News Physiol Sci 15: 275-279, 2000;
1548-9213/00 $5.00
News in Physiological Sciences, Vol. 15, No. 5, 275-279,
October 2000
© 2000 Int. Union Physiol. Sci./Am. Physiol. Soc.
No Role for the Ryanodine Receptor in Regulating Cardiac Contraction?
D. A. Eisner and
A. W. Trafford
D. A. Eisner and A. W. Trafford are in the Unit of Cardiac Physiology, Faculty of Medicine, The University of Manchester, 1.524 Stopford Building, Manchester M13 9PT, UK.
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Abstract
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Cardiac contraction is initiated by Ca2+ leaving the sarcoplasmic reticulum through the ryanodine receptor (RyR). Although opening of the RyR can be modified by various ligands, these have no maintained effect on contraction. We conclude that modulation of the RyR controls sarcoplasmic reticulum Ca2+ content rather than cytoplasmic Ca2+ concentration.
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Introduction
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Contraction of cardiac muscle results from an increase of intracellular Ca2+ concentration ([Ca2+]i) from resting or diastolic levels of 100 nM to a systolic level on the order of 1 µM. Varying the magnitude of this systolic increase of Ca2+ alters the degree of activation of the contractile proteins and therefore force production (4). As shown in Fig. 1A
, the systolic Ca2+ transient has two sources: 1) Ca2+ enters the cell from the extracellular fluid via the L-type Ca2+ current, and 2) Ca2+ is released from an intracellular store, the sarcoplasmic reticulum (SR). In mammalian cardiac muscle, the release from the SR contributes the bulk of the increase of [Ca2+]i. Ca2+ leaves the SR down a concentration gradient through the Ca2+ release channel or ryanodine receptor (RyR). The probability that the channel is open is increased by an increase of the cytoplasmic Ca2+ concentration. This results in the process of Ca2+-induced Ca2+ release (CICR), in which the entry of a small or trigger amount of Ca2+ through the sarcolemmal L-type Ca2+ current results in a local increase of [Ca2+]i, which then opens the RyR, resulting in the release of a much larger amount of Ca2+ from the SR. Recent work using confocal microscopy has led to the suggestion that the elementary event of CICR is a Ca2+ "spark," in which the opening of a single L-type Ca2+ channel results in the opening of one or a small number of RyRs. Further information can be found in recent reviews (5, 11).
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FIGURE 1. A: scheme for excitation-contraction coupling in cardiac muscle. During the action potential, Ca2+ enters the cell through the L-type Ca2+ channel (ICa; a). This produces a local rise of [Ca2+]i near the ryanodine receptor (RyR), causing the RyR to open, thus releasing Ca2+ from the sarcoplasmic reticulum (SR; b). This efflux of Ca2+ activates the myofilaments (MF; c). Relaxation is produced by Ca2+ being returned to the SR via the Ca2+-ATPase (d) and being pumped out of the cell via the Na+/Ca2+ exchange (e). For simplicity, the sarcolemmal Ca2+-ATPase is not shown. B: Ca2+ flux balance in a cardiac cell. Traces show intracellular Ca2+ concentration ([Ca2+]i), membrane current (Im) and integrated Ca2+ movement (sarcolemmal Ca flux). The inset shows the tail current on an expanded scale. A 100-ms duration pulse was applied from –40 to 0 mV. The membrane current trace shows the L-type Ca2+ current on depolarization and the Na+/Ca2+ exchange current on repolarization. The Ca2+ movement trace shows the calculated changes of total cell Ca2+ calculated by integrating the Ca2+ and Na+/Ca2+ exchange current traces. Note that the Ca2+ efflux has been corrected to allow for the fact that a fraction of the efflux is via the sarcolemmal Ca2+-ATPase.
