INTRODUCTION

TOP ABSTRACT INTRODUCTION CA2+-INDUCED CA2+ RELEASE VOLTAGE-SENSITIVE RELEASE... REGULATION OF VSRM BY... CONDITIONS REQUIRED FOR... MECHANISM OF EC COUPLING... ROLE OF VSRM IN... SUMMARY REFERENCES

CONTRACTION IN THE HEART is initiated by a transient rise in intracellular free Ca²⁺. The cardiac action potential triggers this Ca²⁺ transient, which rapidly rises and decays with each cardiac cycle. The events that couple depolarization of the sarcolemma to elevation of Ca²⁺ and initiation of contraction are referred to as excitation-contraction coupling (EC coupling). Several mechanisms of EC coupling have been proposed for cardiac muscle, and some of these are controversial. This article reviews various mechanisms of EC coupling, their characteristics, and evidence for their roles in EC coupling.

	CA2+-INDUCED CA2+ RELEASE

TOP ABSTRACT INTRODUCTION CA2+-INDUCED CA2+ RELEASE VOLTAGE-SENSITIVE RELEASE... REGULATION OF VSRM BY... CONDITIONS REQUIRED FOR... MECHANISM OF EC COUPLING... ROLE OF VSRM IN... SUMMARY REFERENCES

Many studies of EC coupling conducted in the 1970s and 1980s established that depolarization of the cell membrane triggered cardiac contraction (reviewed in Refs. 29, 36). Depolarization also activates inward Ca²⁺ current, and several hypotheses relating activation of Ca²⁺ current to contraction were proposed (2, 28, 53, 56). The role of Ca²⁺ influx was explored further by Fabiato (15-17) in studies that were conducted on "skinned" myocardial cells (i.e., cells that have had their sarcolemma removed by mechanical or chemical means). Fabiato (15-17) showed that rapid application of Ca²⁺-containing solutions to skinned myocytes could trigger release of Ca²⁺ from intracellular stores. Fabiato also showed that Ca²⁺ release could be blocked by ryanodine, an agent that disrupts sarcoplasmic reticulum (SR) release channels (ryanodine receptors) and that is used widely to identify involvement of SR Ca²⁺ release in contraction (3, 64, 82). These studies (15-17) showed that SR Ca²⁺ release in the heart can be triggered by a rise in Ca²⁺ concentration in the vicinity of ryanodine receptors. The process by which a small amount of Ca²⁺ triggers release of SR Ca²⁺ has been called Ca²⁺-induced Ca²⁺ release (CICR) (14).

Although Fabiato's studies showed that CICR occurred in response to application of Ca²⁺ to skinned myocytes, it was essential to determine whether CICR also occurred in cells when the sarcolemma was intact. This was shown in experiments on intact ventricular myocytes in which the initial small increase in free Ca²⁺ (trigger Ca²⁺) was provided by photolysis of "caged" (chelated) Ca²⁺ (54, 86). As in skinned myocytes, this initial small rise in Ca²⁺ triggered a much larger rise in intracellular free Ca²⁺, which was released from the SR. Thus CICR can initiate release of SR Ca²⁺ in both skinned and intact cardiac myocytes.

Hypothetically, the source of trigger Ca²⁺ for CICR under physiological conditions could be intracellular or extracellular. Evidence for an essential role of extracellular Ca²⁺ in initiation of CICR contractions comes from a study by Nabauer et al. (52). This study demonstrated that SR Ca²⁺ release in ventricular myocytes does not occur in the absence of extracellular Ca²⁺. This suggests either that influx of Ca²⁺ from the extracellular space may provide the trigger that activates CICR, or that Ca²⁺ must be present at an extracellular site on the cell for initiation of contraction.

The specific routes of Ca²⁺ entry that initiate CICR have been investigated. Many studies provide evidence that influx of Ca²⁺ through L-type Ca²⁺ channels is an important trigger for CICR. For example, voltage-clamp studies (1, 3, 4, 12, 13, 43) demonstrate that CICR is graded by the magnitude of peak L-type Ca²⁺ current (I_Ca-L). In these studies, Ca²⁺ transients or contractions were initiated by test steps to different membrane potentials. The magnitudes of Ca²⁺ transients (Fig. 1A) and contractions were proportional to the magnitudes of I_Ca-L initiated by the same test steps. Therefore, the relationship between Ca²⁺ transients (or contractions) and membrane potential was bell shaped, like the current-voltage (I-V) relationship for I_Ca-L (Fig. 1B). At potentials where I_Ca-L was minimal, contractions and transients also were negligible. In addition, Nabauer et al. (52) showed that in the absence of extracellular Ca²⁺, Na⁺ influx through Ca²⁺ channels did not initiate SR Ca²⁺ release. Because of these observations, it is generally accepted that I_Ca-L is the primary trigger for CICR in cardiac cells.

View larger version (14K): [in this window] [in a new window]   Fig. 1.   Ca2+ transients initiated by Ca2+-induced Ca2+ release (CICR) are proportional to the magnitude of inward Ca2+ current. A: recordings of Ca2+ transients (top) and verapamil-sensitive currents (bottom) initiated by steps from 40 mV to different membrane potentials in a voltage-clamped guinea pig ventricular myocyte. Voltages of the test steps are indicated below the respective current traces. B: plots of amplitudes of Ca2+ transients (top) and of verapamil-sensitive current (bottom) as a function of the voltage-clamp test steps. [From Beuckelmann and Wier (4)]

T-type Ca²⁺ channels provide a second route of influx of Ca²⁺ through the sarcolemma. Several studies (78, 94) demonstrate that influx of Ca²⁺ by this route also can trigger CICR. However, these studies indicated that T-type Ca²⁺ current is a very weak trigger for CICR and probably does not contribute substantially to EC coupling in ventricular myocytes.

The Na⁺/Ca²⁺ exchanger (NaCa_EX) also has been proposed as a trigger for CICR. Normally, the NaCa_EX is believed to extrude Ca²⁺ equal to the amount that enters the cell through voltage-gated channels during each cardiac cycle (7). However, at positive potentials the direction of exchange can reverse, and Ca²⁺ can enter by this route and induce slow, ramplike contractions (69, 71, 83). Contractions initiated by this mechanism may remain large or even increase in amplitude with voltage steps to very positive potentials. These slow, ramplike contractions persist in the presence of ryanodine, which suggests that they are activated directly by Ca²⁺ entering through the sarcolemma rather than by release of SR Ca²⁺ (69, 71). However, a number of studies (26, 38, 55, 88, 89) provide evidence that Ca²⁺ influx through reverse NaCa_EX also may trigger SR Ca²⁺ release via CICR. In these studies, phasic contractions and Ca²⁺ transients attributed to CICR coupled to NaCa_EX were observed when reverse NaCa_EX was enhanced by high (10-20 mM) intracellular Na⁺ levels.

