Rationale Soluble guanylate cyclase (sGC) heme iron, in its oxidized state (Fe3+), is desensitized to nitric oxide (NO) and limits cyclic guanosine 3′, 5′-monophosphate (cGMP) production needed for downstream activation of PKG-dependent signaling and blood vessel dilation. Objective While reactive oxygen species are known to oxidize the sGC heme iron, the basic mechanism(s) governing sGC heme iron recycling to its NO-sensitive, reduced state, remain poorly understood. Methods and Results Oxidant challenge studies show vascular smooth muscle cells have an intrinsic ability to reduce oxidized sGC heme iron and form protein-protein complexes between cytochrome b5 reductase 3 (Cyb5R3), also known as methemoglobin reductase, and oxidized sGC. Genetic knockdown and pharmacological inhibition in VSMCs reveal Cyb5R3 expression and activity is critical for NO-stimulated cGMP production and vasodilation. Mechanistically, we show Cyb5R3 directly reduces oxidized sGC required for NO sensitization as assessed by biochemical, cellular, and ex vivo assays. Conclusions Together, these findings identify new insights into NO-sGC-cGMP signaling and reveal Cyb5R3 as the first identified physiological sGC heme iron reductase in VSMCs, serving as a critical regulator of cGMP production and PKG dependent signaling.

Aim: To test the hypothesis that diminished vascular nitric oxide availability might explain the inability of individuals with chronic heart failure (CHF) to maintain the microvascular PO 2 's (PO 2mv µ O 2 delivery-to-uptake ratio) seen in healthy animals. Methods: We superfused sodium nitroprusside (SNP; 300 lm), KrebsHenseleit (control, CON) and l-nitro arginine methyl ester (l-NAME; 1.5 mm) onto the spinotrapezius muscle and measured PO 2mv by phosphorescence quenching in female Sprague-Dawley rats (n ¼ 26) at rest and during twitch contractions (1 Hz). Seven rats served as controls (Sham) while CHF was induced by myocardial infarction. CHF rats were grouped as moderate (MOD; n ¼ 15) and severe CHF (SEV; n ¼ 4) according to morphological data and baseline PO 2mv . Results: In contrast to Sham and MOD, l-NAME did not affect the PO 2mv response (dynamics and steady-state) of SEV when compared with CON. SNP restored the PO 2mv profile of SEV to that seen in Sham animals during CON. Specifically, the effect of l-NAME expressed as D(l-NAME -CON) were: Baseline PO 2mv [in mmHg, DSham ¼ )7.0 AE 1.6 (P < 0.05); DSEV ¼ )1.2 AE 2.1], end-contractions PO 2mv [in mmHg, DSham ¼ )5.0 AE 1.0 (P < 0.05); DSEV ¼ )2.5 AE 0.5] and time constant of PO 2mv decrease [in s, DSham ¼ )6.5 AE 3.0 (P < 0.05); DSEV ¼ )3.2 AE 1.8]. Conclusion: These data provide the first direct evidence that the pathological profiles of PO 2mv associated with severe CHF can be explained, in part, by a diminished vascular NO availability.

