Introduction: There is an ambivalent relationship between the spleen and the heart in myocardial infarction (Circ Res 124; 2019; 26-8). Sympathetic activation of the spleen initially increases myocardial inflammatory infiltration but resolves it later by initiating a myocardial healing response. Vagal activation of the spleen induces the release of cardioprotective factors: Perfusion of an isolated rat spleen with the vagomimetic carbachol resulted in the release of factors which then reduced infarct size (IS) in an isolated perfused rat heart.

Aim: To characterize cardioprotection of spleen-derived factors, with a focus on their cholinergic action.

Methods: Rat spleens and hearts were isolated and buffer-perfused at constant pressure. Isolated spleens were either challenged by infusion of saline (as control) or the vagomimetic carbachol (500 pmol/L), and splenic effluate (SEff; 100 mL) was sampled after saline infusion (SEff_saline) or carbachol infusion (SEff_carbachol), respectively. Cell-free SEff was prepared by centrifugation and filtration. SEff_saline or SEff_carbachol was infused into isolated hearts which were then subjected to 30 min global ischemia and 120 min reperfusion (I/R). In subsets of experiments, the hearts were pretreated with the nicotinic receptor antagonist hexamethonium (50 µmol/L) or the muscarinic receptor antagonist atropine (100 pmol/L). IS was demarcated by triphenyltetrazolium chloride staining and calculated as percent of ventricular mass. To study the relevance of spleen-derived protection on the cardiomyocyte level, adult rat ventricular cardiomyocytes were prepared. Cells were incubated for 30 minutes with SEff_saline or SEff_carbachol, respectively, and then subjected to 30 min hypoxia/ 5 min reoxygenation (H/R) or exposed to SEff_saline or SEff_carbachol for 35 minutes as time control (TC). Since nicotinic and muscarinic receptors are also expressed on cardiomyocytes, all experiments were repeated after pretreatment with hexamethonium (1 µmol/L) or atropine (100 nmol/L). Cardiomyocyte viability was quantified before (baseline) and after H/R or TC, as the percent fraction of rod-shaped, unstained (5% trypan blue) cells over all cells. The estimated final concentration of carbachol in the SEff_carbachol of ≤10 pmol/L and the SEff_saline did neither reduce IS in isolated perfused rat hearts nor improve cardiomyocyte viability in isolated rat cardiomyocytes.

Results: With SEff_saline, IS was 34±7%. With SEff_carbachol, IS was reduced to 19±8%. Hexamethonium or atropine had no impact on IS per se in hearts with SEff_saline (33±8%; 34±6%), but attenuated the cardioprotection by SEff_carbachol (27±10%; 26±10%; Figure A). The viability of cardiomyocytes subjected to H/R was better preserved with SEff_carbachol than with SEff_saline or saline alone. Again, this protection was attenuated by hexamethonium or atropine (Figure B, insert).

Conclusion: The cardioprotection by spleen-derived factors, which were released in response to the vagomimetic carbachol, is mediated through nicotinic and muscarinic receptors within the heart. Apparently, this protection does not need to involve neuronal structures within the myocardium – nicotinic and muscarinic receptors on cardiomyocytes per se mediate protection.