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(Circulation Research. 2002;91:594.)
© 2002 American Heart Association, Inc.

Cellular Biology

Quantitative Assessment of the SR Ca²⁺ Leak-Load Relationship

Thomas R. Shannon, Kenneth S. Ginsburg, Donald M. Bers

From the Department of Physiology, Loyola University Chicago.

Correspondence to Thomas R. Shannon, Department of Physiology, Loyola University Chicago, 2160 S First Ave, Maywood, IL 60153. E-mail tshanno{at}lumc.edu

	Abstract

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Increased diastolic SR Ca²⁺ leak (J_leak) could depress contractility in heart failure, but there are conflicting reports regarding the J_leak magnitude even in normal, intact myocytes. We have developed a novel approach to measure SR Ca²⁺ leak in intact, isolated ventricular myocytes. After stimulation, myocytes were exposed to 0 Na⁺, 0 Ca²⁺ solution ±1 mmol/L tetracaine (to block resting leak). Total cell [Ca²⁺] does not change under these conditions with Na⁺-Ca²⁺ exchange inhibited. Resting [Ca²⁺]_i declined 25% after tetracaine addition (126±6 versus 94±6 nmol/L; P<0.05). At the same time, SR [Ca²⁺] ([Ca²⁺]_SRT) increased 20% (93±8 versus 108±6 µmol/L). From this Ca²⁺ shift, we calculate J_leak to be 12 µmol/L per second or 30% of the SR diastolic efflux. The remaining 70% is SR pump unidirectional reverse flux (backflux). The sum of these Ca²⁺ effluxes is counterbalanced by unidirectional forward Ca²⁺ pump flux. J_leak also increased nonlinearly with [Ca²⁺]_SRT with a steeper increase at higher load. We conclude that J_leak is 4 to 15 µmol/L cytosol per second at physiological [Ca²⁺]_SRT. The data suggest that the leak is steeply [Ca²⁺]_SRT-dependent, perhaps because of increased [Ca²⁺]_i sensitivity of the ryanodine receptor at higher [Ca²⁺]_SRT. Key factors that determine [Ca²⁺]_SRT in intact ventricular myocytes include (1) the thermodynamically limited Ca²⁺ gradient that the SR can develop (which depends on forward flux and backflux through the SR Ca²⁺ ATPase) and (2) diastolic SR Ca²⁺ leak (ryanodine receptor mediated).

Key Words: Ca²⁺ cycling • membrane transport • sarcoplasmic reticulum • ryanodine receptors • excitation-contraction coupling

	Introduction

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Much is known about Ca²⁺-induced Ca²⁺ release (CICR) and sarcoplasmic reticulum (SR) Ca²⁺ uptake in cardiac myocytes. SR Ca²⁺-pump function is usually described as a unidirectional uptake mechanism (J_pumpF) and this can adequately describe Ca²⁺ transport during twitch relaxation and [Ca²⁺]_i decline.^1,2 Much less is known about how diastolic SR Ca²⁺ fluxes, especially SR Ca²⁺ leak flux (J_leak) and SR Ca²⁺ pump backflux (J_pumpR), alter excitation-contraction coupling (ECC). These important fluxes directly affect the SR Ca²⁺ content ([Ca²⁺]_SRT), and free intra-SR [Ca²⁺] ([Ca²⁺]_SR) and can thus powerfully modulate ECC. Notably, the fractional SR Ca²⁺ release depends very steeply on [Ca²⁺]_SRT, such that small changes in [Ca²⁺]_SRT can cause large changes in SR Ca²⁺ release during ECC.^3,4