Contractions also can be triggered by CICR in response to rapid elevation of intracellular Na⁺ following Na⁺ influx through voltage-gated Na⁺ channels (34, 35, 39). When this occurs, it is believed that the sudden rise in intracellular Na⁺ may initiate CICR by promoting influx of Ca²⁺ via reverse NaCa_EX (34, 35, 39). However, initiation of Ca²⁺ release by this mechanism has not been observed under all experimental conditions (6, 71, 77). These differing observations may reflect the relative inefficiency of the NaCa_EX as a trigger for CICR (79) as well as differences in the transmembrane gradients of Na⁺ and Ca²⁺ under different experimental conditions.

The concept of CICR holds that influx of a small amount of Ca²⁺ will activate release of a larger quantity of Ca²⁺ by opening ryanodine receptors in the SR membrane. However, CICR is intrinsically a positive feedback system that leads to a paradox (81). It is logical that Ca²⁺ released by these ryanodine receptors would recruit other nearby ryanodine receptors, and that this secondary activation of Ca²⁺ release might become regenerative and activate even more distant ryanodine receptors. This positive feedback system would be expected to continue until all ryanodine receptors throughout the cell are activated. Thus one would predict a maximal release of SR Ca²⁺ whenever depolarization initiates Ca²⁺ influx, regardless of the magnitude of the trigger. However, many studies show that the magnitude of Ca²⁺ release initiated by I_Ca-L is proportional to the peak amplitude of this current. This paradox has led to formulation of a concept known as local control theory (8, 44, 81). According to this theory, Ca²⁺ release from ryanodine receptors is controlled by Ca²⁺ influx through immediately adjacent L-channels and not by a generalized rise in the concentration of intracellular free Ca²⁺ throughout the cytosol (9, 45, 65). Thus activation of a single L-channel might cause a localized flux of Ca²⁺ sufficient to fully activate a group of neighboring ryanodine receptors. However, this activation does not spread to distant ryanodine receptors. Each L-channel and its functionally associated ryanodine receptors would constitute a release unit. Gradation of contraction by magnitude of whole cell I_Ca-L would reflect the number of release units activated, rather than a change in Ca²⁺ flux through individual ryanodine receptors.

Evidence for local activation of Ca²⁺ release and release units comes from experiments with laser scanning confocal microscopy in cells loaded with Ca²⁺-sensitive fluorescent dyes. Cheng et al. (11) first demonstrated that SR Ca²⁺ release in cardiac myocytes can occur as quanta, which they called "Ca²⁺ sparks." Sparks are discrete foci of increased fluorescence in response to interaction of released Ca²⁺ with fluorescent dye. Ca²⁺ sparks appear to originate near specialized Ca²⁺ release regions (junctional regions between SR and t tubules) both in cardiac myocytes (5, 59, 70) and in skeletal muscle (67). Sparks exhibit characteristic amplitudes, widths, and durations (10, 24, 46). These properties are stochastic and therefore do not vary with the stimulus magnitude. Thus sparks may represent the fundamental release units proposed in local control theory. Ca²⁺ transients are thought to be generated by the sum of numerous sparks (8, 10, 44, 65), and the magnitude of the Ca²⁺ transient would reflect the number of release units activated.

Local control theory can explain the bell-shaped dependence of contractions and Ca²⁺ transients on the magnitude of I_Ca-L at different membrane potentials (8, 44). At negative membrane potentials at which I_Ca-L and contraction first appear, only a small number of L-channels are activated. Therefore, only a small number of sparks, or release units, will be activated and contraction will be weak (Fig. 2). As voltage steps are made progressively more positive, the number of activated L-channels increases, and this causes corresponding increases in the number of release units and the amplitude of contraction. Eventually, at more positive potentials, contractions decrease in amplitude, even though essentially all L-channels are activated. This is thought to occur when the driving force for Ca²⁺ influx declines and the magnitude of I_Ca-L through individual channels decreases (8, 44). As membrane potential moves toward the reversal potential for I_Ca-L, the number of L-channels generating current sufficient to activate a release unit decreases. Therefore, there is a decrease in the number of release units activated and the magnitude of contraction declines. Thus the bell-shaped contraction-voltage relation for CICR coupled to I_Ca-L reflects the number of release units recruited at different membrane potentials.

View larger version (28K): [in this window] [in a new window]   Fig. 2.   Relationship between the voltage dependence of Ca2+ sparks and local control theory. A: relationship between the number of local Ca2+ transients (nLCT, also called Ca2+ sparks) and time after initiation of voltage-clamp steps to different membrane potentials. The probability of LCT occurring was highest near the beginning of each 200-ms test step and declined markedly by the end of the step. The late LCT shown for the test step to +70 mV occurred upon repolarization. B: relationship between the peak nLCT evoked by a given step and the membrane potential (Vp) of the test step. The frequency of LCT demonstrated a bell-shaped relationship to Vp, which resembles the bell-shaped relationship between Ca2+ transients and test step voltage shown in Fig. 1. C: probability of a single L-channel opening resulting in a LCT. Solid line, open probability of L-type Ca2+ channels (Po,L) as a function of Vp. Filled circles, relationship between peak nLCT and Vp (B) normalized to the maximum nLCT observed with Vp = +10 mV. Open squares, normalized nLCT divided by Po,L for the corresponding Vp. This measurement is interpreted as the probability of a LCT occurring at different membrane potentials (Pi,LCT). The relationship of Pi,LCT to Vp is indicated by the broken line. Thus the probability of an L-channel opening initiating a Ca2+ spark is very high with test steps to negative membrane potentials, because the driving force for Ca2+ entry is high and the single channel current would be large. However, Pi,LCT declines with test steps to more positive membrane potentials, where the driving force for Ca2+ entry is small, and the current associated with L-channel opening becomes small. Reprinted with permission from: Lopez-Lopez JR, Shacklock PS, Balke CW, and Weir WG. Local calcium transients triggered by single L-type calcium channel currents in cardiac cells. [Science 268: 1042-1045, 1995 (45). Copyright 1995 American Association for the Advancement of Science]

Although sparks have been proposed as a fundamental release unit for SR Ca²⁺ release, some studies have presented evidence for Ca²⁺ transients in the absence of Ca²⁺ sparks. For example, Lipp and Niggli (40) demonstrate SR Ca²⁺ release without sparks in response to "flash-photolysis" of caged Ca²⁺ in cardiac myocytes. Lopez-Lopez et al. (45) also observed Ca²⁺ transients without Ca²⁺ sparks when Ca²⁺ transients were initiated by NaCa_EX. However, this study did not determine whether these Ca²⁺ transients involved Ca²⁺ release as well as Ca²⁺ influx. Ca²⁺ release in the absence of sparks also may play an important role in mammalian skeletal muscle. Transients characterized by a rapid diffuse rise in Ca²⁺ without visible sparks have been reported in adult rat skeletal muscle (75). In amphibian skeletal muscle, Ca²⁺ sparks accompany Ca²⁺ transients; however, Ca²⁺ sparks can be abolished with tetracaine without eliminating the Ca²⁺ transient (76). These observations indicate that sparks are not required for initiation of Ca²⁺ transients in skeletal muscle. There also is evidence that quantal release events smaller than sparks may occur in cardiac and skeletal muscle. Lipp and Niggli (40, 41) and Tsugorka et al. (84) measured SR Ca²⁺ release events (called "quarks"), which are 5 to 10 times smaller than Ca²⁺ sparks in cardiac and skeletal muscles. Thus it would appear that several fundamental release units or modes may exist, including sparks, quarks, and even finer release units. The relationship between these alternate release modes and local control theory is unknown and will require further study with very high resolution measurements.

