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(Circulation Research. 2000;87:1087.)
© 2000 American Heart Association, Inc.

Review

Integrative Analysis of Calcium Cycling in Cardiac Muscle

D. A. Eisner, H. S. Choi, M. E. Díaz, S. C. O’Neill, A. W. Trafford

From the Unit of Cardiac Physiology, University of Manchester, UK.

Correspondence to D.A. Eisner, Unit of Cardiac Physiology, University of Manchester, 1.524 Stopford Bldg, Oxford Rd, Manchester M13 9PT, UK. E-mail Eisner{at}man.ac.uk

	Abstract

Top Abstract Introduction Ca2+ Buffering Sites at Which CICR... What Regulates SR Ca2+... Modulation of the RyR Integration of SR Release,... Stability and Alternans Conclusions References

 
Abstract—The control of intracellular calcium is central to regulation of contractile force in cardiac muscle. This review illustrates how analysis of the control of calcium requires an integrated approach in which several systems are considered. Thus, the calcium content of the sarcoplasmic reticulum (SR) is a major determinant of the amount of Ca²⁺ released from the SR and the amplitude of the Ca²⁺ transient. The amplitude of the transient, in turn, controls Ca²⁺ fluxes across the sarcolemma and thence SR content. This control of SR content influences the response to maneuvers that modify, for example, the properties of the SR Ca²⁺ release channel or ryanodine receptor. Specifically, modulation of the open probability of the ryanodine receptor produces only transient effects on the Ca²⁺ transient as a result of changes of SR content. These interactions between various Ca²⁺ fluxes are modified by the Ca²⁺ buffering properties of the cell. Finally, we predict that, under some conditions, the above interactions can result in instability (such as alternans) rather than ordered control of contractility.

Key Words: excitation-contraction coupling • sarcoplasmic reticulum • ryanodine receptor

	Introduction

Top Abstract Introduction Ca2+ Buffering Sites at Which CICR... What Regulates SR Ca2+... Modulation of the RyR Integration of SR Release,... Stability and Alternans Conclusions References

 
Contraction of cardiac muscle is initiated by an increase of [Ca²⁺]_i. The magnitude of this rise of Ca²⁺ or "systolic Ca²⁺ transient" must be controlled to produce a constant cardiac output, must increase to increase the force of contraction of the heart, and may fall in heart failure. The purpose of this article is to discuss the steps responsible for this control. We also point out that focusing attention on any of the individual steps that control Ca²⁺ can be misleading; a full understanding of the control of contraction requires considering how the various steps are integrated.

The reader is referred to recent reviews for general aspects of cardiac excitation-contraction coupling.^{1 2} Much recent work shows that Ca²⁺ is released from the sarcoplasmic reticulum (SR) through a specialized release channel, the ryanodine receptor (RyR), via the process of Ca²⁺-induced Ca²⁺ release (CICR). The entry of a small amount of ("trigger") Ca²⁺ through the sarcolemmal L-type Ca²⁺ current (I_Ca) produces a localized increase of [Ca²⁺]_i in the small space between the surface and SR membranes.^{3 4 5} This then increases the open probability of the RyR, resulting in the efflux of Ca²⁺ from the SR into the cytoplasm. Obviously, for the heart to function as a pump, it must relax as well as contract. Relaxation is initiated by a reduction of [Ca²⁺]_i produced either by pumping back into the SR by the SR Ca²⁺-ATPase (SERCA) or out of the cell, largely by the sarcolemmal Na⁺-Ca²⁺ exchange.

Therefore, each time the heart contracts, Ca²⁺ enters the cytoplasm both from the extracellular fluid and from the SR. For the heart to be in a steady state it is essential that, during each cardiac cycle, exactly that amount of Ca²⁺ that had entered from outside the cell is pumped back out and that which is released by the SR is returned. In the rest of this article, we will show that this requirement for Ca²⁺ flux balance has significant implications for the regulation of contraction. However, before dealing with this regulation, we will address a few comments to the properties of intracellular Ca²⁺ buffers, because these determine the magnitude of the changes of free Ca²⁺ produced by a given Ca²⁺ flux.

