ABC | Volume 110, Nº1, January 2018

Original Article Melatonin-Induced Protective Effects on Cardiomyocytes Against Reperfusion Injury Partly Through Modulation of IP3R and SERCA2a Via Activation of ERK1 Shunying Hu, Pingjun Zhu, Hao Zhou, Ying Zhang, Yundai Chen Chinese PLA General Hospital, Beijing – China Mailing Address: Shunying Hu • 28 Fuxing Road, 100853, Beijing – China E-mail: hsylily@163.com Manuscript received January 24, 2017, revised manuscript July 31, 2017, accepted September 22, 2017 DOI: 10.5935/abc.20180008 Resumo Background: Melatonin is a neuroendocrine hormone synthesizedprimarily by the pineal gland that is indicated to effectively prevent myocardial reperfusion injury. It is unclear whether melatonin protects cardiac function from reperfusion injury by modulating intracellular calcium homeostasis. Objective: Demonstrate that melatonin protect against myocardial reperfusion injury through modulating IP3R and SERCA2a to maintain calcium homeostasis via activation of ERK1 in cardiomyocytes. Methods: In vitro experiments were performed using H9C2 cells undergoing simulative hypoxia/reoxygenation (H/R) induction. Expression level of ERK1, IP3R and SERCA2a were assessed by Western Blots. Cardiomyocytes apoptosis was detected by TUNEL. Phalloidin-staining was used to assess alteration of actin filament organization of cardiomyocytes. Fura-2 /AM was used to measure intracellular Ca 2+ concentration. Performing in vivo experiments, myocardial expression of IP3R and SERCA2a were detected by immunofluorescence staining using myocardial ischemia/ reperfusion (I/R) model in rats. Results: In vitro results showed that melatonin induces ERK1 activation in cardiomyocytes against H/R which was inhibited by PD98059 (ERK1 inhibitor). The results showed melatonin inhibit apoptosis of cardiomyocytes and improve actin filament organization in cardiomyocytes against H/R, because both could be reversed by PD98059. Melatonin was showed to reduce calcium overload, further to inhibit IP3R expression and promote SERCA2a expression via ERK1 pathway in cardiomyocytes against H/R. Melatonin induced lower IP3R and higher SERCA2a expression in myocardium that were reversed by PD98059. Conclusion: melatonin-induced cardioprotection against reperfusion injury is at least partly through modulation of IP3R and SERCA2a to maintain intracellular calcium homeostasis via activation of ERK1. (Arq Bras Cardiol. 2018; 110(1):44-51) Keywords: Melatonin; Myocardial Reperfusion; Cardiac Myocytes; Myocardial Infarction; Heart Failure. Introduction Myocardial ischemia-reperfusion injury typically arises in patients presenting with acute ST-segment elevationmyocardial infarction (STEMI), in whom the most effective therapeutic intervention for reducing acute myocardial ischemic injury and limiting the size of myocardial infarction (MI) is timely and effective myocardial reperfusion therapy. 1 However, the process of myocardial reperfusion can itself induce further myocardial reperfusion injury. 1-4 Myocardial reperfusion injury weakens the benefit of reperfusion therapy and brings to patients larger MI size, more severe heart failure and worse prognosis. Restoration of cardiac circulation is accompanied by cell damages and death (lethal reperfusion injury), reperfusion arrhythmias, myocardial stunning, and no‑reflow phenomenon. Lethal reperfusion injury (cardiomyocyte death induced by reperfusion) is a key therapeutic target with anticipated significant impact on the patient’s prognosis. 1-6 Melatonin (N-acetyl-5-methoxytryptamine) is a neuroendocrine hormone, which is synthesized primarily by the pineal gland. 7,8 Melatonin presents profound protective effects against myocardial ischemia-reperfusion injury through antioxidant actions. 9-18 Ca 2+ overload is the primary stimulator to ischemia/reperfusion injury and induce cardiomyocytes apoptosis in ischemia/reperfusion condition. It is unclear whether melatonin protects cardiac function from reperfusion injury by modulating intracellular calcium homeostasis. Yeung et al. 19 suggested that melatonin is cardioprotective against chronic hypoxia-induced myocardial injury because it improves calcium handling in the sarcoplasmic reticulum (SR) of cardiomyocytes via an antioxidant mechanism. However, theevidenceaboutmelatonin’s effect andunderlying mechanism on Ca 2+ overload under acute ischemia/reperfusion is rare. The cardiac inositol 1,4,5-trisphosphate receptors (IP3R) and sarcoplasmic reticulum Ca 2+ -ATPase (SERCA2a) are key mediators for intracellular calcium handling, contractility and 44