	VOLTAGE-SENSITIVE RELEASE MECHANISM

TOP ABSTRACT INTRODUCTION CA2+-INDUCED CA2+ RELEASE VOLTAGE-SENSITIVE RELEASE... REGULATION OF VSRM BY... CONDITIONS REQUIRED FOR... MECHANISM OF EC COUPLING... ROLE OF VSRM IN... SUMMARY REFERENCES

In cardiac muscle, Ca²⁺ sparks and local control theory can explain how CICR generates contractions and Ca²⁺ transients, which are proportional to I_Ca-L. However, it has become apparent that SR Ca²⁺ release and contraction are not always proportional to magnitude of I_Ca-L. Studies in isolated cardiac myocytes demonstrate an additional mechanism for SR Ca²⁺ release in which the magnitude of Ca²⁺ transients and contraction are graded by membrane potential rather than the magnitude of I_Ca-L (18, 20, 22, 29, 31, 96). This mechanism can initiate SR Ca²⁺ release and contraction when CICR coupled to I_Ca-L, Na current (I_Na), or NaCa_EX is inhibited (18, 20, 22, 27, 31, 96). Because this mechanism is graded by membrane voltage rather than current, it has been named the voltage-sensitive release mechanism (VSRM) (29, 31).

Both CICR and the VSRM are triggered by depolarization. The VSRM is first activated at relatively negative membrane potentials (near 60 mV), whereas CICR coupled to I_Ca-L is first activated when significant I_Ca-L appears (near 30 mV). Under physiological conditions, when contraction is triggered by action potentials, both mechanisms would be activated and likely contribute to initiation of contraction. However, experimentally it is possible to activate CICR and the VSRM separately with voltage-clamp techniques (18, 31). Experiments in which these mechanisms are activated separately demonstrated that phasic contractions can be triggered by either CICR or the VSRM (Fig. 3). Interestingly, when cells are activated with long depolarizations, the VSRM also triggers sustained Ca²⁺ release and sustained contraction (20). In contrast, CICR results in only a phasic contraction, which terminates with a characteristic time course even if Ca²⁺ influx is prolonged (72).

View larger version (12K): [in this window] [in a new window]   Fig. 3.   Separation of voltage-sensitive release mechanism (VSRM) and CICR components of excitation-contraction coupling with voltage-clamp techniques. Top: schematic of the voltage-clamp protocol, which consisted of conditioning pulses followed by a 4-s step to a postconditioning potential of 65 mV and then two sequential test steps to 40 and 0 mV. Thick bar, period during which drugs were applied with a computer-controlled rapid solution switching device. A: phasic contractions (top trace) initiated by the VSRM and CICR were elicited by the test steps to 40 and 0 mV, respectively. L-type Ca2+ current (ICa-L) occurred with the step to 0 mV (bottom trace). B: rapid application of 100 µM Cd2+ abolished ICa-L and the CICR contraction elicited by the step to 0 mV. Both the phasic and the sustained component of the VSRM remained when CICR was inhibited with Cd2+. [From Ferrier et al. (20)]

The VSRM is characterized by electrophysiological and pharmacological properties that allow it to be distinguished from other mechanisms of EC coupling (18-20, 22, 27, 31, 47, 48, 62, 93, 96). Voltage-clamp studies demonstrate that, in contrast to CICR coupled to I_Ca-L, the voltage dependence of the VSRM is sigmoidal (Fig. 4A). Phasic contractions and Ca²⁺ transients initiated when the VSRM is activated first appear with voltage steps to approximately 60 mV, reach a plateau near 20 mV, and then remain relatively constant with voltage steps as positive as +80 mV (18, 22, 31, 96). Inward Ca²⁺ current measured in the same experiments exhibits a typical bell-shaped I-V relationship, which peaks near 0 mV and declines toward zero or reverses as voltage steps approach +60 mV (Fig. 4B). Thus the magnitude of contractions initiated by the VSRM clearly is not proportional to the amplitude of inward current, and contractions remain maximal at membrane potentials near or beyond the reversal potential for I_Ca-L. However, when test steps to the same voltages were initiated from a conditioning potential of 40 mV at which the VSRM is inactivated, the contraction-voltage relationship in the same cells became bell shaped and proportional to I_Ca-L (Fig. 4, A and B). These curves are typical of CICR as reported in studies by others (Fig. 1B) (1, 4, 12, 13, 18, 22, 31, 43, 96). Thus within the same cell one can demonstrate CICR, which is directly dependent on the magnitude of I_Ca-L, and the VSRM, which is independent of the amplitude of I_Ca-L. Therefore, because these relationships to I_Ca-L are mutually exclusive, one must postulate that CICR and the VSRM are mediated by different mechanisms.

View larger version (28K): [in this window] [in a new window]   Fig. 4.   Activation and inactivation properties of the VSRM are distinct from those of CICR and ICa-L. A and B: mean contraction-voltage (A) and current-voltage relations (B) were recorded from rat ventricular myocytes voltage clamped with high-resistance microelectrodes (n = 12). Plots show magnitudes of contractions and Ca2+ currents activated by 200-ms depolarizing steps to different potentials from a postconditioning potential of either 40 or 65 mV. When the postconditioning potential was 40 mV the VSRM was inactivated, and the contraction-voltage relationship was bell shaped and typical of CICR. When the postconditioning potential was 65 mV, both CICR and the VSRM are available for activation, and the contraction-voltage relationship became sigmoidal. In addition, activation was shifted to more negative potentials, and the maximum amplitude of contractions increased dramatically. Current-voltage relations were affected very little by the change in postconditioning potential from 40 to 65 mV. C: schematic of the voltage-clamp protocol utilized to examine steady-state inactivation of currents or contractions. The membrane was clamped to different conditioning voltages (VPC) between the last conditioning pulse and a test step. A test step to 35 mV was used to assess the availability of the VSRM. A test step to 0 mV was used to assess availability of ICa-L. D: mean steady-state inactivation curves for contractions initiated by the VSRM and inward ICa-L recorded in rat ventricular myocytes. The half-inactivation voltages for the VSRM (53.2 ± 0.4 mV) and for ICa-L (25.3 ± 0.9 mV) were significantly different (P < 0.001, n = 9). [Panel D from Howlett et al. (31)]

