Chen W, London R, Murphy E, Steenbergen C
Department of Pathology, Duke University Medical Center, Durham, NC 27710, USA.
Circ Res. 1998 Nov 2;83(9):898-907. doi: 10.1161/01.res.83.9.898.
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 (DeltaGATP) and the energy required for maintenance of the SR Ca2+ gradient (DeltaGCa2+SR) during physiological and pathological manipulations that alter DeltaGATP 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 DeltaGATP, and the increase in the SR Ca2+ gradient was matched to the increase in DeltaGATP; DeltaGATP increased from 58.3+/-0.5 to 60.4+/-1.0 kJ/mol (P<0.05), and DeltaGCa2+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 DeltaGATP 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 DeltaGATP, 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 DeltaGATP, and this should also have an effect on SR Ca2+ handling. During 30 minutes of ischemia, DeltaGATP decreased by 12 kJ/mol, but the decrease in DeltaGCa2+SR was 16 kJ/mol, greater than would be predicted by the fall in DeltaGATP 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 DeltaGATP and the fall in DeltaGCa2+SR during ischemia. We found that preconditioning attenuated the fall in DeltaGCa2+SR during ischemia; the fall in DeltaGCa2+SR was of comparable magnitude to the fall in DeltaGATP, 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 DeltaGATP and kinetic regulation, which can alter the relationship between DeltaGATP and DeltaGCa2+SR.
心肌收缩力取决于钙离子从肌浆网（SR）的释放和摄取。SR基质与胞质溶胶之间的钙离子梯度（SR钙离子梯度）由SR钙离子ATP酶利用ATP水解产生的自由能来维持。SR钙离子ATP酶的活性不仅取决于细胞的能量状态，还受到诸如受磷蛋白等SR蛋白的动力学调节。为了评估SR钙离子梯度的热力学和动力学调节的重要性，我们研究了在改变ATP水解自由能（ΔGATP）和受磷蛋白磷酸化状态的生理和病理操作过程中，ATP水解产生的能量（ΔGATP）与维持SR钙离子梯度所需的能量（ΔG Ca2+SR）之间的关系。我们使用先前开发的19F核磁共振方法来测量Langendorff灌注兔心脏SR中的游离钙离子浓度。我们发现添加丙酮酸或异丙肾上腺素会导致左心室舒张末压升高和SR中钙离子浓度升高。丙酮酸增加了ΔGATP，并且SR钙离子梯度的增加与ΔGATP的增加相匹配；ΔGATP从58.3±0.5增加到60.4±1.0 kJ/mol（P<0.05），ΔG Ca2+SR从47.1±0.3增加到48.5±0.1 kJ/mol（P<0.05）。相比之下，尽管存在异丙肾上腺素时ΔGATP从58.3±0.5下降到55.8±0.6 kJ/mol，但SR钙离子梯度仍增加。因此，数据表明SR钙离子梯度可通过增加ΔGATP而升高，丙酮酸的正性肌力作用可以通过改善能量相关的SR钙离子处理来解释，而异丙肾上腺素的结果与消除受磷蛋白对肌浆网/内质网钙离子ATP酶活性的动力学限制一致，这使得SR钙离子梯度更接近其热力学极限。缺血会降低ΔGATP，这也应该对SR钙离子处理产生影响。在30分钟的缺血过程中，ΔGATP下降了12 kJ/mol，但ΔG Ca2+SR的下降为16 kJ/mol，大于由ΔGATP下降所预测的值，并且与SR钙离子释放增加和SR钙离子循环增加一致。由于据报道缺血预处理会在随后的持续缺血期间减少SR钙离子循环，我们研究了缺血预处理是否会影响缺血期间ΔGATP下降与ΔG Ca2+SR下降之间的关系。我们发现预处理减弱了缺血期间ΔG Ca2+SR的下降；ΔG Ca2+SR的下降幅度与ΔGATP的下降幅度相当，并且这与再灌注期间功能恢复的显著改善相关。数据表明存在由ΔGATP对SR钙离子梯度的热力学调节以及动力学调节，这可以改变ΔGATP与ΔG Ca2+SR之间的关系。
