Study of functional coupling among membrane excitation, contraction and mitochondria in cardiac myocyte.

我们测定了Rhod-2AM负载后大鼠心室肌细胞线粒体基质中Ca^<2+>的变化。用含有300 nM Ca^<2+ >和0 mM Na+的浴溶液灌流可增加Rhod-2的强度。这种增加可被钙离子单向转运体抑制剂钌红完全消除，这支持了钙离子单向转运体是线粒体的主要钙离子内流途径。将浴槽溶液改变为含有0 Ca^2+和6 mM Na^+的溶液，诱导了Rhod-2信号的衰减，而在没有Na^+的情况下仅观察到轻微的衰减。因此，Na^+/Ca^2+交换（mNCX）是心肌线粒体Ca^2+外流的主要机制。mNCX对线粒体膜电位（Δ N）的依赖性一直存在争议。我们在与上述相同的实验方案中使用TMRE测量了通过mNCX诱导Ca^2+流出的Δ。但TMRE荧光强度无明显变化。为了进一步研究Δ Ca依赖性，在用BK通道开放剂NS 1619、H^+离子载体FCCP或去除线粒体底物（丙酮酸、琥珀酸和苹果酸）去极化线粒体膜的条件下，测定Ca^2+流出率。Ca^2+流出率与对照组相似。基于上述实验数据，我们重建了由Ca ^2+单向转运体、mNCX和一个假定的Ca^2+缓冲液组成的线粒体Ca^2+动力学的计算机模型，并将其纳入心肌兴奋-收缩耦合的综合模型（京都模型）。模型分析表明，线粒体内NADH的变化是由ATP水解产物ADP和无机磷酸盐的增加以及Ca^2+激活线粒体ATP酶引起的，这种变化依赖于搏动频率。

We measured Ca^<2+> of mitochondria matrix in rat ventricular myocytes, which were permeabilized with saponin after loading of Rhod-2AM. Superfusion with a bath solution containing 300 nM Ca^<2+ >and 0 mM Na+ increased the Rhod-2 intensity. The increase was completely abolished by ruthenium red, an inhibitor of Ca^<2+> uniporter, supporting that the Ca^<2+> uiniporter is the main Ca^<2+> influx pathway of mitochondria. Changing the bath solution to that containing 0 Ca^<2+> and 6 mM Na^+ induced a decay of Rhod-2 signal, while only a slight decay was observed without Na^+. Therefore, the Na^+/Ca^<2+> exchange (mNCX) is the main Ca^<2+> efflux mechanism of cardiac mitochondria. The dependence of mNCX on mitochondrial membrane potential (ΔΨ) has been controversial. We measured ΔΨ using TMRE during the same experimental protocol as above inducing Ca^<2+> efflux via mNCX. However, nosignificant change in TMRE fluorescence was observed. To further study the ΔΨ dependence, the Ca^<2+> efflux rate was measured under the condition that the mitochondrial membrane was depolarized by NS 1619 ( an opener of BK channel), FCCP (H^+ ionophore) or removal of mitochondrial substrates (pyruvate, succinate and malate). The Ca^<2+> efflux rate was similar to that measured in the control condition. The above experimental data indicate that Ca^<2+> efflux through mNCX is almost independent of ΔΨ.Based on the above experimental date, we reconstructed a computer model of mitochondria Ca^<2+> dynamics, which consists of Ca^<2+> uniporter, mNCX and a putative Ca^<2+> buffer, and incorporated it into a comprehensive model of cardiac excitation-contraction coupling (Kyoto model). The model analysis indicated that the NADH change, which is dependent on beating frequency, is caused by the increases of ADP and inorganic phosphate (products of ATP hydrolysis) and Ca^<2+> activation of mitochondrial dehydrogenases.