The VSRM also can be distinguished from other mechanisms of EC coupling by inactivation characteristics. Phasic contractions induced by the VSRM exhibit steady-state inactivation. This can be observed when initiation of these contractions is preceded by depolarization to different membrane potentials (Fig. 4, C and D) (20, 22, 30, 31, 96). Full availability of phasic VSRM contractions is seen when activation steps are made from membrane potentials near or negative to 65 mV, whereas complete inactivation occurs with steps near or positive to 35 mV. The voltage dependence of inactivation can be described by a Boltzmann function with a half-inactivation voltage of approximately 50 mV (Fig. 4D). These inactivation properties allow the VSRM to be distinguished from CICR coupled to I_Ca-L, because I_Ca-L exhibits a half-inactivation potential near 25 mV (Fig. 4D) (31). The inactivation properties of the VSRM also serve to distinguish VSRM contractions from contractions initiated by reverse NaCa_EX. NaCa_EX does not inactivate, thus contractions initiated by reverse NaCa_EX can be elicited by depolarizing steps starting from membrane potentials at which the VSRM is inactivated (37, 42, 55, 88, 89).

The activation and inactivation properties of the VSRM also provide the basis for separating phasic contractions initiated by the VSRM and CICR as shown in Fig. 3 (18, 31). When cells are depolarized by a step from 65 mV to 40 mV, the contraction or Ca²⁺ transient that is generated is initiated almost entirely by the VSRM. This occurs because the VSRM activates at more negative membrane potentials than CICR coupled to I_Ca-L. However, when activation steps are made from 40 to 0 mV, the VSRM is largely inactivated, whereas I_Ca-L is inactivated only slightly. Thus activation steps initiated from a membrane potential of 40 mV elicit phasic contractions or Ca²⁺ transients generated almost entirely by CICR.

The VSRM also initiates a sustained component of Ca²⁺ release and contraction. Although the sustained component is typically smaller than the phasic component of the VSRM, it shows a sigmoidal voltage dependence similar to the phasic component and remains maximal positive to 20 mV (Fig. 5, A and B) (20). In contrast to phasic VSRM contractions and transients, sustained responses persist as long as depolarization is maintained, even for depolarizations lasting many seconds. Thus the sustained component does not show inactivation in response to maintained depolarization. Sustained contractions and transients terminate promptly on repolarization to potentials near the resting membrane potential. If cells are not repolarized abruptly, but stepwise, the sustained component also declines in a stepwise manner (Fig. 5, C and D). The decline of the sustained component shows a voltage dependence that is identical to the voltage dependence of activation (20). Thus the sustained component of the VSRM exhibits activation and deactivation that follow the same relationship with membrane potential (Fig. 5, B and D). The voltage dependence of the sustained component of the VSRM allows the VSRM to grade sustained Ca²⁺ release and contraction reversibly between 80 and 20 mV. Because deactivation of the sustained component of the VSRM is graded by membrane potential, changes in action potential duration could affect the time course of relaxation. Furthermore, full relaxation may be prevented if the cell does not fully repolarize.

View larger version (28K): [in this window] [in a new window]   Fig. 5.   VSRM generates sustained Ca2+ transients that show activation and deactivation with identical voltage dependence. Sequence of voltage steps is in the direction of the arrows (A, C). A: guinea pig ventricular myocytes were depolarized to different potentials with the voltage-clamp protocol (top). Ca2+ transients were detected with fura-2. Progressively more positive activation steps elicited sustained Ca2+ transients (middle), which increased in amplitude until a maximum was reached. Currents are shown at bottom. B: activation curve derived by plotting mean amplitudes of sustained Ca2+ transients as a function of test potential. F, peak fluorescence; F0 baseline fluorescence. C: voltage-clamp protocol used to investigate deactivation (top). Ca2+ transients and currents elicited by this protocol are shown by the middle and bottom traces. D: deactivation curve derived by plotting the amplitude of the sustained Ca2+ transient as a function of the repolarization voltage. The deactivation curve was very similar to the activation curve shown in B. The half-maximal voltage (Vh) was determined from Boltzmann fits and was similar for activation and deactivation of the sustained component of the VSRM. [Adapted from Ferrier et al. (20)]

Pharmacological characteristics also distinguish the VSRM from other mechanisms of EC coupling. Much of the pharmacological profile for the VSRM has emerged from experiments designed to explore how the VSRM functions. Many of these studies compared effects of different agents on the VSRM and CICR in experiments in which both of these mechanisms were activated sequentially. Contractions and transients initiated by CICR are inhibited by agents that block I_Ca-L (Fig. 3) (20, 22, 27, 30, 31, 47, 96). In contrast, VSRM contractions and/or transients still can be elicited when CICR is blocked by Cd²⁺, Ni²⁺, or nifedipine (20, 22, 27, 30, 31, 47, 96). Because Ni²⁺ also blocks the T-type Ca²⁺ current (49, 85), persistence of the VSRM in the presence of Ni²⁺ also indicates that the T-type Ca²⁺ current does not trigger the VSRM (20, 27). Further evidence excluding the T-type current comes from studies in adult rat ventricular myocytes, in which T-type Ca²⁺ current cannot be detected (85), but in which VSRM contractions can be observed (27, 31). The lack of inhibition of the VSRM by Ni²⁺ also serves to distinguish the VSRM from NaCa_EX. VSRM Ca²⁺ transients can be elicited in the presence of 2-5 mM Ni²⁺ and/or 0 mM intracellular Na⁺ (20, 27, 96), both of which inhibit contractions and transients initiated by NaCa_EX (32, 37, 55, 71, 88).

Inward Na⁺ or Ca²⁺ current carried by Na⁺ channels also can initiate CICR under certain conditions (34, 66). However, the VSRM is not inhibited by substitution of extracellular Na⁺ with choline or sucrose, or by block of I_Na with tetrodotoxin or lidocaine (18, 31, 47), which would block CICR secondary to Na⁺ or Ca²⁺ influx through Na⁺ channels (34, 66). These findings demonstrate that VSRM contractions and Ca²⁺ transients are not initiated by ion fluxes through Na⁺ channels.