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Ca2+ influx and efflux are equal over the cardiac cycle
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For the heart to function as a pump, it must relax as well as contract. This relaxation requires that Ca2+ be removed from the cytoplasm either by reuptake into the SR (by the SR Ca2+-ATPase; Fig. 1A
) or by pumping out of the cell (by the combined actions of the sarcolemmal Ca2+-ATPase and Na+/Ca2+ exchange; Fig. 1A). It is important to note that, for the heart to be in a steady state, the amount of Ca2+ that enters the cytoplasm on each beat must exactly equal that which is removed. Furthermore, the amount released from the SR must balance that taken back up into the SR and that entering the cell from outside must be pumped back out of the cell. This cellular Ca2+ balance is demonstrated in Fig. 1B. Ca2+ entry occurs during the depolarizing pulse via the L-type Ca2+ current. The magnitude of this entry can be measured by integrating the current and, in this case, corresponds to 3.5 µmol/l. The efflux from the cell can be measured from the Na+/Ca2+ exchange current on repolarization (after correcting for the electroneutral Ca2+-ATPase; for methods and so forth see Ref. 9). The cumulative integral shows that efflux equals influx. As will be reviewed below, this condition of cellular Ca2+ balance has important consequences for maneuvers that affect systolic [Ca2+]i.
The above description provides an overview of the origin of the systolic increase of [Ca2+]i. It does not, however, address the physiologically important question of how the magnitude of the transient is regulated to control cardiac output. The following are among the potential control points: 1) The magnitude of the Ca2+ influx. The larger this is the greater the trigger will be, leading to increased RyR opening. 2) The properties of the RyR. In particular, the dependence of the opening probability of the RyR on the trigger [Ca2+]i is important. 3) The Ca2+ content of the SR. This will determine how much Ca2+ is released when a given number of RyRs open. The importance of 1 and 3 are well established; both an increase of the magnitude of the L-type Ca2+ current and of SR Ca2+ content increase systolic Ca2+. Indeed, the magnitude of the systolic Ca2+ transient depends very steeply on SR Ca2+ content (3). In contrast, as will be shown below, the effects of 2 are much more complicated.
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The regulation of SR Ca2+ content ultimately depends on the control of surface membrane Ca2+ fluxes: control by release of Ca2+ from the SR
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Given that SR Ca2+ content affects the size of the systolic Ca2+ transient, it is important that the SR Ca2+ content is regulated. It is therefore important to understand the mechanisms controlling the SR Ca2+ content. The immediate control point is the balance of Ca2+ uptake into the SR and Ca2+ release. Thus maneuvers that stimulate the SR Ca2+-ATPase (such as phosphorylation of phospholamban) will increase the SR Ca2+ content, whereas stimulation of Ca2+ release will decrease the content. It should also be noted that sarcolemmal Ca2+ fluxes play a major but indirect role in the control of the SR Ca2+ content. If the SR is emptied, then the rate and extent of refilling are both increased by electrical stimulation (9).
At first sight, it might seem obvious that stimulation increases the rate of refilling of the SR. One would expect that the Ca2+ entry produced by each depolarization would lead to an increase of cell and therefore SR Ca2+ content. The situation is, however, more complex than this. The experiment illustrated in Fig. 2 measures the Ca2+ influx and efflux produced by each pulse during refilling. Immediately after removal of caffeine, the systolic Ca2+ transient is very small, presumably due to the reduced SR Ca2+ content. This small Ca2+ transient is accompanied by an entry of Ca2+ on the Ca2+ current, which is larger than that observed in the steady state (when the systolic Ca2+ transient has recovered). The increase of Ca2+ entry is presumably due to the fact that the smaller Ca2+ transient results in less Ca2+-dependent inactivation of the L-type Ca2+ current (1). In addition, the efflux of Ca2+ on the first transient is less than that in the steady state, because the small Ca2+ transient produces a reduced activation of the Na+/Ca2+ exchange. The data in this figure show that, when caffeine is initially removed, the Ca2+ entry on the L-type Ca2+ current is larger than the efflux on the exchanger. This results in a net gain of Ca2+ by the cell and therefore by the SR. As the systolic Ca2+ transient increases in size, the influx gradually decreases and the efflux increases until they are once more in balance. This result therefore shows two important negative feedback systems. An increase of the Ca2+ transient results in 1) a decrease of Ca2+ entry into the cell and 2) an increase of efflux such that the SR Ca2+ content will decrease. This, in turn, will decrease the systolic Ca2+ transient. Therefore, the effect of systolic Ca2+ on sarcolemmal Ca2+ fluxes serves to control the SR Ca2+ content.