	Ca2+ Buffering

Top Abstract Introduction Ca2+ Buffering Sites at Which CICR... What Regulates SR Ca2+... Modulation of the RyR Integration of SR Release,... Stability and Alternans Conclusions References

 
The magnitude of the systolic rise of [Ca²⁺]_i depends not only on the magnitude of the fluxes but, in addition, on the Ca²⁺ buffering power of the cell. Buffering properties of cardiac cells have been measured in a variety of ways. Relatively direct measurements can be made in permeabilized cells,⁶ but because some cytoplasmic constituents will have been lost, there is a need for data from intact cells. The general approach used is to estimate the change of total Ca²⁺ (Ca_T) by integrating sarcolemmal Ca²⁺ fluxes (under conditions where the SR does not contribute) and compare this with the change of [Ca²⁺]_i ([Ca²⁺]_i) obtained with a fluorescent indicator. One method measures the increase of [Ca²⁺]_i produced by a given I_Ca.⁷ The use of pulses that activate different amounts of I_Ca therefore allows a buffer curve to be produced. An alternative and more rapid method uses caffeine to release Ca²⁺ from the SR, resulting in an increase of [Ca²⁺]_i, which decays as Ca²⁺ is pumped out of the cell on Na⁺-Ca²⁺ exchange. The integral of the Na⁺-Ca²⁺ exchange current (after correction for the electroneutral Ca²⁺-ATPase) gives a measure of the total amount of Ca²⁺ pumped out of the cell, and the change of free Ca²⁺ is obtained from a fluorescent indicator.⁸ One caffeine application then gives an entire curve (see Figure 3B). This method also has the advantage that it provides a quantitative measure of SR content.⁹ To a first approximation, the buffering in the cell can be described with a single K_d, although for more precise work, note should be taken of the fact that the overall buffering is made up of contributions from various components with different K_ds.^{10 11 12} These include troponin and calmodulin as well as membrane binding sites. Values for the overall K_d of 0.5 to 1 µmol/L and for the maximum capacity (B_max) of 100 to 200 µmol/L accessible cell water have been found.^{6 8 11} It is important to remember that the buffering power is not constant but decreases as [Ca²⁺]_i increases. The buffering power at any value of [Ca²⁺]_i can be defined as follows: ß=Ca_T/[Ca²⁺]_i=(K_dxB_max)/K_d+[Ca²⁺]_i)².

View larger version (19K): [in this window] [in a new window]   Figure 3. Figure 3. Quantitative relationships between the various Ca2+ fluxes. A, Dependence of the amplitude of the total (•) and free () Ca2+ transients on SR Ca2+ content. The curves through the data are of the form y=axxn. n=2.1 for the total and 5.2 for the free curves. B, Dependence of free (ordinate) on total (abscissa) Ca2+. C, Dependence of Ca2+ efflux on the amplitude of the Ca2+ transient. D, Dependence of Ca2+ efflux on SR Ca2+ content. n=3.3. All data were obtained from the experiment illustrated in Figure 2 (rat myocyte). Data are modified from Reference 30.

This equation means that ß decreases with increasing [Ca²⁺]_i and therefore that a given increase of total cytoplasmic Ca²⁺ will produce a greater increase of [Ca²⁺]_i as [Ca²⁺]_i increases. For example, with a K_d of 0.6 µmol/L and a B_max of 175 µmol/L accessible cell water, the value of ß changes from 214 at a diastolic [Ca²⁺]_i of 100 nmol/L to 41 at a systolic [Ca²⁺]_i of 1 µmol/L and 16 at 2 µmol/L [Ca²⁺]_i. This [Ca²⁺]_i dependence of buffering power is responsible for the fact that increasing the amplitude of the Ca²⁺ transient increases the rate constant of decay.¹³ Furthermore, as will be described later, it also has important effects on regulation of systolic Ca²⁺.

	Sites at Which CICR May Be Regulated

Top Abstract Introduction Ca2+ Buffering Sites at Which CICR... What Regulates SR Ca2+... Modulation of the RyR Integration of SR Release,... Stability and Alternans Conclusions References

 
The amount of Ca²⁺ released from the SR for a given entry on I_Ca depends on at least 2 factors, as follows: (1) the properties of the RyR, in particular the relationship between [Ca²⁺]_i and the open probability of the RyR, and (2) the Ca²⁺ content of the SR and thence the Ca²⁺ release flux when a channel opens. In the remainder of this article, we will discuss the contributions of these 2 factors.

	What Regulates SR Ca2+ Content?

