Regional Oxidative Stress Disrupts the Normal Propagation of Voltage Waves and Promotes Reentry in Monolayers of Cardiac Myocytes

Abstract

It has been suggested that the oxidative stress due to ischemic injury plays an important role in arrhythmogenesis. Reactive oxygen species can activate the mitochondrial inner membrane anion channel causing mitochondrial depolarization, which can lead to arrhythmias as we previously showed using experimental and computational models. In particular, our previous experiments showed that coverslip-induced ischemia leads to oxidation of GSH/GSSG couple. Here, we study the effects of regional depletion of GSH pool on mitochondrial function and propagation of the voltage wave in monolayers of cardiac myocytes. Neonatal rat ventricular myocytes were isolated and cultured on fibronectin-coated plastic coverslips (D = 2.1 cm) for 5-7 days. Mitochondrial inner membrane potential was observed using potentiometric fluorescent dye, TMRM, in the dequench mode (2 μM). Optical mapping was used to record the changes in sarcolemmal membrane potential using voltage-sensitive fluorescent dye, di-4-ANEPPS (5 μM), as the voltage waves elicited by a stimulation pulse train, propagated through the monolayer. A thiol oxidizing agent, diamide (1 mM) was locally perfused in the center part (D = 0.5 cm) of the monolayer, while the rest of the coverslip received normal Tyrode's solution. The resulting GSH oxidation led to mitochondrial depolarization (left figure). Action potential amplitude decrease was followed by complete inexcitability in 20 minutes and reentry occurred in 2 out of 3 monolayers (right figure). There was no incidence of reentry in control. The results emphasize the role of oxidative stress in mitochondrial dysfunction and the following post-ischemic arrhythmias. This work was supported by NIH's R01HL105216 and R37HL54598 (B.O'R.) and AHA's 14POST20000018 (S.S.).