Hemoglobin βCys93 is essential for cardiovascular function and integrated response to hypoxia

Abstract

Oxygen delivery by Hb is essential for vertebrate life. Three amino acids in Hb are strictly conserved in all mammals and birds, but only two of those, a His and a Phe that stabilize the heme moiety, are needed to carry O2. The third conserved residue is a Cys within the β-chain (βCys93) that has been assigned a role in S-nitrosothiol (SNO)-based hypoxic vasodilation by RBCs. Under this model, the delivery of SNO-based NO bioactivity by Hb redefines the respiratory cycle as a triune system (NO/O2/CO2). However, the physiological ramifications of RBC-mediated vasodilation are unknown, and the apparently essential nature of βCys93 remains unclear. Here we report that mice with a βCys93Ala mutation are deficient in hypoxic vasodilation that governs blood flow autoregulation, the classic physiological mechanism that controls tissue oxygenation but whose molecular basis has been a longstanding mystery. Peripheral blood flow and tissue oxygenation are decreased at baseline in mutant animals and decline excessively during hypoxia. In addition, βCys93Ala mutation results in myocardial ischemia under basal normoxic conditions and in acute cardiac decompensation and enhanced mortality during transient hypoxia. Fetal viability is diminished also. Thus, βCys93-derived SNO bioactivity is essential for tissue oxygenation by RBCs within the respiratory cycle that is required for both normal cardiovascular function and circulatory adaptation to hypoxia.

Keywords: S-nitrosohemoglobin; S-nitrosylation; hypoxic vasodilation; nitric oxide.

Fig. 1.

Diminished viability in the absence of βC93. Embryonic/fetal viability is greatly diminished in βC93A and γβC93A mice versus γβC93 control mice: The average litter size is reduced by ∼50%. Data are presented as mean ± SD; n = 28–56; *P < 0.001 by one-way ANOVA.

Fig. 2.

Diminished SNO generation, vasorelaxation, and blood flow in the absence of βC93. (A) The quantity of endogenously S-nitrosylated Hb (SNO-Hb) does not differ significantly in RBCs obtained from C57BL/6J, βC93A, γβC93, or γβC93A mice. Data are presented as mean ± SD; n = 6–12; P < 0.05 by one-way ANOVA. (B) Basal S-nitrosylation of Hbα, Hbβ and Hbγ in βC93A, γβC93, and γβC93A mice as well as in C57BL/6J mice. Ascorbate is omitted as a control for specificity of the SNO-RAC procedure. (C) Deoxygenation of RBCs from γβC93 (control) but not γβC93A or βC93A mice in the presence of GSH formed GSNO. Data are shown as mean ± SEM; n = 3 mice per strain. (D) Basal blood flow and (E) basal pO₂ in the gastrocnemius muscle of mice breathing room air demonstrate that tissue perfusion and oxygenation are deficient in βC93A and γβC93A mice versus γβC93 control mice. Data are presented as mean ± SEM; n = 18–23. (F) Representative bioassays of aortic rings (derived from eNOS^−/− mice) to assess vasodilation at 1% O₂ by RBCs from γβC93 mice and isogenic γβC93A mice. GSH (1 mM) was added before contraction with phenylephrine (PE). (G) GSH-mediated potentiation of vasorelaxation does not differ between wild-type C57BL/6J and γβC93 RBCs but is eliminated in RBCs from βC93A and γβC93A mice. Data are presented as mean ± SEM; n = 4–5; (H) Hypoxic vasodilation by RBCs from γβC93A versus γβC93 mice was significantly diminished in bioassays of endothelium-intact wild-type aortic rings. Data are presented as mean ± SEM; n = 14–17. *P < 0.05 by one-way ANOVA in C–E and G and by Student's t test in H.

Fig. 3.

Autoregulation of blood flow and reactive hyperemia are dependent on βC93. (A) Representative continuous recordings of muscle blood flow during progressive hypoxia in γβC93 and γβC93A mice. (B) Blood flow is significantly compromised in βC93A and γβC93A mice versus γβC93 control mice. (C) The rate of change of blood flow upon transition to lower FiO₂ is significantly greater in βC93A and γβC93A mice than in γβC93 mice. (D) Muscle pO₂ was significantly lower in βC93A and γβC93A mice than in γβC93 mice. In B, C, and D, data are presented as mean ± SEM; n = 18–23; *P < 0.05 by one-way ANOVA for differences between βC93A and γβC93A mice versus γβC93 mice. (E) Enhancement of flow-mediated perfusion (blood flow) in gastrocnemius muscle is deficient in βC93A and γβC93A mice versus γβC93 mice. Data are presented as mean ± SEM; n = 14–23; *P < 0.05 by one-way ANOVA.

Fig. 4.

Impaired hemodynamic and cardiac responses to hypoxia in βCys93-deficient mice. (A–D) Invasive hemodynamic monitoring. Changes in (A) mean blood pressure, (B) rate of change of left ventricular pressure (dP/dt) (diastolic is shown), (C) cardiac output, and (D) stroke work during progressive hypoxia [FiO₂ of 0.21 (room air), 0.10, and 0.05]. Declines in all measures (A–D) are significantly greater in βC93A and γβC93A mice versus γβC93 control mice. (E and F) Echocardiography demonstrates that ventricular function is significantly compromised during progressive global hypoxia in βC93A and γβC93A mice versus γβC93 mice (see Fig. S3 for additional measures). (E) Left ventricular end systolic volume. (F) Representative recordings from mice at FiO₂ = 0.21 and FiO₂ = 0.05 that illustrate left ventricular dilation in βC93A and γβC93A versus γβC93 control mice. Data are quantified in Fig. S3. (In F, the vertical scale bar represents 2 mm, and the horizontal scale bar represents 100 ms.) Data in A–E are presented as mean ± SEM; n = 9–12; *P < 0.05 by one-way ANOVA for differences between βC93A and γβC93A mice versus γβC93 mice; ^†P < 0.05 by one-way ANOVA for differences between γβC93A mice versus γβC93 mice.

Fig. 5.

Cardiac ischemia and myocardial injury in βCys93-deficient mice. (A) Representative electrocardiographic recordings in a γβC93 (control) mouse (Upper) and a γβC93A mouse (Lower) at FiO₂ = 0.21 and FiO₂ = 0.05. T- and ST-waves are indicated. (B) During transient progressive hypoxia, ST-wave elevation (and hyperacute T-waves), indicative of acute ischemic injury, are significantly greater and far more frequent in βC93A and γβC93A mice than in γβC93 control mice (see also Fig. S4). (C) At FiO₂ = 0.21 (room air), T-wave amplitude is significantly reduced in βC93A and γβC93A mice versus γβC93 mice, indicative of myocardial ischemia. In B and C data are presented as mean ± SEM; n = 13–21 mice; *P < 0.05 by one-way ANOVA for differences between βC93A and γβC93A mice versus γβC93 mice; ^†P < 0.05 by one-way ANOVA for differences between βC93A mice versus γβC93 mice.

Fig. 6.

Increased hypoxia-induced mortality in βCys93-deficient mice. βC93A and γβC93A mice are significantly more likely than γβC93 (isogenic control) mice to die during or following brief hypoxic challenge, i.e., respiration at FiO₂ = 0.05. n = 27–35 per strain as indicated; *P < 0.05 by Fisher’s exact test.