Regulation of the Ca²⁺ Gradient Across the Sarcoplasmic Reticulum in Perfused Rabbit Heart

A ¹⁹F Nuclear Magnetic Resonance Study

From the Department of Pathology (W.C., C.S.), Duke University Medical Center, Durham, NC, and LSB (R.L.) and LMC (E.M.) National Institute of Environmental Health Sciences, Research Triangle Park, NC.

Correspondence to Dr Charles Steenbergen, Department of Pathology, Box 3712, Duke University Medical Center, Durham, NC 27710.

	Abstract

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Abstract—Myocardial contractility depends on Ca²⁺ release from and uptake into the sarcoplasmic reticulum (SR). The Ca²⁺ gradient between the SR matrix and the cytosol (SR Ca²⁺ gradient) is maintained by the SR Ca²⁺-ATPase using the free energy available from hydrolysis of ATP. The activity of the SR Ca²⁺-ATPase is not only dependent on the energy state of the cell but is also kinetically regulated by SR proteins such as phospholamban. To evaluate the importance of thermodynamic and kinetic regulation of the SR Ca²⁺ gradient, we examined the relationship between the energy available from ATP hydrolysis (G_ATP) and the energy required for maintenance of the SR Ca²⁺ gradient (G_Ca²⁺SR) during physiological and pathological manipulations that alter G_ATP and the phosphorylation state of phospholamban. We used our previously developed ¹⁹F nuclear magnetic resonance method to measure the ionized [Ca²⁺] in the SR of Langendorff-perfused rabbit hearts. We found that addition of either pyruvate or isoproterenol resulted in an increase in left ventricular developed pressure and an increase in [Ca²⁺]_SR. Pyruvate increased G_ATP, and the increase in the SR Ca²⁺ gradient was matched to the increase in G_ATP; G_ATP increased from 58.3±0.5 to 60.4±1.0 kJ/mol (P<0.05), and G_Ca²⁺SR increased from 47.1±0.3 to 48.5±0.1 kJ/mol (P<0.05). In contrast, the increase in the SR Ca²⁺ gradient in the presence of isoproterenol occurred despite a decline in G_ATP from 58.3±0.5 to 55.8±0.6 kJ/mol. Thus, the data indicate that the SR Ca²⁺ gradient can be increased by an increase in G_ATP, and that the positive inotropic effect of pyruvate can be explained by improved energy-linked SR Ca²⁺ handling, whereas the results with isoproterenol are consistent with removal of the kinetic limitation of phospholamban on the activity of the sarcoplasmic/endoplasmic reticulum Ca²⁺-ATPase, which allows the SR Ca²⁺ gradient to move closer to its thermodynamic limit. Ischemia decreases G_ATP, and this should also have an effect on SR Ca²⁺ handling. During 30 minutes of ischemia, G_ATP decreased by 12 kJ/mol, but the decrease in G_Ca²⁺SR was 16 kJ/mol, greater than would be predicted by the fall in G_ATP and consistent with increased SR Ca²⁺ release and increased SR Ca²⁺ cycling. Because ischemic preconditioning is reported to decrease SR Ca²⁺ cycling during a subsequent sustained period of ischemia, we examined whether ischemic preconditioning affects the relationship between the fall in G_ATP and the fall in G_Ca²⁺SR during ischemia. We found that preconditioning attenuated the fall in G_Ca²⁺SR during ischemia; the fall in G_Ca²⁺SR was of comparable magnitude to the fall in G_ATP, and this was associated with a significant improvement in functional recovery during reperfusion. The data suggest that there is both thermodynamic regulation of the SR Ca²⁺ gradient by G_ATP and kinetic regulation, which can alter the relationship between G_ATP and G_Ca²⁺SR.

Key Words: sarcoplasmic reticulum • ¹⁹F NMR spectroscopy • Ca²⁺ transport

	Introduction

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The sarcoplasmic reticulum (SR) plays an important role in regulation of mammalian cardiac muscle contraction.^{1 2 3} The SR Ca²⁺ content available for release is an important determinant of contractile state. Because the uptake of two Ca²⁺ ions into the SR consumes one ATP, it is not surprising that contractility is linked to the energetic status of the cell. It is generally agreed that the Ca²⁺ gradient between the SR matrix and the cytosol is maintained by the SR Ca²⁺-ATPase, which operates at 75% to 85% of the theoretical limit, on the basis of the free energy available from ATP hydrolysis (G_ATP).^{4 5} However, the Ca²⁺ gradient across the SR is likely to be regulated kinetically as well as thermodynamically.⁶ Recent data from phospholamban (PLB) knockout mice are consistent with kinetic regulation of [Ca²⁺]_SR, because PLB null mice have twice the total SR Ca²⁺ content as wild-type mice, with no apparent increase in G_ATP.⁷ In addition, recent studies have suggested that increased SR Ca²⁺ cycling during ischemia or metabolic inhibition may contribute to myocyte injury.^{8 9 10} Despite the importance of SR Ca²⁺ transport, the regulation of the Ca²⁺ gradient across the SR membrane has not been examined in the intact beating heart, because of the lack of suitable methods for measuring the free Ca²⁺ concentration in the SR ([Ca²⁺]_SR).

