Melatonin as a therapy in cardiac ischemia-reperfusion injury: Potential mechanisms by which MT2 activation mediates cardioprotection

Abstract

Introduction: Previous studies reported the beneficial effects of pretreatment with melatonin on the heart during cardiac ischemia/reperfusion (I/R) injury. However, the effects of melatonin given after cardiac ischemia, as well as its comparative temporal effects are unknown. These include pretreatment, during ischemia, and at the onset of reperfusion. Also, the association between melatonin receptors and cardiac arrhythmias, mitochondrial function and dynamics, autophagy, and mitophagy during cardiac I/R have not been investigated.

Objectives: We tested two major hypotheses in this study. Firstly, the temporal effect of melatonin administration exerts different cardioprotective efficacy during cardiac I/R. Secondly, melatonin provides cardioprotective effects via MT2 activation, leading to improvement in cardiac mitochondrial function and dynamics, reduced excessive mitophagy and autophagy, and decreased cardiac arrhythmias, resulting in improved LV function.

Methods: Male rats were subjected to cardiac I/R, and divided into 4 intervention groups: vehicle, pretreatment with melatonin, melatonin given during ischemia, and melatonin given at the onset of reperfusion. In addition, either a non-specific melatonin receptor (MT) blocker or specific MT2 blocker was given to rats.

Results: Treatment with melatonin at all time points alleviated cardiac I/R injury to a similar extent, quantified by reduction in infarct size, arrhythmia score, LV dysfunction, cardiac mitochondrial dysfunction, imbalance of mitochondrial dynamics, excessive mitophagy, and a decreased Bax/Bcl2 ratio. In H9C2 cells, melatonin increased %cell viability by reducing mitochondrial dynamic imbalance and a decrease in Bax protein expression. The cardioprotective effects of melatonin were dependent on MT2 activation.

Conclusion: Melatonin given before or after ischemia exerted equal levels of cardioprotection on the heart with I/R injury, and its beneficial effects on cardiac arrhythmias, cardiac mitochondrial function and dynamics were dependent upon the activation of MT2.

Keywords: Cardiac I/R injury; Cardiomyocyte death; Left ventricular function; Melatonin; Melatonin receptors; Mitochondria.

Graphical abstract

Fig. 1

The effects of melatonin administration at different time points and the roles of melatonin receptors on myocardial infarct size and cell death pathways during cardiac I/R injury. (A) Representative and quantitative analysis of myocardial infarct size by Evan blue/TTC staining (n = 5–6/group); (B) cleaved-caspase3/total-caspase3 (n = 5–6/group); (C) Bax/Bcl2 ratio (n = 5–6/group); (D) Beclin1/Actin (n = 5–6/group); (E) LC3II/Actin (n = 5–6/group); (F) p62/Actin (n = 5–6/group). *p < 0.05 vs. vehicle. Abbreviations: I/R: ischemia reperfusion injury; AAR: area at risk; I: ischemic area; R: remote area; P-Mel: pretreatment with melatonin; I-Mel: melatonin administration during myocardial ischemia; R-Mel: melatonin administration at onset of reperfusion; MelLuz: melatonin and Luzindole administration; MelDot: melatonin and 4-PPDOT administration.

Fig. 2

The effects of melatonin administration at different time points and the roles of melatonin receptors on LV function during cardiac I/R injury. (A) Heart rate (n = 5–6/group); (B) Stroke volume (n = 5–6/group); (C) Left ventricular end systolic pressure (n = 5–6/group); (D) Left ventricular end diastolic pressure (n = 5–6/group); (E) + dP/dt max (n = 5–6/group); (F) -dP/dt min (n = 5–6/group). *p < 0.05 vs. its respective baseline. Abbreviations: P-Mel: pretreatment with melatonin; I-Mel: melatonin administration during myocardial ischemia; R-Mel: melatonin administration at onset of reperfusion; MelLuz: melatonin and Luzindole administration; MelDot: melatonin and 4-PPDOT administration.

Fig. 3

The effects of melatonin administration at different time points and the roles of melatonin receptors on cardiac arrhythmias and gap junction function during cardiac I/R injury. (A) Quantitative analysis of arrhythmia score based on the criteria of Curtis and Walker (n = 5–6/group); (B) p-Cx43^ser368/Cx43 (n = 5–6/group). *p < 0.05 vs. vehicle. Abbreviations: I: ischemic area; R: remote area; P-Mel: pretreatment with melatonin; I-Mel: melatonin administration during myocardial ischemia; R-Mel: melatonin administration at onset of reperfusion; MelLuz: melatonin and Luzindole administration; MelDot: melatonin and 4-PPDOT administration.

Fig. 4

The effects of melatonin administration at different time points and the roles of melatonin receptors on cardiac mitochondrial function during cardiac I/R injury. (A) Mitochondrial ROS level (n = 5–6/group); (B) Mitochondrial membrane potential changes (n = 5–6/group); (C) Mitochondrial swelling (n = 5–6/group); (D) Representative images of mitochondrial morphology taken by transmission electron microscopy (n = 5–6/group). *p < 0.05 vs. the remote area, †p < 0.05 vs ischemic area of the vehicle group. Abbreviations: P-Mel: pretreatment with melatonin; I-Mel: melatonin administration during myocardial ischemia; R-Mel: melatonin administration at onset of reperfusion; MelLuz: melatonin and Luzindole administration; MelDot: melatonin and 4-PPDOT administration; ROS: reactive oxygen species.

Fig. 5

The effects of melatonin administration at different time points and the roles of melatonin receptors on cardiac mitochondrial dynamics and mitophagy during cardiac I/R injury. (A) p-Drp1/Drp1 (n = 5–6/group); (B) Mfn1/Actin (n = 5–6/group); (C) Mfn2/Actin (n = 5–6/group); (D) OPA1/Actin (n = 5–6/group); (E) PINK1/Actin (n = 5–6/group); (F) Parkin/Actin (n = 5–6/group). *p < 0.05 vs. vehicle. Abbreviations: I: ischemic area; R: remote area; P-Mel: pretreatment with melatonin; I-Mel: melatonin administration during myocardial ischemia; R-Mel: melatonin administration at onset of reperfusion; MelLuz: melatonin and Luzindole administration; MelDot: melatonin and 4-PPDOT administration; Drp1: dynamin related protein 1; Mfn: mitofusin, OPA: optic atrophy protein.

Fig. 6

The effects of melatonin administration and the roles of its receptors on cell viability, survival, apoptosis, and mitochondrial dynamics in H9C2 cells subjected to H/R injury. (A) %cell viability (n = 5/group); (B) p-ERK/ERK (n = 5/group); (C) Bax/Actin (n = 5/group); (D) p-Drp1^ser616/Drp1 (n = 5/group); (E) Mfn2/Actin (n = 5/group). *p < 0.05 vs. vehicle; †p < 0.05 vs melatonin. Abbreviations: H/R: hypoxia/reoxygenation; Veh: vehicle; Mel: melatonin; MT: melatonin receptor; Drp1: dynamin related protein; Mfn: mitofusin.