Background/Purpose: For decades, research in the field of cardioprotection has focused primarily on cardiomyocytes, with the particular aim of reducing infarct size. However, microvascular damage during acute myocardial infarction, independent of infarct size, determines patient outcome. Remote ischemic conditioning (RIC), brief cycles of ischemia and reperfusion (I/R) in tissues distant from the heart, triggers the release of humoral factors that confer cardioprotection. This protective effect of RIC can be transferred from conditioned donors to naïve ex vivo heart preparations subjected to I/R protocols by infusing plasma dialysates or washed platelets. Not only platelets, but also red blood cells (RBC)s and the RBC-derived nitric oxide (NO) play a role in cardioprotection, i.e. in isolated perfused rat hearts with I/R, they reduce myocardial infarct size without a pre-treatment.
Methods: Healthy volunteers (3 females, 10 males; age 27 ± 5 years) underwent RIC (3x5/5 min upper-arm I/R at 200 mmHg). Venous blood samples were collected before and 60 minutes after RIC. RBCs were separated by centrifugation, and 4x washed with saline. To investigate the role of RBC nitric oxide synthase (NOS) activity and RBC-derived NO bioavailability on the RBC-mediated effects, aliquots of all RBC samples were preincubated during the washing steps with arginase or oxygenated hemoglobin. Washed RBCs (5×10⁴/mL) were infused into isolated, perfused rat hearts for 8 minutes, followed by a 2-minute washout period. Saline infusion served as control. After 30 minutes of left anterior descending coronary artery occlusion and 120 minutes of reperfusion, the area at risk was demarked using patent blue dye, microvascular injury (area of no-reflow) was visualized with thioflavin-S, and infarct size was determined using triphenyltetrazolium chloride. Area of no-reflow and infarct size were quantified and expressed as a percent of the area at risk.
Results: Infarct size and no-reflow were 45±9% and 9±4%, respectively, following saline treatment. Compared to saline, RBC infusion before RIC reduced infarct size to 34±11%, but not the area of no-reflow (12±6%). In contrast, RBC infusion after RIC not only further reduced infarct size to 20±9%, but also decreased the area of no-reflow to 5±2%. The per se reduction of infarct size was lost when RBCs were preincubated with either arginase or oxyhemoglobin (44±8% and 44±3%, respectively), area of no-reflow was not affected (13±6% and 14±7%, respectively). However, the RBC-mediated dual role of cardioprotection with RIC was still evident in presence of arginase and oxyhemoglobin, infarct size was reduced to 32±12% and 29±10%, and area of no-reflow to 6±3% and 8±5%, respectively.
Conclusion: In line with prior studies, RBCs reduced infarct size per se in isolated perfused hearts and this protection was RBC-NO dependent. RIC enhanced RBC's cardioprotective potential and extended it to the coronary microcirculation. The RIC-mediated reduction of infarct size and no-reflow, however, was RBC-NO independent. RBCs may serve as a unique novel target for delivering protection to the microcirculation. Future experiments aim to substantiate the results and to characterize the NO-independent, RBC-dependent cardioprotective mediators.
