M3 Subtype of Muscarinic Acetylcholine Receptor Promotes Cardioprotection via the Suppression of miR-376b-5p

Zhenyu Pan,Yong Zhang,Shu Wang,Jing Xie,Yuhua Fan,Ning Wang,Rong Huo,Zhimin Du,Yunlong Hou,Yan Liu,Hanping Qi,Kai Fan,Yueping Guo,Feng Guo,Hua Zhao,Karl-Wilhelm Koch

doi:10.1371/journal.pone.0032571

Abstract

The M3 subtype of muscarinic acetylcholine receptors (M3-mAChR) plays a protective role in myocardial ischemia and microRNAs (miRNAs) participate in many cardiac pathophysiological processes, including ischemia-induced cardiac injury. However, the role of miRNAs in M3-mAChR mediated cardioprotection remains unexplored. The present study was designed to identify miRNAs that are involved in cardioprotective effects of M3-mAChR against myocardial ischemia and elucidate the underlying mechanisms. We established rat model of myocardial ischemia and performed miRNA microarray analysis to identify miRNAs involved in the cardioprotection of M3-mAChR. In H9c2 cells, the viability, intracellular free Ca2+ concentration ([Ca2+]i), intracellular reactive oxygen species (ROS), miR-376b-5p expression level, brain derived neurophic factor (BDNF) and nuclear factor kappa-B (NF-κB) levels were measured. Our results demonstrated that M3-mAChR protected myocardial ischemia injury. Microarray analysis and qRT-PCR revealed that miR-376b-5p was significantly up-regulated in ischemic heart tissue and the M3-mAChRs agonist choline reversed its up-regulation. In vitro, miR-376b-5p promoted H2O2-induced H9c2 cell injuries measured by cells viability, [Ca2+]i and ROS. Western blot and luciferase assay identified BDNF as a direct target of miR-376b-5p. M3-mAChR activated NF-κB and thereby inhibited miR-376b-5p expression. Our data show that a novel M3-mAChR/NF-κB/miR-376b-5p/BDNF axis plays an important role in modulating cardioprotection. MiR-376b-5p promotes myocardial ischemia injury possibly by inhibiting BDNF expression and M3-mAChR provides cardioprotection at least partially mediated by the downregulation of miR-376b-5p through NF-κB. These findings provide new insight into the potential mechanism by which M3-mAChR provides cardioprotection against myocardial ischemia injury.

Highlights

Ischemic heart disease is the leading cause of death in the industrialized world [1]
To determine whether miRNAs are involved in M3-mAChR mediated cardioprotection, we performed miRNA microarray analysis and identified 12 differentially upregulated miRNAs upon myocardial ischemia, among which 10 miRNAs were differentially downregulated by M3-mAChR agonist choline (Table 1)
For the first time, we found that miR-376b5p inhibited the expression of brain derived neurophic factor (BDNF) and miR-376b-5p promoted H2O2-induced H9c2 cells injury, indicating that miR-376b-5p promoted myocardial ischemia injury possibly by inhibiting the expression of BDNF

Summary

Introduction

Ischemic heart disease is the leading cause of death in the industrialized world [1]. Myocardial ischemia could induce unstable angina, arrhythmia, myocardial infarction or its neopathy, leading to sudden cardiac death [2,3,4]. While many drugs failed to efficaciously treat cardiac death with myocardial ischemia and myocardial infarction [5], recent studies have shown that M3 subtype of muscarinic acetylcholine receptors (M3-mAChR) in the heart have cardioprotective effect against myocardial ischemia [6,7,8,9,10]. Several miRNAs including miRNA-320 [15], miRNA-21 [16] and miRNA494 [17] are involved in myocardial ischemia. The present study aimed to explore the contribution of miRNAs to cardioprotective effects mediated by M3-mAChR against myocardial ischemia. Our results revealed for the first time that miR-376b-5p increased myocardial ischemia injury and was involved in M3-mAChR’s cardioprotection

Methods

Results

Conclusion