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Circulation Research. 1998;83:898-907

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(Circulation Research. 1998;83:898-907.)
© 1998 American Heart Association, Inc.


Original Contributions

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

A 19F Nuclear Magnetic Resonance Study

Weina Chen, Robert London, Elizabeth Murphy, , Charles Steenbergen

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
up arrowTop
*Abstract
down arrowIntroduction
down arrowMaterials and Methods
down arrowResults
down arrowDiscussion
down arrowReferences
 
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 ({Delta}GATP) and the energy required for maintenance of the SR Ca2+ gradient ({Delta}GCa2+SR) during physiological and pathological manipulations that alter {Delta}GATP 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 {Delta}GATP, and the increase in the SR Ca2+ gradient was matched to the increase in {Delta}GATP; {Delta}GATP increased from 58.3±0.5 to 60.4±1.0 kJ/mol (P<0.05), and {Delta}GCa2+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 {Delta}GATP 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 {Delta}GATP, 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 {Delta}GATP, and this should also have an effect on SR Ca2+ handling. During 30 minutes of ischemia, {Delta}GATP decreased by 12 kJ/mol, but the decrease in {Delta}GCa2+SR was 16 kJ/mol, greater than would be predicted by the fall in {Delta}GATP 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 {Delta}GATP and the fall in {Delta}GCa2+SR during ischemia. We found that preconditioning attenuated the fall in {Delta}GCa2+SR during ischemia; the fall in {Delta}GCa2+SR was of comparable magnitude to the fall in {Delta}GATP, 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 {Delta}GATP and kinetic regulation, which can alter the relationship between {Delta}GATP and {Delta}GCa2+SR.


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


*    Introduction
up arrowTop
up arrowAbstract
*Introduction
down arrowMaterials and Methods
down arrowResults
down arrowDiscussion
down arrowReferences
 
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 ({Delta}GATP).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 {Delta}GATP.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 Kd 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 Kd 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 {approx}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 {Delta}GATP, 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 {Delta}GATP 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
up arrowTop
up arrowAbstract
up arrowIntroduction
*Materials and Methods
down arrowResults
down arrowDiscussion
down arrowReferences
 
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 ({approx}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 {approx}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 Pi and phosphocreatine (PCr) peaks. The Pi, 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 Pi, 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 Kd (such as in the cytosol under basal conditions). However, for Ca2+ measurements above the Kd (all measurements of SR Ca2+), the corrections due to pH and Mg2+ are negligible. Briefly, the Kd is 65 µmol/L, {delta}0 is the measured shift difference between the 6F and the 5F resonance peak of TF-BAPTA, {delta}1 (5.13) is the shift difference in the presence of EGTA at pH 12, {delta}2+{delta}3 (13.41) and {delta}4 (8.53) are the shifts caused by protonation, {delta}5 (14.83) is the shift difference with excess Ca2+, and {delta}6+{delta}7 (11.21) and {delta}8 (7.83) correspond to the shift difference due to binding of single or two Mg2+, respectively. Because of intermediate exchange,11 {delta}5=14.83 (for [Ca2+]>7 µmol/L) or {delta}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 {approx}3 mmol/L in our previous study.17

Calculation of Free Energy ({Delta}G) for SR Ca2+-ATPase
We calculated the {Delta}G required for the SR Ca2+-ATPase using the following equation4 : {Delta}GCa2+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 ({Delta}GATP)
The high-energy phosphates, Pi, and pHi 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 {Delta}GATP: {Delta}GATP={Delta}G0+RT ln ({[ADP]fx[Pi]f}/[ATP]f), with {Delta}G0=-30.5 kJ/mol. By thermodynamic convention, values for {Delta}G0 and {Delta}GATP are negative for exergonic reactions, but after calculating {Delta}GATP, 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+]xKck=10-0.87 pHi+8.31.

The PCr and Pi 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 Pi 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, Pi, 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 ({Delta}GCa2+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 ({Delta}GATP)
To examine how substrates, isoproterenol, and ischemia affect {Delta}GATP, 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 {Delta}GATP, 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, {Delta}GCa2+SR, and contractility after addition of pyruvate, isoproterenol, or ischemia. The level of statistical significance was P<0.05.


*    Results
up arrowTop
up arrowAbstract
up arrowIntroduction
up arrowMaterials and Methods
*Results
down arrowDiscussion
down arrowReferences
 
Typical 19F NMR spectra of TF-BAPTA–loaded hearts are shown in Figure 1Down. 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 {approx}5 ppm corresponds to a time-averaged cytosolic free Ca2+ concentration ([Ca2+]c) of {approx}600 nmol/L. The resonance peak at {approx}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



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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 {Delta}GATP and {Delta}GCa2+SR
As shown in Table 1Down, 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).