Top Abstract Introduction Ca2+ Buffering Sites at Which CICR... What Regulates SR Ca2+... Modulation of the RyR Integration of SR Release,... Stability and Alternans Conclusions References

 
Much attention has been focused on the effects of directly modulating the activity of SERCA and, in particular, its interaction with the inhibitory accessory protein phospholamban. Phosphorylation of phospholamban relieves the inhibition of SERCA, thereby stimulating its activity.^{14 15} This occurs, for example, during sympathetic stimulation of ß-receptors resulting in an enhanced SR Ca²⁺ content and increased rate of decay of the Ca²⁺ transient.^{16 17} As would be expected, animals deficient in phospholamban (phospholamban knockout mice) have elevated SR Ca²⁺ contents,¹⁸ Ca²⁺ transients with faster rates of decay than controls, and a smaller response to ß stimulation.^{19 20}

Autoregulation of SR Ca²⁺ Content
Another important factor that controls the SR Ca²⁺ content is the level of cytoplasmic Ca²⁺. The higher the [Ca²⁺]_i, the greater the rate of Ca²⁺ pumping into the SR. In addition, Ca²⁺ release from the SR influences sarcolemmal Ca²⁺ fluxes.²¹ This can be seen experimentally if the SR Ca²⁺ content is altered. In the experiment illustrated in Figure 1, the SR had initially been emptied by exposure to 10 mmol/L caffeine. When stimulation was begun, the Ca²⁺ transient was initially very small but then recovered over the next minute as the SR refilled with Ca²⁺ (Figure 1A). Figure 1B shows expanded records of these Ca²⁺ transients. Trace b was recorded in the steady state. Accompanying the large Ca²⁺ transient is Ca²⁺ entry via the L-type Ca²⁺ current and efflux on the Na⁺-Ca²⁺ exchange on repolarization (Figure 1C). It is clear that the entry via the L-type Ca²⁺ current balances the efflux on Na⁺-Ca²⁺ exchange. In other words, in the steady state, Ca²⁺ entry equals efflux. A very different result is seen for the first stimulus (a). The Ca²⁺ entry is larger than in the steady state and the efflux smaller. Therefore, instead of being in Ca²⁺-flux balance, the cell gains Ca²⁺. The Ca²⁺ movements on each pulse are shown in the lower panels of Figure 1A. The larger Ca²⁺ currents when the SR is empty and the decrease in size on refilling result from decreased Ca²⁺-induced inactivation of the L-type Ca²⁺ current.^{22 23 24} The increase of efflux on Na⁺-Ca²⁺ exchange with increased systolic Ca²⁺ simply represents the fact that the rate of Ca²⁺ pumping by Na⁺-Ca²⁺ exchange increases with an increase in the amplitude of the systolic Ca²⁺ transient. The third panel of Figure 1A shows that, on starting stimulation, there is a net gain of Ca²⁺ on each pulse and that this gain disappears in the steady state. Finally, the bottom panel sums the net influx on each pulse. This shows that, by the end of the period shown, the cell had gained 80 µmol/L as a result of stimulation.

View larger version (24K): [in this window] [in a new window]   Figure 1. Figure 1. Relationship between SR Ca2+ content and sarcolemmal Ca2+ fluxes. A, Time course of original data. Top trace shows measurements of [Ca2+]i. The cell was held at -40 mV, and 100-ms-duration depolarizing pulses were applied to 0 mV at 0.5 Hz. Caffeine (10 mmol/L) had been applied until 10 seconds before the record began, to empty the SR. The second panel shows Ca2+ influx (via the L-type Ca2+ current) and efflux on each pulse (measured as shown for specimen records of panel B). The third panel shows net Ca2+ entry (influx-efflux), and the bottom panel, calculated cumulative gain of Ca. B, Specimen records from transients a and b showing (from top to bottom) [Ca2+]i, membrane current, and calculated net sarcolemmal Ca2+ movement. These Ca2+ movements were calculated by integrating the currents after making allowance for the stoichiometry (charges per Ca2+ transported) and allowing for the fact that some of the Ca2+ efflux is produced by the electroneutral sarcolemmal Ca-ATPase. Data were obtained from a ferret myocyte (adapted from Reference 21). C, Expanded tail currents.

This mechanism, which we have previously referred to as "autoregulation,"²⁵ therefore provides a simple way to control the Ca²⁺ content of the SR. An increase of SR Ca²⁺ content will result in greater release, thereby decreasing Ca²⁺ entry into and increasing efflux out of the cell. This will tend to decrease SR content toward the initial level. The effectiveness or "gain" of this system will depend on the relationship between SR Ca²⁺ content and sarcolemmal fluxes. The greater the slope of this relationship, the more tightly controlled will be the SR content. However, as discussed later in this article, there may be reasons for not wanting too steep a relationship.

