Mitochondrial membrane potential modulates regulation of mitochondrial Ca2+in rat ventricular myocytes

Abstract

Although recent studies focused on the contribution of mitochondrial Ca²⁺to the mechanisms of ischemia-reperfusion injury, the regulation of mitochondrial Ca²⁺under pathophysiological conditions remains largely unclear. By using saponin-permeabilized rat myocytes, we measured mitochondrial membrane potential (ΔΨ_m) and mitochondrial Ca²⁺concentration ([Ca²⁺]_m) at the physiological range of cytosolic Ca²⁺concentration ([Ca²⁺]_c; 300 nM) and investigated the regulation of [Ca²⁺]_mduring both normal and dissipated ΔΨ_m. When ΔΨ_mwas partially depolarized by carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP, 0.01–0.1 μM), there were dose-dependent decreases in [Ca²⁺]_m. When complete ΔΨ_mdissipation was achieved by FCCP (0.3–1 μM), [Ca²⁺]_mremained at one-half of the control level despite no Ca²⁺influx via the Ca²⁺uniporter. The ΔΨ_mdissipation by FCCP accelerated calcein leakage from mitochondria in a cyclosporin A (CsA)-sensitive manner, which indicates that ΔΨ_mdissipation opened the mitochondrial permeability transition pore (mPTP). After FCCP addition, inhibition of the mPTP by CsA caused further [Ca²⁺]_mreduction; however, inhibition of mitochondrial Na⁺/Ca²⁺exchange (mitoNCX) by a Na⁺-free solution abolished this [Ca²⁺]_mreduction. Cytosolic Na⁺concentrations that yielded one-half maximal activity levels for mitoNCX were 3.6 mM at normal ΔΨ_mand 7.6 mM at ΔΨ_mdissipation. We conclude that 1) the mitochondrial Ca²⁺uniporter accumulates Ca²⁺in a manner that is dependent on ΔΨ_mat the physiological range of [Ca²⁺]_c; 2) ΔΨ_mdissipation opens the mPTP and results in Ca²⁺influx to mitochondria; and 3) although mitoNCX activity is impaired, mitoNCX extrudes Ca²⁺from the matrix even after ΔΨ_mdissipation.