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Background: Many patients with heart failure with preserved ejection fraction (HFpEF) have metabolic syndrome and develop exercise-induced pulmonary hypertension (EIPH). Increases in pulmonary vascular resistance in patients with HFpEF portend a poor prognosis; this phenotype is referred to as combined pre-and post-capillary PH (CpcPH). Therapeutic trials for EIPH and CpcPH have been disappointing, suggesting the need for strategies that target upstream mechanisms of disease. This work reports novel rat EIPH models and mechanisms of pulmonary vascular dysfunction centered around the transcriptional repression of the soluble guanylate cyclase (sGC) enzyme in pulmonary artery smooth muscle cells (PAVSMCs). Methods: We used obese ZSF-1 leptin-receptor knock-out rats (HFpEF model), obese ZSF-1 rats treated with SU5416 to stimulate resting PH (Obese+sugen, CpcPH model), and Lean ZSF-1 rats (controls). Right and left ventricular hemodynamics were evaluated via implanted-catheters during treadmill exercise. PA function was evaluated using MRI and myography. Overexpression of NFYA, a transcriptional-enhancer of sGCβ1, was performed by PA delivery of adeno-associated-virus 6 (AAV6). Treatment groups received SGLT2 inhibitor Empagliflozin in drinking water. PAVSMCs from rats and humans were cultured with Palmitic acid, Glucose, and Insulin (PGI) to induce metabolic-stress. Results: Obese rats showed normal resting right ventricular systolic pressures (RVSP) which significantly increased during exercise, modeling EIPH. Obese+sugen rats showed anatomical PA remodeling and developed elevated RVSP at rest, which was exacerbated with exercise, modeling CpcPH. Myography and MRI during dobutamine-challenge revealed PA functional impairment of both obese groups. PAs of obese rats produced reactive oxygen species (ROS) and decreased sGCβ1 expression. Mechanistically, cultured PAVSMCs from obese rats, humans with diabetes or treated with PGI, showed increased mitochondrial-ROS, which enhanced miR-193b-dependent RNA-degradation of NFYA, resulting in decreased sGCβ1-cGMP signaling. Forced NYFA expression by AAV6 delivery increased sGCβ1 levels and improved exercise-PH in Obese+sugen rats. Treatment of Obese+sugen rats with Empagliflozin improved metabolic syndrome, reduced mitochondrial ROS and miR-193b levels, restored NFYA/sGC activity, and prevented EIPH. Conclusions: In HFpEF and CpcPH models, metabolic syndrome contributes to pulmonary vascular dysfunction and EIPH through enhanced ROS and miR-193b expression, which down-regulates NFYA-dependent sGCβ1 expression. AAV-mediated NFYA overexpression and SGLT2 inhibition restores NFYA-sGCβ1-cGMP signaling and ameliorates EIPH.

Sickle cell disease (SCD) is associated with intravascular hemolysis and oxidative inhibition of nitric oxide (NO) signaling. BAY 54-6544 is a small-molecule activator of oxidized soluble guanylate cyclase (sGC), which, unlike endogenous NO and the sGC stimulator, BAY 41-8543, preferentially binds and activates heme-free, NO-insensitive sGC to restore enzymatic cGMP production. We tested orally delivered sGC activator, BAY 54-6544 (17 mg/kg/d), sGC stimulator, BAY 41-8543, sildenafil, and placebo for 4-12 weeks in the Berkeley transgenic mouse model of SCD (BERK-SCD) and their hemizygous (Hemi) littermate controls (BERK-Hemi). Right ventricular (RV) maximum systolic pressure (RVmaxSP) was measured using micro right-heart catheterization. RV hypertrophy (RVH) was determined using Fulton's index and RV corrected weight (ratio of RV to tibia). Pulmonary artery vasoreactivity was tested for endothelium-dependent and -independent vessel relaxation. Right-heart catheterization revealed higher RVmaxSP and RVH in BERK-SCD versus BERK-Hemi, which worsened with age. Treatment with the sGC activator more effectively lowered RVmaxSP and RVH, with 90-day treatment delivering superior results, when compared with other treatments and placebo groups. In myography experiments, acetylcholine-induced (endothelium-dependent) and sodium-nitroprusside-induced (endothelium-independent NO donor) relaxation of the pulmonary artery harvested from placebo-treated BERK-SCD was impaired relative to BERK-Hemi but improved after therapy with sGC activator. By contrast, no significant effect for sGC stimulator or sildenafil was observed in BERK-SCD. These findings suggest that sGC is oxidized in the pulmonary arteries of transgenic SCD mice, leading to blunted responses to NO, and that the sGC activator, BAY 54-6544, may represent a novel therapy for SCD-associated pulmonary arterial hypertension and cardiac remodeling.