimulation
together with other neurotransmitters; and 2) as a
metabolite, ATP is released from cardiac cells exposed to hypoxia
(203). A possible pathway for the release in the
extracellular medium is the PKA-activated anion channel
(871). During a limited time, [ATP] may reach the micromolar concentration range and induce multiple functional changes
in cardiac cells (1027). In the extracellular space, it is
rapidly degraded to adenosine by ectonucleotidases. B) RECEPTORS AND COUPLING.
The effects of ATP occur via stimulation of specific
P2 receptors. At least five subtypes of
P2 receptors can be distinguished, among which the
P2x and P2y are the most important
for cardiac cells. The P2x receptor molecule should be
mentioned separately because the molecule expressed in vas deferens is
itself a NSC channel, permeable to Na+, Ca2+,
and K+. The structure resembles the inward-rectifying
K+ channel (1008). Whether the
P2x receptor forms a NSC channel in heart is not
known. Other P2 receptors (P2y) are
linked directly to ion channels via a Gs protein
(ICaL, Refs. 853, 1180;
IKs, Ref. 668) or act indirectly via
stimulation of adenylate cyclase (IKATP)
(31), activation of PLC and secondarily of PKC
(ICaL) (852), or possibly
activation of tyrosine kinase (649). C) EFFECTS OF [ATP]O ON CHANNELS.
The effect of [ATP]o is usually of short duration
because ATP is rapidly broken down in the extracellular medium.
channels has not been elucidated.
A NSC channel is transiently activated by [ATP]o in
atrial cells of the frog (301), atrial cells of the
rabbit, atrial and ventricular cells from the guinea pig
(411, 752), and ventricular cells of the rat
(1180). The current carries bivalent as well as monovalent
ions. Its Erev is close to zero, and its
current-voltage relation is linear or slightly inward rectifying
(899). At the present time, it is not clear whether
activation occurs directly by binding to the P2x
channel (1008) or indirectly by activation of a
Cl/HCO3 exchanger, intracellular
acidification, and release of Ca2+ or after phosphorylation
of extracellular proteins (see Ref. 1027).
D) ELECTROPHYSIOLOGICAL EFFECTS OF [ATP]o. Changes in electrophysiological properties may be different in ischemic versus nonischemic conditions. In normal conditions, the overall initial effect of [ATP]o in most preparations is depolarization, due to activation of the INSC and the ICl with concomitant activation of INa and ICa (especially T-type ICa). These effects seem to overrule the smaller stimulation of K+ currents. The depolarization may be conducive to the generation of spontaneous activity (1027). In ischemic conditions, with cells partly depleted of [ATP], stimulation of adenylate cyclase by [ATP]o may further deplete local subsarcolemmal [ATP] and activate IKATP (31). This will lead to hyperpolarization and shortening of the action potential. The [ATP]o may thus cause opposite changes in healthy tissue and in ischemic cells. In vivo, the effect is of short duration, because ATP is rapidly broken down to adenosine.
2. Adenosine and ACh
A) RELEASE OF ADENOSINE AND ACETYLCHOLINE.
Adenosine is generated either intracellularly or extracellularly.
Extracellularly it is formed from ATP under the influence of
ectonucleotidases. In the cell it is normally formed from AMP under the
action of 5'-nucleotidase but again rapidly synthesized to AMP under
the influence of adenosine kinase (203). Because ATP is
consumed in this high turnover process, the cycle may be regarded as
futile; there are, however, some advantages to it. When oxygen tension
is reduced below a critical threshold, adenosine kinase becomes
inhibited, and adenosine genesis is amplified and released from the
cell, possibly via the PKA-activated anion channel (871). The high concentration reached permits rapid
signaling, causing for instance vasodilation that counteracts the
hypoxic stimulus.
B) RECEPTORS AND COUPLING. The effect of adenosine occurs via stimulation of P1 purinergic receptors. In the group of P1 receptors, four subtypes have been described, of which A1 is the cardiac receptor. The effect of ACh occurs via stimulation of M2 receptors (M1 receptors are also present in the rat and may be responsible for the stimulatory effect at high concentrations (878; but see Ref. 658).
