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

Am J Physiol Heart Circ Physiol. 2024 Jul 1;327(1):H70-H79. doi: 10.1152/ajpheart.00211.2024. Epub 2024 May 3.


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 nonneuronal cholinergic cardiac system (NNCCS) in cardiomyocytes has been described as an additional source of ACh. To investigate whether the NNCCS mediates cardioprotection in the absence of vagal and ICNS activation, we used a reductionist approach of isolated adult rat ventricular cardiomyocytes without neuronal cells, using hypoxic preconditioning (HPC) as a protective stimulus. Adult rat ventricular cardiomyocytes were isolated, the absence of neuronal cells was confirmed, and 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 at baseline and after HPC+H/R or H/R. 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 and nicotinic ACh receptor (m- and nAChR) antagonists were added during HPC or during H/R. 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, respectively). The 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%). In conclusion, activation of the NNCCS is involved in cardiomyocyte protection; HPC increases intra- and extracellular ACh during H/R, and m- and nAChRs are causally involved in HPC's cardiomyocyte protection during H/R. The interplay between upstream ICNS activation and NNCCS activation in myocardial cholinergic metabolism and cardioprotection needs to be investigated in future studies.NEW & NOTEWORTHY The intracardiac nervous system is considered to be involved in ischemic conditioning's cardioprotection through the release of acetylcholine (ACh). However, we demonstrate that hypoxic preconditioning (HPC) protects from hypoxia/reoxygenation injury and increases intra- and extracellular ACh during hypoxia in isolated adult ventricular rat cardiomyocytes. HPC's protection involves cardiomyocyte muscarinic and nicotinic ACh receptor activation. Thus, besides the intracardiac nervous system, a nonneuronal cholinergic cardiac system may also be causally involved in cardiomyocyte protection by ischemic conditioning.

Keywords: acetylcholine; cardioprotection; intracardiac nervous system; isolated adult ventricular rat cardiomyocytes; nonneuronal cholinergic cardiac system.

MeSH terms

  • Acetylcholine* / metabolism
  • Acetylcholine* / pharmacology
  • Animals
  • Cell Hypoxia
  • Cell Survival
  • Cells, Cultured
  • Ischemic Preconditioning, Myocardial
  • Male
  • Muscarinic Antagonists / pharmacology
  • Myocardial Reperfusion Injury* / metabolism
  • Myocardial Reperfusion Injury* / pathology
  • Myocardial Reperfusion Injury* / physiopathology
  • Myocardial Reperfusion Injury* / prevention & control
  • Myocytes, Cardiac* / metabolism
  • Non-Neuronal Cholinergic System
  • Rats
  • Rats, Sprague-Dawley
  • Receptors, Muscarinic / metabolism


  • Acetylcholine
  • Receptors, Muscarinic
  • Muscarinic Antagonists