In the present study, we characterize diastolic SR Ca²⁺ fluxes. During diastole, [Ca²⁺]_i declines to low levels as Ca²⁺ is transported into the SR by J_pumpF and out of the cell (mainly via Na⁺-Ca²⁺ exchange).⁵ Thus, [Ca²⁺]_SR rises as [Ca²⁺]_i declines (Figure 1A). This rise increases both passive J_leak and Ca²⁺ pump–mediated J_pumpR. Two schools of thought describe diastolic SR Ca²⁺ flux as either (1) a strict forward pump versus leak balance (J_pumpF=J_leak without appreciable J_pumpR, Figure 1B)^1,2,6 or (2) approaching a thermodynamic equilibrium where J_pumpF=J_pumpR (with little J_leak, Figure 1C).^7–10 In both cases, the systolic rise in [Ca²⁺]_i stimulates net uptake of Ca²⁺ by the SR, which raises [Ca²⁺]_SR. In the first scenario, this rise in [Ca²⁺]_SR causes J_leak to rise until it equals J_pumpF (which falls as [Ca²⁺]_i declines but is still 10% to 20% of V_max at diastolic [Ca²⁺]_i). In this case, J_pumpR is not considered and several ECC models or analyses have assumed a large SR Ca²⁺ leak (20 µmol/L cytosol per second).^1,2 Flux balance in this scenario requires this large resting J_leak and a large ATP consumption to simply retain constant diastolic SR Ca²⁺.

View larger version (39K): [in this window] [in a new window]   Figure 1. A, Theoretical curves describing [Ca2+]i and [Ca2+]SRT during a typical contraction. As Ca2+ is taken up, a [Ca2+] gradient builds across the SR membrane enhancing SR Ca2+ efflux, either by passive leak through spontaneous RyR openings (Jleak) or by a unidirectional reverse flux through the SR Ca2+ pump (JpumpR). B, Theoretical profile of a pure pump-leak balance. With release, leak declines and the pump rate rises due to the fall of [Ca2+]SR and the rise of [Ca2+]i, respectively. As SR uptake progresses, Jleak rises and Jpump (ie, JpumpF with little or no JpumpR present) falls until the two are equal at steady state. C, Flux profile of an alternative hypothesis where backflux accounts for a high percentage of SR Ca2+ efflux at steady state. In this case, JpumpF=JpumpR+Jleak, where Jleak is small.

The second scenario (Figure 1C) is based on measurements showing very low SR Ca²⁺ leak rates in intact myocytes⁷ and the appreciation that the SR Ca²⁺ pump is reversible (it can actually completely reverse and make ATP).^11,12 The pump can generate a [Ca²⁺] gradient where [Ca²⁺]_SR/[Ca²⁺]_i can approach a thermodynamic limit of 7000.^8,10 In this case (Figure 1C), most of the diastolic J_pumpF is balanced by J_pumpR, which rises with [Ca²⁺]_SR during [Ca²⁺]_i decline (with little J_leak required).^8–12 Note that J_pumpR cannot exceed J_pumpF in an intact cell, but as it approaches J_pumpF, it could create a steady-state balance where there is almost no net pump flux (J_pump), just enough to be counterbalanced by a very small J_leak. This would require very little ATP consumption to retain diastolic [Ca²⁺]_SRT but would make [Ca²⁺]_SRT quite sensitive to energetic state (eg, G_ATP).

The actual value of J_leak in intact ventricular myocytes is controversial and estimates have ranged from 0.3 µmol/L cytosol per second^7,9 to 5 to 30 µmol/L cytosol per second.¹³ Models that do not consider J_pumpR require J_leak 20 µmol/L cytosol per second to compensate for diastolic J_pumpF.^1,2 Some of our data have more strongly favored values at the lower end of this J_leak range.^7–9 However, blocking J_leak with tetracaine can substantially raise SR Ca²⁺ content,^6,14,15 an effect that would be most consistent with relatively large J_leak. One factor that makes this an especially important issue is that if J_leak limits SR Ca²⁺ load, then it is energetically costly. It has also been suggested that during heart failure an increase in diastolic SR Ca²⁺ leak might be responsible (in part) for reducing SR Ca²⁺ content, thereby playing an important direct role in depressed cardiac contractility in heart failure.¹⁶

In the present study, we use a novel quantitative approach to directly address the roles of J_pumpF, J_pumpR, and J_leak in determining SR Ca²⁺ content in intact resting rabbit ventricular myocytes. Moreover, we examine the relative roles of J_pumpR and J_leak as a function of SR Ca²⁺ load itself. On block of J_leak by tetracaine, we measure both the fall in [Ca²⁺]_i and the complementary rise in [Ca²⁺]_SRT under conditions where transsarcolemmal Ca²⁺ fluxes are prevented. We find that J_leak depends very steeply on [Ca²⁺]_SR, and this may explain some of the discrepancies in this field.

