REGULATION OF MITOCHONDRIAL ENERGETICS BY INTRACELLULAR IONS

心脏做功的增加会引起线粒体耗氧量的迅速增加和 满足能量需求的ATP合成。这种反应的机制被认为涉及刺激途径中多个位置的氧化磷酸化。通常提出的两种机制包括：(1)通过ATP消耗产物(ADP，PI)提高线粒体电子传递和ATP合成的速率；(2)在Krebs循环水平上，钙离子上游刺激NADH的产生。在完整的心肌中，缺血和再灌流改变了离子稳态和线粒体功能，但对线粒体整体控制的影响尚未被研究。本项目的目的是确定细胞内阳离子在完整心肌和分离心肌细胞能量供需匹配机制中的作用，并确定这些过程是如何被缺血和再灌注改变的。具体地说，我们将使用新的荧光技术来研究细胞内钙离子、钠离子和钾离子相互依赖的作用如何调节线粒体对正常和缺血后心肌和细胞负荷变化的生物能量反应。测定心肌兴奋收缩偶联过程中钙离子从细胞质间隙向线粒体基质转运的动力学，确定线粒体钙离子内流与能量代谢刺激的偶联作用。我们将检验这一假设，即细胞内钠离子在缺血期间急剧增加，在改变细胞内钙离子变化的生物能量反应中发挥关键作用。我们还将研究线粒体钙激活K+(MitoKca)通道是否因线粒体基质钙离子的生理变化而被激活，从而改变能量反应。这些数据将被整合到新开发的心肌细胞综合计算模型中，作为了解离子稳态改变和缺血对完整心肌的电生理学、力产生和钙处理特性的影响的框架。结果将与蛋白质组学和生化分析以及将要进行的代谢控制实验进行比较 在计划项目的其他组成部分中使用相同的动物模型。阐明离子稳态对供需匹配的贡献将使我们能够合理地设计治疗策略，以应对缺血后心脏的收缩衰竭和心律失常。

An increase in cardiac work evokes a rapid increase in mitochondrial oxygen consumption and ATP synthesis to meet the energetic demand. The mechanism of this response is thought to involve stimulation of oxidative phosphorylation at multiple sites in the pathway. Two commonly proposed mechanisms include i) enhancement of the rate of mitochondrial electron transport and ATP synthesis by the products of ATP consumption (ADP, Pi), and ii) upstream stimulation of NADH production by Ca2+ at the level of the Krebs cycle. In intact cardiac muscle, ischemia and reperfusion alters both ion homeostasis and mitochondrial function, but the effects on global mitochondrial control have not been investigated. The goal of this project is to characterize the role of intracellular cations in the mechanism of energy supply and demand matching in intact cardiac muscle and isolated cardiomyocytes and to determine how these processes are modified by ischemia and reperfusion. Specifically, we will employ novel fluorescence techniques to examine how the interdependent actions of intracellular Ca2+, Na , and K+ modulate the mitochondrial bioenergetic response to a change in workload in normal and post-ischemic cardiac muscles and cells. The dynamics of Ca2+ transport from the cytoplasmic space to the mitochondrial matrix during cardiac excitation-contraction coupling will be measured, and the coupling of mitochondrial Ca2+ influx to the stimulation of energy metabolism will be determined. We will test the hypothesis that intracellular Na+, which increases dramatically during ischemia, plays a key role in modifying the bioenergetic response to changes in intracellular Ca2+. We will also examine whether mitochondrial Ca2+-activated K+ (mitoKca) channels are activated in response to physiological changes in mitochondrial matrix Ca2+, thus modifying the energetic response. This data will be integrated into a newly developed comprehensive computational model of the cardiac cell as a framework for understanding the effects of altered ion homeostasis and ischemia on the electrophysiology, force production, and Ca2+ handling properties of intact cardiac muscle. The results will be compared with the proteomic and biochemical analyses, and metabolic control experiments to be carried out using the same animal model in the other components of the Program Project. Elucidating the contribution of ion homeostasis to supply and demand matching will permit us to rationally design therapeutic strategies for coping with contractile failure and arrhythmogenesis in the post-ischemic heart.