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FIGURE 2. SR refilling and sarcolemmal fluxes interact. A: experimental data showing Indo-1 records in response to stimulation with voltage clamp pulses (from –40 to 0 mV). At the start of the record, caffeine (10 mM) was removed and the SR was empty. B: specimen records of membrane current (top) and calculated Ca2+ gain for the 1st and 9th pulse (bottom). The Na+/Ca2+ exchange current tails are shown on an expanded scale below. Note that on the steady-state (9th) pulse, influx and efflux are equal. In contrast, on the 1st pulse influx is much larger than efflux. C: Ca2+ movements during a similar experiment on a different cell. Top: Ca2+ movements on the Ca2+ current (•) and Na+/Ca2+ exchange ( ). Middle: calculated gain per pulse (Ca2+ current minus Na+/Ca2+ exchange). Bottom: calculated cumulative gain of Ca2+ as a function of time. The x-axis for all data is the number of the stimulus (stimuli applied at 0.33 Hz). Data taken from Ref. 9.
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Two interesting conclusions can be drawn from the above result. The first is that without the feedback of SR Ca2+ release onto the sarcolemmal fluxes, it would be very hard to regulate cell Ca2+ balance. Any small imbalance between Ca2+ entry and efflux across the sarcolemma would persist, and the cell would either gain or lose Ca2+. Although mechanisms exist to regulate the rate of the SR Ca2+-ATPase, these cannot compensate for a net imbalance of Ca2+ flux into the cell. The second point comes from a comparison with the control of endoplasmic reticulum Ca2+ content in nonexcitable cells. This involves the process of capacitative Ca2+ influx, in which the state of filling of the endoplasmic reticulum controls a dedicated Ca2+ entry across the surface membrane (for review see Ref. 2). Such a mechanism would not be effective in cardiac muscle, because it would be swamped by the large sarcolemmal fluxes. Instead, a similar function is obtained by allowing Ca2+ release from the SR to regulate the L-type Ca2+ current and the Na+/Ca2+ exchange.
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The properties of RyR do not regulate systolic Ca2+
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As well as being regulated by cytoplasmic Ca2+ concentration, the properties of RyR can be influenced by a variety of other agents and conditions (for review see Refs. 7 and 8). Pharmacologically, caffeine increases the open probability and the local anesthetic tetracaine decreases it. Of particular note under physiological conditions is the fact that the opening probability is enhanced by phosphorylation and cADP ribose. A decrease of ATP concentration or acidification (as occur during ischemia) reduce the open probability, whereas an increase of the concentration of free radicals (as happens on reperfusion) will increase the open probability of the RyR. Finally, it has also been suggested that one explanation for the changes of the systolic Ca2+ transient in cardiac hypertrophy and failure is an effect on the coupling between the L-type Ca2+ current and the RyR, leading to a decrease of open probability (6). To investigate the role of modulation of the RyR, we have carried out experiments in which the SR Ca2+ content, sarcolemmal fluxes, and systolic [Ca2+]i were measured. We find that pharmacological modification of the RyR has no steady state effect on contraction (10). This is demonstrated in Fig. 3, which shows the effects of applying a low concentration of caffeine to increase the open probability of the RyR. However, the effect on systolic Ca2+ is purely transient and, in the steady state, the contraction in the presence of caffeine is the same magnitude as that under control conditions. On removal of caffeine, there is an undershoot before contraction recovers. The explanation of this transient effect is that, as shown above, in the steady state, Ca2+ efflux must equal Ca2+ influx. Since caffeine has no effect on Ca2+ influx, in the steady state it cannot affect the efflux. Because the efflux is determined by the magnitude of the systolic Ca2+ transient, this requires that the amplitude of the systolic Ca2+ transient be unaffected by manipulation of the RyR. The transient response arises because caffeine initially increases the fraction of the SR Ca2+ content that is released. This therefore increases the efflux of Ca2+ from the cell and results in a decrease of SR Ca2+ content. This, in turn, decreases the amplitude of the next systolic Ca2+ transient. In the steady state in caffeine, the Ca2+ transient has the same amplitude as the control one as a result of the opposing effects of an increase in the fractional release of Ca2+ and a decrease of SR Ca2+ content.
