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

我们测定了负载Rhod-2AM后被皂苷通透的大鼠心室肌细胞线粒体基质中的钙离子。用含300 nM Ca~(2+)和0 mM Na~+的浴液灌流可增加Rhod-2的强度。这种增加可被钙单转运体的抑制剂Ru红完全消除，支持钙转运体是线粒体主要的钙内流途径。当浴液中含有0 Ca~(2+)和6 mM Na~(2+)时，Rhod-2信号有明显的衰减，而未加Na~(2+)时仅有轻微的衰减。因此，Na~(++)/Ca~(2+)~(2+)交换(MNCX)是心肌线粒体Ca~(2+)外流的主要机制。线粒体膜电位(ΔΨ)对mNCX的依赖性一直存在争议。我们用薄层磁共振仪测定了ΔΨ，实验方法与上述相同，通过mNCX诱导Ca~(2+)外流。然而，TMRE的荧光没有明显变化。为进一步研究ΔΨ依赖性，用BK通道开放剂NS1619、H+离子载体FCCP或去除线粒体底物(丙酮酸、琥珀酸、苹果酸)去极化线粒体膜，测定线粒体膜Ca~(2+)外流速率。Ca~(2+)流出速率与对照条件下测得的结果相近。在上述实验数据的基础上，我们重建了线粒体钙动力学的计算机模型，该模型由单转运体、单转运体和一个假定的钙离子缓冲液组成，并将其纳入心肌兴奋-收缩偶联的综合模型(ΔΨ模型)。模型分析表明，NADH的变化依赖于敲击频率，是由于线粒体脱氢酶的ADP和无机磷(ATP水解产物)增加以及Ca~(2+)和Gt~(2+)激活所致。

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.