Original article
Cardiac arrhythmias induced by glutathione oxidation can be inhibited by preventing mitochondrial depolarization

https://doi.org/10.1016/j.yjmcc.2009.11.011Get rights and content

Abstract

We have previously proposed that the heterogeneous collapse of mitochondrial inner membrane potential (ΔΨm) during ischemia and reperfusion contributes to arrhythmogenesis through the formation of metabolic sinks in the myocardium, wherein clusters of myocytes with uncoupled mitochondria and high KATP current levels alter electrical propagation to promote reentry. Single myocyte studies have also shown that cell-wide ΔΨm depolarization, through a reactive oxygen species (ROS)-induced ROS release mechanism, can be triggered by global depletion of the antioxidant pool with diamide, a glutathione oxidant. Here we examine whether diamide causes mitochondrial depolarization and promotes arrhythmias in normoxic isolated perfused guinea pig hearts. We also investigate whether stabilization of ΔΨm with a ligand of the mitochondrial benzodiazepine receptor (4′-chlorodiazepam; 4-ClDzp) prevents the formation of metabolic sinks and, consequently, precludes arrhythmias. Oxidation of the GSH pool was initiated by treatment with 200 μM diamide for 35 min, followed by washout. This treatment increased GSSG and decreased both total GSH and the GSH/GSSG ratio. All hearts receiving diamide transitioned from sinus rhythm into ventricular tachycardia and/or ventricular fibrillation during the diamide exposure: arrhythmia scores were 5.5 ± 0.5; n = 6 hearts. These arrhythmias and impaired LV function were significantly inhibited by co-administration of 4-ClDzp (64 μM): arrhythmia scores with diamide + 4-ClDzp were 0.4 ± 0.2 (n = 5; P < 0.05 vs. diamide alone). Imaging ΔΨm in intact hearts revealed the heterogeneous collapse of ΔΨm beginning 20 min into diamide, paralleling the timeframe for the onset of arrhythmias. Loss of ΔΨm was prevented by 4-ClDzp treatment, as was the increase in myocardial GSSG. These findings show that oxidative stress induced by oxidation of GSH with diamide can cause electromechanical dysfunction under normoxic conditions. Analogous to ischemia–reperfusion injury, the dysfunction depends on the mitochondrial energy state. Targeting the mitochondrial benzodiazepine receptor can prevent electrical and mechanical dysfunction in both models of oxidative stress.

Introduction

Sudden death from cardiac arrhythmias is a leading cause of mortality in the industrialized world. Despite advances in treatment, a paucity of information exists regarding the sub-cellular events that set the stage for arrhythmias at the organ level, thus limiting treatment strategies aimed at prevention of arrhythmogenesis.

The oxidative stress elicited by ischemia reaches a zenith during early reperfusion and is commonly associated with electromechanical dysfunction [1], [2]. In isolated myocytes, we have previously shown that oxidative stress (elicited by either laser flash or substrate deprivation) can lead to lability in action potential duration [3], [4]. The underlying cause of action potential heterogeneity was subsequently found to be due to oscillations in mitochondrial membrane potential (ΔΨm), which couple directly to cellular excitability via the activity of sarcolemmal ATP-sensitive potassium channels [3]. Treatment with ligands of the mitochondrial benzodiazepine receptor (mBzR) which are known to inhibit mitochondrial inner membrane anion channel (IMAC) activity (see [5] for review), stopped the ΔΨm oscillations in single cells, stabilizing ΔΨm in the polarized state [3]. Consistent with the hypothesis that rapid ΔΨm loss could activate sarcolemmal KATP channels and cause arrhythmias in the whole heart, 4-chlorodiazepam (4-ClDzp) successfully prevented reperfusion arrhythmias in intact guinea pig [6] and rabbit [7] hearts.

We have also shown that overwhelming the cellular antioxidant defenses with diamide, which chemically oxidizes the glutathione pool [8], leads to an accumulation of ROS in mitochondria of isolated cardiomyocytes. The accumulation of ROS ultimately reached a threshold level, resulting in oscillations in ΔΨm and NADH [9]. Similar to previous observations [3], 4-ClDzp prevented or reversed the oscillations induced by diamide treatment [9], providing further evidence that oxidative stress elicits oscillations in ΔΨm via the cyclical activation of IMAC.

In the intact heart, we recently observed oscillations in ΔΨm following diamide treatment [10]. Administration of diamide to the heart caused a gradual oxidation of the glutathione pool, ultimately leading to increased ROS production and ΔΨm collapse, resembling hearts exposed to ischemia/reperfusion [10]. However, we did not test whether diamide could induce arrhythmias, and if 4-ClDzp could prevent the collapse in ΔΨm evoked by diamide.

In light of our previous observations, here we examine whether overwhelming the cellular antioxidant defenses with diamide could induce arrhythmias in the intact heart under normoxic conditions. We also subjected hearts to a well-established model of oxidative stress, global ischemia and reperfusion, to show that these two distinct models of oxidative stress cause arrhythmias via the activity of the mBzR. We hypothesized that blocking the mBzR with 4-ClDzp would prevent the diamide-induced collapse in ΔΨm and prevent arrhythmias in guinea pig hearts.

Section snippets

Animals

Adult male guinea pigs (age 4–6 months) were housed in an animal facility with food and water provided ad libitum. All experiments were performed with prior approval from the Institutional Animal Care and Use Committee at both the Johns Hopkins University School of Medicine and East Carolina University, and were in accordance with guidelines established by the American Physiological Society.

Experimental protocol

On each day of the experiment, animals were injected with pentobarbital (35 mg kg 1, ip injection) and

Myocardial glutathione content

Diamide treatment led to a significant decrease in total glutathione (GSHt; Fig. 1A; P < 0.05), and 4-ClDzp did not prevent the decreased GSHt. There were no differences in GSHt between diamide and diamide + 4-ClDzp groups (ANOVA). Administration of diamide resulted in a significant increase in GSSG (Fig. 1B, P < 0.05), and the presence of 4-ClDzp prevented the GSSG increase. The GSH/GSSG ratio in all diamide-treated hearts was significantly lower after diamide, and the decrease in GSH/GSSG was not

Discussion

In this study, we hypothesized that overwhelming the antioxidant defenses with diamide in normoxic perfused hearts would trigger mitochondrial criticality [15], leading to lethal arrhythmias in isolated hearts. Diamide treatment mediated the collapse of ΔΨm, increased arrhythmias, and initiated mechanical dysfunction. A ligand of the mBzR, 4'-chlorodiazepam (4-ClDzp), prevented the collapse in ΔΨm evoked by diamide and protected guinea pig hearts from electromechanical dysfunction. The

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