Effects of 4'-chlorodiazepam on cellular excitation-contraction coupling and ischaemia-reperfusion injury in rabbit heart

Abstract

Aims: Recent evidence indicates that the activity of energy-dissipating ion channels in the mitochondria can influence the susceptibility of the heart to ischaemia-reperfusion injury. In this study, we describe the effects of 4'-chlorodiazepam (4-ClDzp), a well-known ligand of the mitochondrial benzodiazepine receptor, on the physiology of both isolated cardiomyocytes and intact hearts.

Methods and results: We used current- and voltage-clamp methods to determine the effects of 4-ClDzp on excitation-contraction coupling in isolated rabbit heart cells. At the level of the whole heart, we subjected rabbit hearts to ischaemia/reperfusion in order to determine how 4-ClDzp influenced the susceptibility to arrhythmias and contractile dysfunction. In isolated rabbit cardiomyocytes, 4-ClDzp evoked a significant reduction in the cardiac action potential that was associated with a decrease in calcium currents and peak intracellular calcium transients. In intact perfused normoxic rabbit hearts, 4-ClDzp mediated a dose-dependent negative inotropic response, consistent with the observation that 4-ClDzp was reducing calcium influx. Hearts that underwent 30 min of global ischaemia and 30 min of reperfusion were protected against reperfusion arrhythmias and post-ischaemic contractile impairment when 4-ClDzp (24 microM) was administered throughout the protocol or as a single bolus dose given at the onset of reperfusion. In contrast, hearts treated with cyclosporin-A, a classical blocker of the mitochondrial permeability transition pore, were not protected against reperfusion arrhythmias.

Conclusion: The findings indicate that the effects of 4-ClDzp on both mitochondrial and sarcolemmal ion channels contribute to protection against post-ischaemic cardiac dysfunction. Of clinical relevance, the compound is effective when given upon reperfusion, unlike other pre-conditioning agents.

Figure 1

Effects of 4′-chlorodiazepam (4-ClDzp) on rabbit ventricular action potential duration. (A) Representative action potential tracings approximating the mean action potential morphology in the study. (B) Mean time to 50 and 90% repolarization in myocytes used in the study (n = 16 cells for each group); *P < 0.05 vs. control (paired t-test).

Figure 2

Transmembrane currents and calcium transients recorded from rabbit cardiomyocytes before and after the addition of 24 μM 4′-chlorodiazepam (4-ClDzp). (A) Representative current tracing from the same myocytes before (black line) and after (blue line) exposure to 4′-chlorodiazepam. (B) Current density for rabbit myocytes (n = 12) at 50 and 150 ms after a test pulse to 10 mV without (black bars) and with (blue bars) 4′-chlorodiazepam. (C) Representative calcium transients from the same cell in the absence (black line) and presence (blue line) of 4′-chlorodiazepam. (D) Current-voltage plot for calcium currents in the study. (E) Indo-1-mediated intracellular calcium transient recordings from the same cell under control (black line) and 4′-chlorodiazepam (blue line) conditions. (F) Peak calcium concentrations in rabbit cardiomyocytes with and without the addition of 4′-chlorodiazepam. *P < 0.05 vs. myocytes under control conditions .

Figure 3

Dose-response curve describing the negative inotropic effect of various concentrations of 4′-chlorodiazepam (4-ClDzp) on left ventricular developed pressure (LVDP) in intact rabbit hearts. Animal numbers were n = 7, 4, 6, and 1 for 0, 12, 24, and 64 μM 4′-chlorodiazepam.

Figure 4

Representative left ventricular pressure tracings and ECG waveforms for the last 5 s of reperfusion in (A) control hearts, (B) hearts receiving 24 μM 4′-chlorodiazepam (4-ClDzp) beginning 10 min prior to ischaemia through the duration of reperfusion, (C) hearts receiving a bolus of 4′-chlorodiazepam at the onset of reperfusion, and (D) hearts receiving 0.2 μM cyclosporin-A 10 min prior to ischaemia through the duration of reperfusion .

Figure 5

Arrhythmia scores tabulated for the entire 30 min reperfusion period for hearts in the study. *P < 0.05 vs. control hearts (ANOVA with Dunnett’s t-test) .

Figure 6

(A) Left ventricular end-diastolic pressure for rabbit hearts during the 30 min ischaemic period. ⁺P < 0.05 for 24 μM 4′-chlorodiazepam (4-ClDzp) when compared with control (repeated measures ANOVA). (B) Left ventricular developed pressure (LVDP) for the 30 min reperfusion period for hearts receiving 4′-chlorodiazepam either prior to ischaemia (24 μM 4′-chlorodiazepam) or as a bolus at the onset of reperfusion. *P < 0.05 for both 4′-chlorodiazepam groups when compared with control (repeated measures ANOVA) .