	Materials and Methods

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All chemicals were from Sigma Chemical Co, except as indicated. 0 Na⁺, 0 Ca²⁺ normal tyrode (NT) had Li⁺ substituted for Na⁺ and 10 mmol/L EGTA with no added Ca²⁺. A more detailed description of the solutions and protocols can be found in the online data supplement (see http://www.circresaha.org).

Experimental Protocol
Experiments were conducted in accordance with the Guide for the Use of Experimental Animals at Loyola University Medical Center and conformed to the Guide for the Care and Use of Laboratory Animals published by the NIH (publication No. 85-23, revised 1985). New Zealand White rabbit (Myrtle’s Rabbitry, Inc, Thompson Station, Tenn) ventricular myocytes were isolated as described previously.¹⁷ All experiments were performed at room temperature using the general protocol shown in Figure 2. Resting [Ca²⁺]_i and [Ca²⁺]_SRT were measured using fluo-3 fluorescence in isolated myocytes in the presence and absence of J_leak. Cells were stimulated at least 20 times at 0.5 Hz or the indicated frequency in 2 mmol/L Ca²⁺ normal Tyrode to bring the cellular Ca²⁺ content to steady state. After the last pulse, the superfusate was rapidly switched to 0 Na⁺, 0 Ca²⁺ NT ±1 mmol/L tetracaine. Na⁺-Ca²⁺ exchange, the primary Ca²⁺ influx and efflux mechanism at rest, was therefore blocked so that little or no Ca²⁺ entered or left the resting cell. In the control condition, [Ca²⁺]_i was monitored while 0 Na⁺, 0 Ca²⁺ solution without tetracaine was perfused for a minimum of 30 seconds, then 10 mmol/L caffeine was added to cause SR Ca²⁺ release. The difference between the basal and peak total cytosolic [Ca²⁺] ([Ca²⁺]_T) in the presence of caffeine is therefore [Ca²⁺]_SRT.

View larger version (36K): [in this window] [in a new window]   Figure 2. Protocol used to measure Jleak. The SR is loaded by pacing at 0.5 Hz, then solution is rapidly switched to 0 Na+, 0 Ca2+ NT. After at least 30 seconds, [Ca2+]SRT is measured with 10 mmol/L caffeine. Inclusion of 1 mmol/L tetracaine in the 0 Na+, 0 Ca2+ NT causes steady-state [Ca2+]i to drop and [Ca2+]SRT to rise (ie, a shift of Ca2+ from cytosol to SR). Peaks of caffeine transients were the same indicating that total cellular [Ca2+] was the same in both cases.

In the test condition, 0 Na⁺, 0 Ca²⁺ NT was perfused with tetracaine added. Under this condition, J_leak is blocked and the measured changes in the steady-state condition were used to determine the magnitude of the three relevant SR fluxes: J_pumpF, J_pumpR, and J_leak.

Calculation of Cellular SR Fluxes
SR Ca²⁺ fluxes were calculated as described in the online methods. Briefly, at steady state, the leak rate is equal to the net influx through the SR Ca²⁺ pump:

We calculated or inferred relevant SR Ca²⁺ pump parameters when the leak was 0 in the presence of tetracaine. At this point, J_pumpF=J_pumpR (ie, J_pump=0). We then applied these parameters to the situation where leak was unblocked to calculate the net influx through the SR Ca²⁺ pump and therefore the net efflux through passive SR Ca²⁺ leak, which balances it.

