Activation of the non-neuronal cholinergic cardiac system by hypoxic preconditioning protects isolated adult cardiomyocytes from hypoxia/reoxygenation injury

Abstract

Background: Activation of the vagus nerve mediates cardioprotection and attenuates myocardial ischemia/reperfusion (I/R) injury. In response to vagal activation, acetylcholine (ACh) is released from the intracardiac nervous system (ICNS) and activates intracellular cardioprotective signaling cascades. Recently, however, a non-neuronal cholinergic cardiac system (NNCCS) in cardiomyocytes has been described as an additional source of ACh. Aim: To investigate whether the NNCCS mediates cardioprotection in absence of vagal and ICNS activation. For that, we used a reductionist approach of isolated adult rat ventricular cardiomyocytes in absence of neuronal cells with hypoxic preconditioning (HPC) as protective stimulus. Methods: Adult rat ventricular cardiomyocytes were isolated, absence of neuronal cells was confirmed, HPC was induced by 10/20 min hypoxia/reoxygenation (H/R) before subjection to 30/5 min H/R to simulate I/R injury. Cardiomyocyte viability was assessed by trypan blue staining. Intra- and extracellular ACh was quantified using liquid chromatography-coupled mass spectrometry at baseline, after HPC, after hypoxia, and after reoxygenation, respectively. In a subset of experiments, muscarinic/nicotinic ACh receptor (mAChR/nAChR) antagonists were added during HPC or during H/R. Results: Cardiomyocyte viability at baseline (69±4%) was reduced by H/R (10±3%). With HPC cardiomyocyte viability was preserved after H/R (25±6%). Intra- and extracellular ACh increased during hypoxia, HPC further increased both intra- and extracellular ACh (from 0.9±0.7 to 1.5±1.0 nmol/mg; from 0.7±0.6 to 1.1±0.7 nmol/mg). Addition of mAChR and nAChR antagonists during HPC had no impact on HPC´s protection, however protection was abrogated when antagonists were added during H/R (cardiomyocyte viability after H/R: 23±5%; 13±4%). Conclusion: Activation of the NNCCS is involved in cardiomyocyte protection: HPC increases intra- and extracellular ACh during H/R, and m-/nAChRs are causally involved in HPC´s cardiomyocyte protection during H/R. The interplay between upstream ICNS activation and the NNCCS activation to myocardial cholinergic metabolism and cardioprotection needs to be investigated in future studies.