A1 and M2 receptors are linked to the effector in more than one way (scheme 3).
|
-receptor stimulation (accentuated
antagonism) (286, 401). Withdrawal of ACh
under those conditions causes transient increases in
ICa. Inhibitory effects also occur via
activation of NO synthase (NOS3) (645, 792,
895) and secondary increase in cGMP. Guanosine
3',5'-cyclic monophosphate has been proposed to activate PDE II and
reduce the cAMP concentration (681, 792) or
activate PKG with phosphorylation and reduced activity of the effector
(600, 680, 947, but see Ref. 365 for a facilitatory effect via PKG). In
accord with the PKG hypothesis, NO donors have been shown to reduce
ICa in the presence of nonhydrolyzable
cAMP, whereas the effect disappears with block of guanylate cyclase or
of PKG (1048). Consistent with the proposal of PDE II
activation, a fall in cAMP concentration has been demonstrated in the
SAN, secondary to NO production upon cholinergic receptor activation;
the effect was absent when NO synthase was inhibited (371). It should be added, however, that low
concentrations of cGMP have been found to inhibit PDE III in mammalian
cells and thus enhance the concentration of cAMP (740).
Inhibition of PDE III has also been implied in the rebound stimulation
of ICa upon withdrawal of ACh
(1064). Guanosine 3',5'-cyclic monophosphate may not only
affect PDE or PKG but exert a direct activating effect, similar to
cAMP, e.g., on the If current
(223). In this context, it should be mentioned that
exogenous NO can enhance the If current and
increase the rate of beating (705); this effect probably occurs via a direct stimulatory effect of cGMP on the channel protein
(219).
The G protein involved in the activation of NOS3 is not known.
Stimulation of the synthase, however, may be secondary to
IP3 formation, which may cause an increase of free
[Ca2+] and activation of the enzyme. Formation of
IP3 probably results from PLC stimulation (see Ref.
643).
C) EFFECTS OF ADENOSINE AND ACETYLCHOLINE ON CHANNELS.
In general, the effects of adenosine and ACh are opposite to
-receptor stimulation, but exceptions to this rule exist.
background current induced by
isoproterenol (793).
Acetylcholine or muscarinic stimulation causes the activation curve of
If to shift in the negative direction
(891). This effect occurs at concentrations much smaller
(Kd 20 nM) than necessary to open the KACh
channel (0.5 µM, Ref. 221) or to inhibit
ICa (1173) (Fig. 21). In the
SAN, ACh has an effect on its own; in secondary pacemakers like the
Purkinje fibers, it only reverses the effect of -receptor
stimulation (144). Adenosine exerts an effect similar to
ACh via an analogous pathway (1174).
Gap junction conductance is decreased by carbachol or cGMP, an effect
opposite to that of catecholamines or cAMP (572,
955).
Among the K+ currents,
IKACh and IKATP
are activated, whereas IK1 and
IKs are inhibited. Adenosine and ACh
efficiently activate the IKATP in
ventricles (469, 531, 540,
969) and the IKACh in the SAN,
AVN, atria, and ventricles (55). For both types of
currents, the link is direct via a G protein. The direct coupling is
not universally present in mammalian ventricles but exists in the human
(95, 558), rat (676), ferret
(95), and dog (1142). In addition to a direct
coupling, the increase in IKATP also occurs
indirectly via activation of PKC and is supposed to play a role in
preconditioning (439, 608, 619,
1062). At first sight, stimulation of PKC however has two
opposing effects. It inhibits maximum activity of KATP but at the same
time reduces the slope or Hill coefficient from 2.2 to 1.0 (609). From the fall in slope, activation of the channel
at millimolar [ATP] was predicted and later confirmed
(610). The effect is antagonized by protein kinase
inhibitors or phosphatases.
