Regulation of the Ca 2+ Gradient Across the Sarcoplasmic Reticulum in Perfused Rabbit Heart

Abstract

Abstract —Myocardial contractility depends on Ca 2+ release from and uptake into the sarcoplasmic reticulum (SR). The Ca 2+ gradient between the SR matrix and the cytosol (SR Ca 2+ gradient) is maintained by the SR Ca 2+ -ATPase using the free energy available from hydrolysis of ATP. The activity of the SR Ca 2+ -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 Ca 2+ gradient, we examined the relationship between the energy available from ATP hydrolysis (ΔG ATP ) and the energy required for maintenance of the SR Ca 2+ gradient (ΔG Ca 2+ SR ) during physiological and pathological manipulations that alter ΔG ATP and the phosphorylation state of phospholamban. We used our previously developed 19 F nuclear magnetic resonance method to measure the ionized [Ca 2+ ] 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 [Ca 2+ ] SR . Pyruvate increased ΔG ATP , and the increase in the SR Ca 2+ gradient was matched to the increase in ΔG ATP ; ΔG ATP increased from 58.3±0.5 to 60.4±1.0 kJ/mol ( P <0.05), and ΔG Ca 2+ SR increased from 47.1±0.3 to 48.5±0.1 kJ/mol ( P <0.05). In contrast, the increase in the SR Ca 2+ gradient in the presence of isoproterenol occurred despite a decline in ΔG ATP from 58.3±0.5 to 55.8±0.6 kJ/mol. Thus, the data indicate that the SR Ca 2+ gradient can be increased by an increase in ΔG ATP , and that the positive inotropic effect of pyruvate can be explained by improved energy-linked SR Ca 2+ handling, whereas the results with isoproterenol are consistent with removal of the kinetic limitation of phospholamban on the activity of the sarcoplasmic/endoplasmic reticulum Ca 2+ -ATPase, which allows the SR Ca 2+ gradient to move closer to its thermodynamic limit. Ischemia decreases ΔG ATP , and this should also have an effect on SR Ca 2+ handling. During 30 minutes of ischemia, ΔG ATP decreased by 12 kJ/mol, but the decrease in ΔG Ca 2+ SR was 16 kJ/mol, greater than would be predicted by the fall in ΔG ATP and consistent with increased SR Ca 2+ release and increased SR Ca 2+ cycling. Because ischemic preconditioning is reported to decrease SR Ca 2+ cycling during a subsequent sustained period of ischemia, we examined whether ischemic preconditioning affects the relationship between the fall in ΔG ATP and the fall in ΔG Ca 2+ SR during ischemia. We found that preconditioning attenuated the fall in ΔG Ca 2+ SR during ischemia; the fall in ΔG Ca 2+ SR was of comparable magnitude to the fall in ΔG 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 Ca 2+ gradient by ΔG ATP and kinetic regulation, which can alter the relationship between ΔG ATP and ΔG Ca 2+ SR .