Clin Res Cardiol (2021) DOI DOI https://doi.org/10.1007/s00392-021-01843-w

Cardiomyocyte-intrinsic cholinergic system mediates cardioprotection by ischemic preconditioning.

H. R. Lieder1, F. Braczko1, G. Heusch1, P. Kleinbongard1

1Institut für Pathophysiologie, Universitätsklinikum Essen, Essen;

Introduction: Brief episodes of myocardial ischemia/reperfusion preceding more sustained ischemia reduce lethal ischemia/reperfusion injury in all species tested so far, including humans. Although such ischemic preconditioning (IPC) is established for more than 3 decades, the underlying signaling mechanisms are unclear in detail. A recruitment of the intrinsic cardiac nervous system and the release of acetylcholine have been proposed as mechanisms of IPC: the ganglionic blocker hexamethonium attenuates the infarct size reduction by IPC in isolated perfused rat hearts, and exogenous acetylcholine is cardioprotective and activates intracellular IPC pathways. We have now addressed the role of cardiomyocyte-derived acetylcholine in isolated adult ventricular cardiomyocytes from buffer-perfused rat hearts and subjected them to hypoxia and reoxygenation (H/R) without and with prior IPC. To antagonize nicotinic, muscarinic, and α7-nicotinic cholinergic receptors, hexamethonium, atropine and α-bungarotoxine were added, respectively. Methods: Hearts from male Lewis rats were isolated, buffer-perfused and subjected to IPC (3x5 min/ 5 min cycles of global zero-flow ischemia/reperfusion) or control. Cardiomyocytes were incubated for 30 min ±hexamethonium (1 µmol/L), ±atropine or ±α-bungarotoxine (100 nmol/L, both), respectively, subjected to 30 min/ 5 min H/R and compared to cardiomyocytes exposed to normoxia for 35 min. In a second set of experiments, cardiomyocytes were isolated from naïve hearts, again incubated without or with the respective blockers and subjected to IPC by 10 min/ 20 min H/R or normoxia for 30 min before undergoing 30 min/ 5 min H/R or normoxia for 35 min. Cardiomyocyte viability was quantified with trypan blue as the fraction of viable over all cells. Results: With IPC, the viability of cardiomyocytes subjected to H/R was better preserved than without IPC (Figure A). Blockade of nicotinic, muscarinic or α7-nicotinic acetylcholine receptors attenuated the protection by IPC to the same extent (Figure A, insert). In cardiomyocytes with IPC by H/R, the viability was better preserved than with control (Figure B). The protection was again attenuated by blockade of nicotinic, muscarinic, or α7-nicotinic acetylcholine receptors (Figure B, insert). Conclusion: An intrinsic cardiomyocyte cholinergic system is causally involved in cardioprotection by IPC. Figure: Data are normally distributed and presented as means ± standard deviations. BTX: α-bungarotoxine; hexa: hexamethonium; H/R: hypoxia/reoxygenation; IPC: ischemic preconditioning. A: Viability of cardiomyocytes isolated from isolated perfused hearts without or with prior IPC; *p<0.05 vs. without IPC; †p<0.05 vs. IPC; one-way ANOVA with Fisher‘s least significant differences post-hoc tests. B: Viability of cardiomyocytes isolated from naÏve hearts and without or with IPC by H/R; *p<0.05 vs. without IPC; †p<0.05 vs. IPC by H/R; one-way ANOVA with Fisher‘s least significant differences post-hoc tests.

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