	Results

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J_leak Determination Under Steady-State Conditions
Isolated rabbit myocytes were stimulated to steady state at 0.5 Hz, and [Ca²⁺]_i was monitored in 0 Na⁺, 0 Ca²⁺ NT with and without tetracaine for at least 30 seconds, followed by caffeine application to measure [Ca²⁺]_SRT. When we performed this protocol, resting [Ca²⁺]_i declined to 75% of control when tetracaine was added (126±6 versus 94±6 nmol/L, P<0.05; Figures 2 and 3A). Therefore, [Ca²⁺]_T declined. The peak of the subsequent caffeine transient, however, remained the same, indicating that the sum of the [Ca²⁺]_T and [Ca²⁺]_SRT was unchanged with and without tetracaine, and that there was no differential transport of Ca²⁺ into the mitochondria or out of the cell (eg, through the sarcolemmal Ca²⁺ pump). These results are in agreement with Bassani and Bers,¹⁸ where no loss of SR [Ca²⁺] is observed after up to 5 minutes in 0 Na⁺, 0 Ca²⁺ solution in rabbit or rat ventricular myocytes. Since the peak [Ca²⁺]_i in caffeine was the same but the baseline [Ca²⁺]_i was decreased, [Ca²⁺]_SRT was increased with tetracaine to 120% of control (93±8 versus 108±6 µmol/L, Figure 3A). This indicates that there is a shift of Ca²⁺ from the cytosol to the SR. The data clearly show that J_leak is large enough to have a measurable effect on the diastolic [Ca²⁺]_i and [Ca²⁺]_SRT in the isolated intact myocyte, confirming results from other groups.^6,14,15

View larger version (40K): [in this window] [in a new window]   Figure 3. A, Tetracaine-dependent decrease in [Ca2+]i and increase in [Ca2+]SRT (P<0.05; t test). B, [Ca2+]SRT vs Jleak relationship (Equations 3 and 4) before and after the shift of Ca2+ from cytosol to SR with 1 mmol/L tetracaine. Each curve represents the theoretical [Ca2+]SRT vs Jleak relationship from the averaged data without tetracaine (upper curve) and with tetracaine. The arrows represent Jleak at those values (ie, 0 µmol/L cytosol per second with tetracaine and 12 µmol/L cytosol per second without tetracaine).

This Ca²⁺ shift from the cytosol to the SR is the natural result of blocking the ryanodine receptor (RyR) (ie, J_leak), which causes the SR Ca²⁺ influx (J_pumpF) to become greater than the efflux (now only J_pumpR). Ca²⁺ is therefore transported from the cytosol to the SR causing [Ca²⁺]_i, and therefore J_pumpF, to decline and at the same time, [Ca²⁺]_SR, and therefore J_pumpR, to rise until the net SR Ca²⁺ flux (J_SR) is once again zero and steady state is achieved. The greater the magnitude of J_leak in the absence of tetracaine, the greater the shift of Ca²⁺ with tetracaine and the greater the differences in both [Ca²⁺]_i and [Ca²⁺]_SRT.

We have previously shown⁸ that resting SR Ca²⁺ fluxes can be described in the following manner:

where B_max-SR and K_d-SR are the usual Michaelis parameters for intra-SR Ca²⁺ binding, K_mf and K_mr are the K_m values for forward and reverse unidirectional fluxes through the pump, respectively, k_leak is the rate constant for J_leak, V_max and H are the maximum Ca²⁺-pump influx (and backflux) rate and the Hill coefficient, respectively. Values for the relevant parameters are in a table in the online data supplement.

The curves in Figure 3B show the dependence of [Ca²⁺]_SRT on J_leak (using Equations 3 and 4) at the two mean steady-state [Ca²⁺]_i values measured with and without tetracaine. The lower curve ([Ca²⁺]_i=95 nmol/L) is with tetracaine where J_leak=0. Therefore the numerator in Equation 3 must be zero since J_SR=0 at steady state. In the following case:

at this, the thermodynamically limiting [Ca²⁺] gradient.¹⁰

Thus, when J_leak=0 at steady state, [Ca²⁺]_SRT depends directly on [Ca²⁺]_i in a highly predictable manner (Equations 4 and 5) where J_pumpF must equal J_pumpR. As J_leak increases in the absence of tetracaine, the [Ca²⁺]_SRT declines and [Ca²⁺]_i rises. The upper curve is for [Ca²⁺]_i=126 nmol/L and the measured [Ca²⁺]_SRT is 86% of the value at J_leak=0. From this, we infer that J_leak=12 µmol/L per second in the absence of tetracaine.

Using this analysis, we determine J_leak to be 12 µmol/L per second or 30% of the total diastolic efflux from the SR. The remaining 70% is accounted for by J_pumpR.

