Dysfunctional RBCs in anemia mitigate the hypertension in red blood cell eNOS deficient mice.

Background: Red blood cells (RBCs) are known to contain functionally active endothelial nitric oxide synthase (eNOS), which maintains blood pressure (BP) independent of endothelial NOS. We recently showed that eNOS expressed in RBCs (RBC-eNOS) is cardioprotective during myocardial infarction (MI). The functional role of RBC-eNOS in anemia is not understood. In this study, we assessed the functional consequences of RBC-eNOS or endothelial cell eNOS (EC-eNOS) deletion on hemodynamics and heart function in anemia. 

Methods: Anemia was induced in RBC- and EC-eNOS knockout (KO) and respective control mice. In a separate group of these mice, MI was induced by left anterior descending artery (LAD) ligation. Endothelial function was assessed using in vivo flow-mediated dilation (FMD) measurements and ex vivo wire myograph measurements. Millar catheter was used to assess hemodynamic parameters before and after anemia. Cardiac function was assessed using echocardiography at baseline, 3 days after anemia and 24h post-AMI. Infarct sizes 24 h after AMI were assessed using Triphenyl Tetrazolium Chloride (TTC) staining. The analysis of the nitric oxide (NO) pool was performed by using chemiluminescence detection (CLD) analyzer and ENO-30.

Results: Endothelial function assessment using in vivo FMD revealed that RBC-eNOS KO mice show preserved FMD responses whereas these responses were significantly decreased in EC-eNOS KO mice. The assessment of endothelium-dependent relaxation responses using wire myograph experiments in the isolated aortic rings revealed that anemic RBC-eNOS KO mice show preserved relaxation responses whereas they are abolished in anemic EC-eNOS KO mice compared to their respective anemic control mice. Blood pressure measurements using millar catheter showed that systolic and diastolic blood pressure were significantly increased in anemic EC-eNOS KO mice whereas they were unaltered in RBC-eNOS KO mice compared to their respective anemic control mice. Echocardiography analysis of the heart at baseline, 3 days after anemia, and 24h post-MI showed that heart function was preserved in all genotypes. TTC staining of infarcted hearts showed a significantly increased infarct area in RBC-eNOS KO mice compared to control mice. 

Conclusion: Our results demonstrate that in anemia the cardioprotective properties of RBC-eNOS are retained during MI without further consequences on the vascular system. The basal hypertension phenotype is compensated in RBC-eNOS KO mice after anemia which is accompanied by unchanged plasma NO metabolites.