Blockade of Melatonin Receptors Abolishes Its Antiarrhythmic Effect and Slows Ventricular Conduction in Rat Hearts

Abstract

Melatonin has been reported to cause myocardial electrophysiological changes and prevent ventricular tachycardia or fibrillation (VT/VF) in ischemia and reperfusion. We sought to identify electrophysiological targets responsible for the melatonin antiarrhythmic action and to explore whether melatonin receptor-dependent pathways or its antioxidative properties are essential for these effects. Ischemia was induced in anesthetized rats given a placebo, melatonin, and/or luzindole (MT1/MT2 melatonin receptor blocker), and epicardial mapping with reperfusion VT/VFs assessment was performed. The oxidative stress assessment and Western blotting analysis were performed in the explanted hearts. Transmembrane potentials and ionic currents were recorded in cardiomyocytes with melatonin and/or luzindole application. Melatonin reduced reperfusion VT/VF incidence associated with local activation time in logistic regression analysis. Melatonin prevented ischemia-related conduction slowing and did not change the total connexin43 (Cx43) level or oxidative stress markers, but it increased the content of a phosphorylated Cx43 variant (P-Cx43³⁶⁸). Luzindole abolished the melatonin antiarrhythmic effect, slowed conduction, decreased total Cx43, protein kinase Cε and P-Cx43³⁶⁸ levels, and the IK1 current, and caused resting membrane potential (RMP) depolarization. Neither melatonin nor luzindole modified INa current. Thus, the antiarrhythmic effect of melatonin was mediated by the receptor-dependent enhancement of impulse conduction, which was associated with Cx43 phosphorylation and maintaining the RMP level.

Keywords: conduction velocity; connexin-43; melatonin; post-ischemic arrhythmias; potassium current; rat heart; sodium current.

Figure 1

Methods: Panel (A): Schematic presentation of the study design. The tested substances were administered just before reperfusion (protocol a) and in baseline (protocol b). After in vivo electrophysiological study, the heart was rapidly excised to be used for the measurements of oxidative stress parameters, Western blot analysis of Cx43, its variant P-Cx43³⁶⁸, and protein kinase C Epsilon (PKCƐ), as well as for in vitro electrophysiological study (placebo group only) in protocols a and b, respectively. Panel (B): The lead terminals’ surface of the recording plate (4 × 4 mm, 64 leads, 2 stimulating electrodes). Electrical step symbol indicates a pacing site. Panel (C): View of the in vivo heart preparation with the recording plate positioned on the left ventricle. Panel (D): Representative examples of the baseline activation map and the electrograms recorded at sites a, b, and c, which were used for calculation of the longitudinal and transverse conduction velocity. “St” indicates a pacing artifact.

Figure 2

Electrophysiological parameters in the left ventricle at reperfusion with the infusion of placebo (Control), melatonin (Mel), luzindole (Luz), and melatonin + luzindole (Mel + Luz) prior to reperfusion (see protocol a). Bar graphs depict average AT at reperfusion (panel (A)) and DOR at reperfusion (panel (B)). Data are presented as mean ± SEM. Panel (C) depicts the incidence of ventricular tachycardia (VT) and ventricular fibrillation (VF). * p < 0.05 vs. control.

Figure 3

Representative left ventricular isochrone activation maps and unipolar electrograms at the specified sites (a, b, c) during electrical pacing of the control- and luzindole-treated rats. Electrical step symbol indicates the site of pacing. Note the longer periods (maximal values on scales) needed for activation spread across the mapped area with luzindole application.

Figure 4

Conduction velocity (CV) in the left ventricle in the control, melatonin, luzindole, and melatonin + luzindole groups (mean ± SEM). Melatonin prevents ischemia CV slowing. All tested substances were infused at baseline (see protocol b in Figure 1). Bar graphs depict the longitudinal (CV_L, panel (A)) and transverse (CV_T, panel (B)) at the baseline state. Linear graphs depict the changes in the CV_L (panel (C)) and the CV_T (panel (D)) at ischemia and reperfusion. * p < 0.05 vs. control.

Figure 5

Effect of melatonin on the levels of superoxide dismutase activity (SOD, panel (A)), 4-hydroxynonenal adducts content (4-HNE, panel (B)), total antioxidant capacity (TAC, panel (C)), and total glutathione content (GSH, panel (D)). No significant differences were found.

Figure 6

Patch-clamp studies of the IK1 (panels (A,B)) and INa (panels (C,D)) ionic currents in the control cardiomyocytes and with the application of luzindole. Voltage–current curves (panels (A,C)) and peak current densities (panels (B,D)) for both currents are presented. Luzindole caused a decrease in the IK1 current and no effect on INa. Comparison of peak current densities of IK1 in the control and luzindole groups for outward potassium current (upper) and inward potassium current (lower) (panel (B)). Comparison of voltage–current curves (panel (C)) and peak current densities (panel (D)) of INa in the control and luzindole groups; no significant differences were observed. Effect of luzindole on action potential in the current-clamp mode (representative recordings, panel (E)). * p < 0.0005 vs. control.

Figure 7

Immunoblot demonstration and protein levels of myocardial Cx43 (A), functional P-Cx43³⁶⁸ variant (B), and PKCε (C) in experimental rats. Note the tendency of melatonin to increase these protein levels, while luzindole abolished this effect. Cx43—connexin 43, P-Cx43³⁶⁸—Connexin 43 variant phosphorylated at serine 368, PKCε- protein kinase C Epsilon, * p < 0.05 vs. Control. Data are presented as means ± SD, n = 6 per group.