J_leak as a Function of [Ca²⁺]_SRT
The data above account for leak under only one loading condition (pacing at 0.5 Hz). However, Ca²⁺ spark frequency increases with SR Ca²⁺ load and since the gain of ECC and fractional release both increase as steep, nonlinear functions of [Ca²⁺]SRT, it might be expected that diastolic release (ie, J_leak) will vary substantially with total SR [Ca²⁺]. We therefore tested the hypothesis that there is a higher relative J_leak at higher [Ca²⁺]_SRT.

The same general protocol described above was used (Figure 2). [Ca²⁺]_SRT was altered by varying the protocol before the switch to 0 Na⁺, 0 Ca²⁺ solution ±1 mmol/L tetracaine. The SR was emptied of Ca²⁺ in all cases by addition of caffeine in 0 Ca²⁺ NT (allowing Na⁺-Ca²⁺ exchange to extrude the SR Ca²⁺). When the SR was allowed to load passively in NT for 30 seconds to 5 minutes, [Ca²⁺]_SRT (without tetracaine) stayed relatively low (Figure 4). Stimulation once during reloading gave a slightly higher [Ca²⁺]_SRT and pacing at increasing frequencies gave progressively higher loads above this point.

View larger version (44K): [in this window] [in a new window]   Figure 4. [Ca2+]SRT without tetracaine at each loading protocol. The lowest SR Ca2+ load was achieved in myocytes allowed to load at rest. The next was achieved by a single contraction. Higher loads were achieved at higher pacing frequencies.

There is a distinct advantage to using this protocol to determine the load dependence of J_leak. Obviously, we expect J_leak to rise at least to some extent as [Ca²⁺]_SRT goes up. This is simply because the amount of Ca²⁺ available for leak is increased (ie, the SR Ca²⁺ gradient, and therefore the driving force, is increased). However, the leak may also increase because [Ca²⁺]_SR (and therefore [Ca²⁺]_SRT) affects the leak process itself. Increased [Ca²⁺]_SR may increase the P_o of the RyR as shown in bilayers.^19,20 The key is that any increase in J_leak strictly due to an increase in the SR Ca²⁺ gradient will be linearly related to [Ca²⁺]_SR. However, this relationship may be highly nonlinear if there is an effect on RyR gating. This technique, therefore, discriminates between J_leak changes due to an increase in SR Ca²⁺ gradient and those due to altered leak gating (ie, an effect on the leak rate constant, k_leak, Equation 3).

We measured J_leak as in Figure 2 at different SR Ca²⁺ loads. As [Ca²⁺]_SRT (without tetracaine) rose, the application of tetracaine caused larger decreases in resting [Ca²⁺]_i and, at the same time, larger increases in [Ca²⁺]_SRT (Figure 5). Therefore, more Ca²⁺ shifted from the cytosol to the SR when tetracaine was included in the protocol (Figure 5). Since this shift rises with J_leak, the data show that J_leak does, indeed, rise with [Ca²⁺]_SRT, even without further analysis. When the measurements are analyzed quantitatively (Figure 6), it can be seen that J_leak rises in a highly nonlinear manner with a particularly steep increase at higher loads (still within the physiological range, Figure 6A). J_leak, therefore, is between 4 and 15 µmol/L cytosol per second, depending on the [Ca²⁺]_SRT (and [Ca²⁺]_SR), within physiological limits. Figure 6B represents the leak as a fraction of the total SR Ca²⁺ efflux (ie, in a manner that is independent of the V_max, which we chose for the SR Ca²⁺ pump). Note that even when [Ca²⁺]_SRT is high, J_leak is still only approaching the level of J_pumpR.

View larger version (21K): [in this window] [in a new window]   Figure 5. Changes in [Ca2+]SRT and [Ca2+]i with tetracaine addition. When tetracaine is included in the protocol, [Ca2+]i decreases and [Ca2+]SRT increases. The magnitude of these changes increases as [Ca2+]SRT (without tetracaine) increases. This suggests that Jleak increases as [Ca2+]SRT without tetracaine increases. Lines through data are modified exponentials.

