title: Acetylcholine Attenuates Hypoxia/Reoxygenation Injury by Inducing Mitophagy Through PINK1/Parkin Signal Pathway in H9c2 Cells.
abstract: Acetylcholine (ACh) protected against cardiac injury via promoting autophagy and mitochondrial biogenesis, however, the involvement of mitophagy in ACh-elicited cardioprotection remains unknown. In the present study, H9c2 cardiomyocytes were subjected to hypoxia/reoxygenation (H/R) and ACh treatment during reoxygenation. Mitophagy markers PTEN-induced kinase 1 (PINK1) and Parkin translocation were examined using western blot and confocal fluorescence microscopy. Mitochondrial membrane potential and reactive oxygen species (ROS) were detected with fluorescence staining. We found that H/R-treated cells exhibited reduced levels of PINK1 and Parkin in mitochondria, accompanied with decreased autophagy flux (reduced LC3-II/LC3-I and increased p62). Conversely, ACh increased PINK1 and Parkin translocation to mitochondria and enhanced autophagy proteins. Confocal imaging of Parkin and MitoTracker Green-labeled mitochondria further confirmed ACh-induced mitochondrial translocation of Parkin, which was reversed by M2 receptor antagonist methoctramine and M2 receptor siRNA, suggesting ACh could induce mitophagy by M2 receptor after H/R. Mitophagy inhibitor 3-methaladenine abolished ACh-induced mitoprotection, manifesting as aggravated mitochondrial morphology disruption, ATP and membrane potential depletion, increased ROS overproduction, and apoptosis. Furthermore, PINK1/Parkin siRNA attenuated the protective effects of ACh against ATP loss and oxidative stress due to mitochondrial-dependent injury. Taken together, ACh promoted mitochondrial translocation of PINK1/Parkin to stimulate cytoprotective mitophagy via M2 receptor, which may provide beneficial targets in the preservation of cardiac homeostasis against H/R injury.
authors.affiliation: Departmentof Pharmacology, Xi'an Jiaotong University Health Science Center, Xi'an, Shaanxi, P.R. China.
authors.forename: Lei
authors.initials: L
authors.lastname: Sun
authors.affiliation: Departmentof Pharmacology, Xi'an Jiaotong University Health Science Center, Xi'an, Shaanxi, P.R. China.
authors.forename: Mei
authors.initials: M
authors.lastname: Zhao
authors.affiliation: Departmentof Pharmacology, Xi'an Jiaotong University Health Science Center, Xi'an, Shaanxi, P.R. China.
authors.forename: Yang
authors.initials: Y
authors.lastname: Yang
authors.affiliation: Departmentof Pharmacology, Xi'an Jiaotong University Health Science Center, Xi'an, Shaanxi, P.R. China.
authors.forename: Run-Qing
authors.initials: RQ
authors.lastname: Xue
authors.affiliation: Departmentof Pharmacology, Xi'an Jiaotong University Health Science Center, Xi'an, Shaanxi, P.R. China.
authors.forename: Xiao-Jiang
authors.initials: XJ
authors.lastname: Yu
authors.affiliation: Key Laboratory of Biomedical Information Engineering of Ministry of Education, School of Life Science and Technology, Xi'an Jiaotong University, Xi'an, Shaanxi, P.R. China.
authors.forename: Jian-Kang
authors.initials: JK
authors.lastname: Liu
authors.affiliation: Departmentof Pharmacology, Xi'an Jiaotong University Health Science Center, Xi'an, Shaanxi, P.R. China.
authors.forename: Wei-Jin
authors.initials: WJ
authors.lastname: Zang
mesh_terms: D000109:Acetylcholine / Q000494:pharmacology*; D000818:Animals; D015687:Cell Hypoxia / Q000187:drug effects; D002460:Cell Line; D019610:Cytoprotection / Q000187:drug effects; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism; D063306:Mitophagy / Q000187:drug effects*; D010100:Oxygen / Q000494:pharmacology*; D011494:Protein Kinases / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000378:metabolism; D051381:Rats; D043585:Receptor, Muscarinic M2 / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects*; D044767:Ubiquitin-Protein Ligases / Q000378:metabolism*; D000099202:PTEN-Induced Putative Kinase
chemical_list: D000109:Acetylcholine; D010100:Oxygen; D011494:Protein Kinases; D034741:RNA, Small Interfering; D043585:Receptor, Muscarinic M2; D044767:Ubiquitin-Protein Ligases; D000099202:PTEN-Induced Putative Kinase; C111567:parkin protein