Essential role of hemoglobin beta-93-cysteine in posthypoxia facilitation of breathing in conscious mice

Abstract

When erythrocyte hemoglobin (Hb) is fully saturated with O2, nitric oxide (NO) covalently binds to the cysteine 93 residue of the Hb β-chain (B93-CYS), forming S-nitrosohemoglobin. Binding of NO is allosterically coupled to the O2 saturation of Hb. As saturation falls, the NO group on B93-CYS is transferred to thiols in the erythrocyte, and in the plasma, forming circulating S-nitrosothiols. Here, we studied whether the changes in ventilation during and following exposure to a hypoxic challenge were dependent on erythrocytic B93-CYS. Studies were performed in conscious mice in which native murine Hb was replaced with human Hb (hB93-CYS mice) and in mice in which murine Hb was replaced with human Hb containing an alanine rather than cysteine at position 93 on the Bchain (hB93-ALA). Both strains expressed human γ-chain Hb, likely allowing a residual element of S-nitrosothiol-dependent signaling. While resting parameters and initial hypoxic (10% O2, 90% N2) ventilatory responses were similar in hB93-CYS mice and hB93-ALA mice, the excitatory ventilatory responses (short-term potentiation) that occurred once the mice were returned to room air were markedly diminished in hB93-ALA mice. Further, short-term potentiation responses were virtually absent in mice with bilateral transection of the carotid sinus nerves. These data demonstrate that hB93-CYS plays an essential role in mediating carotid sinus nerve-dependent short-term potentiation, an important mechanism for recovery from acute hypoxia.

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

“…We confirmed that total Hb concentration and blood O 2 content of the mutant strains are unchanged from wild type at baseline (room air), as reported ( Fig. S2 A and B) (19,20), and that plasma nitrite levels, a direct measure of circulatory NO production by endothelial nitric oxide synthase (eNOS), was similar in all strains (Fig. S2C).…”

“…Enhanced S-nitrosylation of a closely spaced, alternative Cys in the absence of a preferred site also has been observed in other SNO-proteins (32), including invertebrate Hb (33), and a similar shift in the site of other regulatory posttranslational modifications when preferred sites are mutated is well documented. In addition, although γ-subunits reportedly constitute only ∼1% of Hb in γβC93 and γβC93A mice (20) (percentages that we have confirmed qualitatively; Fig. S1), Hbγ concentrations nonetheless far exceed endogenous amounts of SNO-Hb, and all strains will, in fact, retain some SNO-(γ)Cys93.…”

“…In γβC93 and γβC93A mice (genetically matched with the exception of βCys93), RBCs also contained the human γ (fetal)-subunit, which was introduced to enhance fetal viability but remains a minor species (Fig. S1) (20), consistent with measurements of O 2 affinity that indicate minimal changes in mutant mice (19); in all three strains, RBCs retain the murine γ-subunit. βCys93 was replaced with Ala in βC93A and γβC93A mice.…”

“…homozygous mutation of human βCys93 (which minimally affects traditional Hb functions) has never been reported, and no other mutation of Hb (or any other protein of which we are aware) is known to cause myocardial ischemia in normoxia. Recently, it has been shown that the ventilatory response to hypoxia is abnormal in βC93A-mutant mice, implicating SNO-Hb in the central control of breathing (15,20). Further, SNO-Hb levels in humans increase with ascent to altitude and are correlated strongly with exercise performance under hypoxia (34).…”

Hypoxia-Induced Changes in Protein S-Nitrosylation in Female Mouse Brainstem

“…First, the expression of HVR and shortterm potentiation (STP) are absent in conscious mice with bilateral carotid sinus nerve transection (39), supporting a role of carotid body primary glomus cells/ chemoafferents in these responses. As such, the potential release of S-nitrosothiols from eNOS-positive glomus cells (11) and subsequent activation of chemoafferent terminals may contribute to the initiation of HVR, whereas S-nitrosothiols generated from red blood cell hemoglobin (see subsequent text) do not appear to be vital for initiation of the HVR (39). Second, ventilatory roll-off during the hypoxic challenge is absent in conscious GSNOR 1/2 or GSNO 2/2 mice (11), clearly suggesting that roll-off is due to enhanced catabolism of GSNO in the carotid body and/or central structures.…”