View larger version (25K): [in this window] [in a new window]   Figure 6. Jleak increases as [Ca2+]SRT and free SR [Ca2+] ([Ca2+]SR) increase. Lines through the data are modified exponentials. A, Jleak is calculated from data in Figure 5 as described in the online data supplement. B, Jleak is expressed as a fraction of total SR Ca2+ efflux (JpumpR+Jleak). This makes the determination less dependent on our value for Vmax. C, The leak rate constant, kleak, varies with both [Ca2+]SRT and [Ca2+]SR indicating that the leak rate constant depends on [Ca2+]SR (ie, that [Ca2+]SR has an effect on the release process itself).

The highly nonlinear nature of the relationship between J_leak and [Ca²⁺]_SR (Figures 6A and 6B) indicates that J_leak is increasing not just because the SR free [Ca²⁺] gradient increases but also because the P_o of the RyR increases as well. This is further demonstrated by the observed rise in k_leak as a function of [Ca²⁺]_SRT and [Ca²⁺]_SR (Figure 6C), as this parameter is entirely gradient independent and would otherwise be constant (Equation 3).

	Discussion

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Experimental Technique
We have described a new quantitative technique to assess J_leak in intact isolated myocytes. Even without the detailed analysis, the technique has the advantage that all of the results are paired and J_leak is directly related to the amount of Ca²⁺ shift from cytosol to SR.

We also determined that the sarcolemmal Ca²⁺ pump and mitochondrial Ca²⁺ uptake do not remove significant amounts of Ca²⁺ from the cytosol during the holding period in 0 Na⁺, 0 Ca²⁺ NT (where Na⁺-Ca²⁺ exchange is inhibited, as previously shown¹⁸). This interpretation is explained by 2 factors: (1) These mechanisms are known to be extremely slow relative to Na⁺-Ca²⁺ exchange, the major efflux pathway,² and (2) The peaks of the caffeine transients with and without tetracaine are the same.

This last point deserves further explanation. The difference in [Ca²⁺]_SRT with and without tetracaine results from a decrease in baseline [Ca²⁺]_i, not a difference in peak [Ca²⁺]_i with caffeine. The caffeine peaks are the same because, regardless of whether tetracaine is present in the 0 Na⁺, 0 Ca²⁺ NT solution, the total cellular Ca²⁺ has not changed during the holding period. Any net transport into a compartment other than the cytosol or the SR (eg, extracellular) should change the caffeine transient peak. This was not observed.

Magnitude of the SR Ca²⁺ Leak
The measured J_leak is RyR-dependent. The only source of diastolic Ca²⁺ flux from the SR (other than backflux) that has been directly observed in cardiac myocytes is from the RyR, typically in the form of Ca²⁺ sparks. These elementary units of SR Ca²⁺ release are observed by confocal microscopy using a Ca²⁺-sensitive fluorescent dye. It is possible that flux through the RyR at rest can explain nearly all of the J_leak. A resting J_leak of 10 µmol/L cytosol per second requires about 50 Ca²⁺ sparks per second in the cell (or 2 sparks/pL per second). This is a typical resting Ca²⁺ spark frequency in ventricular myocytes²¹ and depending on the spark current (3 to 20 pA^22,23) could explain the entire resting J_leak of Ca²⁺ from the SR. In addition, direct measurement of SR Ca²⁺ leak rate in permeabilized myocytes in the presence of ruthenium red (to block RyR) and thapsigargin (to block the SR Ca²⁺ pump) was insignificant compared with other fluxes.²⁴ This indicates that any non-RyR–mediated leak is insignificant. Also consistent with this hypothesis, [Ca²⁺]_SR within SR vesicles approaches the thermodynamic limit of the SR Ca²⁺ pump when RyRs are blocked by ruthenium red.¹⁰

The Controversy
Determining J_leak and its SR Ca²⁺ load dependence in intact cardiac myocytes was a major goal of this study. Results from previous attempts to measure J_leak directly in isolated myocytes are mixed. In one example, Bassani and Bers⁷ determined J_leak by measuring the rate of SR Ca²⁺ loss with the SR Ca²⁺ pump completely blocked. [Ca²⁺]_SRT decreased with a of 385 seconds, giving an initial J_leak of 0.3 µmol/L cytosol per second at [Ca²⁺]_SRT 100 µmol/L cytosol (in both rat and rabbit). This is a very low value. Because J_pumpF 25 µmol/L cytosol per second at resting [Ca²⁺]_i must equal J_leak+J_pumpR at steady state, this implies that J_pumpR is nearly equal J_pumpF at rest.