We recently described a new method for measuring [Ca²⁺]_SR in perfused rabbit heart loaded with the acetoxymethyl ester of 1,2-bis(2-amino-5,6-difluorophenoxy)ethane-N,N,N',N'-tetraacetic acid (TF-BAPTA). TF-BAPTA has a high K_d for Ca²⁺ (65 µmol/L) and combines both a large shift sensitivity and fast-intermediate exchange kinetics at typical magnetic field strengths.^{11 12} Such an indicator offers the potential for simultaneous determinations of Ca²⁺ concentrations in different cellular compartments. Furthermore, because of the high K_d value, TF-BAPTA is able to measure the high free [Ca²⁺] that is present in the SR. We reported previously that in the isolated beating rabbit heart, the time-averaged [Ca²⁺]_SR is 1 mmol/L, a value in good agreement with estimates obtained using calsequestrin binding constants.^{6 13 14}

The goal of the present study is to investigate the regulation of the SR Ca²⁺ gradient, specifically the relationship between the energy state of the cell and the SR Ca²⁺ gradient under a variety of physiological and pathological conditions. The energy state of the heart can be modulated by addition of pyruvate to the glucose-containing perfusate.^{15 16} Pyruvate increases G_ATP, and this would be expected to increase the thermodynamic driving force for the SR Ca²⁺ pump, to accentuate the SR Ca²⁺ gradient, and to increase the availability of Ca²⁺ for release during each cardiac cycle. We investigated whether the positive inotropic effect observed with isoproterenol is accompanied by an increase in SR [Ca²⁺] and whether this altered the relationship between the thermodynamic driving force for the SR Ca²⁺-ATPase and the SR Ca²⁺ gradient. Furthermore, recent studies have suggested that ischemia may promote SR Ca²⁺ efflux, leading to SR Ca²⁺ cycling, which may contribute to ischemic injury. We therefore also examined the relationship between G_ATP and the SR Ca²⁺ gradient during ischemia, and we examined whether cardioprotective interventions such as ischemic preconditioning would improve SR Ca²⁺ handling during ischemia.

	Materials and Methods

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Isolated Rabbit Heart Preparation
Male New Zealand White rabbits (1 to 1.6 kg) were used and received humane care in accordance with the Guide for the Care and Use of Laboratory Animals, published by the US National Institutes of Health (NIH publications No. 8523, revised 1985). Rabbits were anesthetized by intravenous injection of pentobarbitone (100 mg) into a marginal ear vein. The heart was excised rapidly, and the aorta was cannulated. Retrograde perfusion was begun under constant pressure (100 cm H₂O). The nonrecirculating perfusate was a Krebs-Henseleit buffer containing (in mmol/L) NaCl 120, KCl 4.7, MgSO₄ 1.2, KH₂PO₄ 1.2, CaCl₂ 1.25, NaHCO₃ 25, and glucose 11. The buffer was maintained at pH 7.4 by aerating with a mixture of 95% O₂/5% CO₂, at a temperature of 37°C.

Hearts were placed in a 30-mm nuclear magnetic resonance (NMR) tube. After 10 minutes of control perfusion, loading with 1000 mL of 3.5 µmol/L of the acetoxymethyl ester of TF-BAPTA was started. With typical flow rates of 30 to 50 mL/min, loading took about 20 minutes. To monitor contractility, a latex balloon was inserted into the left ventricle. The balloon was inflated to give an end-diastolic pressure of 5 to 10 cm H₂O. As observed in our previous studies,^{4 12} TF-BAPTA loading did not cause a significant reduction in contractility. Global normothermic ischemia was created by cross-clamping the perfusate inflow line.

NMR Measurements
¹⁹F NMR measurements were performed on a Varian 400 wide-bore NMR spectrometer at 376.27 MHz at 37°C. We shimmed on the proton signal from the unbathed heart, and we routinely obtained a nonspinning line width at one-half height of 0.25 ppm. Spectra, which were not gated to the cardiac cycle, were acquired every 5 minutes using 0.26-second intervals between scans with a pulse of 40° (20 µs). The spectral width was ±7060 Hz, and 4K data points were collected. The free-induction decay was multiplied by an exponential function corresponding to a 100-Hz line broadening before Fourier transformation.

³¹P NMR measurements were also performed on a Varian wide-bore NMR spectrometer at 161.9 MHz at 37°C. Spectra were acquired using a 2-second interval between scans with a pulse width of 70°. The spectral width was ±3603 Hz, and 4K data points were collected. The free-induction decay was multiplied by an exponential function corresponding to a 20-Hz line broadening before Fourier transformation. The pH was determined from the chemical shift difference between the intracellular P_i and phosphocreatine (PCr) peaks. The P_i, PCr, and ATP concentrations were determined by spectral integration. The integrated peaks were corrected by multiplication with saturation factors of 1.3, 1.39, and 1.08 for P_i, PCr, and ß-ATP, respectively; these factors were calculated by comparison of the peak integrals obtained with repetition times of 2 and 20 seconds.