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Table 1. Calculation of Free Energy for Ca2+-ATPase and ATP Hydrolysis With Addition of Pyruvate and Isoproterenol

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 ({Delta}GCa2+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 2Down 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 2Down) results in a decrease in Pi 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 2Down) showed similar trends. Using the values in Table 2Down and the measured pH values, we calculated the {Delta}GATP in the glucose-perfused hearts to be 58.3 kJ/mol (Table 1Up), 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 {Delta}GATP 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 1Up). The difference between {Delta}GATP and the free energy required for the SR Ca2+-ATPase ({Delta}GCa2+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 {Delta}GATP.



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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.


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Table 2. Metabolites in Glucose and Glucose+Pyruvate Hearts

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/dtmax 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 ({Delta}GATP), 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 {Delta}GATP and {Delta}GCa2+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 {Delta}GATP and {Delta}GCa2+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/dtmax (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 3Down). As illustrated in Table 1Up, 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 {Delta}GCa2+SR with addition of isoproterenol (see Table 1Up). However, this increase in {Delta}GCa2+SR with addition of isoproterenol occurs despite a fall in available energy ({Delta}GATP) (see Table 1Up). 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 {Delta}GATP. Indeed, after addition of isoproterenol, the difference between {Delta}GATP and {Delta}GCa2+SR is decreased (see Table 1Up). 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.



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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 1Up. 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 {Delta}GATP and {Delta}GCa2+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 {Delta}GATP and the SR Ca2+ gradient. As shown in Table 3Down, in nonpreconditioned hearts during ischemia, the decline in {Delta}GCa2+SR exceeds the decline in {Delta}GATP, and thus the difference between {Delta}GATP and {Delta}GCa2+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 3Down), 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 3Down, 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 {Delta}GCa2+SR was higher in preconditioned (38.7 kJ/mol) than in nonpreconditioned hearts (33.6 kJ/mol). This difference in {Delta}GCa2+SR occurs even though after 15 to 20 minutes of ischemia, there are no significant differences in {Delta}GATP between preconditioned and nonpreconditioned hearts (Table 3Down). The metabolite concentrations used to calculate {Delta}GATP are presented in Table 4Down. At 25 to 30 minutes of ischemia, the difference between {Delta}GATP and {Delta}GCa2+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 {Delta}GCa2+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 {Delta}GATP.


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Table 3. Calculation of Free Energy for Ca2+-ATPase and ATP Hydrolysis During Ischemia in Nonpreconditioned and Preconditioned Hearts


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Table 4. Metabolites in Preconditioned and Nonpreconditioned Ischemic Hearts


*    Discussion
up arrowTop
up arrowAbstract
up arrowIntroduction
up arrowMaterials and Methods
up arrowResults
*Discussion
down arrowReferences
 
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 {approx}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 {Delta}GCa2+SR and {Delta}GATP 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 {Delta}GATP and SR Ca2+ increased, providing an example of a proportionate increase in {Delta}GATP and {Delta}GCa2+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 {Delta}GATP.

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 {Delta}GATP is {approx}58 kJ/mol and the {Delta}GCa2+SR is {approx}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 {Delta}GCa2+SR, such that the difference between {Delta}GATP and {Delta}GCa2+SR is similar in glucose and pyruvate hearts. In contrast, {Delta}GCa2+SR increases in the presence of isoproterenol, despite a decline in global {Delta}GATP. Thus, the difference between {Delta}GATP and {Delta}GCa2+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 {Delta}GCa2+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 {Delta}GATP, 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 {Delta}GATP, 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 {Delta}GATP, 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 3Up). 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 ({Delta}GATP), and therefore there may not be a true basal value for [Ca2+]SR. The variation in [Ca2+]SR parallels the variation in {Delta}GATP. For this reason, we have time-matched the measurements of {Delta}GATP 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 Kd for Ca2+ that is far removed from the measured [Ca2+]SR. TF-BAPTA binding to Ca2+ is most sensitive to Ca2+ levels near the Kd (65 µmol/L). At Ca2+ concentrations far above the Kd, 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 ({approx}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 Kd, none is currently available.44 TF-BAPTA has a relatively high Kd (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 {approx}30% at the start of systole, but this decrease is very brief, with {approx}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 {Delta}GCa2+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 {Delta}GATP. In contrast, the positive inotropic effect of isoproterenol is associated with increased [Ca2+]SR but with a decline in global {Delta}GATP. 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 {Delta}GCa2+SR compared with nonpreconditioned ischemic hearts, without any apparent difference in {Delta}GATP. 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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up arrowAbstract
up arrowIntroduction
up arrowMaterials and Methods
up arrowResults
up arrowDiscussion
*References
 

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