Although, the VSRM is not inhibited when I_Na is blocked by lidocaine or tetrodotoxin, or when cells are exposed to Na⁺-free solutions, the VSRM is inhibited by the local anesthetic tetracaine (47). This effect can be observed in myocytes in which I_Na already has been inhibited either by lidocaine and/or by tetrodotoxin (20, 47, 96). Thus the effect of tetracaine appears not to be related to its ability to block Na⁺ channels. Tetracaine also is believed to act on ryanodine receptors (57, 58), and it is possible that tetracaine may inhibit the VSRM by an action at this site. When tetracaine is applied with a rapid solution changing device, it selectively blocks the VSRM without blocking CICR (47). The selective block of the VSRM by tetracaine can be used to evaluate the contribution of the VSRM to contractions initiated by voltage steps that recruit both CICR and the VSRM. Inhibition of the VSRM with tetracaine reduces the magnitude of contraction by ~50% at voltages that would maximally activate both CICR and the VSRM (Fig. 6). At more positive potentials, corresponding to the overshoot and plateau of the action potential, the fraction of contraction inhibited by tetracaine is even larger. Thus the component of the contraction-voltage relationship inhibited by tetracaine appears to be identical to that removed by voltage inactivation of the VSRM (Fig. 4). These observations indicate that the VSRM contributes substantially to EC coupling, even in the presence of CICR.

View larger version (19K): [in this window] [in a new window]   Fig. 6.   Selective block of the VSRM with tetracaine demonstrates the contribution of the VSRM to the contraction-voltage relationship. Tetracaine was applied rapidly, 3 s in advance of test steps to avoid effects on sarcoplasmic reticulum (SR) Ca2+ content. Na currents were blocked with lidocaine throughout the experiments. A: contraction-voltage relationships before and after application of tetracaine. In the presence of tetracaine, the contraction-voltage relationship became bell shaped and proportional to inward Ca2+ current (B). In addition, tetracaine reduced the maximum peak contraction and shifted the activation curve in the positive direction. * Significantly different from control (P < 0.05). B: current-voltage (I-V) relationships in the absence and presence of tetracaine. Tetracaine had little effect on the I-V relationship. [From Mason and Ferrier (47)]

Contractions and transients can be initiated by the VSRM when I_Ca-L is blocked. This suggests that the Ca²⁺ transient likely originates entirely from release of intracellular stores of Ca²⁺. Release of SR Ca²⁺ through ryanodine receptors can be assessed with ryanodine. At low concentrations (micromolar), ryanodine is believed to render the SR leaky, thereby depleting SR Ca²⁺ stores (50, 64, 73). However, experiments with ryanodine demonstrated that unexpectedly low concentrations of ryanodine (30 nM) blocked the VSRM (18). Furthermore, block of the VSRM occurred at concentrations that did not deplete the SR of Ca²⁺ and that had little or no effect on contractions initiated by CICR coupled to I_Ca-L (48). These observations confirmed that ryanodine receptors are essential for the operation of the VSRM and also indicated that a high affinity binding site for ryanodine may be associated with this mechanism. Selective blockade of the VSRM by very low concentrations of ryanodine also raises an intriguing possibility that the VSRM and CICR utilize separate populations of ryanodine receptors.

The preceding discussion focuses on characteristics of phasic VSRM contractions and transients. Sustained transients and contractions initiated by the VSRM show the same pharmacology as phasic VSRM contractions and are inhibited by tetracaine and ryanodine, but not by Cd²⁺, Ni²⁺, tetrodotoxin, or lidocaine (Figs. 3 and 7) (20). These electrophysiological and pharmacological properties add further evidence that the VSRM and CICR are different and distinct mechanisms for EC coupling.

View larger version (18K): [in this window] [in a new window]   Fig. 7.   Sustained component of the VSRM shows the same pharmacological properties as the phasic component of the VSRM. A: effects of ryanodine (1 µM) on contractions elicited by sequential steps to 40 and 0 mV (voltage-clamp protocol at left). Ryanodine abolished both the phasic and sustained contractions elicited by the VSRM but had much less effect on the CICR contraction. B: sustained component of the VSRM is not inhibited by 2 mM Ni2+. Inset: voltage-clamp protocol. [Adapted from Ferrier et al. (20)]

	REGULATION OF VSRM BY PHOSPHORYLATION

TOP ABSTRACT INTRODUCTION CA2+-INDUCED CA2+ RELEASE VOLTAGE-SENSITIVE RELEASE... REGULATION OF VSRM BY... CONDITIONS REQUIRED FOR... MECHANISM OF EC COUPLING... ROLE OF VSRM IN... SUMMARY REFERENCES

Phosphorylation pathways play a major role in regulation of the strength of cardiac contraction. The VSRM is a target for phosphorylation by at least two major routes: the adenylyl cyclase-protein kinase A (AC/PKA) pathway and the Ca²⁺-calmodulin-dependent kinase (CaM kinase) pathway. Therefore, phosphorylation of the VSRM may play a role in regulation of cardiac strength.

The importance of phosphorylation in activation of the VSRM first was recognized in experiments with patch pipettes (22, 96). The VSRM is readily demonstrable when cells are studied with high-resistance microelectrodes, which minimize intracellular dialysis with electrode solution. However, when cells were dialyzed with conventional intracellular patch pipette solutions, the VSRM was not observed (22, 27, 96). Under these conditions, contraction-voltage relations were bell shaped as expected for CICR, even when activation steps were made from near the resting membrane potential to ensure that the VSRM was not inactivated. Subsequent experiments demonstrated that addition of either 8-bromo-cAMP or calmodulin (Fig. 8) to the pipette solution restored activation of the VSRM (22, 27, 96).

View larger version (23K): [in this window] [in a new window]   Fig. 8.   Calmodulin (CaM) restores activation of the VSRM in experiments with patch pipettes. A: voltage-clamp protocol. B: only the CICR contraction was demonstrable in cells dialyzed with standard potassium-based patch pipette solution. C: addition of CaM to the patch pipette solution allowed activation of the VSRM in addition to CICR. D: effect of CaM was prevented by KN-62, a specific inhibitor of CaM kinase. [From Zhu and Ferrier (96)]

The component of EC coupling activated by these agents was identified as the VSRM by its electrophysiological and pharmacological properties. Responses that appeared in the presence of calmodulin or 8-bromo-cAMP exhibited half-inactivation voltages typical of the VSRM (approximately 50 mV) were resistant to blockade of I_Ca-L by Cd²⁺ and were inhibited by tetracaine (22, 96). Furthermore, the presence of either 8-bromo-cAMP or calmodulin in the intracellular solution resulted in sigmoidal contraction-voltage (Fig. 9) or Ca²⁺ transient-voltage relations (22, 96). Activation of the VSRM by either agent also increased the maximum amplitude of contractions or Ca²⁺ transients at membrane potentials corresponding to the overshoot and plateau of the action potential. Thus the component of EC coupling supported by 8-bromo-cAMP or calmodulin had characteristics identical to the VSRM in myocytes studied with high-resistance microelectrodes.

