The dual role of red blood cells in cardioprotection: reduction of myocardial infarct size and coronary no-reflow in isolated perfused rat hearts

Background/Purpose Cardioprotection studies have focused primarily on cardiomyocytes, i.e. infarct size reduction. However, also the extent of microvascular injury determines the patient's prognosis. Remote ischemic conditioning (RIC), induced by brief cycles of ischemia/reperfusion (I/R) in tissues remote from the heart, induces the release of humoral cardioprotective factors. RIC’s protection is transferable from RIC-conditioned donors to ex vivo naive heart preparations via infusion of blood plasma-dialysates or washed platelets. Previous studies demonstrated that the infusion of red blood cells (RBCs) into ex vivo isolated heart preparations reduced infarct size per se via RBC nitric oxide synthase dependent mechanisms. Here, we aim to address RBC's role in cardioprotection by RIC and focus on infarct size and microvascular injury. Methods Healthy volunteers underwent RIC (three cycles of 5/5 minute upper-arm I/R). Blood samples were collected before and after RIC. Plasma and RBCs were separated by centrifugation. RBCs were repeatedly washed with saline ±arginase or ±oxygenated hemoglobin to address the role of RBC nitric oxide synthase activity and RBC-derived nitric oxide bioavailability. Plasma-dialysates, prepared to control for RIC’s humoral cardioprotective transfer, and washed RBCs (5×10⁴/mL) were infused into isolated rat hearts before 30-minute left anterior descending coronary occlusion followed by 120-minute reperfusion. Saline infusion served as control. The area at risk was demarcated by patent blue, microvascular injury (no-reflow) by thioflavin-S, and infarct size by triphenyl-tetrazolium-chloride. No-reflow and infarct size were quantified and calculated as percent of area at risk. Results Infarct size/no-reflow was 45±9%/9±4% with saline and 43±5%/9±3% with dialysate infusion before RIC. Dialysate after RIC reduced infarct size to 34±8% but had no effect on no-reflow (9±4%). In comparison to saline and dialysate, RBC infusion before RIC reduced infarct size to 34±11% but did not affect no-reflow (12±6%). However, RBC infusion after RIC reduced infarct size further to 20±9% and also decreased no-reflow to 5±2% (Figure 1). Both, RBC preincubation with arginase or oxyhemoglobin increased infarct size before RIC to 44±8% and 44±13% but had no impact on no-reflow (13±6% and 14±7%). Although infarct size was larger compared to RBC’s without arginase or oxyhemoglobin preincubation, RBCs after RIC still reduced infarct size to 32±12% and 29±10% and no-reflow to 6±3% and 8±5% (Figure 2). Conclusion RBCs reduced infarct size per se. However, RIC enhanced RBC's cardioprotective potential and extended it to the coronary microcirculation. RBC-mediated infarct size reduction was in part dependent on RBC-derived nitric oxide, whereas RIC’s reduction of microvascular injury is independent of RBC nitric oxide synthase activity. Thus, RBCs may serve as a unique novel target for delivering protection to the microcirculation.