The above focus on the control of sarcolemmal fluxes by SR content does not mean that control of the SERCA by, for example, phosphorylation of phospholamban is unimportant, but rather that such control mechanisms will simply adjust the set point produced by this autoregulation. It is worthwhile noting that the phospholamban mechanism itself cannot control SR content, as, with the exception of the results of one study,²⁶ phosphorylation depends on parameters other than SR content such as the concentration of cAMP and consequent activation of protein kinase A. In contrast, autoregulation senses the SR content via effects on the amplitude of the systolic Ca²⁺ transient. In this sense, this mechanism carries out a function similar to that of "capacitative" control of Ca²⁺ entry in nonexcitable cells. In capacitative entry, when the endoplasmic reticulum Ca²⁺ content decreases, a signal is produced to increase Ca²⁺ influx across the surface membrane of the cell. This increases Ca²⁺ entry via a channel known as I_CRAC (Ca²⁺ release–activated Ca²⁺ channel). The Ca²⁺ flux through this channel is somehow increased by depletion of the Ca²⁺ content of the endoplasmic reticulum (for review see Barritt²⁷ ). It may be that the large magnitude of the transmembrane Ca²⁺ fluxes in the heart makes modulation of I_Ca and the Na⁺-Ca²⁺ exchange a more suitable strategy than I_CRAC.

Figure 1A shows that increasing the SR Ca²⁺ content (bottom panel) results in a large increase of the amplitude of the systolic Ca²⁺ transient (top). This is emphasized in Figure 3A, which shows that the amount of Ca²⁺ released from the SR increases steeply with increased content such that, at higher contents, a greater fraction of the SR content is released.^{7 21 28 29 30} This steep dependence has 2 consequences, as follows: (1) it makes it imperative that SR content be controlled and (2) it means that an increase of SR Ca²⁺ content is an effective positive inotropic maneuver.

The above considerations mean that, in the steady state, the systolic Ca²⁺ transient must be of a magnitude to produce a Ca²⁺ efflux that exactly balances the Ca²⁺ influx into the cell. This can explain the effects of many inotropic maneuvers without any knowledge of the internal mechanisms of the cell. Thus, an increase of the L-type Ca²⁺ current will increase Ca²⁺ influx. In the steady state, this will require an increased Ca²⁺ transient to support an increased efflux. Catecholamines accelerate the rate of decay of the systolic Ca²⁺ transient.¹⁶ This acceleration will leave less time for the surface membrane to remove calcium, and therefore a larger Ca²⁺ transient is required to support the same Ca²⁺ efflux.

Diastolic Ca²⁺ Fluxes
In the examples presented above, the cell is in calcium balance at the end of systole. It is, however, possible that there may be a net flux of Ca²⁺ during the systolic period that is compensated by an equal and opposite flux during diastole. An example is provided by the effects of cardiac glycosides. These have long been known to increase the magnitude of the systolic Ca²⁺ transient.^{31 32 33} This results from an increase of intracellular Na⁺ concentration and consequent effects on Na⁺-Ca²⁺ exchange leading to an increase of SR Ca²⁺ content.^{34 35} One might expect that this would be due to a decreased ability of Na⁺-Ca²⁺ exchange to remove Ca²⁺ requiring a larger Ca²⁺ transient to produce the same efflux. It appears, however, that the cell loses Ca²⁺ during systole in strophanthidin (as the larger Ca²⁺ transient increases Ca²⁺ efflux and decreases influx) and that this is compensated for by increased diastolic Ca²⁺ entry presumably on Na⁺-Ca²⁺ exchange.³⁶ This emphasizes the potential importance of the diastolic as well as the systolic period. Longer-term³⁷ modulation must also be considered. For example, altering the rate of stimulation can produce gradual changes of contraction that have been linked to changes of intracellular Na⁺ concentration. Such longer-term effects will alter the steady states referred to above by altering SR content.

Limitations on SR Ca²⁺ Content as the Only Regulator of Systolic Ca²⁺
One unanswered question is the extent to which SR Ca²⁺ content can be increased to increase the force of contraction. There is presumably a maximum level of SR Ca²⁺ content. It has been suggested that the maximum SR free Ca²⁺ concentration is limited only by the energy available from ATP.¹² In contrast to this result, agents that inhibit opening of the RyR increase the SR content,³⁸ suggesting that leak of Ca²⁺ out of the SR may limit the maximum content. Furthermore, the maximum content also appears to be limited by spontaneous Ca²⁺ release (waves) occurring at high Ca²⁺ loads.^{39 40} Whatever the origin of the maximum SR content, its existence means that mechanisms in addition to an increase of SR content are required to increase the magnitude of the systolic Ca²⁺ transient.