View larger version (23K): [in this window] [in a new window]   Fig. 9.   Addition of 8-bromo-cAMP (8-Br-cAMP) to patch pipette solutions dramatically enhances the contribution of the VSRM to contraction but causes only modest stimulation of CICR contractions. A: contraction-voltage relationships determined when the VSRM was available for activation (postconditioning potential 65 mV). In the absence of 8-Br-cAMP, the contraction-voltage relationship was bell shaped. Inclusion of 8-Br-cAMP in the pipette solution resulted in a contraction-voltage relationship characteristic of the VSMR. 8-Br-cAMP caused the contraction-voltage relationship to become sigmoidal, markedly increased the amplitudes of contractions, and shifted activation negatively. B: inclusion of 8-Br-cAMP in the pipette increased ICa-L primarily near the peak of the I-V relationship. C: relationship between contraction and current determined from a postconditioning potential of 65 mV in the absence of 8-Br-cAMP was similar to those in F. However, addition of 8-Br-cAMP eliminated the proportionality between contraction and current. D: effects of 8-Br-cAMP on contraction-voltage relations elicited when the VSRM was inactivated (postconditioning potential 40 mV). Contraction-voltage relations were bell shaped and increased modestly when 8-Br-cAMP was included in the pipette. E: I-V relations corresponding to experiments illustrated in D. F: plot of contraction amplitudes as a function of corresponding currents. The relationship between contraction and current was not changed by addition of 8-Br-cAMP to the pipette solution. Thus CICR contractions remained proportional to magnitude of current and there was no evidence for a significant change in gain. [Adapted from Ferrier et al. (22)]

The observation that dialysis of myocytes with patch pipettes inhibits the VSRM unless 8-bromo-cAMP or calmodulin are added to the intracellular solution suggests that dialysis can disrupt phosphorylation pathways essential for activation of the VSRM. Evidence supporting this interpretation comes from experiments with specific kinase inhibitors (e.g., Fig. 8D). When the VSRM is supported by calmodulin, the CaM kinase inhibitor KN-62 abolishes VSRM responses; however, the PKA inhibitor H-89 is without effect (96). In contrast, when the VSRM is supported by addition of 8-bromo-cAMP to the patch pipette solution, H-89 inhibits the responses (22), whereas KN-62 is without effect (96). These observations suggest that calmodulin and 8-bromo-cAMP restore activation of the VSRM by activation of the CaM kinase and PKA pathways, respectively. Evidence that these pathways normally regulate activation of the VSRM comes from experiments with nondialyzed cells studied with high-resistance microelectrodes (96). In these experiments, inhibition of either PKA or CaM kinase individually resulted in about 50% inhibition of the VSRM. Simultaneous superfusion of the myocytes with both H-89 and KN-62 virtually abolished the VSRM. These observations indicate that these phosphorylation pathways play a critical role in regulation of the VSRM in cardiac muscle.

Although 8-bromo-cAMP and calmodulin both activated the VSRM, they had very different effects on maximum inward I_Ca-L. The effects of 8-bromo-cAMP were accompanied by an increase in maximum peak inward I_Ca-L (22). In contrast, calmodulin increased VSRM contractions and transients without increasing the magnitude of I_Ca-L or contractions initiated by CICR (96). In either case, block of I_Ca-L eliminated CICR contractions but had virtually no effect on the amplitude of contractions activated by the VSRM. Thus activation of the VSRM by these agents was independent of effects on I_Ca-L.

In the absence of 8-bromo-cAMP, when the conditioning potential was 65 mV to allow activation of the VSRM, both the I-V relationship for I_Ca-L and the contraction-voltage relationship were bell shaped (Fig. 9, A and B). When contraction amplitude is plotted as a function of magnitude of I_Ca-L, it is clear that contraction is proportional to the magnitude of I_Ca-L (Fig. 9C). However, addition of 8-bromo-cAMP to the patch pipette solution caused an increase in the amplitude of contraction, and the contraction-voltage relationship became sigmoidal (Fig. 9A). In contrast, the I-V relationship for I_Ca-L remained bell shaped, although the amplitude of current was increased by 8-bromo-cAMP (Fig. 9B). If one plots the amplitude of contraction as a function of magnitude of I_Ca-L, it is apparent that amplitude of contraction is unrelated to magnitude of I_Ca-L in the presence of 8-bromo-cAMP when the VSRM is available (Fig. 9C).

When the VSRM was inactivated by a conditioning potential of 40 mV, a different picture emerged. Although addition of 8-bromo-cAMP to patch pipette solutions increased the amplitude of I_Ca-L and CICR contractions, CICR contractions remained proportional to the magnitude of current and still exhibited bell-shaped contraction-voltage relationships (Fig. 9, D and E). The increase in CICR contractions appeared to be related directly to the increased magnitude of I_Ca-L, because the relationship between the amplitudes of contraction and current (gain) was unchanged by 8-bromo-cAMP (Fig. 9F) (22). Thus activation of the VSRM by 8-bromo-cAMP occurred without disruption of the graded relationship between CICR contractions and I_Ca-L in the same cells. Therefore, activation of the VSRM in response to inclusion of 8-bromo-cAMP in the intracellular solution cannot be explained by a marked increase in gain of CICR resulting in a "hair-trigger" for contraction (91).

Experiments that demonstrated that cAMP could restore VSRM contractions and Ca²⁺ transients in dialyzed myocytes utilized 8-bromo-cAMP. This analog of cAMP is resistant to hydrolysis by phosphodiesterases (51). A recent study concluded that there was no evidence for the VSRM in myocytes dialyzed with pipette solutions containing Tris-cAMP (60). Tris-cAMP is readily hydrolyzable by phosphodiesterases (51). We hypothesized that Tris-cAMP was ineffective in activating the VSRM because it would be rapidly broken down by phosphodiesterases. In experiments to examine whether different analogs of cAMP would support the VSRM, we found that two phosphodiesterase-resistant analogs, 8-bromo- and dibutyryl-cAMP, activated the VSRM (21). However, unsubstituted cAMP (Tris- or Na-salt), which is hydrolyzable by phosphodiesterase, was ineffective in activating the VSRM (21). To confirm the role of phosphodiesterase in these observations, the effect of 3-isobutyl-1-methylxanthine (IBMX), a nonspecific phosphodiesterase inhibitor was examined (21). In myocytes dialyzed with standard intracellular solutions not containing either cAMP or calmodulin, IBMX allowed activation of the VSRM (21). Therefore, it would appear that phosphodiesterase plays an important role in regulating activation of the VSRM.

