Rationale: The signal transduction of remote ischemic conditioning is still largely unknown. Objective: Characterization of neurohumoral signal transfer and vago-splenic axis in remote ischemic preconditioning (RIPC). Methods and Results: Anesthetized pigs were subjected to 60 minutes of coronary occlusion and 180 minutes of reperfusion (placebo+ischemia/reperfusion [PLA+I/R]). RIPC was induced by 4×5/5 minutes of hindlimb I/R 90 minutes before coronary occlusion (RIPC+I/R). Arterial blood samples were taken after placebo or RIPC before I/R. In subgroups of pigs, bilateral cervical vagotomy, splenectomy, or splenic denervation were performed before PLA+I/R or RIPC+I/R, respectively. In pigs with RIPC+I/R, infarct size (percentage of area at risk) was less than in those with PLA+I/R (23±12% versus 45±8%); splenectomy or splenic denervation abrogated (splenectomy+RIPC+I/R: 38±15%; splenic denervation+RIPC+I/R: 43±5%), and vagotomy attenuated (vagotomy+RIPC+I/R: 36±11%) RIPC protection. RIPC increased phosphorylation of STAT3 (signal transducer and activator of transcription 3) in left ventricular biopsies taken at early reperfusion. Splenectomy or splenic denervation, but not vagotomy, abolished this increased phosphorylation. In rats with vagotomy, splenectomy, or splenic denervation, RIPC (3×5/5 minutes of hindlimb occlusion/reperfusion) or placebo was performed, respectively. Hearts were isolated, saline perfused, and subjected to 30/120-minute global I/R. With RIPC, infarct size (percentage of ventricular mass) was less (20±7%) than with placebo (37±6%), and vagotomy, splenectomy, or splenic denervation abrogated RIPC protection (38±12%, 36±9%, and 36±7%), respectively. Rat spleens were isolated, saline perfused, and splenic effluate (SEff) was sampled after infusion with carbachol (SEff carbachol ) or saline (SEff saline ). Pig plasma or SEff was infused into isolated perfused rat hearts subjected to global I/R. Infarct size was less with infusion of RIPC+I/R plasma + (24±6%) than with PLA+I/R plasma (40±8%), vagotomy+PLA+I/R plasma (39±11%), splenectomy+PLA+I/R plasma (35±8%), vagotomy+RIPC+I/R plasma (40±9%), splenectomy+RIPC+I/R plasma (33±9%), or splenic denervation+RIPC+I/R plasma (39±8%), respectively. With infusion of SEff carbachol , infarct size was less than with infusion of SEff saline (24 [19–27]% versus 35 [32–38]%). Conclusions: Activation of a vago-splenic axis is causally involved in RIPC cardioprotection.

Remote ischemic perconditioning (RPER) during ongoing myocardial ischemia reduces infarct size. The signal transduction of RPER's cardioprotection is still largely unknown. Anesthetized pigs were therefore subjected to RPER by 4 × 5 min/5 min of hindlimb ischemia-reperfusion during 60 min of coronary occlusion before 3 h of reperfusion. Pigs without RPER served as placebo (PLA). The phosphorylation of Akt and ERK [reperfusion injury salvage kinase (RISK) pathway] and STAT3 [survivor activating factor enhancement (SAFE) pathway] in the area at risk was determined by Western blot analysis. Wortmannin/U0126 or AG490 was used for pharmacological RISK or SAFE blockade, respectively. Pig plasma/plasma dialysate sampled after RPER or PLA, respectively, was transferred to isolated rat and mouse hearts subjected to 30 min/120 min of global ischemia-reperfusion. Mitochondria were isolated from rat hearts at early reperfusion. Isolated mouse cardiomyocytes were subjected to 1 h of hypoxia/5 min of reoxygenation without and with prior plasma dialysate incubation. RPER reduced infarct size in pigs to 21 ± 15% versus 44 ± 9% in PLA (percentage of the area at risk, mean ± SD, P < 0.05) and increased STAT3 phosphorylation at early reperfusion. AG490 but not RISK blockade abolished the protection. RPER plasma/plasma dialysate reduced infarct size in rat (22 ± 3% of ventricular mass vs. 40 ± 11% with PLA plasma, P < 0.05) and mouse (29 ± 4% vs. 63 ± 8% with PLA plasma dialysate, P < 0.05) hearts and improved mitochondrial function (e.g., increased respiration, ATP formation, and calcium retention capacity and decreased reactive oxygen species formation). RPER dialysate also improved the viability of mouse cardiomyocytes after hypoxia/reoxygenation. RISK or SAFE blockade each abrogated these beneficial effects. NEW & NOTEWORTHY Remote ischemic perconditioning salvages the myocardium in patients with acute infarction. We identified a signal transduction with humoral transfer and STAT3 activation in pigs and an involvement of reperfusion injury salvage kinases and STAT3 in rat and mouse hearts, along with better cardiomyocyte viability and mitochondrial function.

