Loss of cardiomyocyte CYB5R3 impairs redox equilibrium and causes sudden cardiac death.

Sudden cardiac death (SCD) in patients with heart failure (HF) is allied with an imbalance in reduction and oxidation (redox) signaling in cardiomyocytes; however, the basic pathways and mechanisms governing redox homeostasis in cardiomyocytes are not fully understood. Here, we show that cytochrome b5 reductase 3 (CYB5R3), an enzyme known to regulate redox signaling in erythrocytes and vascular cells, is essential for cardiomyocyte function. Using a conditional cardiomyocyte-specific CYB5R3-knockout mouse, we discovered that deletion of CYB5R3 in male, but not female, adult cardiomyocytes causes cardiac hypertrophy, bradycardia, and SCD. The increase in SCD in CYB5R3-KO mice is associated with calcium mishandling, ventricular fibrillation, and cardiomyocyte hypertrophy. Molecular studies reveal that CYB5R3-KO hearts display decreased adenosine triphosphate (ATP), increased oxidative stress, suppressed coenzyme Q levels, and hemoprotein dysregulation. Finally, from a translational perspective, we reveal that the high-frequency missense genetic variant rs1800457, which translates into a CYB5R3 T117S partial loss-of-function protein, associates with decreased event-free survival (~20%) in Black persons with HF with reduced ejection fraction (HFrEF). Together, these studies reveal a crucial role for CYB5R3 in cardiomyocyte redox biology and identify a genetic biomarker for persons of African ancestry that may potentially increase the risk of death from HFrEF.

FoxO4 controls sGCß transcription in vascular smooth muscle.

Nitric oxide (NO) binds soluble guanylyl cyclase ß (sGCß) to produce cGMP and relax vascular smooth muscle cells (SMCs) needed for vasodilation. Although the regulation of NO-stimulated sGC activity has been well characterized at the posttranslational level, the mechanisms that govern sGC transcription remain incompletely understood. Recently, we identified Forkhead box subclass O (FoxO) transcription factors as essential for expression of sGC; however, the specific FoxO family member responsible for the expression of sGCß in SMC remains unknown. Using FoxO shRNA knockdown adenovirus treatment in rat aortic SMCs, we show that FoxO1 or FoxO3 knockdown causes greater than twofold increases in Gucy1a3 and Gucy1b3 mRNA expression, without changes in NO-dependent cGMP production or cGMP-dependent phosphorylation. FoxO4 knockdown produced a 50% decrease in Gucy1a3 and Gucy1b3 mRNA with 70% loss of sGCα and 50% loss of sGCß protein expression. Knockdown of FoxO4 expression decreased cGMP production and downstream protein kinase G-dependent phosphorylation more than 50%. Triple FoxO knockdown exacerbated loss of sGC-dependent function, phenocopying previous FoxO inhibition studies. Using promoter luciferase and chromatin immunoprecipitation assays, we find that FoxO4 acts as a transcriptional activator by directly binding several FoxO DNA motifs in the promoter regions of GUCY1B3 in human aortic SMCs. Collectively, our data show FoxO4 is a critical transcriptional regulator of sGCß expression in SMC.NEW & NOTEWORTHY One of the key mechanisms of vascular smooth muscle cell (SMC) dilation occurs through nitric oxide (NO)-dependent induction of soluble guanylyl cyclase (sGC) by means of its ß-subunit. Herein, we are the first to identify Forkhead box subclass O protein 4 (FoxO4) as a key transcriptional regulator of GUCY1B3 expression, which codes for sGCß protein in human and animal SMCs. This discovery will likely have important implications for the future usage of antihypertensive and vasodilatory therapies which target NO production, sGC, or FoxO transcription factors.

Targeting Pulmonary Endothelial Hemoglobin α Improves Nitric Oxide Signaling and Reverses Pulmonary Artery Endothelial Dysfunction.

Pulmonary hypertension is characterized by pulmonary endothelial dysfunction. Previous work showed that systemic artery endothelial cells (ECs) express hemoglobin (Hb) α to control nitric oxide (NO) diffusion, but the role of this system in pulmonary circulation has not been evaluated. We hypothesized that up-regulation of Hb α in pulmonary ECs contributes to NO depletion and pulmonary vascular dysfunction in pulmonary hypertension. Primary distal pulmonary arterial vascular smooth muscle cells, lung tissue sections from unused donor (control) and idiopathic pulmonary artery (PA) hypertension lungs, and rat and mouse models of SU5416/hypoxia-induced pulmonary hypertension (PH) were used. Immunohistochemical, immunocytochemical, and immunoblot analyses and transfection, infection, DNA synthesis, apoptosis, migration, cell count, and protein activity assays were performed in this study. Cocultures of human pulmonary microvascular ECs and distal pulmonary arterial vascular smooth muscle cells, lung tissue from control and pulmonary hypertensive lungs, and a mouse model of chronic hypoxia-induced PH were used. Immunohistochemical, immunoblot analyses, spectrophotometry, and blood vessel myography experiments were performed in this study. We find increased expression of Hb α in pulmonary endothelium from humans and mice with PH compared with controls. In addition, we show up-regulation of Hb α in human pulmonary ECs cocultured with PA smooth muscle cells in hypoxia. We treated pulmonary ECs with a Hb α mimetic peptide that disrupts the association of Hb α with endothelial NO synthase, and found that cells treated with the peptide exhibited increased NO signaling compared with a scrambled peptide. Myography experiments using pulmonary arteries from hypoxic mice show that the Hb α mimetic peptide enhanced vasodilation in response to acetylcholine. Our findings reveal that endothelial Hb α functions as an endogenous scavenger of NO in the pulmonary endothelium. Targeting this pathway may offer a novel therapeutic target to increase endogenous levels of NO in PH.