M₃ subtype of muscarinic acetylcholine receptor inhibits cardiac fibrosis via targeting microRNA-29b/beta-site app cleaving enzyme 1 axis

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 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-kappa B) 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 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. M-3-mAChR activated NF-kappa B and thereby inhibited miR-376b-5p expression. Our data show that a novel M-3-mAChR/NF-kappa B/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-kappa B. These findings provide new insight into the potential mechanism by which M-3-mAChR provides cardioprotection against myocardial ischemia injury.