Letter by Reynolds et al Regarding Article, "Hemoglobin β93 Cysteine Is Not Required for Export of Nitric Oxide Bioactivity From the Red Blood Cell".

Circulation. 2019;140:e758–e759. DOI: 10.1161/CIRCULATIONAHA.119. 041389 e758 James D. Reynolds, PhD Richard T. Premont, PhD Jonathan S. Stamler, MD To the Editor: Sun et al1 claim that “Red blood cells (RBCs) export nitric oxide (NO) bioactivity independently of SNO formation (S-nitrosylation) at β93 cysteine of hemoglobin (Hb). ” Their conclusion is surprising given strong published evidence to the contrary. Mice deficient in βCys93 have significant impairment in RBC export of NO bioactivity not only measured directly but also as impaired ability to effect hypoxic vasodilation in vitro, and by profound deficits in micro-vascular blood flow and tissue oxygenation in vivo2; notably, βCys93Ala is the only mammalian mutation known to cause myocardial ischemia at baseline. Sun et al do not contradict these findings because their experiments do not test the same effects, and the experiments they do perform are not relevant to the physiology of RBCs or their governance of tissue oxygenation (ie, classic physiology relating blood flow to tissue oxygenation as described by Guyton3). Instead, the authors conflate: (1) extreme pharmacology with physiology, (2) endothelial with RBC-mediated vasodilation, and (3) vessel diameter with micro-vascular blood flow and tissue oxygenation. Sun et al study extreme pharmacology, not physiology. In their only experiment using native RBCs, the authors in fact find that mutant βCys93Ala-RBCs significantly impair recovery from cardiac ischemia but choose to deemphasize this finding. Instead, they massively preload RBCs with NO donors (1000-fold over endogenous NO); the authors might as well use NO donors without RBCs, because RBCs export the donors (and bioactive derivatives) and thus dilate blood vessels independently of Hb or pO2. 3,4 The same concern for artifact applies to experiments with millimolar NO (1000-fold excess), which is well-known to inactivate physiological SNO-Hb–mediated vasorelaxation,3 and to platelet experiments with 100-fold excess nitrite, which have no physiological or clinical corollary. The authors also pharmacologically amplify RBC–endothelial NO synthase with an arginase inhibitor in 95% oxygen. However, SNO-Hb is inactive at high pO2, and blood flow governing tissue oxygenation is independent of endothelial NO synthase.3–5 We have tested this arginase inhibitor at high and low pO2 and it does not promote vasodilation by RBCs! Thus, there is no basis to implicate hypoxic export of NO bioactivity, let alone Hb βCys93. Finally, in their only in vivo experiment, the authors measure local vasodilation of 40–60 μm arterioles, which is almost certainly endothelium-dependent and involves multiple mediators (no evidence is presented for involvement of RBCs or NO export). Hypoxic relaxations by RBC-βCys93, which govern tissue oxygenation,2,3 are related to composite changes in blood flow across the entire vascular unit (miles! ). RBC-mediated effects that are linked to tissue pO2 include much smaller endothelial NO synthase–deficient vessels (note: because micro-vascular blood flow is impaired in βCys93A mutants,2 “comparable dilation” would actually represent significant impairment). © 2019 American Heart Association, Inc. LETTER TO THE EDITOR