Calculation of [Ca²⁺]_SR
The Ca²⁺-insensitive fluorine in the 6-position (6F) of TF-BAPTA is set at 0 ppm. The fluorine in the 5-position (5F) shifts upon Ca²⁺ complexation, and as described previously,^{11 12} the shift difference between the 6F and 5F resonance peaks of TF-BAPTA can be used to calculate ionized [Ca²⁺], by using the following equation¹¹ :

The derivation of the equation has been described previously in detail.¹¹ This equation corrects for changes in pH and Mg²⁺, which can have a measurable effect on the calculated Ca²⁺ when free Ca²⁺ is well below the K_d (such as in the cytosol under basal conditions). However, for Ca²⁺ measurements above the K_d (all measurements of SR Ca²⁺), the corrections due to pH and Mg²⁺ are negligible. Briefly, the K_d is 65 µmol/L, ₀ is the measured shift difference between the 6F and the 5F resonance peak of TF-BAPTA, ₁ (5.13) is the shift difference in the presence of EGTA at pH 12, ₂+₃ (13.41) and ₄ (8.53) are the shifts caused by protonation, ₅ (14.83) is the shift difference with excess Ca²⁺, and ₆+₇ (11.21) and ₈ (7.83) correspond to the shift difference due to binding of single or two Mg²⁺, respectively. Because of intermediate exchange,¹¹ ₅=14.83 (for [Ca²⁺]>7 µmol/L) or ₅=11.44 (for [Ca²⁺]<7 µmol/L). The pH and [Mg²⁺] were measured as 7.2 (see Results) and 1 mmol/L for normoxic perfused hearts,¹⁷ respectively. The pH values during 25 to 30 minutes of ischemia were measured as 5.93 in nonpreconditioned hearts and as 6.28 in preconditioned hearts. [Mg²⁺] during ischemia was measured to be 3 mmol/L in our previous study.¹⁷

Calculation of Free Energy (G) for SR Ca²⁺-ATPase
We calculated the G required for the SR Ca²⁺-ATPase using the following equation⁴ : G_Ca²⁺SR=2RT ln([Ca²⁺]_SR/[Ca²⁺]_c), where R and T are the gas constant and temperature, respectively, and we assume no membrane potential.¹⁸

Calculation of Free Energy for ATP Hydrolysis (G_ATP)
The high-energy phosphates, P_i, and pH_i were monitored by ³¹P NMR. Hearts were snap-frozen, and ATP and total creatine contents were measured enzymatically after perchloric acid extraction to allow for quantitation of the ³¹P NMR spectra.¹⁹

The following equation was used to calculate G_ATP: G_ATP=G₀+RT ln ({[ADP]_fx[P_i]_f}/[ATP]_f), with G₀=-30.5 kJ/mol. By thermodynamic convention, values for G₀ and G_ATP are negative for exergonic reactions, but after calculating G_ATP, we refer to the values in the text as absolute values. The [ATP]_f/[ADP]_f ratio was calculated by assuming that the creatine kinase reaction is at equilibrium: [ATP]/[ADP]={[PCr] K'_ck}/[Cr]. The apparent equilibrium constant (K'_ck) was calculated according to the following equation²⁰ : K'_ck=[H⁺]xK_ck=10^{-0.87 pHi+8.31}.

The PCr and P_i contents were obtained by comparing the NMR peak area to that of the basal ß-ATP peak, after correcting for NMR saturation, and assuming that the PCr and P_i peak are entirely cytosolic. The ß-ATP peak was quantified by comparison to the ATP measured enzymatically in the snap-frozen extracts. Creatine (Cr) content was obtained by subtraction of PCr content (measured by NMR) from total creatine content, which was measured enzymatically. PCr, P_i, and Cr contents were converted to concentrations by assuming that these metabolites were entirely cytosolic and that cytosolic volume equaled 2.3 mL/g dry weight.

Protocols
SR Ca²⁺ Gradient (G_Ca²⁺SR)
To determine the effect of substrates, isoproterenol, and ischemia on SR Ca²⁺, TF-BAPTA–loaded hearts were perfused with control perfusate (containing 11 mmol/L glucose) for a 15-minute stabilization period followed by an additional 15-minute period during which control (pretreatment) spectra were acquired. The perfusate was then modified (1 mmol/L pyruvate [n=6] for 15 minutes, 100 nmol/L isoproterenol [n=5] for 15 minutes), or the hearts were subjected to an ischemia protocol (30 minutes of global ischemia [n=4] or a preconditioning protocol followed by 30 minutes of ischemia [n=5]), and spectra were acquired. The preconditioning protocol consisted of 4 cycles of 5 minutes of ischemia separated by 5 minutes of reperfusion before the final 30 minutes of sustained ischemia.

Energetics (G_ATP)
To examine how substrates, isoproterenol, and ischemia affect G_ATP, hearts were placed in the NMR magnet, and ³¹P NMR spectra were acquired. At the end of the study, hearts were freeze-clamped using tongs precooled with liquid nitrogen for assessment of ATP and total creatine. One group of controls (n=8) was time-matched to pyruvate- (n=4) and isoproterenol- (n=4) treated hearts. Control hearts were frozen at the end of a 30-minute perfusion. Pyruvate hearts were frozen after 15 minutes of control and 15 minutes of pyruvate perfusion, and isoproterenol hearts were frozen after 15 minutes of control and 15 minutes of isoproterenol perfusion. A second group of controls (n=3) was time-matched to nonpreconditioned ischemic (n=4) and preconditioned ischemic (n=5) hearts. Nonpreconditioned ischemic hearts and preconditioned ischemic hearts were frozen at the end of 30 minutes of sustained ischemia.

