Protection of Mouse Cardiomyocytes by Plasma‐Dialysate from Volunteers Undergoing Remote Ischemic Conditioning Through STAT3 Signaling

Background Brief cycles of ischemia/reperfusion in a tissue or organ remote from the heart reduce myocardial infarct size (IS) in all species tested so far, including humans. Such remote ischemic conditioning (RIC) releases cardioprotective factors into the circulating blood. Plasma-dialysate from healthy volunteers undergoing RIC reduces IS in isolated perfused rodent hearts. Such protection is abrogated by the signal transducer and activator of transcription (STAT)3 inhibitor stattic. Whether or not such protection and its signaling are related to cardiomyocytes or other cellular components of the myocardium, is not established so far. Aim To characterize RIC's humoral protection on the cardiomyocyte level. Methods Healthy volunteers (4 males/ 4 females; 35±15 years) were subjected to RIC by 3 cycles of 5 min forearm ischemia/5 min reperfusion. Venous blood samples were taken before and 30 min after the RIC protocol, and a plasma-dialysate (12-14 kDa; dialysis tubing; dialysis over night at 4°C against normoxic buffer in a 1:10 volume ratio) was prepared. To confirm cardioprotection by humoral factors released by RIC, plasma-dialysate was infused into isolated rat hearts perfused at constant-pressure, before hearts were subjected to global-zero flow ischemia and reperfusion. IS was demarcated with triphenyl tetrazolium chloride staining and calculated as percent of ventricular mass. Infusion of plasma-dialysate after RIC protected, and IS was less after vs. before RIC: 20±6% vs. 39±9%. Ventricular mouse cardiomyocytes were isolated by enzymatic digestion and incubated with either normoxic buffer or plasma-dialysate for a 30 min baseline before being subjected to 50 min hypoxia and 5 min reoxygenation. Hypoxia was induced by exposing cardiomyocytes to iso-osmolar, glucose-free, pH 6.5 buffer. Reoxygenation was induced by normoxic hypo-osmolar (250 mOsm/L), glucose-containing (15 mmol/L) reoxygenation buffer. For time control, cardiomyocytes were incubated with normoxic buffer for 85 min. In subgroups, experiments were repeated under STAT3 blockade (stattic, 1 µmol/l). Cardiomyocyte viability was expressed as the percentage of rod-shaped, trypan-blue unstained cardiomyocytes over the total number of cells. Results Plasma-dialysate after RIC preserved viability better than plasma-dialysate before RIC (Figure). Incubation with stattic had no impact on cardiomyocyte viability per se, but abrogated the protection by plasma dialysate after RIC (Figure). Conclusion RIC´s humoral transfer of protection occurs at the cardiomyocyte level and causally involves STAT3.