title: M3 subtype of muscarinic acetylcholine receptor promotes cardioprotection via the suppression of miR-376b-5p.
abstract: The M(3) subtype of muscarinic acetylcholine receptors (M(3)-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 M(3)-mAChR mediated cardioprotection remains unexplored. The present study was designed to identify miRNAs that are involved in cardioprotective effects of M(3)-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 M(3)-mAChR. In H9c2 cells, the viability, intracellular free Ca(2+) concentration ([Ca(2+)]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 M(3)-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 M(3)-mAChRs agonist choline reversed its up-regulation. In vitro, miR-376b-5p promoted H(2)O(2)-induced H9c2 cell injuries measured by cells viability, [Ca(2+)]i and ROS. Western blot and luciferase assay identified BDNF as a direct target of miR-376b-5p. M(3)-mAChR activated NF-κB and thereby inhibited miR-376b-5p expression. Our data show that a novel M(3)-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 M(3)-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 M(3)-mAChR provides cardioprotection against myocardial ischemia injury.
authors.affiliation: Department of Pharmacology, Key Laboratory of Cardiovascular Research, Ministry of Education, Harbin Medical University, Harbin, Heilongjiang, People's Republic of China.
authors.forename: Zhenyu
authors.initials: Z
authors.lastname: Pan
authors.forename: Yueping
authors.initials: Y
authors.lastname: Guo
authors.forename: Hanping
authors.initials: H
authors.lastname: Qi
authors.forename: Kai
authors.initials: K
authors.lastname: Fan
authors.forename: Shu
authors.initials: S
authors.lastname: Wang
authors.forename: Hua
authors.initials: H
authors.lastname: Zhao
authors.forename: Yuhua
authors.initials: Y
authors.lastname: Fan
authors.forename: Jing
authors.initials: J
authors.lastname: Xie
authors.forename: Feng
authors.initials: F
authors.lastname: Guo
authors.forename: Yunlong
authors.initials: Y
authors.lastname: Hou
authors.forename: Ning
authors.initials: N
authors.lastname: Wang
authors.forename: Rong
authors.initials: R
authors.lastname: Huo
authors.forename: Yong
authors.initials: Y
authors.lastname: Zhang
authors.forename: Yan
authors.initials: Y
authors.lastname: Liu
authors.forename: Zhimin
authors.initials: Z
authors.lastname: Du
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D002794:Choline / Q000494:pharmacology; D015536:Down-Regulation; D005786:Gene Expression Regulation; D007511:Ischemia / Q000473:pathology; D008297:Male; D035683:MicroRNAs / Q000378:metabolism*; D008954:Models, Biological; D009203:Myocardial Infarction / Q000473:pathology; D017202:Myocardial Ischemia / Q000473:pathology; D032383:Myocytes, Cardiac / Q000473:pathology; D016328:NF-kappa B / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D017382:Reactive Oxygen Species / Q000378:metabolism; D043587:Receptor, Muscarinic M3 / Q000235:genetics* / Q000378:metabolism*
chemical_list: D035683:MicroRNAs; D016328:NF-kappa B; D017382:Reactive Oxygen Species; D043587:Receptor, Muscarinic M3; D002794:Choline; D002118:Calcium