Statistics
Values are expressed as mean±SEM. Statistical analysis was performed using a Systat 5 program. An independent t test was used for comparisons for G_ATP, phosphorus metabolites, and functional recovery after 30 minutes of ischemia in preconditioned and nonpreconditioned hearts, and a paired t test was used to determine differences in [Ca²⁺]_SR, G_Ca²⁺SR, and contractility after addition of pyruvate, isoproterenol, or ischemia. The level of statistical significance was P<0.05.

	Results

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Typical ¹⁹F NMR spectra of TF-BAPTA–loaded hearts are shown in Figure 1. Ionized free Ca²⁺ can be measured from the shift difference between the Ca²⁺-insensitive 6F resonance and the Ca²⁺-sensitive 5F resonance of TF-BAPTA. If there are compartments with different Ca²⁺ concentrations, there will be separate resonance peaks for each compartment. The resonance peak at 5 ppm corresponds to a time-averaged cytosolic free Ca²⁺ concentration ([Ca²⁺]_c) of 600 nmol/L. The resonance peak at 14 ppm corresponds to the free [Ca²⁺] in the SR, because it behaves as expected in the presence of the SR Ca²⁺ release channel activator (caffeine), the SR Ca²⁺-ATPase inhibitor (cyclopiazonic acid), and perfusion with high K⁺.⁴

View larger version (20K): [in this window] [in a new window]   Figure 1. Typical 19F NMR spectra from a heart perfused with glucose followed by perfusion with glucose+pyruvate. The spectra are obtained by using 0.26-second intervals between scans with a pulse of 40° (20 µs). The spectral width was ±7060 Hz, and 4K data points were collected. With the addition of pyruvate to the glucose perfusate, the resonance corresponding to the SR shifted upfield (from 14.16 to 14.31 ppm), indicating an increase in [Ca2+] in the SR (from 0.94 to 1.22 mmol/L).

Effects of Pyruvate on G_ATP and G_Ca²⁺SR
As shown in Table 1, in hearts perfused with glucose as the sole exogenous substrate, the time-averaged [Ca²⁺]_SR is 0.94 mmol/L, calculated from the shift difference of 14.16±0.04 ppm. After the addition of 1 mmol/L of pyruvate, the shift difference increased to 14.31±0.01 ppm (P<0.05), corresponding to a [Ca²⁺]_SR of 1.22 mmol/L, a value significantly higher than that observed in the hearts perfused with glucose alone (P<0.05).

Martin et al²¹ found that there was no detectable change in diastolic [Ca²⁺]_c when pyruvate was added to glucose as an exogenous substrate. Previous work²² using fluorescent indicators to measure cytosolic [Ca²⁺] in isolated cardiac myocytes, in which rigorous calibrations were performed, has demonstrated that diastolic [Ca²⁺]_c in control beating myocytes is in the range of 100 to 200 nmol/L. Using a constant value for diastolic [Ca²⁺]_c of 100 nmol/L, we calculated the free energy required for the SR Ca²⁺-ATPase (G_Ca²⁺SR) to be 47.1 kJ/mol for glucose-perfused hearts and 48.5 kJ/mol (P<0.05) for hearts perfused with glucose+pyruvate. Thus, pyruvate significantly increases the free energy required for the SR Ca²⁺-ATPase.

Figure 2 illustrates the ³¹P NMR spectra recorded from a heart perfused with glucose for 15 minutes followed by perfusion with glucose+pyruvate. Addition of pyruvate (Figure 2) results in a decrease in P_i and an increase in PCr but no measurable change in ATP. The intracellular pH did not change after addition of pyruvate (7.19±0.02 versus 7.19±0.01 in control glucose-perfused hearts). Studies of snap-frozen myocardium (Table 2) showed similar trends. Using the values in Table 2 and the measured pH values, we calculated the G_ATP in the glucose-perfused hearts to be 58.3 kJ/mol (Table 1), consistent with the reported values in the range of 55 to 60 kJ/mol.^{23 24} As expected from previous studies,^{15 16 25 26} the addition of pyruvate increased G_ATP to 60.4 kJ/mol (P<0.05), which would increase the free energy available for the uptake of Ca²⁺, leading to an increased SR/cytosol Ca²⁺ gradient, as observed (Table 1). The difference between G_ATP and the free energy required for the SR Ca²⁺-ATPase (G_Ca²⁺SR) is similar in glucose-perfused hearts (11.2 kJ/mol) compared with glucose+pyruvate-perfused hearts (11.9 kJ/mol). Thus, the increase in the SR Ca²⁺ gradient appears to parallel the increase in G_ATP.

View larger version (21K): [in this window] [in a new window]   Figure 2. Typical 31P NMR spectra from a heart perfused for 15 minutes with glucose followed by perfusion for 15 minutes with glucose+pyruvate. The spectra are obtained by using 2-second intervals between scans with a pulse of 70° (30 µs). Compared with the control glucose perfusion, addition of pyruvate results in an increase in PCr and a decline in Pi. SP indicates sugar monophosphate; PDE phosphodiester.

