Mitochondrial depolarization underlies delay in permeability transition by preconditioning with isoflurane: roles of ROS and Ca2+.

During reperfusion, the interplay between excess reactive oxygen species (ROS) production, mitochondrial Ca²⁺ overload, and mitochondrial permeability transition pore (mPTP) opening, as the crucial mechanism of cardiomyocyte injury, remains intriguing. Here, we investigated whether an induction of a partial decrease in mitochondrial membrane potential (ΔΨ_m) is an underlying mechanism of protection by anesthetic-induced preconditioning (APC) with isoflurane, specifically addressing the interplay between ROS, Ca²⁺, and mPTP opening. The magnitude of APC-induced decrease in δΨ_m was mimicked with the protonophore 2.4-dinitrophenol (DNP), and the addition of pyruvate was used to reverse APC- and DNP-induced decrease in ΔΨ_m. In cardiomyocytes. ΔΨ_m, ROS, mPTP opening, and cytosolic and mitochondrial Ca²⁺ were measured using confocal microscope, and cardiomyocyte survival was assessed by Trypan blue exclusion. In isolated cardiac mitochondria, antimycin A-induced ROS production and Ca²⁺ uptake were determined spectrofluorometrically. In cells exposed to oxidative stress, APC and DNP increased cell survival, delayed mPTP opening, and attenuated ROS production, which was reversed by mitochondrial repolarization with pyruvate. In isolated mitochondria, depolarization by APC and DNP attenuated ROS production, but not Ca²⁺ uptake. However, in stressed cardiomyocytes, a similar decrease in ΔΨ_m attenuated both cytosolic and mitochondrial Ca²⁺ accumulation. In conclusion, a partial decrease in ΔΨ_m underlies cardioprotective effects of APC by attenuating excess ROS production, resulting in a delay in mPTP opening and an increase in cell survival. Such decrease in ΔΨ_m primarily attenuates mitochondrial ROS production, with consequential decrease in mitochondrial Ca²⁺ uptake. [ABSTRACT FROM AUTHOR]