Acetylcholine exerts cytoprotection against hypoxia/reoxygenation-induced apoptosis, autophagy and mitochondrial impairment through both muscarinic and nicotinic receptors.

Acetylcholine (ACh) has been shown to exert cardioprotection against myocardial ischemia/reperfusion (I/R) injury. However, whether ACh exerts its cardioprotection predominantly through the activation of muscarinic or nicotinic ACh receptors is not fully understood. We investigated the effects of hypoxia/reoxygenation (H/R) in the presence or absence of ACh receptor agonists in H9c2 cells. Cells (2.5 × 105 cells/well) were incubated in the hypoxic chamber with the ischemic solution (30 min) followed by reoxygenation (120 min) with the normal media. ACh or nicotinic ACh receptor agonist (GTS21) was applied 5 min prior to hypoxia, during hypoxia or at reoxygenation onset. Cell viability, apoptosis, ER stress, mitochondrial dynamics and biogenesis were determined. H/R significantly decreased cell viability and mitochondrial biogenesis and increased apoptosis, ER stress, mitochondrial fission and autophagic flux compared with the control. ACh and GTS21 significantly increased cell viability via reducing apoptosis, autophagy, and ER stress. However, ACh and GTS21 increased mitochondrial fusion when applied before or during hypoxia. During reoxygenation onset, only ACh increased mitochondrial biogenesis. Co-treatment with atropine reversed the beneficial effects of ACh and GTS21. Our findings demonstrated that ACh exerted cytoprotection against H/R-induced apoptosis, autophagy and mitochondrial impairment through the activation of both muscarinic and nicotinic receptors.