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.These studies were supported by NIH grants R35 HL 161177 (to ACS), R01 HL 133864 (to ACS), R01 HL 128304 (to ACS), R41 HL15098 (to GS), R01 GM 122091 (to PHT), GM125944 (to FJS), R01 DK112854 (to FJS), R21 NS112787 (to MF), NS121706 (to YLW), EB023507 (to YLW), F31 HL149241 (to HMS), and F31 HL151173 (to JCG). Support was also provided by American Heart Association grants 19EIA34770095 (to ACS), AHA 18CDA34140024 (to YLW), and 19PRE34380152 (to NTC); the Spanish Ministry of Health (grant FIS PI17-01286); Junta de Andalucía BIO-177 and the FEDER Funding Program from the European Union and CIBERER (U729)-ISCIII (to PN); Department of Defense W81XWH1810070 (to YLW); and Vitalant. This research was supported in part by the University of Pittsburgh Center for Research Computing through the resources provided. Specifically, this work used the HTC cluster, which is supported by NIH award number S10OD028483.Peer reviewe

Treatment With Treprostinil and Metformin Normalizes Hyperglycemia and Improves Cardiac Function in Pulmonary Hypertension Associated With Heart Failure With Preserved Ejection Fraction

Objective: Pulmonary hypertension (PH) due to left heart disease (group 2), especially in the setting of heart failure with preserved ejection fraction (HFpEF), is the most common cause of PH worldwide; however, at present, there is no proven effective therapy available for its treatment. PH-HFpEF is associated with insulin resistance and features of metabolic syndrome. The stable prostacyclin analog, treprostinil, is an effective and widely used Food and Drug Administration-approved drug for the treatment of pulmonary arterial hypertension. While the effect of treprostinil on metabolic syndrome is unknown, a recent study suggests that the prostacyclin analog beraprost can improve glucose intolerance and insulin sensitivity. We sought to evaluate the effectiveness of treprostinil in the treatment of metabolic syndrome-associated PH-HFpEF. Approach and Results: Treprostinil treatment was given to mice with mild metabolic syndrome-associated PH-HFpEF induced by high-fat diet and to SU5416/obese ZSF1 rats, a model created by the treatment of rats with a more profound metabolic syndrome due to double leptin receptor defect (obese ZSF1) with a vascular endothelial growth factor receptor blocker SU5416. In high-fat diet-exposed mice, chronic treatment with treprostinil reduced hyperglycemia and pulmonary hypertension. In SU5416/Obese ZSF1 rats, treprostinil improved hyperglycemia with similar efficacy to that of metformin (a first-line drug for type 2 diabetes mellitus); the glucose-lowering effect of treprostinil was further potentiated by the combined treatment with metformin. Early treatment with treprostinil in SU5416/Obese ZSF1 rats lowered pulmonary pressures, and a late treatment with treprostinil together with metformin improved pulmonary artery acceleration time to ejection time ratio and tricuspid annular plane systolic excursion with AMPK (AMP-activated protein kinase) activation in skeletal muscle and the right ventricle. Conclusions: Our data suggest a potential use of treprostinil as an early treatment for mild metabolic syndrome-associated PH-HFpEF and that combined treatment with treprostinil and metformin may improve hyperglycemia and cardiac function in a more severe disease

Cardiomyocyte BRAF and type 1 RAF inhibitors promote cardiomyocyte and cardiac hypertrophy in mice in vivo

The extracellular signal-regulated kinase 1/2 (ERK1/2) cascade promotes cardiomyocyte hypertrophy and is cardioprotective, with the three RAF kinases forming a node for signal integration. Our aims were to determine if BRAF is relevant for human heart failure, whether BRAF promotes cardiomyocyte hypertrophy, and if Type 1 RAF inhibitors developed for cancer (that paradoxically activate ERK1/2 at low concentrations: the “RAF paradox”) may have the same effect. BRAF was upregulated in heart samples from patients with heart failure compared with normal controls. We assessed the effects of activated BRAF in the heart using mice with tamoxifen-activated Cre for cardiomyocyte-specific knock-in of the activating V600E mutation into the endogenous gene. We used echocardiography to measure cardiac dimensions/function. Cardiomyocyte BRAFV600E induced cardiac hypertrophy within 10 d, resulting in increased ejection fraction and fractional shortening over 6 weeks. This was associated with increased cardiomyocyte size without significant fibrosis, consistent with compensated hypertrophy. The experimental Type 1 RAF inhibitor, SB590885, and/or encorafenib (a RAF inhibitor used clinically) increased ERK1/2 phosphorylation in cardiomyocytes, and promoted hypertrophy, consistent with a “RAF paradox” effect. Both promoted cardiac hypertrophy in mouse hearts in vivo, with increased cardiomyocyte size and no overt fibrosis. In conclusion, BRAF potentially plays an important role in human failing hearts, activation of BRAF is sufficient to induce hypertrophy, and Type 1 RAF inhibitors promote hypertrophy via the “RAF paradox”. Cardiac hypertrophy resulting from these interventions was not associated with pathological features, suggesting that Type 1 RAF inhibitors may be useful to boost cardiomyocyte function

Adrenergic stimulation promotes T-wave alternans and arrhythmia inducibility in a TNF-α genetic mouse model of congestive heart failure

T-wave alternans (TWA) is a proarrhythmic repolarization instability that is common in congestive heart failure (CHF). Although transgenic mice are commonly used to study the mechanisms of arrhythmogenesis in CHF, little is known about the dynamics of TWA in these species. We hypothesized that TWA is present in a TNF-α model of CHF and can be further promoted by adrenergic stimulation. We studied 16 TNF-α mice and 12 FVB controls using 1) in vivo intracardiac electrophysiological testing and 2) ambulatory telemetry during 30 min before and after an intraperitoneal injection of isoproterenol. TWA was examined using both linear and nonlinear filtering applied in the time domain. In addition, changes in the mean amplitude of the T wave and area under the T wave were computed. During intracardiac electrophysiological testing, none of the animals had TWA or inducible arrhythmias before the injection of isoproterenol. After the injection, sustained TWA and inducible ventricular tachyarrhythmias were observed in TNF-α mice but not in FVB mice. In ambulatory telemetry, before the isoproterenol injection, the cardiac cycle length (CL) was longer in TNF-α mice than in FVB mice (98 ± 9 and 88 ± 3 ms, P = 0.04). After the injection of isoproterenol, the CL became 8% and 6% shorter in TNF-α and FVB mice (P < 10−4); however, the 2% difference between the groups in the magnitude of CL changes was not significant. In TNF-α mice, the magnitude of TWA was 1.5–2 times greater than in FVB mice both before and after the isoproterenol injection. The magnitude of TWA increased significantly after the isoproterenol injection compared with the baseline in TNF-α mice (P = 0.003) but not in FVB mice. The mean amplitude of the T wave and area under the T wave increased 60% and 80% in FVB mice (P = 0.006 and 0.009) but not in TNF-α mice. In conclusion, TWA is present in a TNF-α model of CHF and can be further promoted by adrenergic stimulation, along with the enhanced susceptibility for ventricular arrhythmias