Inhibition of IK1 (559) and
IKs (748) occurs when the
currents have been upgraded by -receptor stimulation and increase in
cAMP (accentuated antagonism).
D) ELECTROPHYSIOLOGICAL EFFECTS OF ADENOSINE AND ACETYLCHOLINE. In contrast to ATP, the effect of adenosine and ACh results in hyperpolarization and shortening of the action potential, slowing of the rate of diastolic depolarization in pacemaker cells, and inhibition of EAD and DAD (55, 927). In general, adenosine and ACh are considered to act as cardioprotective and stabilizing agents. Mice overexpressing A1 receptors show an increased resistance against ischemia evidenced by a longer time to development of contracture and improved functional recovery upon reperfusion (648). The electrophysiological changes are the result of K+ current activation (KACh and or KATP) and inhibition of ICa, If, and ICl. In the SAN, the rate is slowed and pacemaker shifts may occur (53, 1086). In AVN cells, the action potential is inhibited resulting in conduction slowing or block. In the clinical context, this blocking effect is used to suppress paroxysmal supraventricular tachycardia (226).
As antiadrenergic agents adenosine and ACh suppress catecholamine-induced early and late afterdepolarizations (927), and reduce inward background current (792, 793). This effect may play an anti-arrhythmic role during ischemia and reperfusion. A protective effect of vagal stimulation on reperfusion arrhythmias has been demonstrated in cats (1190). Stimulation of M2 receptors with oxotremorine markedly reduced the occurrence of ventricular fibrillation in a feline model subjected to acute ischemia and stimulation of the left ganglion stellate (1190). Animals selected for their elevated vagal tone were less susceptible to ventricular fibrillation upon acute ischemia (170). Indirect inhibitory effects via adenylate cyclase are of functional importance in the presence of-receptor stimulation
(accentuated antagonism) (286, 401).
Withdrawal of ACh under those conditions causes transient increases in
ICa, lengthening of the action potential duration, and induction of DAD and spontaneous activity
(438, 698, 1060).
In contrast to the stabilizing effects described above, high
concentrations of ACh induce a TTX-insensitive Na+
current in guinea pig ventricular myocytes (658,
896). The current reverses at 25 mV in normal Tyrode
solution, suggesting selectivity for Na+.
M2 receptor activation with consequent stimulation of
PLC, IP3 formation, and activation of PKC is a
possible scheme (658). The result is an increase of
[Na+]i (822) and
[Ca2+]i (557) and an
enhancement of Ca2+ transients and contraction
(785).
3. Synopsis
Release of ATP during metabolic inhibition is increased. Because of its rapid metabolization in the extracellular space, its functional importance is limited. Information on the coupling between receptor and effector is incomplete. Adenosine 5'-triphosphate stimulates INSC and ICl, facilitates ICaT, and activates some K+ currents (IKACh, IKs, and IKATP). Prolongation as well as shortening of the action potential have been described. Shortening may become important during ischemia.
Adenosine release as well as vagal reflexes are enhanced during
ischemia. A1 receptors (adenosine) and
M2 receptors (ACh) are linked to their effectors
directly via a G protein or indirectly via changes in the activity of
PLC, NO synthase, and adenylate cyclase. In general, the effects are
opposite to -receptor stimulation. More specifically,
ICa, ICl,
If, and gap junction conductance are
negatively affected, whereas activation occurs for
IKACh and IKATP. Electrophysiologically adenosine and
ACh cause hyperpolarization, shortening of the action potential,
slowing of diastolic depolarization, and inhibition of EAD and DAD.
