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.

Linking Endoplasmic Reticular Stress and Alternative Splicing.

RNA splicing patterns in antibody-secreting cells are shaped by endoplasmic reticulum stress, ELL2 (eleven-nineteen lysine-rich leukemia gene 2) induction, and changes in the levels of snRNAs. Endoplasmic reticulum stress induces the unfolded protein response comprising a highly conserved set of genes crucial for cell survival; among these is Ire1, whose auto-phosphorylation drives it to acquire a regulated mRNA decay activity. The mRNA-modifying function of phosphorylated Ire1 non-canonically splices Xbp1 mRNA and yet degrades other cellular mRNAs with related motifs. Naïve splenic B cells will activate Ire1 phosphorylation early on after lipopolysaccharide (LPS) stimulation, within 18 h; large-scale changes in mRNA content and splicing patterns result. Inhibition of the mRNA-degradation function of Ire1 is correlated with further differences in the splicing patterns and a reduction in the mRNA factors for snRNA transcription. Some of the >4000 splicing changes seen at 18 h after LPS stimulation persist into the late stages of antibody secretion, up to 72 h. Meanwhile some early splicing changes are supplanted by new splicing changes introduced by the up-regulation of ELL2, a transcription elongation factor. ELL2 is necessary for immunoglobulin secretion and does this by changing mRNA processing patterns of immunoglobulin heavy chain and >5000 other genes.

RNA Splicing in the Transition from B Cells to Antibody-Secreting Cells: The Influences of ELL2, Small Nuclear RNA, and Endoplasmic Reticulum Stress.

In the transition from B cells to Ab-secreting cells (ASCs) many genes are induced, such as ELL2, Irf4, Prdm1, Xbp1, whereas other mRNAs do not change in abundance. Nonetheless, using splicing array technology and mouse splenic B cells plus or minus LPS, we found that induced and "uninduced" genes can show large differences in splicing patterns between the cell stages, which could influence ASC development. We found that ∼55% of these splicing changes depend on ELL2, a transcription elongation factor that influences expression levels and splicing patterns of ASC signature genes, genes in the cell-cycle and N-glycan biosynthesis and processing pathways, and the secretory versus membrane forms of the IgH mRNA. Some of these changes occur when ELL2 binds directly to the genes encoding those mRNAs, whereas some of the changes are indirect. To attempt to account for the changes that occur in RNA splicing before or without ELL2 induction, we examined the amount of the small nuclear RNA molecules and found that they were significantly decreased within 18 h of LPS stimulation and stayed low until 72 h. Correlating with this, at 18 h after LPS, endoplasmic reticulum stress and Ire1 phosphorylation are induced. Inhibiting the regulated Ire1-dependent mRNA decay with 4u8C correlates with the reduction in small nuclear RNA and changes in the normal splicing patterns at 18 h. Thus, we conclude that the RNA splicing patterns in ASCs are shaped early by endoplasmic reticulum stress and Ire1 phosphorylation and later by ELL2 induction.

Cytochrome b5 Reductase 3 Modulates Soluble Guanylate Cyclase Redox State and cGMP Signaling.

RATIONALE: Soluble guanylate cyclase (sGC) heme iron, in its oxidized state (Fe3+), is desensitized to NO and limits cGMP production needed for downstream activation of protein kinase G-dependent signaling and blood vessel dilation. OBJECTIVE: Although 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 that vascular smooth muscle cells have an intrinsic ability to reduce oxidized sGC heme iron and form protein-protein complexes between cytochrome b5 reductase 3, also known as methemoglobin reductase, and oxidized sGC. Genetic knockdown and pharmacological inhibition in vascular smooth muscle cells reveal that cytochrome b5 reductase 3 expression and activity is critical for NO-stimulated cGMP production and vasodilation. Mechanistically, we show that cytochrome b5 reductase 3 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 cytochrome b5 reductase 3 as the first identified physiological sGC heme iron reductase in vascular smooth muscle cells, serving as a critical regulator of cGMP production and protein kinase G-dependent signaling.