Diazoxide opens the mitochondrial permeability transition pore and alters Ca2+ transients in rat ventricular myocytes.

Abstract

The mitochondrial K(ATP) channel (mitoK(ATP)) has been implicated as an end effector or trigger of ischemic preconditioning (IP). Although a mitoK(ATP) opener, diazoxide, mimics IP, mechanisms for the cardioprotective action remain unclear. We measured Ca2+ transients (CaTs) and mitochondrial inner membrane potential (Deltapsi(m)) with confocal microscopy and the fluorescent probes fluo-4 and tetramethylrhodamine ethyl ester perchlorate in rat ventricular myocytes. Diazoxide increased the amplitudes and diastolic levels of CaTs dose dependently. The effects of diazoxide on CaTs were inhibited by the mitoK(ATP) antagonist sodium 5-hydroxydecanoic acid (100 micromol/L), whereas application of diazoxide caused little change in Deltapsi(m). After sarcoplasmic reticulum function was disabled with ryanodine and thapsigargin, the effects of diazoxide on CaTs were still observed. The opening of the mitochondrial permeability transition pore was monitored with fluorescent calcein. Diazoxide accelerated the leakage of calcein from mitochondrial matrix (16% of control; P<0.05), and this effect was inhibited by cyclosporin A (2 micromol/L). Cyclosporin A also abolished the effects of diazoxide on CaTs. Diazoxide oxidized flavoprotein fluorescence reversibly, and this effect was partially blunted by cyclosporin A (by 24%; P<0.05). We conclude that in rat ventricular myocytes, diazoxide modulates the opening of the mitochondrial permeability transition pore, resulting in an increase in CaTs independent of the changes in Deltapsi(m). The action of diazoxide on the mitochondrial permeability transition pore also affects the mitochondrial redox state.