	CONDITIONS REQUIRED FOR ACTIVATION OF VSRM

TOP ABSTRACT INTRODUCTION CA2+-INDUCED CA2+ RELEASE VOLTAGE-SENSITIVE RELEASE... REGULATION OF VSRM BY... CONDITIONS REQUIRED FOR... MECHANISM OF EC COUPLING... ROLE OF VSRM IN... SUMMARY REFERENCES

The VSRM is demonstrable in cells from different species, including guinea pigs, rats, hamsters, rabbits, and humans (18, 25, 27, 30, 31, 87). Furthermore, the characteristics of the VSRM are essentially identical in these studies, despite the use of different myocyte isolation procedures in different laboratories (18, 25, 27, 30, 31, 87). Early studies in multicellular preparations at body temperature also reported initiation of contraction at membrane potentials negative to those at which significant I_Ca-L is activated (e.g., 2, 23; and reviewed in 29, 36). Contractions initiated in these studies may have included activation of the VSRM; however, the mechanism of these contractions was not clearly identified. Subsequent to these studies in multicellular preparations, isolated myocytes became available for experimentation. However, evidence for the VSRM was not observed in these early studies in isolated myocytes. The properties of the VSRM described in this review likely explain why this mechanism was not detected in older studies in myocytes. In the past, EC coupling studies typically were conducted at room temperature with patch pipettes and with protocols in which holding or conditioning potentials near 40 mV were utilized (29). Clearly experiments conducted with patch pipettes, in the absence of calmodulin or a nonhydrolyzable analog of cAMP, would not have activated this mechanism. The VSRM also is inhibited in experiments conducted at room temperature (19, 29). Similarly, the VSRM would be inactivated in experiments that utilized holding or conditioning potentials near 40 mV, because of the steady-state inactivation properties of phasic VSRM contractions. In addition, the VSRM is much more sensitive to conditioning pulses (amplitude and frequency) than CICR (31, 62, 95). Thus it has become clear from recent studies that, to study the VSRM, one must avoid inactivation with depolarized conditioning potentials, use physiological temperature (37°C), provide a substrate for phosphorylation when experiments are conducted with patch pipettes, and precede test steps with conditioning pulses of sufficient amplitudes and durations to activate the VSRM. The use of high-resistance electrodes minimizes cell dialysis with electrode solution and eliminates the need for exogenous cAMP or calmodulin required with patch pipettes.

Most early studies in isolated myocytes were conducted under conditions that would not allow activation of the VSRM. Therefore, early studies with isolated myocytes only investigated CICR and concluded that this was the only mechanism for cardiac EC coupling. Because of this, the existence of the VSRM is controversial. It is extremely important that attempts to verify the existence of the VSRM are conducted under conditions that have been demonstrated to allow its activation. A recent study by Piacentino et al. (60) concluded that the VSRM could be explained by CICR in feline ventricular myocytes. However, there is no evidence in this study that the mechanism which we have identified as the VSRM was ever activated. Piacentino et al. (60) conducted their study two ways. In one case, cells were dialyzed with patch pipettes containing Tris-cAMP, which is a hydrolyzable analog of cAMP. Ferrier et al. (21) demonstrate that hydrolyzable analogs will not activate the VSRM. Thus the VSRM would not have been activated under these conditions. In other experiments, Piacentino et al. (60) voltage-clamped myocytes with patch pipettes that did not contain cAMP. Extracellular application of 50 nM isoproterenol was used in an attempt to activate the VSRM. However, the contraction-voltage relations remained bell shaped and were very sensitive to Cd²⁺, as expected for CICR. Thus it is doubtful that the VSRM was activated under the experimental conditions employed by Piacentino et al. (60). Because there was no evidence that the VSRM was activated in this study, it is not clear how one can conclude that the VSRM can be explained by CICR.

	MECHANISM OF EC COUPLING FOR VSRM

TOP ABSTRACT INTRODUCTION CA2+-INDUCED CA2+ RELEASE VOLTAGE-SENSITIVE RELEASE... REGULATION OF VSRM BY... CONDITIONS REQUIRED FOR... MECHANISM OF EC COUPLING... ROLE OF VSRM IN... SUMMARY REFERENCES

The mechanism by which the VSRM is coupled to membrane potential is not clear. Although VSRM contractions are not proportional to I_Ca-L, activation of the VSRM requires extracellular Ca²⁺ (18). However, the VSRM can be activated in the presence of as little as 50 µM extracellular Ca²⁺ (18). Thus it is not known whether Ca²⁺ entry is required for activation of the VSRM, or alternatively, whether Ca²⁺ is necessary at some extracellular binding site. If Ca²⁺ influx is required to trigger the VSRM, this mechanism must be different from, and coexist with, conventional CICR. The influx must be extremely small, and there can be little, if any, proportionality between the magnitude of Ca²⁺ entry and the magnitude of contractions or Ca²⁺ transients. If this Ca²⁺ entry occurs through voltage-gated channel openings, it cannot be the same as or as large as that entering through L-channels, because summation of L-channel openings gives rise to whole cell I_Ca-L. This is precluded because the VSRM is independent of the magnitude of I_Ca-L and persists when the I_Ca-L is blocked by Cd²⁺. Furthermore, this conductance must be inactivated with a conditioning potential of 40 mV, despite the fact that I_Ca-L and CICR persist from this potential. This hypothetical explanation for the VSRM would require the operation of two mechanisms for CICR in the same cell at the same time: one with ultra-high gain for the VSRM, and one with low gain for conventional CICR graded by I_Ca-L. To postulate that CICR can activate the VSRM, it will be necessary to address these paradoxes experimentally.

The sustained component of the VSRM also is difficult to explain by conventional CICR. Phasic CICR contractions terminate with a characteristic time course, even when inactivation of I_Ca-L is slowed greatly by a Ca²⁺ channel agonist (72). This observation has led to the hypothesis that ryanodine receptors enter a refractory or inactivated state, during which CICR is no longer possible. This property of CICR is not compatible with the occurrence of sustained Ca²⁺ release observed with the VSRM. This problem also would need to be resolved to explain the VSRM in terms of CICR.

Alternatively, Ca²⁺ influx may not be required for activation of the VSRM. In this case, one must propose that the VSRM is coupled to changes in membrane potential through some voltage-dependent event separate from Ca²⁺ permeation. This might be gating charge movement of a channel moiety, as in skeletal muscle (33), or it could be a biochemical link such as release of a second messenger or activation of a phosphorylation step (61, 92). It is not clear whether one would expect a biochemical link to show a voltage dependence, which is described by a Boltzmann function, like that of the VSRM. However, charge movement in voltage-gated ion channels, including the voltage sensor for EC coupling in skeletal muscle, does follow a Boltzmann distribution and would be compatible with the voltage dependence of the VSRM.

Ca²⁺ transients initiated by the VSRM resemble those observed in skeletal muscle (63, 74). Skeletal muscle transients are characterized by an initial rapid phase, which exhibits inactivation, followed by a sustained component, which does not inactivate (Fig. 10) (68, 74). The configuration of these transients is similar to that of transients observed in cardiac myocytes when the VSRM is available (Fig. 5A). The configurations of the Ca²⁺ transients in skeletal muscle are believed to reflect changes in efflux of Ca²⁺ from the SR (Fig. 10). The sustained component of skeletal muscle responses is believed to reflect opening of ryanodine receptors by a voltage sensor. The initial phasic component occurs in response to recruitment of adjacent ryanodine receptors through intracellular CICR (63). Thus CICR provides a multiplier or amplification system for the voltage sensor component and causes a phasic Ca²⁺ release that terminates with a time course that is independent of the sustained component (63, 68). One may speculate that similar mechanisms may account for phasic and sustained components of the VSRM observed in cardiac myocytes. Clearly additional studies will be required to test this possibility.

