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

Abstract

-During reperfusion, the interplay between excess reactive oxygen species (ROS) production, mitochondrial Ca 2ϩ 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 (⌬⌿ m) is an underlying mechanism of protection by anesthetic-induced preconditioning (APC) with isoflurane, specifically addressing the interplay between ROS, Ca 2ϩ , and mPTP opening. The magnitude of APCinduced decrease in ⌬⌿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 ⌬⌿ m. In cardiomyocytes, ⌬⌿ m, ROS, mPTP opening, and cytosolic and mitochondrial Ca 2ϩ were measured using confocal microscope, and cardiomyocyte survival was assessed by Trypan blue exclusion. In isolated cardiac mitochondria, antimycin A-induced ROS production and Ca 2ϩ 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 Ca 2ϩ uptake. However, in stressed cardiomyocytes, a similar decrease in ⌬⌿m attenuated both cytosolic and mitochondrial Ca 2ϩ accumulation. In conclusion, a partial decrease in ⌬⌿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 ⌬⌿m primarily attenuates mitochondrial ROS production, with consequential decrease in mitochondrial Ca 2ϩ uptake.cardioprotection; oxidative stress; mitochondria; reactive oxygen species DAMAGE DURING ISCHEMIA and reperfusion (I/R) of the heart involves complex processes where the cellular machinery itself becomes a source of deleterious mediators of injury. Such processes include excessive production of mitochondrial reactive oxygen species (ROS) and cellular Ca 2ϩ overload (6, 37), which trigger opening of the mitochondrial permeability transition pore (mPTP) (16, 19), a critical event in the transition towards cell death (18). The mPTP opening instantly dissipates mitochondrial membrane potential (⌬⌿ m ), ceases mitochondrial ATP production, and initiates cell death pathways (6).Studies suggest that most of the cell death occurs during reperfusion (51, 53), which can be attenuated with antioxidants (52), indicating an important role of ROS. In cardiomyocytes, ROS are primarily generated at complexes I and III of the mitochondrial electron transport chain (50). During I/R, accumulation of cytosolic Ca 2ϩ , which drives accumulation of mitochondrial Ca 2ϩ (45), is primarily attributed to insufficient ATP production and derangement of intracellular ion homeostasis (37). It is suggested that excess ROS production and mitochondrial Ca 2ϩ overload are mutually de...

Ischemia/Reperfusion Injury Protection by Mesenchymal Stem Cell Derived Antioxidant Capacity

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