The mitochondrial origin of postischemic arrhythmias

Abstract

Recovery of the mitochondrial inner membrane potential (DeltaPsi(m)) is a key determinant of postischemic functional recovery of the heart. Mitochondrial ROS-induced ROS release causes the collapse of DeltaPsi(m) and the destabilization of the action potential (AP) through a mechanism involving a mitochondrial inner membrane anion channel (IMAC) modulated by the mitochondrial benzodiazepine receptor (mBzR). Here, we test the hypothesis that this mechanism contributes to spatiotemporal heterogeneity of DeltaPsi(m) during ischemia-reperfusion (IR), thereby promoting abnormal electrical activation and arrhythmias in the whole heart. High-resolution optical AP mapping was performed in perfused guinea pig hearts subjected to 30 minutes of global ischemia followed by reperfusion. Typical electrophysiological responses, including progressive AP shortening followed by membrane inexcitablity in ischemia and ventricular fibrillation upon reperfusion, were observed in control hearts. These responses were reduced or eliminated by treatment with the mBzR antagonist 4'-chlorodiazepam (4'-Cl-DZP), which blocks depolarization of DeltaPsi(m). When applied throughout the IR protocol, 4'-Cl-DZP blunted AP shortening and prevented reperfusion arrhythmias. Inhibition of ventricular fibrillation was also achieved by bolus infusion of 4'-Cl-DZP just before reperfusion. Conversely, treatment with an agonist of the mBzR that promotes DeltaPsi(m) depolarization exacerbated IR-induced electrophysiological changes and failed to prevent arrhythmias. The effects of these compounds were consistent with their actions on IMAC and DeltaPsi(m). These findings directly link instability of DeltaPsi(m) to the heterogeneous electrophysiological substrate of the postischemic heart and highlight the mitochondrial membrane as a new therapeutic target for arrhythmia prevention in ischemic heart disease.

Figure 1

Blockade of mitochondrial oscillations and stabilization of the cellular AP by 4′-Cl-DZP. Freshly isolated cardiomyocytes were loaded with TMRM (100 nM) at 37°C, and APs were recorded under whole-cell current-clamp conditions on the stage of the microscope as described in Methods. (A) The reversible effect of acutely added 4′-Cl-DZP (32 μM) on mitochondrial ΔΨ _m oscillations. (B) Mitochondrial oscillations in ΔΨ _m and the sarcolemmal APD were triggered after a highly localized laser flash (3 minutes before the train of oscillating APDs shown in this panel). APs evoked by brief current injections were recorded during the oscillations. Previously, we showed that during a synchronized cell-wide depolarization-repolarization cycle, the AP shortens in synchrony with fast mitochondrial depolarization (4). (C) During the APD oscillations, the cell becomes inexcitable when ΔΨ _m is fully depolarized (remaining upward spikes are from the stimulus only). (D) After addition of 64 μM 4′-Cl-DZP, a stable AP is restored and ΔΨ _m oscillations are suppressed.

Figure 2

Protection against ischemia-induced APD shortening by 4′-Cl-DZP. (A) Ischemia-induced APD shortening in control hearts and hearts treated with varying concentrations of 4′-Cl-DZP. (B) Representative APs from a control and a 100 μM 4′-Cl-DZP–treated heart recorded at various intervals during the ischemia protocol. At baseline, APD of control and 100 μM 4′-Cl-DZP–treated hearts were comparable.

Figure 3

Ischemia-induced APD shortening and representative APs recorded from hearts pretreated with 4.6 μM FGIN-1-27 (top panel), 0.2 μM CsA (middle panel), and 10 μM GLIBEN (bottom panel) compared with control untreated hearts.

Figure 4

Effects on ischemia-induced AP amplitude and upstroke velocity. (A) Progressive reduction in the APA during the first 10 minutes of ischemia in control, FGIN-1-27–, and 4′-Cl-DZP–treated hearts. FGIN-1-27–treated hearts exhibited a more enhanced reduction of APA compared with control and 4′Cl-DZP–treated hearts. (B) Comparison of normalized APA and dF/dt after 10 minutes of ischemia compared with preischemic baseline perfusion in untreated control hearts and hearts treated with FGIN-1-27, 4′-Cl-DZP, GLIBEN, or a combination of FGIN-1-27 and GLIBEN. *P < 0.05 vs. control; ^†P < 0.05 vs. FGIN-1-27. (C) Representative raw traces of dF/dt in control hearts and hearts treated with FGIN-1-27, 4′-Cl-DZP, GLIBEN, or a combination of FGIN-1-27 and GLIBEN.

Figure 5

Metabolic sink/block as a mechanism of conduction failure and arrhythmias. (A) Sequential isopotential contour maps recorded every 1.2 ms that display the level of membrane potential (color coded) across 464 epicardial sites simultaneously. Red indicates depolarized membrane potential, and blue indicates resting membrane potential. These maps demonstrate the sequential spread of activation across the epicardium of a representative guinea pig heart treated with FGIN-1-27 at 11 minutes of ischemia. Under these conditions, the wavefront fails to propagate across the entire mapping field (i.e., metabolic sink/block). (B) Representative AP traces (A to B, location marked on first contour map) recorded at 11 minutes of ischemia (left) and 10 minutes of reperfusion (right) indicating presence of conduction block (left) and arrhythmias with electrical silence at the same sites of conduction block (right).

Figure 6

Effects of drugs on post-ischemic arrhythmias. (A) Incidence of reperfusion-related arrhythmias in all groups. (B) Representative AP traces recorded in a control heart (left) and a heart pretreated with 4′-Cl-DZP (right) at various time points during IR, demonstrating the protection of 4′-Cl-DZP against reperfusion arrhythmias.

Figure 7

Post-ischemic AP recovery and arrhythmias. (A) Representative APs during recovery upon reperfusion in control, 4′-Cl-DZP–, CsA-, and FGIN-1-27–treated hearts. (B) Recovery of APD after 5 minutes of reperfusion as a percentage of baseline APD in hearts treated with 64 μM 4′-Cl-DZP and various concentrations of CsA, indicating that the optimal concentration was 0.2 μM. (C) Plot of the recovery of APD upon reperfusion normalized to the baseline APD before ischemia in 4′-Cl-DZP– and CsA-treated hearts.