Such a low J_leak would hardly affect diastolic [Ca²⁺]_SRT. Ginsburg et al⁹ tested this hypothesis by measuring maximal steady-state [Ca²⁺]_SRT in rabbit cardiac myocytes under voltage clamp with SR pump stimulation or inhibition. If [Ca²⁺]_SRT is limited by J_leak, it should be sensitive to these maneuvers. Slowing the pump with thapsigargin or accelerating it with isoproterenol resulted in nearly the same maximum [Ca²⁺]_SRT as control, implying that the pump operates close to its thermodynamic limit against a small leak.

However, not all results agree with this interpretation. For instance, two groups^6,14,15 found that tetracaine block of J_leak in rat ventricular myocytes nearly doubled [Ca²⁺]_SRT, an unexpected effect if the leak were very small. These data conflict with the conclusions above⁹ and imply that altering J_leak in cardiac ventricular cells may affect ECC through alteration of [Ca²⁺]_SRT.

We used a variation on the tetracaine technique above with Na⁺-Ca²⁺ exchange blocked (Figure 2). Thus, the cell effectively becomes a closed system preventing significant sarcolemmal Ca²⁺ flux. This has at least 2 effects: (1) it makes more quantitative measurements possible (a critical improvement) and (2) it prevents SR Ca²⁺ loading or loss that can occur through Na⁺-Ca²⁺ exchange. The data clearly show that J_leak has measurable effects on [Ca²⁺]_SRT within ventricular myocytes. The magnitude of J_leak after pacing at 0.5 Hz was 12 µmol/L per second or 30% of the total efflux rate. The rest is J_pumpR, emphasizing the significance of this flux as well.

Load Dependence of the Leak: The Resolution?
The [Ca²⁺]_SRT dependence of J_leak also complicates accurate leak measurement. This was emphasized by recent work using permeabilized myocytes where resting [Ca²⁺]_i was clamped.^13,25 Ca²⁺ spark frequency increased as [Ca²⁺]_SRT (and [Ca²⁺]_SR) rose at constant [Ca²⁺]_i (making it clear that [Ca²⁺]_SR alters diastolic RyR gating in situ). Quantitatively, this measurement is complicated by uncertainty about the flux though a single spark (3 to 20 pA)^22,23 and because Ca²⁺ spark frequency is often much higher in permeabilized myocytes.²⁵

In this study, we measured the [Ca²⁺]_SRT dependence of J_leak in intact, unpermeabilized myocytes, thus avoiding the dialysis of potentially important proteins and other substances from the cytosol. We hypothesized that the leak would rise in a nonlinear fashion with increasing steepness at higher [Ca²⁺]_SR. The basis for this hypothesis is related to our ECC study³ of the relationship between fractional release versus [Ca²⁺]_SRT and [Ca²⁺]_SR. We found that both ECC and fractional SR Ca²⁺ release increased sharply as a function of either [Ca²⁺]_SRT or [Ca²⁺]_SR especially at high [Ca²⁺]_SRT. Because the increased fractional release is most likely because of enhanced [Ca²⁺]_i sensitivity of the RyR at high [Ca²⁺]_SR,^19,20 we expected a similar relationship between SR [Ca²⁺] and resting J_leak.

Our measurement of J_leak as a function of [Ca²⁺]_SRT was consistent with the ECC data. J_leak rose in a highly nonlinear manner with a particularly steep increase at higher loads (Figure 6). The magnitude was between 4 and 15 µmol/L cytosol per second, depending on [Ca²⁺]_SRT, within the physiological range.

This relationship presents a possible resolution to the apparent dichotomous results of J_leak large enough to limit [Ca²⁺]_SRT^6,13 and those of a low leak, which has little effect on [Ca²⁺]_SRT.^7,9 High J_leak may occur at higher [Ca²⁺]_SRT and the [Ca²⁺]_SRT will be more sensitive to J_leak at these loads. Note that higher [Ca²⁺]_SRT does not mean overloaded (eg, causing Ca²⁺ waves or spontaneous contractions), and the leak may have a significant effect on normal diastolic [Ca²⁺]_SRT and be manifest as increased spark frequency.

