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Previous findings have shown that acetylcholine (ACh) decreased hypoxia-induced tumor necrosis factor alpha (TNF α) production, thus protected against cardiomyocyte injury. However, whether and how ACh affects TNF α-induced endoplasmic reticulum (ER) stress and cell apoptosis remain poorly defined. This study was aimed at determining the effect of ACh in H9c2 cells after TNF α stimulation. Presence of ER stress was verified using the ER stress protein markers glucose regulatory protein 78 (GRP78) and C/EBP homologous protein (CHOP). Cell apoptosis was shown by caspase-3 activation and terminal deoxynucleotidyl transferase mediated dUTP-biotin nick end labeling. Exogenously administered ACh significantly decreased these TNF α-induced changes. Moreover, when the cells were exposed to nonspecific muscarinic receptor (M AChR) inhibitor atropine, methoctramine (M2 AChR inhibitor) or the epidermal growth factor receptor (EGFR) inhibitor AG1478, the cardioprotection elicited by ACh was diminished. Furthermore, the above effects were also blocked by M2 AChR or EGFR siRNA, indicating that EGFR transactivation by M2 AChR may be the major pathway responsible for the benefits of ACh. In addition, LY294002, a phosphatidylinositol-3-kinase (PI3K) inhibitor, displayed the similar trends as AG1478, suggesting that PI3K/Akt signaling may be the downstream of EGFR in ACh-elicited anti-apoptotic property. Together, these data indicate that EGFR-PI3K/Akt signaling is involved in M2 AChR-mediated ER apoptotic pathway suppression and the subsequent survival of H9c2 cardiomyocytes. We have identified a novel pathway underlying the cardioprotection afforded by ACh., (© 2014 Wiley Periodicals, Inc.)

Apoptosis of cardiomyocytes plays an important role in the development of cardiovascular diseases (CVD). Numerous studies have shown that generation of reactive oxygen species (ROS) induced by the renin-angiotensin system (RAS) is involved in this pathological process. Recent studies also suggested that acetylcholine (ACh) prevented the hypoxia-induced apoptosis of mouse ES cells by inhibiting the ROS production. However, whether ACh can inhibit the action of angiotensin II (Ang II) and subsequently prevent CVD development remains unclear. In this study, H9c2 cells were stimulated by 10(-6) M Ang II for 24 h with or without 10(-5) M ACh, 10(-5) M ACh + 10(-4) M atropine respectively. The results demonstrated that Ang II increased apoptosis index by fourfold (vs. the control group, P < 0.01), which were significantly diminished by ACh. However, the atropine (ACh receptor [AChR] inhibitor) treatment blocked the protective effect of ACh. Subsequently, Ang II significantly increases the expression and activity of NADPH oxidase so that ROS production is increased by sevenfold (vs. control group, P < 0.01). The activity and expression of caspase-3 along with the Bax/Bcl2 ratio and the levels of p38 mitogen activated protein kinase (MAPK) phosphorylation also appeared to follow a similar trend. Furthermore, we observed that ACh could reduce up-regulation of AT1 receptor expression induced by Ang II. However, all these effects of ACh were inhibited by atropine. In conclusion, ACh prevents Ang II-induced H9c2 cells apoptosis through down-regulation of the AT1 receptor and inhibition of ROS-mediated p38 MAPK activation as well as regulation of Bcl-2, Bax and caspase-3.

Recent findings have reported that up-regulation of tumor necrosis factor-alpha (TNF-α) induced by myocardial hypoxia aggravates cardiomyocyte injury. Acetylcholine (ACh), the principle vagal neurotransmitter, protects cardiomyocytes against hypoxia by inhibiting apoptosis. However, it is still unclear whether ACh regulates TNF-α production in cardiomyocytes after hypoxia. The concentration of extracellular TNF-α was increased in a time-dependent manner during hypoxia. Furthermore, ACh treatment also inhibited hypoxia-induced TNF-α mRNA and protein expression, caspase-3 activation, cell death and the production of reactive oxygen species (ROS) in cardiomyocytes. ACh treatment prevented the hypoxia-induced increase in p38 mitogen-activated protein kinase (MAPK) and c-Jun N-terminal kinase (JNK) phosphorylation, and increased extracellular signal-regulated kinase (ERK) phosphorylation. Co-treatment with atropine, a non-selective muscarinic acetylcholine receptor antagonist, or methoctramine, a selective type-2 muscarinic acetylcholine (M) receptor antagonist, abrogated the effects of ACh treatment in hypoxic cardiomyocytes. Co-treatment with hexamethonium, a non-selective nicotinic receptor antagonist, and methyllycaconitine, a selective alpha7-nicotinic acetylcholine receptor antagonist, had no effect on ACh-treated hypoxic cardiomyocytes. In conclusion, these results demonstrate that ACh activates the M receptor, leading to regulation of MAPKs phosphorylation and, subsequently, down-regulation of TNF-α production. We have identified a novel pathway by which ACh mediates cardioprotection against hypoxic injury in cardiomyocytes. J. Cell. Physiol. 226: 1052-1059, 2011. © 2010 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]