View larger version (27K): [in this window] [in a new window]   Fig. 10.   Phasic and sustained components of Ca2+ release in frog and rat skeletal muscles resembles that observed in cardiac myocytes. Ca2+ was detected with antipyrylazo III. A: representative recordings of Ca2+ transients in frog semitendinosus (left) and rat extensor digitorum longus muscles (right). Transients were initiated by voltage steps from the holding potential to different more positive potentials as indicated in the figure. The transients show a phasic component followed by a sustained component, which terminates promptly with repolarization. B: release flux calculated from the time derivative of the Ca2+ transients in frog (left) and rat (right) muscles. The removal flux was determined from fits to a removal model; (see Ref. 74 for details). Ca2+ flux in both species demonstrated an initial phasic rise followed by a decrease to a constant lower flux. [Reproduced from The Journal of General Physiology, 1996, vol. 107, pp. 1-18 by copyright permission of The Rockefeller University Press (74)]

	ROLE OF VSRM IN HEART DISEASE

TOP ABSTRACT INTRODUCTION CA2+-INDUCED CA2+ RELEASE VOLTAGE-SENSITIVE RELEASE... REGULATION OF VSRM BY... CONDITIONS REQUIRED FOR... MECHANISM OF EC COUPLING... ROLE OF VSRM IN... SUMMARY REFERENCES

The VSRM contributes substantially to contraction in myocytes from normal hearts. However, the VSRM appears to be attenuated in myocytes from two different animal models of congestive heart failure: cardiomyopathic hamsters and rats with heart failure secondary to myocardial infarction. The VSRM contributes substantially to contractile function in myocytes from normal hamsters, much as observed in other species (30). In contrast, contractions initiated by the VSRM are depressed selectively in myocytes from hamsters with a cardiomyopathic genotype, with virtually no change in contractions triggered by CICR (Fig. 11) (30). In rats with heart failure secondary to coronary ligation, the VSRM also is depressed significantly with virtually no change in CICR (80). These studies demonstrate that defects in the VSRM are largely responsible for the decrease in contractile function observed in these two animal models of heart failure. Thus the relative contributions of the VSRM and CICR to EC coupling may be altered in heart diseases characterized by contractile dysfunction.

View larger version (26K): [in this window] [in a new window]   Fig. 11.   Contractile dysfunction in ventricular myocytes from cardiomyopathic hamsters is caused by selective depression of the VSRM. A: contraction-voltage relationships recorded from normal and cardiomyopathic myocytes when the VSRM was inactivated (postconditioning potential 40 mV) were virtually identical. B: I-V relationships determined from 40 mV also were identical in normal and cardiomyopathic cells. C: contraction-voltage relationships recorded when the VSRM was not inactivated (postconditioning potential 60 mV). The contraction-voltage relationship from cardiomyopathic hamsters was significantly depressed relative to the corresponding curve for normal hamsters. * Significantly different from normal (P < 0.05). D: I-V relationships recorded from 60 mV were identical in the two groups. [Adapted from Howlett et al. (30)]

The role of the sustained component of the VSRM in heart disease has not been investigated. Because the sustained component is reversibly graded by voltage, it may alter function in conditions where membrane potential is abnormal. For example, the sustained component of the VSRM might delay relaxation in conditions where the action potential is significantly prolonged, such as cardiac hypertrophy or heart failure (90). In addition, the sustained component might prevent full relaxation in disease conditions, which result in incomplete repolarization.

	SUMMARY

TOP ABSTRACT INTRODUCTION CA2+-INDUCED CA2+ RELEASE VOLTAGE-SENSITIVE RELEASE... REGULATION OF VSRM BY... CONDITIONS REQUIRED FOR... MECHANISM OF EC COUPLING... ROLE OF VSRM IN... SUMMARY REFERENCES

There is increasing evidence that the VSRM and CICR represent two different mechanisms for EC coupling in the heart. We illustrate this possibility in Fig. 12. Both mechanisms likely contribute to initiation of cardiac contraction. These two mechanisms have distinct electrophysiological and pharmacological characteristics that allow them to be distinguished from each other in the same cell. Because of the mutually exclusive nature of many of these characteristics, the VSRM and CICR cannot be explained by a single mechanism. Thus we indicate the VSRM and CICR as two independent mechanisms for release of SR Ca²⁺ (Fig. 12). However, it is unclear whether the VSRM operates a separate population of ryanodine receptors or opens the same population of release channels as CICR. The functional characteristics of the VSRM resemble characteristics associated with Ca²⁺ release coupled to gating charge in skeletal muscle. Therefore, we have indicated a physical link between a channel moiety, functioning as a voltage sensor in the sarcolemma, and the Ca²⁺ release channel in the SR (Fig. 12). However, the molecular identity of the VSRM is not yet known and other mechanisms must be considered. Phosphorylation plays a major role in activation of the VSRM and may allow the VSRM to serve as a major regulatory mechanism for cardiac contraction in normal heart. As suggested in Fig. 12, phosphorylation sites for PKA and CaM kinase may be located on one or more components of the VSRM. At present it would be speculative to suggest what sites would regulate the VSRM. If the VSRM and CICR are separate parallel mechanisms, this allows for separate alterations of either mechanism in disease states. Indeed, the VSRM plays a major role in contractile dysfunction in several models of heart failure and may contribute to impaired contractile performance in heart disease. As our information about the VSRM accumulates, it is becoming clear that this important trigger for cardiac contraction plays a central role in regulation of cardiac contraction in normal and diseased myocardium.

View larger version (71K): [in this window] [in a new window]   Fig. 12.   Schematic illustrating a possible relationship between the VSRM and CICR in cardiac myocytes. The VSRM and CICR are depicted as independent mechanisms, which operate to release Ca2+ from a common Ca2+ store within the SR. CICR is shown with Ca2+ influx occurring through an L-type Ca2+ channel. Incoming Ca2+ binds to an activation site on Ca2+ release channels, which causes these channels to open and release Ca2+. The VSRM is shown as a voltage sensor, based on a channel moiety, which signals Ca2+ release channels to open through a physical link. AC, adenylyl cyclase, which is coupled to various receptors (R) through a stimulatory G protein (Gs). Some possible regulatory phosphorylation sites for protein kinase A (PKA) and CaM kinse (CamK) are indicated on proteins involved in SR Ca2+ release. See text for discussion.