	Modulation of the RyR

Top Abstract Introduction Ca2+ Buffering Sites at Which CICR... What Regulates SR Ca2+... Modulation of the RyR Integration of SR Release,... Stability and Alternans Conclusions References

 
It is well established that the open probability of the RyR can be affected by substances other than cytoplasmic Ca²⁺ concentration.⁴¹ Among the substances that increase the open probability are caffeine,⁴² 2,3-butanedione monoxime (BDM),⁴³ and cADP-ribose.^{44 45} The open probability is also increased by phosphorylation.^{46 47 48} Finally, the RyR is also associated with the FK506 binding protein (FKBP12.6). These FK binding proteins stabilize the RyR and, in their absence, long-lasting subconductance states appear, resulting in increased Ca²⁺ flux.⁴⁹ Dissociation of FKBP12.6 from the RyR is increased by immunophilins such as FK506⁵⁰ or phosphorylation.⁴⁸ The open probability can be decreased by local anesthetics such as tetracaine.^{51 52 53} Of potential relevance to ischemia, the open probability is decreased by acidification⁵⁴ or a decrease of cytoplasmic ATP concentration.⁵⁴ For convenience, we will consider this area in 2 parts, modulation of the RyR during systole and diastole.

Systolic Regulation of the RyR
Many previous studies have suggested that maneuvers or circumstances that affect either the level of expression or the open probability of the RyR will alter systolic Ca²⁺. Thus, the depression of contraction in heart failure has been linked to either a decrease in the number of RyRs^{55 56 57 58 59 60 61} or a change in their properties such that fewer are opened by a given trigger increase of [Ca²⁺]_i.^{62 63} This conclusion does not, however, fit well with results examining the effects of maneuvers that alter the RyR pharmacologically. Figure 2Aa shows that increasing the open probability with a low concentration of caffeine produces a purely transient increase in the amplitude of the systolic Ca²⁺ transient.³⁰ Similarly, BDM produces only a transient increase.^{64 65 66} Other work shows that a decrease of open probability of the RyR produced by either tetracaine⁶⁷ or decreased pH⁶⁸ produces a decrease of systolic [Ca²⁺]_i that is also transient.

View larger version (26K): [in this window] [in a new window]   Figure 2. Figure 2. Changes of sarcolemmal fluxes and SR Ca2+ release during potentiation of CICR by caffeine (500 µmol/L). A, Time course. Traces show the following (from top to bottom): a, [Ca2+]i; b, changes in total Ca; c, net sarcolemmal flux calculated as Ca2+ entry on ICa minus efflux on Na+-Ca2+ exchange; d, fractional Ca2+ efflux, fraction of the total Ca2+ transient that is pumped out of the cell; e, SR Ca2+ content; and f, fractional release, fraction of the SR Ca2+ content that is released. B, Specimen records from a control transient (i) and the first in caffeine (ii). For each transient, records show the following (from top to bottom): [Ca2+]i, membrane current, and calculated movement of Ca2+ across the surface membrane. Note that for transient i, influx=efflux, whereas for transient ii, the efflux is greater than the influx. Data are from a rat myocyte; adapted from Reference 30.

The explanation of the transient effects of modulation of the RyR lies with the interactions between SR content and sarcolemmal fluxes reviewed above. This is shown in Figure 2. The application of caffeine produces a transient increase of systolic [Ca²⁺]_i that decays to resting levels. The steady-state Ca²⁺ transient in caffeine is identical to that of the control.³⁰ The specimen traces of Figure 2B show the accompanying membrane currents and calculated Ca²⁺ fluxes for both the control and the first transient in caffeine. In control conditions (Figure 2Bi), Ca²⁺ influx and efflux are equal and the cell is therefore in Ca²⁺ flux balance. However, when caffeine is applied (Figure 2Bii), the increase of the systolic Ca²⁺ transient results in an increase of Ca²⁺ efflux on Na⁺-Ca²⁺ exchange such that the Ca²⁺ efflux is greater than the influx. This results in a predicted loss of Ca²⁺ from the cell. Panel c of Figure 2A shows the net sarcolemmal Ca²⁺ flux (ie, Ca²⁺ entry minus exit). In control conditions, Ca²⁺ entry and exit are balanced. However, the increase in the amplitude of the Ca²⁺ transient in caffeine results in an increase of Ca²⁺ efflux and thence a net loss of Ca²⁺ from the cell and therefore the SR (panel e).