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FIGURE 3. The effects of potentiating Ca2+-induced Ca2+ release (CICR) on systolic [Ca2+]i, sarcolemmal Ca2+ fluxes, and SR Ca2+ content A: [Ca2+]i. The cell was voltage clamped (holding potential –40mV), and 100-ms duration step depolarizations were applied to 0 mV at 0.5 Hz. Caffeine (500 µM) was applied for the time indicated by the bar above the record. The inset shows Ca2+ transients before (a) and during caffeine application (b). B: pulse-by-pulse analysis of membrane Ca2+ fluxes. Ca2+ influx (•) and Ca2+ efflux ( ) were obtained, respectively, by integrating the L-type Ca2+ current during depolarization and the Na+/Ca2+ exchange tail current on repolarization. C: predicted changes of SR Ca2+ content. The differences between Ca2+ influx and efflux on each pulse were summed to obtain a cumulative net change in cell Ca2+ content. D: [Ca2+]i obtained before caffeine application (a) and from the second pulse in caffeine (b). E: membrane currents. Note the clearly increased Na+/Ca2+ exchange tail current associated with the transient (right) during early caffeine application compared with that at left. F: Ca2+ fluxes calculated by integrating the L-type Ca2+ current (upward deflection) and the Na+/Ca2+ exchange current on repolarization (downward deflection). Data taken from Ref. 10.
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If the modification of the RyR has no steady state effect on systolic Ca2+, why is it regulated?
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The above discussion has shown that modification of the RyR has only a transient effect on systolic Ca2+. The question therefore arises as to why the RyR is regulated. Two possible explanations are illustrated in Fig. 4. This shows a simulation of the effects of increasing the magnitude of the L-type Ca2+ current as occurs, for example, during ß-sympathetic stimulation. This has two effects on excitation-contraction coupling: 1) It will increase the Ca2+ loading of the cell and 2) it will increase the trigger Ca2+, thereby increasing the number of RyRs that open. These two consequences are modeled individually in Fig. 4, A and B. Figure 4A shows the effects of only increasing the loading function. This produces a maintained increase of systolic Ca2+ accompanied by an increase of SR Ca2+ content. The increase of systolic Ca2+ takes time to develop because the SR accumulates Ca2+ on a beat-to-beat basis. The effects of only increasing the trigger are shown in Fig. 4B. Similar to the effects of caffeine, this produces a transient increase of systolic Ca2+ accompanied by a decrease of SR Ca2+ content. There is no maintained effect on systolic Ca2+. The final simulation (Fig. 4C) shows the effect of simultaneously increasing both the loading and trigger functions of the L-type Ca2+ current (as will actually occur). This results in an immediate and maintained increase of systolic Ca2+ with no change of SR Ca2+ content. Coordinated stimulation of both loading and trigger functions is therefore essential if an increase of Ca2+ current is to produce both a rapid onset and a maintained response. Another potential benefit is related to the changes of SR Ca2+ content that accompany the increase of systolic Ca2+. When the loading alone is stimulated, an increase of SR Ca2+ content accompanies the increase of systolic Ca2+. This means that the ATPase has to pump against an increased Ca2+ concentration gradient, and this may become limiting. In addition, excessive filling of the SR results in spontaneous release of Ca2+ from the SR (12), and this may be arrhythmogenic. Further experimental work will be required to see whether, as suggested by the model, SR Ca2+ content is fixed during such simple inotropic interventions.
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FIGURE 4. Computation of the effects of increasing Ca2+ influx via the L-type Ca2+ current. Systolic [Ca2+]i is shown at top, and SR Ca2+ content is shown at bottom (both in arbitrary units). A: effects of increasing the loading function of the L-type Ca2+ current. In this simulation, the ability of the Ca2+ current to trigger Ca2+ release is not changed. This results in a slowly developing but maintained increase of both SR and systolic [Ca2+]i. B: effects of increasing the trigger function only. This produces a transient increase of systolic [Ca2+]i accompanied by a decrease of SR Ca2+ content. C: effects of increasing both loading and trigger. This results in an abrupt and maintained increase of SR Ca2+ content with no change of SR Ca2+ content.
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Conclusion
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The arguments above suggest that, although RyR is essential for normal excitation-contraction coupling in the heart, it does not constitute a locus for regulation of contraction. This is because changes in SR Ca2+ content compensate for effects of modification of the RyR. Rather, modulation of the properties of the RyR may serve to allow rapid changes of contraction and, also, to control the Ca2+ content of the SR.
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References
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Introduction
Ca2+ influx and efflux...