We also examined the effects of addition of pyruvate on the hemodynamics of perfused rabbit hearts. Consistent with previous studies,^{15 25 26} we observed that left ventricular developed pressure (LVDP) was elevated from 110±5 cm H₂O in control hearts (perfused with glucose alone) to 141±7 cm H₂O (P<0.05) in hearts perfused with glucose+pyruvate. Both the +dP/dt_max and -dP/dt increased slightly (+dP/dt increased from 1749±135 to 2042±128 cm H₂O/s, and -dP/dt increased from 1705±136 to 1969±101 cm H₂O/s). There was no significant change in heart rate (170±9 bpm with glucose, 195±16 bpm with the addition of pyruvate). The positive inotropic response to addition of pyruvate is consistent with an increase in available energy (G_ATP), which leads to a thermodynamically driven increase in the SR Ca²⁺ gradient and an increase in the amount of Ca²⁺ available for release by SR Ca²⁺ release channels during the cardiac cycle.

Effects of Isoproterenol on G_ATP and G_Ca²⁺SR
ß-Adrenergic agonists, such as isoproterenol, have well-characterized effects on myocardial contractile function, which have been attributed, at least in part, to increased SR Ca²⁺ uptake and release. This appears to be mediated by kinetic regulation of the SR Ca²⁺-ATPase by phosphorylation of the inhibitory protein PLB. However, it is unclear whether this kinetic regulation would result in an increase in the SR Ca²⁺ gradient or would primarily affect the rate of Ca²⁺ transport and the time required to achieve the steady-state Ca²⁺ gradient. It is also unclear how this would affect the relationship we had previously observed between G_ATP and G_Ca²⁺SR. To address these issues, we measured the effect of isoproterenol on high-energy phosphates and the SR Ca²⁺ gradient. As expected, addition of isoproterenol caused a significant increase in heart rate (154±29% of the basal heart rate of 165±19 bpm), an increase in LVDP (248±27% of the control value of 124±5 cm H₂O), an increase in +dP/dt_max (323±52% of the control value of 1783±309 cm H₂O/s), and an increase in -dP/dt (177±17% of the control value of 1475±247 cm H₂O/s). We measured [Ca²⁺]_SR in TF-BAPTA–loaded hearts, perfused with glucose as a substrate, for 15 minutes before and 15 minutes after addition of 100 nmol/L isoproterenol (see Figure 3). As illustrated in Table 1, in this series of studies, the time-averaged control [Ca²⁺]_SR averaged 1.11 mmol/L and significantly increased to 1.25 mmol/L with addition of 100 nmol/L isoproterenol (P<0.05). O'Rourke et al²⁷ and Martin et al²¹ found that addition of isoproterenol did not alter diastolic Ca²⁺. Using a constant diastolic [Ca²⁺]_c of 100 nmol/L, we calculated an increase in G_Ca²⁺SR with addition of isoproterenol (see Table 1). However, this increase in G_Ca²⁺SR with addition of isoproterenol occurs despite a fall in available energy (G_ATP) (see Table 1). This decline in energetics after addition of isoproterenol is consistent with previous reports.^{15 24} Thus, ß-adrenergic stimulation increases both the rate of Ca²⁺ sequestration and the final SR Ca²⁺ gradient, and this increase cannot be accounted for by an increase in G_ATP. Indeed, after addition of isoproterenol, the difference between G_ATP and G_Ca²⁺SR is decreased (see Table 1). This suggests that isoproterenol allows the SR Ca²⁺ gradient to approach its thermodynamic limit, by relieving the inhibitory effect of PLB on the SR Ca²⁺-ATPase and removing kinetic restrictions on the SR Ca²⁺ gradient.

View larger version (19K): [in this window] [in a new window]   Figure 3. Typical 19F NMR spectra from a heart perfused with glucose followed by perfusion with 100 nmol/L isoproterenol. Pulsing parameters are the same as described in the legend to Figure 1. With the addition of isoproterenol, the resonance corresponding to SR Ca2+ shifted upfield, indicating an increase in [Ca2+] in the SR (from 1.1 to 1.3 mmol/L).

Effects of Ischemia and Preconditioning on G_ATP and G_Ca²⁺SR
Recent studies have suggested that increased SR Ca²⁺ cycling during ischemia may contribute to ischemic injury,^{8 9 10} and one of the protective mechanisms of preconditioning may be to reduce SR-dependent consumption of ATP. To explore these possibilities, the SR Ca²⁺ gradient and the free energy for ATP hydrolysis were measured during ischemia in hearts with and without preconditioning to investigate whether ischemia and/or ischemic preconditioning altered the SR Ca²⁺ gradient. As observed in rat heart,^{28 29} preconditioning resulted in less acidification during the sustained 30 minutes of ischemia (6.28±0.05 measured during 25 to 30 minutes of ischemia) compared with nonpreconditioned ischemic hearts (5.93±0.08, P<0.05). Consistent with previous studies showing that preconditioning is protective, recovery of LVDP (as a percentage of initial LVDP, which was 110±11 cm H₂O) in preconditioned hearts after 30 minutes of ischemia and 30 minutes of reflow was significantly improved (67.4±3.4%) compared with nonpreconditioned hearts (50.4±2.9%, P<0.05).