Aims Female sex has been proposed to be cardioprotective per se. Studies with myocardial ischaemia/reperfusion and infarct size as endpoint have demonstrated cardioprotection in female, castrated male and male pigs. These studies are difficult to compare, given the different pig strains, models, durations of ischaemia and methods of infarct size quantification. The few studies using both female and male pigs reported no differences in infarct size and cardioprotection. We therefore prospectively compared infarct size in Göttingen minipigs undergoing ischaemia/reperfusion (I/R) without and with ischaemic preconditioning (IPC) between female, castrated male and male pigs. Methods and Results In a prospective, randomised approach, 28 Göttingen open-chest, anaesthetised minipigs underwent 60 min ischaemia by distal left anterior descending artery (LAD) occlusion and 180 min reperfusion without and with IPC by 3 cycles of 5 min LAD occlusion/10 min reperfusion. Infarct size with I/R was not different between female, castrated male and male pigs (45±8 vs. 45±13 vs. 41±9% area at risk), as was the reduction in infarct size with IPC (25±11 vs. 30±8 vs. 19±10% area at risk). Also, the area of no-reflow was not different between female, castrated male and male pigs with I/R (57±13 vs. 35±7 vs. 47±26% infarct size) or IPC (4±10 vs.12±20 vs. 0±0% infarct size). Phosphorylation of signal transducer and activator of transcription 3 was increased at 10 min reperfusion by IPC but not by I/R to the same extent in female, castrated male and male pigs (198±30 vs. 230±165 vs. 179±107% of baseline). Conclusion Our data do not support the notion of sex- or castration-related differences in infarct size, coronary microvascular injury and cardioprotection by ischaemic preconditioning. Translational perspective The translation of successful preclinical studies on cardioprotection to the benefit of patients with reperfused myocardial infarction has been difficult. The difficulties have been attributed to confounders such as co-morbidities and co-medications which patients typically have but animals don´t, but also to age and sex. Notably, female sex has been considered as protective per se. We have now, using our established and clinically relevant pig model of reperfused acute myocardial infarction and ischaemic preconditioning as the most robust cardioprotective intervention looked for sex-related differences of infarct size, no-reflow and cardioprotection by ischaemic preconditioning in a prospectively powered approach but found none such difference.

We determined the impact of sex on the magnitude of cardioprotection by local and remote ischemic preconditioning ( IPC and RIPC ) and of ischemic/reperfused peripheral tissue mass on protection by RIPC . Hearts of female and male Lewis rats were excised, perfused with buffer, and underwent either IPC by 3 × 5/5 min global zero‐flow ischemia/reperfusion ( GI /R) or time‐matched perfusion ( TP ) before 30/120 min GI /R. In a second approach, anesthetized female and male Lewis rats underwent RIPC , 3 × 5/5 min ischemia/reperfusion of one or both hindlimbs (1‐ RIPC or 2‐ RIPC ), or placebo. Thirty minutes after the RIPC /placebo protocol, hearts were excised and subjected to GI /R. In female and male hearts, infarct size was less with IPC than with TP before GI /R ( IPC + GI /R female : 12 ± 5%; IPC + GI /R male : 12 ± 7% vs. TP + GI /R female : 33 ± 5%; TP + GI /R male : 37 ± 7%, P < 0.001). With 2‐ RIPC , infarct size was less than with 1‐ RIPC in female and male rat hearts, respectively (2‐ RIPC + GI /R female : 15 ± 5% vs. 1‐ RIPC + GI /R female : 22 ± 7%, P = 0.026 and 2‐ RIPC + GI /R male : 16 ± 5% vs. 1‐ RIPC + GI /R male : 22 ± 8%, P = 0.016). Infarct size after the placebo protocol and GI /R was not different between female and male hearts (36 ± 8% vs. 34 ± 5%). Sex is no determinant of IPC ‐ and RIPC ‐induced cardioprotection in isolated Lewis rat hearts. RIPC ‐induced cardioprotection is greater with greater mass of ischemic/reperfused peripheral tissue.