The transient nature of the response to low concentrations of caffeine therefore arises as follows. In the steady state, Ca²⁺ influx equals Ca²⁺ efflux. Initially in caffeine, the larger Ca²⁺ transient results in greater efflux (to a level greater than the influx), resulting in a net loss of Ca²⁺ from the cell and SR. This, in turn, will decrease the amplitude of the Ca²⁺ transient, thereby decreasing the amount of Ca²⁺ pumped out of the cell. Eventually, a new steady state will be reached at which the Ca²⁺ efflux equals the influx. It is important to realize that, so long as neither the Ca²⁺ influx nor the properties of Na⁺-Ca²⁺ exchange are altered, this steady state can only be reached when the Ca²⁺ transient is the same size as the control one.

Direct measurements of SR Ca²⁺ content show that the decrease of the potentiation of systolic Ca²⁺ produced by caffeine or BDM are, indeed, accompanied by a decrease of SR content.^{30 64 66}

The transient decrease of systolic Ca²⁺ produced by agents that decrease the open probability of the RyR has a similar explanation. In brief, the decrease of the Ca²⁺ transient will decrease the amount of Ca²⁺ that leaves the cell (to a value less than that of the influx). This will then result in an increase of SR content^{67 68} and a consequent increase of systolic Ca²⁺ until both systolic Ca²⁺ and Ca²⁺ efflux return to control levels, accompanied by an increase of SR Ca²⁺ content.

If, for example, stimulation of the RyR has no steady-state effect on the amplitude of systolic Ca²⁺ transient, one should consider why it is regulated. There are 2 possible explanations, which can be understood by considering the inotropic effects of increasing the L-type Ca²⁺ current.^{25 30} (1) Maneuvers that increase the amplitude of I_Ca will increase the loading of the cell, and therefore the SR, with Ca²⁺. This will produce a slowly developing increase of systolic Ca²⁺ and cannot produce a physiologically useful rapid increase of contractility. However, the potentiation of the RyR open probability by the increased "trigger" Ca²⁺ entry will result in a transient increase of systolic Ca²⁺. The combination of a slow (but maintained) increase and a transient effect will result in a rapidly developing and maintained increase of systolic Ca²⁺. (2) As pointed out above, excessive increase of SR content results in spontaneous Ca²⁺ release and also an increase of the gradient against which SERCA pumps. Potentiation of RyR open probability will decrease SR Ca²⁺ content and avoid these problems.

Diastolic Modulation of the RyR
In the above analysis, we assumed that the only effect of modulation of the RyR occurs during systole and, therefore, that any efflux of Ca²⁺ from the SR through the RyR during diastole can be ignored. As reviewed above, low concentrations of caffeine produce a purely transient potentiation of SR Ca²⁺ release. In contrast, high concentrations produce a dose-dependent decrease of systolic [Ca²⁺]_i.^{69 70 71} This is because, at these concentrations, caffeine produces a large increase of SR Ca²⁺ permeability, even at rest, and thereby depletes the SR. Even if all of the SR content is released, the amplitude of the Ca²⁺ transient will still be less than in control.

A recent study has suggested that such an increase of diastolic Ca²⁺ release can account for the decrease of Ca²⁺ transient in heart failure.⁴⁸ It was found that the RyR from failing hearts was hyperphosphorylated. This resulted in increased sensitivity to activating Ca²⁺ and the occurrence of subconductance levels. This will increase the Ca²⁺ leak of the SR and thereby decrease its Ca²⁺ content. This is an attractive explanation for decreased contraction that does not suffer from the problems reviewed above for the idea that either the number of RyR or their systolic activation are modified. Nevertheless, flux balance must still be maintained. This might happen in 2 ways. (1) Increased diastolic leak may slow the rate of relaxation of the systolic Ca²⁺ transient. This is the case for high concentrations of caffeine and arises because the increase of leak makes it harder for the SR to remove Ca²⁺ from the cytoplasm. This leads to a decrease in the rate of decay of the residual Ca²⁺ transient. The decay of the Ca²⁺ transient will also be slowed simply as a consequence of the increased Ca²⁺ buffering at lower [Ca²⁺]_i.¹³ Although the Ca²⁺ transient is smaller than is the case under control conditions, the fact that it lasts longer means that the Na⁺-Ca²⁺ exchange has longer to pump Ca²⁺ out of the cell and therefore the efflux can still equal the influx. (2) An alternative explanation is that there may be an increase of diastolic Ca²⁺. This will support an increased Ca²⁺ efflux from the cell during diastole that will compensate for decreased efflux in systole.