Physiological and Pathophysiological Significance
The mere fact that SR Ca²⁺ load increases when J_leak is blocked provides valuable information about how diastolic fluxes control [Ca²⁺]_SRT and therefore ECC. If the SR Ca²⁺ load is determined to a significant extent by J_leak and, at the same time, J_leak is determined by [Ca²⁺]_SRT, then [Ca²⁺]_SRT is, in effect, inherently self-limiting. Figure 7A demonstrates this by showing the relationship between [Ca²⁺]_SR and [Ca²⁺]_i±J_leak. At higher [Ca²⁺]_i, the higher J_leak prevents the large increases in [Ca²⁺]_SR, which can be seen with J_leak blocked. Figure 7B shows how both J_leak and J_pumpR depend on [Ca²⁺]_SR in Figure 7A. Consider the relationship as a simple cellular feedback loop. As [Ca²⁺]_SRT increases, J_leak increases. As J_leak increases, [Ca²⁺]_SRT decreases. What this means functionally is that one may approach the thermodynamic [Ca²⁺]_SR/[Ca²⁺]_i limit when [Ca²⁺]_SRT is low to moderate. However, at high [Ca²⁺]_SRT, there is an inherent maximal [Ca²⁺]_SRT below the thermodynamic limit (note apparent maximal [Ca²⁺]_SRT in Figures 5 and 6). Anything that shifts this relationship could change the SR Ca²⁺ load for a given diastolic [Ca²⁺]_i.

View larger version (18K): [in this window] [in a new window]   Figure 7. A, [Ca2+]SR in relation to [Ca2+]i both with and without tetracaine. [Ca2+]SR with tetracaine varies linearly with [Ca2+]i (slope=7000, SR thermodynamic limit). [Ca2+]SR without tetracaine is close to this line at low [Ca2+]i but falls away from this thermodynamic limit at higher values as Jleak increases (ie, as kleak increases with [Ca2+]SR and [Ca2+]i). B, JpumpR (backflux) and Jleak (leak) depend on [Ca2+]SR. Curves are based on Equation 3 and kleak as in Figure 6C.

Marx et al¹⁶ suggested that a high persistent diastolic SR Ca²⁺ leak may exist in heart failure myocytes. A high J_leak combined with increased Na⁺-Ca²⁺ exchange and reduced J_pump²⁶ could limit [Ca²⁺]_SRT. The greater ATP consumption resulting from a high rate of pump-leak futile cycling could further exacerbate this effect. Reducing [Ca²⁺]_SRT could have severe effects on ECC and contraction in the failing heart.^3,4 Our data therefore suggest that if J_leak is increased in heart failure, it may reduce [Ca²⁺]_SRT and SR Ca²⁺ release during systole.^3,4

Because J_pumpR also makes up >50% of the total SR Ca²⁺ efflux (Figure 6), it is important to emphasize that J_pumpR might also change under different conditions. In wild-type versus phospholamban (PLB) knockout mice, we found that PLB, in addition to decreasing the rate of SR Ca²⁺ uptake (ie, J_pumpF), also increased J_pumpR. This resulted in a decreased steady-state [Ca²⁺]_SRT and [Ca²⁺]_SR.²⁴ Therefore, PLB phosphorylation may cause an increase in the efficiency of the SR Ca²⁺ pump and thus an increase in the thermodynamic limit of the pump, causing an increase in [Ca²⁺]_SRT.

In summary, our studies show that the two major SR diastolic effluxes, J_pumpR and J_leak, both play a role in determining [Ca²⁺]_SRT (and [Ca²⁺]_SR). Both J_pumpR and/or J_leak are potentially involved in a variety of cardiac disturbances including heart failure.^16,24,27 Moreover, decreasing either or both of these fluxes may enhance ECC,^24,27,28 probably by changing [Ca²⁺]_SR, which has a powerful effect on ECC.^3,4 The proteins that mediate these fluxes may be good potential targets for pharmacological manipulation of the inotropic state, possibly for treatment of cardiac diseases.

	Acknowledgments

 
This study was supported by a Scientist Development Grant, the American Heart Association, and NIH grant HL64098.

Received May 23, 2002; revision received August 30, 2002; accepted August 30, 2002.

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