Quantitative Assessment of the SR Ca2+ Leak-Load Relationship
Increased diastolic SR Ca2+ leak (Jleak) could depress contractility in heart failure, but there are conflicting reports regarding the Jleak 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 Jleak 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. Jleak also increased nonlinearly with [Ca2+]SRT with a steeper increase at higher load. We conclude that Jleak 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).
- Ca2+ cycling
- membrane transport
- sarcoplasmic reticulum
- ryanodine receptors
- excitation-contraction coupling
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 (JpumpF) 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 (Jleak) and SR Ca2+ pump backflux (JpumpR), 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 JpumpF 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 Jleak and Ca2+ pump–mediated JpumpR. Two schools of thought describe diastolic SR Ca2+ flux as either (1) a strict forward pump versus leak balance (JpumpF=Jleak without appreciable JpumpR, Figure 1B)1,2,6⇓⇓ or (2) approaching a thermodynamic equilibrium where JpumpF=JpumpR (with little Jleak, 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 Jleak to rise until it equals JpumpF (which falls as [Ca2+]i declines but is still 10% to 20% of Vmax at diastolic [Ca2+]i). In this case, JpumpR 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 Jleak and a large ATP consumption to simply retain constant diastolic SR Ca2+.
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 JpumpF is balanced by JpumpR, which rises with [Ca2+]SR during [Ca2+]i decline (with little Jleak required).8–12⇓⇓⇓⇓ Note that JpumpR cannot exceed JpumpF in an intact cell, but as it approaches JpumpF, it could create a steady-state balance where there is almost no net pump flux (Jpump), just enough to be counterbalanced by a very small Jleak. This would require very little ATP consumption to retain diastolic [Ca2+]SRT but would make [Ca2+]SRT quite sensitive to energetic state (eg, ΔGATP).
The actual value of Jleak in intact ventricular myocytes is controversial and estimates have ranged from 0.3 μmol/L cytosol per second7,9⇓ to 5 to 30 μmol/L cytosol per second.13 Models that do not consider JpumpR require Jleak ≈20 μmol/L cytosol per second to compensate for diastolic JpumpF.1,2⇓ Some of our data have more strongly favored values at the lower end of this Jleak range.7–9⇓⇓ However, blocking Jleak with tetracaine can substantially raise SR Ca2+ content,6,14,15⇓⇓ an effect that would be most consistent with relatively large Jleak. One factor that makes this an especially important issue is that if Jleak 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 JpumpF, JpumpR, and Jleak in determining SR Ca2+ content in intact resting rabbit ventricular myocytes. Moreover, we examine the relative roles of JpumpR and Jleak as a function of SR Ca2+ load itself. On block of Jleak 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 Jleak depends very steeply on [Ca2+]SR, and this may explain some of the discrepancies in this field.
Materials and Methods
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).
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 Jleak. 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.
In the test condition, 0 Na+, 0 Ca2+ NT was perfused with tetracaine added. Under this condition, Jleak is blocked and the measured changes in the steady-state condition were used to determine the magnitude of the three relevant SR fluxes: JpumpF, JpumpR, and Jleak.
Calculation of Cellular SR Fluxes
We calculated or inferred relevant SR Ca2+ pump parameters when the leak was 0 in the presence of tetracaine. At this point, JpumpF=JpumpR (ie, Jpump=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.
Jleak 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 3⇓A). 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 Jleak 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⇓⇓
This Ca2+ shift from the cytosol to the SR is the natural result of blocking the ryanodine receptor (RyR) (ie, Jleak), which causes the SR Ca2+ influx (JpumpF) to become greater than the efflux (now only JpumpR). Ca2+ is therefore transported from the cytosol to the SR causing [Ca2+]i, and therefore JpumpF, to decline and at the same time, [Ca2+]SR, and therefore JpumpR, to rise until the net SR Ca2+ flux (JSR) is once again zero and steady state is achieved. The greater the magnitude of Jleak 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 Bmax-SR and Kd-SR are the usual Michaelis parameters for intra-SR Ca2+ binding, Kmf and Kmr are the Km values for forward and reverse unidirectional fluxes through the pump, respectively, kleak is the rate constant for Jleak, Vmax 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 Jleak (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 Jleak=0. Therefore the numerator in Equation 3 must be zero since JSR=0 at steady state. In the following case:
at this, the thermodynamically limiting [Ca2+] gradient.10
Thus, when Jleak=0 at steady state, [Ca2+]SRT depends directly on [Ca2+]i in a highly predictable manner (Equations 4 and 5) where JpumpF must equal JpumpR. As Jleak 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 Jleak=0. From this, we infer that Jleak=12 μmol/L per second in the absence of tetracaine.
Using this analysis, we determine Jleak to be 12 μmol/L per second or 30% of the total diastolic efflux from the SR. The remaining 70% is accounted for by JpumpR.
Jleak 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, Jleak) will vary substantially with total SR [Ca2+]. We therefore tested the hypothesis that there is a higher relative Jleak 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.
There is a distinct advantage to using this protocol to determine the load dependence of Jleak. Obviously, we expect Jleak 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 Po of the RyR as shown in bilayers.19,20⇓ The key is that any increase in Jleak 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 Jleak changes due to an increase in SR Ca2+ gradient and those due to altered leak gating (ie, an effect on the leak rate constant, kleak, Equation 3).
We measured Jleak 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 Jleak, the data show that Jleak does, indeed, rise with [Ca2+]SRT, even without further analysis. When the measurements are analyzed quantitatively (Figure 6), it can be seen that Jleak rises in a highly nonlinear manner with a particularly steep increase at higher loads (still within the physiological range, Figure 6A). Jleak, 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 Vmax, which we chose for the SR Ca2+ pump). Note that even when [Ca2+]SRT is high, Jleak is still only approaching the level of JpumpR.
