Activation of a non‐neuronal cholinergic system in rat cardiomyocytes by cardioprotective humoral factors from pigs with remote ischemic conditioning

Brief cycles of ischemia/reperfusion (I/R) in a tissue/organ remote from the heart (remote ischemic conditioning, RIC) reduce myocardial I/R injury. Although operative in all species tested so far, including humans, the translation of RIC into clinical practice has been largely disappointing. Such failure in translation has in part been attributed to incomplete understanding of RIC’s signal transduction. RIC’s cardioprotection can be transferred with plasma between different individuals, even across species. Prior studies have suggested that the intrinsic nervous system is causally involved in RIC’s protection, since the ganglionic blocker hexamethonium attenuated the infarct size reduction in isolated rat hearts in response to plasma taken from different rats after RIC. However, recently a non‐neuronal cardiomyocyte‐based cholinergic system (NNCS) has been identified, and its involvement in the signal transduction of RIC is entirely unclear. We therefor aimed to study whether the NNCS is activated by humoral factor(s) released by RIC. Methods Anesthetized open‐chest minipigs were subjected to 60/180 min occlusion/reperfusion of the left anterior descending coronary artery (n=7, placebo, PLA). RIC was induced by 4×5/5 min hindlimb I/R (n=8) 90 min before coronary occlusion. Arterial blood was sampled after PLA/RIC and plasma separated. Rat ventricular cardiomyocytes were isolated and incubated with PLA or RIC plasma‐dialysate (1:10; cut‐off <12–14 kDa) for 30 min ± hexamethonium (1 μmol/L) or ± atropine (100 nmol/L), respectively, subjected to 30/5 min hypoxia/reoxygenation (H/R) and compared to cardiomyocytes exposed to normoxic buffer (time control, TC) for 35 min. Cardiomyocyte viability was quantified before and after H/R or TC, respectively, as the percent fraction of rod‐shaped, unstained (trypan blue) cells over all cells. Diluted (1:10) PLA or RIC plasma was also infused into isolated perfused rat hearts for 8 min before 30/120 min global ischemia/reperfusion (GI/R) ± hexamethonium (50 μmol/L) or ± atropine (100 nmol/L), respectively. Infarct size (IS) was demarcated by triphenyltetrazolium chloride and calculated as percent of ventricular mass. Results Incubation with RIC plasma‐dialysate preserved cardiomyocyte viability after H/R in comparison to PLA plasma‐dialysate. This protection was attenuated by hexamethonium and by atropine. Hexamethonium or atropine had no impact on cardiomyocyte viability per se (Figure A). In isolated hearts, IS was less with infusion of plasma from pigs with RIC than with that from pigs with PLA. With infusion of PLA plasma, hexamethonium or atropine did not impact on IS per se. The protective effect of RIC plasma, however, was abrogated by hexamethonium and by atropine (Figure B). Conclusion RIC’s humoral transfer of cardioprotection is mediated via an activation of cholinergic receptors on cardiomyocytes and attenuated to the same extent by hexamethonium or by atropine. Whether or not hexamethonium also and additionally attenuates the cardioprotection of RIC at the ganglionic level therefore remains unclear. Support or Funding Information Supported by the German Research Foundation (SFB 1116 B8)Figure legend: A) Viability of rat ventricular cardiomyocytes incubated with placebo (PLA)/remote ischemic conditioning (RIC) plasma‐dialysate after hypoxia (H)/reoxygenation(R), or after normoxic time control (TC). Cardiomyocytes with plasma‐dialysate were incubated with saline, ± hexamethonium (hexa), or ± atropine. Data are means ± standard deviations. †p<0.05 vs. PLA plasma‐dialysate; ‡p<0.05 vs. saline RIC plasma‐dialysate; two‐way ANOVA with Fisher’s least significant differences post‐hoc tests. B) Infarct size in isolated perfused rat hearts (± hexa or ± atropine) with infusion of PLA or RIC plasma and after global ischemia/reperfusion. Data are means ± standard deviations. #p<0.05 vs. PLA plasma; *p<0.05 vs. saline RIC plasma; two‐way ANOVA with Fisher’s least significant differences post‐hoc tests.