Spleen-derived cardioprotective factors from healthy human volunteers activate the non-neuronal cholinergic system of isolated rat cardiomyocytes

The cardioprotective role of the spleen was identified in dogs by Hermann Rein 75 years ago. We confirmed and extended these data by identifying the spleen as a central relay organ for cardioprotection. Spleen-derived circulating factors are released in response to vagal activation induced by remote ischemic conditioning (RIC) in rats, pigs, and humans, as well as by auricular tragus stimulation (ATS) in humans. The release of spleen-derived circulating factors reduced infarct size in isolated perfused rat hearts and preserved cardiomyocyte survival in isolated adult rat cardiomyocytes. So far, the nature of these cardioprotective factor(s) and the induced signaling within the cardiomyocyte is unclear. While there is no doubt that acetylcholine is causally involved in cardioprotection through vagal activation in local ischemic conditioning, the role of vagal impact on the heart was recently challenged for RIC and ATS. However, there is also an endogenous, non-neuronal cholinergic system in cardiomyocytes. In isolated rat cardiomyocytes, hypoxic preconditioning increased acetylcholine release and improved cell viability after simulated ischemia/reperfusion (via hypoxia/reoxygenation). Such protection was blocked by muscarinic (atropine) and nicotinic (hexamethonium) receptor antagonists. The present study aims to investigate whether or not the non-neuronal cholinergic system is also activated through spleen-derived circulating factors released during RIC and ATS. Blood samples were collected from volunteers with prior splenectomy due to traumatic cause (46±9 years; 4F, 4M) and non-splenectomized volunteers, who were matched in age, sex, comorbidities, and medication (52±1 years; 4F, 4M) before and 60 minutes after either RIC (3 cycles of 5-minute upper-arm ischemia followed by 5-minute reperfusion) or ATS (periodically interrupted stimulation at 5 s/5 s; 30 Hz repetition frequency, 200 μs pulse width). Plasma-dialysates (< 12-14 kDa) were prepared and added to isolated adult rat cardiomyocytes, saline served as control. The cardiomyocytes were subsequently subjected to 30 minutes of hypoxia and 5 minutes of reoxygenation (H/R) ± atropine (0.1 µmol/L) or ± hexamethonium (1.0 µmol/L), respectively. Cardiomyocyte viability was assessed by trypan blue staining before and after H/R, and 600–800 cardiomyocytes were counted in each individual preparation. Viability was expressed as the percentage of rod-shaped, unstained isolated cardiomyocytes over the total number of cells. Baseline viability of cardiomyocytes was 66±4%. H/R reduced viability to 12±5%. Plasma-dialysates before RIC or ATS from volunteers with splenectomy and matched volunteers with intact spleen did not affect cardiomyocyte viability after H/R. However, plasma-dialysates after RIC/ATS from matched volunteers with intact spleen preserved cardiomyocyte viability after H/R (30±8%; 24±9%). The addition of atropine or hexamethonium, respectively, as well as their combination, abrogated protection. Plasma-dialysates from volunteers with splenectomy after RIC or ATS did not preserve cardiomyocyte viability (17±6%; 18±7%). Atropine and hexamethonium did not impact on cardiomyocyte viability. In conclusion, human spleen-derived circulating factors appear to activate the cardiomyocyte’s endogenous non-neuronal cholinergic system, thereby contributing to cardioprotection. The involved signaling pathways remain unclear at this point. Funding Source:The M.A. and P.K. acknowledge the support by the German Research Foundation (RTG 2989 P5). P.K. acknowledge the European Union Cost Action METAHEART (CA22169). This abstract was presented at the American Physiology Summit 2026 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.