Acetylcholine mediates AMPK-dependent autophagic cytoprotection in H9c2 cells during hypoxia/reoxygenation injury.

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.