	Integration of SR Release, Buffering, and Sarcolemmal Ca2+ Transport

Top Abstract Introduction Ca2+ Buffering Sites at Which CICR... What Regulates SR Ca2+... Modulation of the RyR Integration of SR Release,... Stability and Alternans Conclusions References

 
The contributions of all of the processes mentioned above can be seen by considering, again, the effects of increasing the open probability of the RyR with caffeine. The data of Figure 2Aa show the transient increase of systolic Ca²⁺ produced by caffeine. The change of cytoplasmic total Ca²⁺ is shown in Figure 2Ab. It is clear that the fractional increase of the amplitude of the free Ca²⁺ transient is larger than that of the total. This (see above) is due to the [Ca²⁺]_i dependence of Ca²⁺ buffering. Figure 2Ac shows the net sarcolemmal Ca²⁺ flux on each pulse. As explained above, this is initially zero as Ca²⁺ influx and efflux are in balance. However, during the onset of the effects of caffeine, there is a net Ca²⁺ efflux. Figure 2Ad shows Ca²⁺ efflux as a fraction of the total Ca²⁺ transient. This is increased but not by as much as the amplitude of the free Ca²⁺ transient (see below). Figure 2Ae shows the calculated SR Ca²⁺ content. This was obtained as follows: sarcolemmal Ca²⁺ flux minus change of total cytoplasmic Ca²⁺. As is emphasized in Figure 2Af, the fraction of SR Ca²⁺ content that is released increases initially in caffeine but then decreases (to a level still higher than that of control) during continued exposure to caffeine. The initial increase is a direct effect of caffeine on the RyR, whereas the subsequent decrease is a consequence of the decrease of SR content. In the steady state, the fraction released is still, however, greater than that of the control, as the same total amount of Ca²⁺ is released from a reduced SR content. This figure therefore shows the complicated consequences of a simple variation of one cellular parameter, the Ca²⁺ sensitivity of the RyR.

The various relationships underlying this behavior are shown in Figure 3. Figure 3A summarizes the dependence of the amplitude of the systolic Ca²⁺ transient on SR Ca²⁺ content. The change of free Ca²⁺ is a much steeper function of SR content than is the total Ca²⁺. The reason for this is shown in Figure 3B; as [Ca²⁺]_i increases, it becomes a very steep function of total Ca²⁺. The Ca²⁺ efflux from the cell is plotted as a function of the amplitude of the free Ca²⁺ transient in Figure 3C. This relationship is tending toward saturation, reflecting the finite K_d of the Na⁺-Ca²⁺ exchange. The graph of Figure 3D shows the dependence of Ca²⁺ efflux on SR content. This therefore involves the following 3 processes: the dependence of the total Ca²⁺ transient on SR content, the buffering properties of the cell, and the dependence of Ca²⁺ efflux on [Ca²⁺]_i. This relationship is less steep than that of [Ca²⁺]_i on SR content because of the flattening effect produced by saturation of Na⁺-Ca²⁺ exchange. The effect of this is visible in Figure 2Ab; the fractional increase of the Ca²⁺ efflux is much less than that of the free Ca²⁺ transient.

In some cases, more than one of the Ca²⁺ regulation processes can be directly affected by a single factor. For example, the activity of both the SR Ca²⁺ ATPase and the RyR are regulated by ATP. During ischemia, therefore, when [ATP] falls, uptake of Ca²⁺ into the SR would be compromised, but inhibition of the RyR might compensate. Some evidence of such effects has been found in intact myocytes from guinea pig hearts in which SR Ca²⁺ content is maintained or rises in metabolic inhibition⁷² and in skinned myocytes from rat hearts in which lowering [ATP] slows reuptake of Ca²⁺ into the SR, while SR Ca²⁺ content is higher than normal.⁷³ It seems likely from these results that lowering [ATP] has more influence on the ability of the SR to store Ca²⁺ through its inhibitory effect on the RyR than through lowering SERCA activity.