We⁴ reported previously that ischemia reduced both the G_ATP and the SR Ca²⁺ gradient. As shown in Table 3, in nonpreconditioned hearts during ischemia, the decline in G_Ca²⁺SR exceeds the decline in G_ATP, and thus the difference between G_ATP and G_Ca²⁺SR increases (15.0 versus 11.1 kJ/mol for control), suggesting kinetic alteration of the SR Ca²⁺ gradient, which is consistent with studies suggesting increased SR Ca²⁺ cycling during ischemia. Intermittent periods of ischemia and reflow (ischemic preconditioning) have been shown to reduce ischemic injury and reduce ATP utilization.³⁰ Examination of the effect of preconditioning on the SR Ca²⁺ gradient during ischemia revealed that [Ca²⁺]_SR in preconditioned hearts rose slightly to 1.8 mmol/L (Table 3), which is similar to what is observed in nonpreconditioned hearts. This demonstrates that a high [Ca²⁺]_SR can be maintained during 25 to 30 minutes of ischemia in both nonpreconditioned and preconditioned hearts. During ischemia [Ca²⁺]_c rises to a level at which it can be measured by cytosolic TF-BAPTA. These measurements show that, compared with nonpreconditioned hearts, preconditioned hearts have a lower [Ca²⁺]_c during ischemia (1.6 versus 3.4 µmol/L in nonpreconditioned hearts at 25 to 30 minutes of ischemia). These measurements showing attenuation of the rise in [Ca²⁺]_c in preconditioned hearts are consistent with previous measurements in rat and rabbit hearts.^{29 31} Thus, as shown in Table 3, by 25 to 30 minutes of sustained ischemia, the SR Ca²⁺ gradient in preconditioned hearts is more than double that found in nonpreconditioned hearts, and the G_Ca²⁺SR was higher in preconditioned (38.7 kJ/mol) than in nonpreconditioned hearts (33.6 kJ/mol). This difference in G_Ca²⁺SR occurs even though after 15 to 20 minutes of ischemia, there are no significant differences in G_ATP between preconditioned and nonpreconditioned hearts (Table 3). The metabolite concentrations used to calculate G_ATP are presented in Table 4. At 25 to 30 minutes of ischemia, the difference between G_ATP and G_Ca²⁺SR is less in preconditioned hearts (10.5 kJ/mol) compared with nonpreconditioned hearts (15.0 kJ/mol). These data suggest that in preconditioned hearts during ischemia, the G_Ca²⁺SR is closer to the thermodynamic limit than is observed in nonpreconditioned hearts, consistent with either an increased efflux of SR Ca²⁺ during ischemia in nonpreconditioned hearts or alternatively a greater decrease in Ca²⁺ uptake than would be necessitated by the fall in G_ATP.

	Discussion

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Cardiac muscle contraction is activated by a combination of Ca²⁺ influx from the extracellular medium, by sarcolemmal L-type Ca²⁺ channels and Na⁺/Ca²⁺ exchange, and Ca²⁺ release from the SR by the SR Ca²⁺ release channels.^{2 3} The latter is controlled by a Ca²⁺-induced Ca²⁺ release mechanism.¹ The SR Ca²⁺ content is determined by the balance between Ca²⁺ release and uptake during the cardiac contraction cycle. The SR Ca²⁺ content available for release is an important determinant of the cytosolic Ca²⁺ transient and contractile state.³² In a previous study,⁴ using a newly developed ¹⁹F NMR method to measure ionized [Ca²⁺] in the SR in the beating rabbit heart, we showed that the time-averaged [Ca²⁺]_SR was 1 mmol/L, a value very similar to diastolic [Ca²⁺]_SR. We also showed that [Ca²⁺]_SR decreases by about 30% at the start of systole. In the present study, we examined the regulation of [Ca²⁺]_SR, specifically the relationship between contractility, the Ca²⁺ gradient across the SR membrane, and the energy state of the cell.

Ca²⁺ is actively transported across the SR membrane against a large chemical gradient by the sarcoplasmic/endoplasmic reticulum Ca²⁺-ATPase (SERCA) using energy derived from ATP hydrolysis. If there were no release or leak of Ca²⁺ from the SR, then a comparison of the G_Ca²⁺SR and G_ATP would indicate the relative efficiency of SERCA, ie, the percentage of the thermodynamic limit that is attained. Kinetic constraints as well as Ca²⁺ efflux could also affect the relative efficiency of SR Ca²⁺ uptake into the SR. Ca²⁺ efflux occurs via well-characterized ryanodine and IP₃ receptors and leak pathways that are unmasked by inhibition of SERCA with thapsigargin.³³ Even when excitation-contraction coupling is blocked by membrane depolarization with high extracellular K⁺, removing the time constraints on SR Ca²⁺ uptake, the SR Ca²⁺ gradient does not reach its thermodynamic limit.⁴ These data are consistent with previous studies, which suggest that the energetic efficiency of SERCA is in the range of 75% to 85%.^{4 5} In our previous study,⁴ SR Ca²⁺ content increased when cytosolic [Ca²⁺] was increased by raising extracellular [K⁺] to 30 mmol/L, but the SR Ca²⁺ gradient did not change significantly. The affinity of SERCA for Ca²⁺ is estimated to be in the range of 250 to 500 nmol/L,^{34 35} and therefore SERCA activity should be increased by the higher cytosolic [Ca²⁺] in hearts arrested with 30 mmol/L extracellular [K⁺]; and because there is no substantial increase in the SR Ca²⁺ gradient, SERCA is either functioning at its maximum efficiency or increased uptake is balanced by increased leak as SR Ca²⁺ increases. In the present study, we increased the driving force for SR Ca²⁺ uptake by increasing the phosphorylation potential without eliminating cytosolic Ca²⁺ transients and without increasing diastolic [Ca²⁺]_c.²¹ When pyruvate was provided as substrate, both G_ATP and SR Ca²⁺ increased, providing an example of a proportionate increase in G_ATP and G_Ca²⁺SR but no significant increase in efficiency.