The highly nonlinear nature of the relationship between Jleak and [Ca2+]SR (Figures 6A and 6B) indicates that Jleak is increasing not just because the SR free [Ca2+] gradient increases but also because the Po of the RyR increases as well. This is further demonstrated by the observed rise in kleak 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).
We have described a new quantitative technique to assess Jleak in intact isolated myocytes. Even without the detailed analysis, the technique has the advantage that all of the results are paired and Jleak 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 Jleak 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 Jleak. A resting Jleak 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 pA22,23⇓) could explain the entire resting Jleak 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
Determining Jleak and its SR Ca2+ load dependence in intact cardiac myocytes was a major goal of this study. Results from previous attempts to measure Jleak directly in isolated myocytes are mixed. In one example, Bassani and Bers7 determined Jleak by measuring the rate of SR Ca2+ loss with the SR Ca2+ pump completely blocked. [Ca2+]SRT decreased with a τ of 385 seconds, giving an initial Jleak 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 JpumpF ≈25 μmol/L cytosol per second at resting [Ca2+]i must equal Jleak+JpumpR at steady state, this implies that JpumpR is nearly equal JpumpF at rest.
Such a low Jleak 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 Jleak, 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 groups6,14,15⇓⇓ found that tetracaine block of Jleak 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 Jleak 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 Jleak has measurable effects on [Ca2+]SRT within ventricular myocytes. The magnitude of Jleak after pacing at 0.5 Hz was 12 μmol/L per second or ≈30% of the total efflux rate. The rest is JpumpR, emphasizing the significance of this flux as well.
Load Dependence of the Leak: The Resolution?
The [Ca2+]SRT dependence of Jleak 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 Jleak 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 Jleak.
Our measurement of Jleak as a function of [Ca2+]SRT was consistent with the ECC data. Jleak 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 Jleak large enough to limit [Ca2+]SRT6,13⇓ and those of a low leak, which has little effect on [Ca2+]SRT.7,9⇓ High Jleak may occur at higher [Ca2+]SRT and the [Ca2+]SRT will be more sensitive to Jleak 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 Jleak 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 Jleak and, at the same time, Jleak 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±Jleak. At higher [Ca2+]i, the higher Jleak prevents the large increases in [Ca2+]SR, which can be seen with Jleak blocked. Figure 7B shows how both Jleak and JpumpR depend on [Ca2+]SR in Figure 7A. Consider the relationship as a simple cellular feedback loop. As [Ca2+]SRT increases, Jleak increases. As Jleak 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.
Marx et al16 suggested that a high persistent diastolic SR Ca2+ leak may exist in heart failure myocytes. A high Jleak combined with increased Na+-Ca2+ exchange and reduced Jpump26 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 Jleak is increased in heart failure, it may reduce [Ca2+]SRT and SR Ca2+ release during systole.3,4⇓
Because JpumpR also makes up >50% of the total SR Ca2+ efflux (Figure 6), it is important to emphasize that JpumpR 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, JpumpF), also increased JpumpR. 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, JpumpR and Jleak, both play a role in determining [Ca2+]SRT (and [Ca2+]SR). Both JpumpR and/or Jleak 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.
This study was supported by a Scientist Development Grant, the American Heart Association, and NIH grant HL64098.
Original received May 23, 2002; revision received August 30, 2002; accepted August 30, 2002.
- ↵Bers DM. Excitation-Contraction Coupling and Cardiac Contractile Force. 2nd ed. Dordrecht, the Netherlands: Kluwer Academic Publishers; 2001.
- ↵Ginsburg KS, Weber CR, Bers DM. Control of maximum sarcoplasmic reticulum Ca load in intact ferret ventricular myocytes: effects of thapsigargin and isoproterenol. J Gen Physiol. 1998; 111: 491–504.
- ↵Takenaka H, Adler PN, Katz AM. Calcium fluxes across the membrane of sarcoplasmic reticulum vesicles. J Biol Chem. 1982; 257: 12649–12656.
- ↵Weber CR, Ginsburg KS, Philipson KD, Shannon TR, Bers DM. Allosteric regulation of Na/Ca exchange current by cytosolic Ca in intact cardiac myocytes. J Gen Physiol. 2001; 117: 119–131.
- ↵Cheng H, Lederer WJ, Cannell MB. Calcium sparks: elementary events underlying excitation-contraction coupling in heart muscle. Science. 1993; 262: 740–744.
- ↵Shannon TR, Chu G, Kranias EG, Bers DM. Phospholamban decreases the energetic efficiency of the sarcoplasmic reticulum Ca pump. J Biol Chem. 2001; 276: 7195–7201.
- ↵Li Y, Kranias EK, Mignery GA, Bers DM. Protein kinase A phosphorylation of the ryanodine receptor does not affect calcium sparks in mouse ventricular myocytes. Circ Res. 2002; 90: 309–316.
- ↵Pogwizd SM, Qi M, Yuan W, Samarel AM, Bers DM. Upregulation of Na+/Ca2+ exchanger expression and function in an arrhythmogenic rabbit model of heart failure. Circ Res. 1999; 85: 1009–1019.
- ↵Prestle J, Janssen PM, Janssen AP, Zeitz O, Lehnart SE, Bruce L, Smith GL, Hasenfuss G. Overexpression of FK506-binding protein FKBP12.6 in cardiomyocytes reduces ryanodine receptor mediated Ca2+ leak from the sarcoplasmic reticulum and increases contractility. Circ Res. 2001; 88: 188–194.