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—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

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