The increased SR [Ca²⁺] observed in pyruvate-perfused hearts would increase the Ca²⁺ available for release from the SR during each contraction cycle. Recent studies have shown that the amount of Ca²⁺ released from the SR increases with increasing SR Ca²⁺ content for a given Ca²⁺ trigger, ie, the fractional SR Ca²⁺ release can be modulated by the SR Ca²⁺ load,³² probably because of the change in the sensitivity of the SR Ca²⁺ release channels. The fraction of SR Ca²⁺ released is affected by both the trigger Ca²⁺ and the SR Ca²⁺ content.³² This increased Ca²⁺ release from the SR is consistent with a recent study showing that pyruvate increases systolic [Ca²⁺]_c and cell shortening in isolated rat ventricular myocytes,²¹ further supporting the conclusion that the positive inotropic effect observed with addition of pyruvate is the result of increased SR Ca²⁺ secondary to an increase in G_ATP.

In contrast to pyruvate, isoproterenol has a positive inotropic effect but does not enhance global energetics. Isoproterenol promotes phosphorylation of PLB, thereby enhancing the activity of the SR Ca²⁺-ATPase, and also increases phosphorylation of the sarcolemmal Ca²⁺ channel, increasing Ca²⁺ entry into the myocyte.^{36 37 38} In hearts perfused with glucose, the energy available from G_ATP is 58 kJ/mol and the G_Ca²⁺SR is 48 kJ/mol. In hearts perfused with pyruvate, the energy available from ATP hydrolysis is increased by 2 kJ/mol, and there is a corresponding increase in the G_Ca²⁺SR, such that the difference between G_ATP and G_Ca²⁺SR is similar in glucose and pyruvate hearts. In contrast, G_Ca²⁺SR increases in the presence of isoproterenol, despite a decline in global G_ATP. Thus, the difference between G_ATP and G_Ca²⁺SR decreases after addition of isoproterenol, which suggests that in the presence of isoproterenol, kinetic stimulation of SERCA results in SERCA operating closer to its thermodynamic limit. Alternatively, the increase in G_Ca²⁺SR could be explained if the local [ADP] in the immediate vicinity of SERCA did not change in parallel with the global increase in [ADP] observed with isoproterenol; this could occur if glycolytic ATP production was preferentially channeled to fuel SR Ca²⁺ uptake as suggested by Xu et al.³⁹

PLB is a small protein, comprising 52 amino acid residues, which is present in cardiac, smooth, and slow-twitch skeletal muscle.³⁶ Dephosphorylated PLB interacts with the SR Ca²⁺-ATPase and inhibits its apparent affinity for Ca²⁺. Phosphorylation of PLB by protein kinases stimulated by isoproterenol reverses PLB inhibition,^{37 38} apparently by disrupting protein-protein interactions between PLB and the Ca²⁺-ATPase.⁴⁰ Chu et al⁷ showed that removal of PLB inhibition of SERCA in PLB null mice caused a 2-fold increase in total SR Ca²⁺ content measured by electron probe microanalysis. Although there was no measurement of free SR [Ca²⁺] in the study of Chu et al,⁷ it is very likely that the increase in total Ca²⁺ is associated with an increase in free SR [Ca²⁺]. This increase in the SR Ca²⁺ gradient seems to occur also without an increase in G_ATP, because Chu et al⁷ reported that the PLB knockout mice had a lower PCr and higher ADP compared with controls. These data on the PLB null mice are consistent with our observations that addition of isoproterenol, which phosphorylates PLB and thereby removes its inhibition, increases [Ca²⁺]_SR. Taken together, these data suggest that increasing the activity of SERCA, even with a decline in global G_ATP, can also lead to an increase in the SR Ca²⁺ gradient, presumably because PLB imposes kinetic limitations on SERCA, causing it to operate further from its thermodynamic limit.

