Melatonin-Induced Protective Effects on Cardiomyocytes Against Reperfusion Injury Partly Through Modulation of IP3R and SERCA2a Via Activation of ERK1

Resumo Background: Melatonin is a neuroendocrine hormone synthesized primarily 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 Ca2+ 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.

摘要：背景：褪黑素（melatonin）是一种主要由松果体合成的神经内分泌激素，被证实可有效预防心肌再灌注损伤（myocardial reperfusion injury）。目前尚不明确褪黑素是否可通过调控细胞内钙稳态（intracellular calcium homeostasis）来减轻再灌注损伤对心功能的损害。目的：阐明褪黑素可通过激活心肌细胞（cardiomyocytes）中的细胞外调节蛋白激酶1（ERK1），调控三磷酸肌醇受体（IP3R）与肌浆网钙ATP酶2a（SERCA2a）以维持钙稳态，从而对抗心肌再灌注损伤。方法：采用模拟缺氧/复氧（hypoxia/reoxygenation，H/R）处理的H9C2细胞开展体外实验。通过蛋白质印迹法（Western Blots）检测ERK1、IP3R及SERCA2a的表达水平；采用脱氧核糖核苷酸末端转移酶介导的缺口末端标记法（TUNEL）检测心肌细胞凋亡情况；采用鬼笔环肽染色（Phalloidin-staining）评估心肌细胞的肌动蛋白丝组织形态改变；采用Fura-2/AM探针检测细胞内钙离子浓度。体内实验则采用大鼠心肌缺血/再灌注（myocardial ischemia/reperfusion，I/R）模型，通过免疫荧光染色检测心肌组织中IP3R与SERCA2a的表达水平。结果：体外实验结果显示，褪黑素可在缺氧/复氧处理的心肌细胞中激活ERK1，该效应可被ERK1抑制剂PD98059阻断。结果表明，褪黑素可对抗缺氧/复氧诱导的心肌细胞凋亡并改善肌动蛋白丝组织形态，且这两种效应均可被PD98059逆转。在缺氧/复氧处理的心肌细胞中，褪黑素可通过ERK1通路降低钙超载，进一步抑制IP3R表达并促进SERCA2a表达。褪黑素可使心肌组织中IP3R表达下调、SERCA2a表达上调，该效应同样可被PD98059逆转。结论：褪黑素介导的抗再灌注损伤心肌保护作用，至少部分是通过激活ERK1，调控IP3R与SERCA2a以维持细胞内钙稳态实现的。