Differentiation between arginase 1 and 2 in regulating tolerance to cardiac ischemia-reperfusion via nitric oxide signaling by red blood cells

Background It is well known that arginase contributes to impaired cardiac tolerance to ischemia-reperfusion via attenuated nitric oxide (NO) signaling by competing for the common substrate, L-arginine with nitric oxide synthase (NOS). Both arginase 1 and 2 isoforms are involved in cardiovascular regulation, but the distribution of the two isoforms is not identical. Due to the lack of specific inhibitors of the two arginase isoforms, separating the roles of the two isoforms in myocardial ischemia-reperfusion has not been possible. Purpose The study aimed to determine the two arginase isoforms regulating cardiac tolerance to ischemia-reperfusion and the specific role of red blood cell (RBC) derived arginase. Methods C57Bl/6 mice were pretreated with arginase (Arg) 1 or 2 anti-sense oligonucleotides (ASO; 50 mg/kg) for 6 weeks, and conditional arginase 1 knockout (Arg1-KO) mice lacking arginase 1 in their endothelial and hematopoietic cells, were studied. For in vivo studies, mice were anesthetized and subjected to 30 min left coronary artery occlusion and 120 min reperfusion. At 15 min before ischemia, mice were given iv: saline, non-specific arginase inhibitor (nor-NOHA; 100 mg/kg), or the NOS inhibitor (L-NMMA; 10 mg/kg). For ex vivo studies, mouse hearts were perfused in a Langendorff set-up, subjected to 40 min global ischemia and 60 min reperfusion. Left ventricular developed pressure (LVDP) was recorded. At the start of ischemia, mouse RBCs were given to the isolated hearts with the NOS inhibitor (L-NAME; 100µM). Animal experiments were performed according to the principles of laboratory animal care (NIH Publication no. 85-23 revised 1985) Results Arg1 ASO reduced Arg1 protein expression and Arg activity in the liver (Fig. 1A and B), and Arg2 ASO reduced Arg 2 expression and Arg activity in the kidney (Fig. 1C and D). Arg1 ASO but not Arg2 ASO improved the recovery LVDP of isolated mouse hearts during reperfusion compared to control hearts (Fig. 1E and F). Administration of RBCs from Arg1 ASO but not Arg2 ASO treated mice improved recovery of LVDP in isolated hearts during reperfusion (Fig. 1G and H). In in vivo experiments, infarct size was smaller in Arg1-KO mice (42±5% of area at risk) and in wild type (WT) mice pretreated with nor-NOHA (45±6%) than in untreated wild type mice (74±12%, P<0.001). Administration of the NOS inhibitor L-NMMA resulted in increased infarct size (69±8%; n=5) in Arg1-KO mice in comparison to control Arg1-KO mice (42±5%, P<0.01; Fig. 2A). Administration of RBCs from Arg1-KO mice to isolated WT hearts resulted in significantly higher recovery LVDP during reperfusion in comparison to hearts receiving RBCs from WT mice, which was blocked by the NOS inhibitor L-NAME (Fig. 2B). Conclusion Arginase 1, but not arginase 2, plays an important role in cardiac ischemic tolerance. Arginase 1 in RBC is a critical determinant of cardiac protection against ischemia-reperfusion injury by regulating NO signaling.