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

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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.