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

Abstract

We have previously proposed that the heterogeneous collapse of mitochondrial inner membrane potential (DeltaPsi(m)) 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 K(ATP) current levels alter electrical propagation to promote reentry. Single myocyte studies have also shown that cell-wide DeltaPsi(m) 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 DeltaPsi(m) 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 microM 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 microM): arrhythmia scores with diamide+4-ClDzp were 0.4+/-0.2 (n=5; P<0.05 vs. diamide alone). Imaging DeltaPsi(m) in intact hearts revealed the heterogeneous collapse of DeltaPsi(m) beginning 20 min into diamide, paralleling the timeframe for the onset of arrhythmias. Loss of DeltaPsi(m) 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.

Figure 1

Content of glutathione in guinea pig hearts. Control bars represent hearts that were perfused under normoxic conditions in the absence of diamide. A. Total myocardial glutathione content (GSH_t); B. Content of oxidized glutathione (GSSG); C. Ratio of GSH/GSSG in hearts; *, P < 0.05 versus untreated (ANOVA).

Figure 1

Content of glutathione in guinea pig hearts. Control bars represent hearts that were perfused under normoxic conditions in the absence of diamide. A. Total myocardial glutathione content (GSH_t); B. Content of oxidized glutathione (GSSG); C. Ratio of GSH/GSSG in hearts; *, P < 0.05 versus untreated (ANOVA).

Figure 2

Representative LV Pressure and ECG from hearts in the study. A. Control heart after 10 minutes of baseline perfusion with simultaneous LV Pressure (red) and ECG (blue). B. LV Pressure and ECG in a heart 6 minutes into the washout period following the diamide treatment showing the transition to VF (blue) with concomitant loss of pump function (red). C. LV Pressure and ECG in a heart 6 minutes into the washout period following diamide treatment plus 64μM 4-ClDzp.

Figure 2

Representative LV Pressure and ECG from hearts in the study. A. Control heart after 10 minutes of baseline perfusion with simultaneous LV Pressure (red) and ECG (blue). B. LV Pressure and ECG in a heart 6 minutes into the washout period following the diamide treatment showing the transition to VF (blue) with concomitant loss of pump function (red). C. LV Pressure and ECG in a heart 6 minutes into the washout period following diamide treatment plus 64μM 4-ClDzp.

Figure 3

Arrhythmia scores for hearts during treatment with diamide and washout in the presence or absence of 64μM 4-ClDzp.

Figure 4

Left ventricular developed pressure (LVDP) for hearts in the study. Hearts were perfused with diamide for 25 minutes in the presence or absence of 64μM 4-ClDzp and then washed out for 15 minutes. LVDP values are normalized to baseline values.

Figure 5

Imaging of ΔΨ_m in intact guinea pig hearts using 2-photon microscopy after exposure to diamide (top) and diamide + 4-ClDzp. Bar is equal to 100μM.

Figure 6

Data from hearts exposed to 30/30 min of ischemia/reperfusion. Representative LV Pressure (red) and ECG (blue) traces for control (A) and 4-ClDzp-treated (B) hearts. C. Arrhythmia scores for hearts exposed to ischemia and reperfusion in the absence or presence of 64μM 4-ClDzp. D. LV Developed Pressure for hearts after 30 minutes of global ischemia.

Figure 6

Data from hearts exposed to 30/30 min of ischemia/reperfusion. Representative LV Pressure (red) and ECG (blue) traces for control (A) and 4-ClDzp-treated (B) hearts. C. Arrhythmia scores for hearts exposed to ischemia and reperfusion in the absence or presence of 64μM 4-ClDzp. D. LV Developed Pressure for hearts after 30 minutes of global ischemia.

Figure 6

Data from hearts exposed to 30/30 min of ischemia/reperfusion. Representative LV Pressure (red) and ECG (blue) traces for control (A) and 4-ClDzp-treated (B) hearts. C. Arrhythmia scores for hearts exposed to ischemia and reperfusion in the absence or presence of 64μM 4-ClDzp. D. LV Developed Pressure for hearts after 30 minutes of global ischemia.