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.

Progressive Reduction in Myocyte Autophagy After Myocardial Infarction in Rabbits: Association with Oxidative Stress and Left Ventricular Remodeling

“…8-OHdG is a sensitive and specific marker of DNA damage induced by oxidative stress [30]. These findings are consistent with a previous report showing that myocyte autophagy was decreased during hypoxia and reoxygenization [31] in association with increased oxidative stress. Reduced myocyte autophagy also occurs in pressure overload-induced myocardial hypertrophy [3,25,26] and aging [32], all of which are associated with increased myocardial oxidative stress [33,34].…”

Myocardial autophagic energy stress responses—macroautophagy, mitophagy, and glycophagy

“…In a Langendorff model of ischemia/reperfusion, inhibition of autophagy with the cell-permeable inhibitor (TATAtg5K130R) neither increased nor reduced infarct size, suggesting that in the absence of pre-or post-conditioning, the beneficial and deleterious effects of autophagy may be balanced (34). Inhibition of autophagic flux increases hypoxia/ reoxygenation injury (109) and blocks the beneficial effects of sevoflurane postconditioning (107). Ischemia/reperfusion injury disrupts autophagosome-lysosome fusion (54), accompanied by a significant increase in Beclin-1.…”

Vagal nerve stimulation improves mitochondrial dynamics via an M3 receptor/CaMKKβ/AMPK pathway in isoproterenol‐induced myocardial ischaemia

“…Furthermore, a previous study from our laboratory showed ACh promotes cell survival via an AMPK‐induced cardiomyocyte autophagy pathway during cardiomyocyte hypoxia/reoxygenation injury 13. Increasing evidence suggests AMPK acts as a hub to bridge mitochondrial dysfunction and IHD 25, 26; however, AMPK's role in mitochondrial dynamics regulation during myocardial ischaemia remains unknown.…”