Recent studies have suggested that SR Ca²⁺ cycling is an important determinant of survival of ischemic or metabolically inhibited myocytes.^{8 9 10} We observed previously that during ischemia, the disparity between the free energy required for the SR Ca²⁺ gradient and the free energy available from ATP hydrolysis is increased.⁴ This would be consistent with increased Ca²⁺ efflux or leak, in agreement with studies reporting that Ca²⁺ release channels are inappropriately opened in the ischemic heart.^{41 42} However, we cannot exclude the alternative explanation for the observed decrease in the SR Ca²⁺ gradient relative to G_ATP, which is that SERCA is inhibited during ischemia. The effects of inhibiting SERCA are difficult to predict; although inhibition of SERCA could accelerate ischemic injury by raising [Ca²⁺]_c, there would be less ATP consumption by this ATPase. Preconditioned ischemic hearts have a more than 2-fold greater Ca²⁺ gradient across the SR membrane compared with nonpreconditioned ischemic hearts, even though the free energy for ATP hydrolysis is not significantly different between preconditioned and nonpreconditioned hearts at 25 to 30 minutes of ischemia (Table 3). These data are entirely consistent with an increase in SR Ca²⁺ efflux during ischemia, which is reduced in preconditioned hearts. Interestingly, Zucchi et al⁴³ have reported that preconditioning is associated with a decline in ryanodine binding sites. A decrease in SR Ca²⁺ release would reduce futile cycling of Ca²⁺ and would reduce ATP utilization, which would preserve ATP for other processes.

We have observed some variability in the baseline values of [Ca²⁺]_SR between experiments that were performed at different times. It is clear from the present study that the value of [Ca²⁺]_SR is a function of metabolic state (G_ATP), and therefore there may not be a true basal value for [Ca²⁺]_SR. The variation in [Ca²⁺]_SR parallels the variation in G_ATP. For this reason, we have time-matched the measurements of G_ATP to measurements of [Ca²⁺]_SR. Also to study the effects of interventions on [Ca²⁺]_SR, a paired analysis was always performed by comparison with the baseline value of [Ca²⁺]_SR for each heart.

A technical limitation of these studies is that the measurements of [Ca²⁺]_SR are made with an indicator that has a K_d for Ca²⁺ that is far removed from the measured [Ca²⁺]_SR. TF-BAPTA binding to Ca²⁺ is most sensitive to Ca²⁺ levels near the K_d (65 µmol/L). At Ca²⁺ concentrations far above the K_d, the indicator is close to saturation with Ca²⁺ so that large changes in Ca²⁺ cause only small shift differences. In the Ca²⁺ concentration range present in the SR (1 mmol/L), approximately 95% of TF-BAPTA is complexed with Ca²⁺. Nevertheless, we are able to measure consistent increases in [Ca²⁺]_SR after pyruvate or isoproterenol administration, with the use of paired analysis. Although it would be preferable to use an indicator with a higher K_d, none is currently available.⁴⁴ TF-BAPTA has a relatively high K_d (65 µmol/L) and a large (10 ppm) shift range, which makes TF-BAPTA one of the best currently available indicators for measuring [Ca²⁺] in the range of 1 mmol/L.

Because measurements of [Ca²⁺]_SR are time-averaged during the cardiac cycle, these values will underestimate diastolic SR [Ca²⁺]. In a previous study,⁴ we gated to the cardiac cycle and showed that SR Ca²⁺ decreases by 30% at the start of systole, but this decrease is very brief, with 40% recovery of [Ca²⁺]_SR 10 ms after the peak of SR Ca²⁺ release. Because of the short duration of the Ca²⁺ transient, we also observed that diastolic SR [Ca²⁺] was very similar to time-averaged [Ca²⁺]_SR. However, with addition of isoproterenol, and to a lesser extent with addition of pyruvate, there is an increase in heart rate that decreases the percentage of the cardiac cycle that is spent between Ca²⁺ transients and therefore increases the contribution of the time intervals when SR [Ca²⁺] is decreased to the time-averaged value of [Ca²⁺]_SR, particularly with isoproterenol, which increased heart rate by 54%. This underestimation may be offset partially by the increased rate of SR Ca²⁺ resequestration at the end of the Ca²⁺ transient in the presence of isoproterenol.

In conclusion, the present study expands on our previous study,⁴ which measured SR [Ca²⁺] and showed that G_Ca²⁺SR was close to thermodynamic equilibrium. The present study further investigates the regulation of the SR Ca²⁺ gradient and demonstrates that the positive inotropic effect observed with addition of pyruvate can be attributed to increased [Ca²⁺]_SR secondary to an increase in G_ATP. In contrast, the positive inotropic effect of isoproterenol is associated with increased [Ca²⁺]_SR but with a decline in global G_ATP. Addition of isoproterenol shifts the SR Ca²⁺ gradient closer to its thermodynamic limit, consistent with removal of the kinetic limitation of PLB on the activity of SERCA. In contrast, during ischemia, the SR Ca²⁺ gradient moves further from thermodynamic equilibrium, consistent with either kinetically enhanced Ca²⁺ efflux or reduced Ca²⁺ uptake into the SR. Furthermore, preconditioned ischemic hearts show a higher G_Ca²⁺SR compared with nonpreconditioned ischemic hearts, without any apparent difference in G_ATP. Thus, compared with nonpreconditioned hearts during ischemia, the SR Ca²⁺-ATPase in preconditioned hearts appears to operate closer to its thermodynamic limit. The data demonstrate that both thermodynamic regulation and kinetic regulation of the SR Ca²⁺ gradient have important effects on contractile function and may be important factors in ischemic injury.

	Acknowledgments

 
Drs C. Steenbergen and W. Chen were supported in part by NIH grant R01-HL39752.

Received September 19, 1997; accepted July 24, 1998.

	References

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