	Stability and Alternans

Top Abstract Introduction Ca2+ Buffering Sites at Which CICR... What Regulates SR Ca2+... Modulation of the RyR Integration of SR Release,... Stability and Alternans Conclusions References

 
The above discussion has described a system whereby an increase of SR Ca²⁺ content leads to an increase of Ca²⁺ efflux and decrease of Ca²⁺ influx that, in turn, compensate for the increased Ca²⁺ content. This autoregulation is a classic negative feedback system. It does, however, involve a delay, as the change of SR Ca²⁺ content on one beat only influences Ca²⁺ fluxes on the next beat. It is well known that delays can cause instability in negative feedback systems. The potential effect of this can be seen qualitatively as follows. Imagine that there is a very steep relationship between SR content and Ca²⁺ efflux. If the cell begins with a large SR content, then the Ca²⁺ transient will result in a large loss of Ca²⁺ from the cell. This will decrease the SR content. The next beat will therefore arise from a depleted SR, resulting in a smaller Ca²⁺ transient and efflux and therefore a net gain of Ca²⁺ by the cell and thence, on the next beat, a large Ca²⁺ transient. If this continues, alternating small and large Ca²⁺ transients will be produced. A simple model of this is shown in Figure 4. Figure 4A shows Ca²⁺ efflux as a function of SR content. This relationship includes the dependence of Ca²⁺ release on SR content, the Ca²⁺ buffering properties of the cytoplasm, and the relationship between [Ca²⁺]_i and Ca²⁺ efflux. For convenience, we represent the overall relationship as follows: efflux=(SR)x(SRⁿ)/K_d+SRⁿ), where the value of n determines the steepness of the curve. Curves are shown for values of n=1, 3, and 6. We assume that the Ca²⁺ influx into the cell per beat is not affected by changes of SR content. This is a simplification of the experimental result (Figure 1). The simulation was begun with an SR content of 100. With n=1, the SR relaxes monotonically to a new level. With n=3, there is a transient oscillation of content before a steady level is reached. However, with n=6, a steady alternans of SR content is produced. Therefore, if the relationship between Ca²⁺ efflux from the cell and SR content is too steep, instability may result. Obviously, if the relationship is shallow, then small changes in influx will result in large changes in SR content. The physiological optimum may therefore be a compromise between these 2 extremes. As shown above (Figure 3), the relationship between SR content and Ca²⁺ efflux from the cell depends on many parameters. It is worth noting that the tendency toward saturation of the Na⁺-Ca²⁺ exchange compensates for that of the Ca²⁺ buffers. It is possible that a linear dependence of Na⁺-Ca²⁺ exchange on [Ca²⁺]_i would result in the sort of instability predicted by Figure 4B.

View larger version (22K): [in this window] [in a new window]   Figure 4. Figure 4. Steep relationship between SR Ca2+ content and efflux may produce alternans. A, Relationship between SR Ca2+ content and Ca2+ efflux. Curves show Ca2+ efflux calculated from the equation efflux=(SR)x(SRn)/(Kd+SRn). The 3 curves correspond to n=1, 3, and 6. Kd is calculated as 50n so that the curves cross at the same point. Horizontal dashed line shows Ca2+ influx per beat and is assumed constant. B, Model predictions. Graphs show SR Ca2+ content as a function of the number of the beat. SR Ca2+ content is initially 100 (off scale at the amplification shown). On each subsequent beat, the cell (and therefore SR) gains 30 via the Ca2+ current and loses an amount of Ca2+ determined by the efflux curves of panel A. From top to bottom, graphs represent values of n=1, 3, and 6. C, Experimental data showing alternans of the amplitude of the Ca2+ transient recorded in a ferret ventricular myocyte.

This alternation of the amplitude of successive Ca²⁺ transients is reminiscent of the phenomenon of mechanical alternans (for review, see Euler⁷⁴ ). An example of an experimentally recorded alternating Ca²⁺ transient is shown in Figure 4C. Of course, other mechanisms may contribute to alternans. For example, it has been suggested that alternans may not involve changes of SR Ca²⁺ content but, rather, of the fraction of Ca²⁺ that is released⁷⁵ (although one would still expect resulting changes of SR content). Nevertheless, an understanding of the factors that normally ensure stability and, in pathological circumstances, allow instability will require taking account of the mechanisms and relationships described in this review.

	Conclusions

Top Abstract Introduction Ca2+ Buffering Sites at Which CICR... What Regulates SR Ca2+... Modulation of the RyR Integration of SR Release,... Stability and Alternans Conclusions References

 
The work reviewed here makes it clear that the cardiac cell has evolved simple but powerful mechanisms to regulate SR content and sarcolemmal fluxes. Ca²⁺ fluxes produced by one pump or channel affect the concentrations seen by others. This means that, when analyzing even the simplest inotropic interventions, it is important to consider these interactions.

	Acknowledgments

 
Work from this laboratory was supported by The British Heart Foundation and The Wellcome Trust.

Received September 26, 2000; revision received October 20, 2000; accepted October 20, 2000.

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