F. Stretch and Volume Changes
1. Existence of stretch
Stretch can take two different forms, depending on whether
it is caused by tension applied to the cell or after an increase in
cell volume due to intracellular hyperosmosis. Stretching of cells
occurs in a passive way during the filling of the ventricles. Stretch
will be pronounced in Purkinje or endocardial cells surviving an
infarct zone. Cells of the ischemic zone will elongate during the
active contraction of viable cells surrounding the infarcted zone. This
type of stretch is characterized by an increase in the longitudinal
dimension but a decrease of the transversal direction. Volume changes
elicited by osmotic forces affect all dimensions of the cell. The
effect may not be the same: in guinea pig ventricular myocytes,
elongation activated a NSC channel while a hyposmotic challenge
stimulated IKs (842). Because
mechanical stability of the cells depends on the integrity of the
cytoskeleton, changes in the microscopic architecture may
determine the electrical changes. As a whole, our knowledge of
stretch-related effects remains deficient. 2. Effect of stretch on channels and exchangers can be direct
or indirect
Indirect changes on channels and exchangers are possible secondary
to activation of membrane enzymes. It is known that stretch rapidly
activates a plethora of second messenger pathways including tyrosine
kinases, mitogen-activated protein kinases, PKC, PLC, PLD, and
PLA2 and causes release of substances such as ANG II, which act as auto- or paracrine factors (821). Mechanical stretch or volume expansion enhances
ICaL (657), causes activation
of NSC and Cl A stretch-activated NSC channel has been described in rat
atrial cells (526), in cultured chick hearts
(815), and guinea pig ventricular cells (313,
841). The channel is permeable to monovalent cations and
Ca2+ (526). In atrial cells, activation is
followed by an increase in [Ca2+]i,
which may trigger atrial natriuretic factor secretion. In neonatal
cells of the rat, another channel is activated and has a permeability
ratio PK/PNa of
3.4 but is impermeable to Ca2+ (188). A volume-activated Cl Swelling activates the IK1 current in chick
embryonic heart (1175) and in feline ventricular cells
(334). The delayed K+ current
IKs is increased in hypotonic solutions in
the guinea pig (734, 798, 842)
and in chick embryonic heart cells (1175)); inhibition
occurs in hypertonic solution (734). Opposite effects occur in the IKr (798).
Pressure applied via the patch electrode enhances the ACh-activated
K+ channel (774), the AA-activated
K+ channel (525), and the KATP channel
(1020, 1022). The
Na+/Ca2+ exchange current is reduced in hypo-
and increased in hyperosmotic conditions (1109). 3. Electrophysiology and arrhythmias upon stretch
From the description of the changes in channel function, it
is clear that inward as well as outward currents are activated. At
negative potentials, the inward currents prevail, whereas at potentials
more positive, outward currents are determining. Stretch or pressure
applied to multicellular preparations or total heart chambers elicits
depolarization of the resting potential while the action potential is
shortened in its early repolarization phase, but lengthened in its
final phase (202, 295, 375,
575, 1010, 1069). In atrial
tissues in vivo, the effective r channels, and enhances outward currents
through the K1, Ks, KACh, KATP, and KAA channels (821,
1089). channel is present in
atrial cells of the rabbit (238, 239,
357), guinea pig (841, 902,
1010, 1012), dog (929,
992), and human (745, 827) and
in cultured cells of the chick embryo heart (1176).
Activation is slow, and a phosphorylation by stretch-activated
tyrosine kinase has been proposed as a possible mechanism
(745).
). [From Firek and Giles (
, Time constant.
In B, protocol was identical except that a brief 20-ms
hyperpolarizing pulse to 
) and 1 µM spermidine (
).
B: normalized currents as a function of membrane potential
in 500 µM spermine (spe), spermidinine (spd), and putrescine (put).
Curves are fitted by Boltzmann functions with electrical distances of
2.64 for spe, 2.21 for spd, 1.69 for put, and 1.12 for
Mg2+. [From Lopatin et al. (
m) of inner mitochondrial membrane
measured by accumulation of tetraphenyl phosphonium in membrane. Arrow
indicates start of respiration by addition of succinate to isolated
mitochondria (rat heart). [Ca2+] was increased from 0.1 µM (i) to 1.0 µM (ii), 3 µM
(iii), and 5 µM (iv); high [Ca2+]
resulted in breakdown of electrical gradient. This effect could be
counteracted by adding ATP (data not shown). [From Duchen et al.
(
