title: Acetylcholine mediates AMPK-dependent autophagic cytoprotection in H9c2 cells during hypoxia/reoxygenation injury.
abstract: BACKGROUND
  Acetylcholine (ACh), a neurotransmitter of vagal nerve, offers tolerance to ischemia/reperfusion injury. Given the regulation of autophagy in cardioprotection, this study was to examine the role of autophagy in ACh-elicited protection against hypoxia/reoxygenation (HR) injury.
  
  
  METHODS
  H9c2 cells were subjected to HR injury. Autophagy was determined by transmission electron microscopy, MDC staining and western blot. MTT kit, LDH and CK release, ATP content and TUNEL assay were used to evaluate cardiomyocytes injury. Atg7 and AMPK knockdown was performed with siRNA transfection.
  
  
  RESULTS
  Following 4, 8, 12 and 16 h reoxygenation, autophagosomes were decreased along with reduced cell viability. ACh during 4 h reoxygenation facilitated autophagy as evidence by increased autophagosomes and MDC labeling autophagic vacuoles. H9c2 cells treated with ACh also underwent a biochemical changes by increased ratio of LC3-II/LC3-I and autophagy flux (decreased p62), while muscarinic receptor antagonist atropine suppressed these effects. Induction of autophagy was correlated with enhanced cell survival and decreased apoptosis. Autophagy inhibition with chloroquine and Atg7 siRNA significantly attenuated ACh-induced cytoprotection. ACh-elicited autophagy activation could be related to increased AMPK phosphorylation and decreased mTOR phosphorylation. AMPK siRNA exhibited an elevation in mTOR phosphorylation and reduced the ratio of LC3-II/LC3-I. Importantly, AMPK knockdown desensitized H9c2 cells to ACh-mediated protection.
  
  
  CONCLUSIONS
  These data provided first evidence that ACh-induced autophagy elicited cytoprotective effects through muscarinic receptor activated-AMPK-mTOR pathway, and suggested a novel mechanism of ACh-induced tolerance against HR injury.
authors.affiliation: Department of Pharmacology, College of Medicine, Xi'an Jiaotong University, Xi'an, Shaanxi, China.
authors.forename: Mei
authors.initials: M
authors.lastname: Zhao
authors.forename: Lei
authors.initials: L
authors.lastname: Sun
authors.forename: Xiao-Jiang
authors.initials: XJ
authors.lastname: Yu
authors.forename: Yi
authors.initials: Y
authors.lastname: Miao
authors.forename: Jin-Jun
authors.initials: JJ
authors.lastname: Liu
authors.forename: Hao
authors.initials: H
authors.lastname: Wang
authors.forename: Jun
authors.initials: J
authors.lastname: Ren
authors.forename: Wei-Jin
authors.initials: WJ
authors.lastname: Zang
mesh_terms: D055372:AMP-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D000109:Acetylcholine / Q000494:pharmacology*; D000818:Animals; D001285:Atropine / Q000494:pharmacology; D001343:Autophagy / Q000187:drug effects*; D000071193:Autophagy-Related Protein 7; D015687:Cell Hypoxia; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D002738:Chloroquine / Q000494:pharmacology; D019610:Cytoprotection / Q000187:drug effects*; D008869:Microtubule-Associated Proteins / Q000378:metabolism; D018727:Muscarinic Antagonists / Q000494:pharmacology; D032383:Myocytes, Cardiac / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000378:metabolism; D051381:Rats; D011976:Receptors, Muscarinic / Q000737:chemistry / Q000378:metabolism; D058570:TOR Serine-Threonine Kinases / Q000378:metabolism; D044764:Ubiquitin-Activating Enzymes / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism
chemical_list: D055372:AMP-Activated Protein Kinases; D000109:Acetylcholine; D001285:Atropine; D000071193:Autophagy-Related Protein 7; D002738:Chloroquine; D008869:Microtubule-Associated Proteins; D018727:Muscarinic Antagonists; D034741:RNA, Small Interfering; D011976:Receptors, Muscarinic; D058570:TOR Serine-Threonine Kinases; D044764:Ubiquitin-Activating Enzymes; C561242:Atg7 protein, rat; C526109:Anxa3 protein, rat