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

Gene-Targeted Mice with the Human Troponin T R141W Mutation Develop Dilated Cardiomyopathy with Calcium Desensitization.

Most studies of the mechanisms leading to hereditary dilated cardiomyopathy (DCM) have been performed in reconstituted in vitro systems. Genetically engineered murine models offer the opportunity to dissect these mechanisms in vivo. We generated a gene-targeted knock-in murine model of the autosomal dominant Arg141Trp (R141W) mutation in Tnnt2, which was first described in a human family with DCM. Mice heterozygous for the mutation (Tnnt2R141W/+) recapitulated the human phenotype, developing left ventricular dilation and reduced contractility. There was a gene dosage effect, so that the phenotype in Tnnt2R141W/+mice was attenuated by transgenic overexpression of wildtype Tnnt2 mRNA transcript. Male mice exhibited poorer survival than females. Biomechanical studies on skinned fibers from Tnnt2R141W/+ hearts showed a significant decrease in pCa50 (-log[Ca2+] required for generation of 50% of maximal force) relative to wildtype hearts, indicating Ca2+ desensitization. Optical mapping studies of Langendorff-perfused Tnnt2R141W/+ hearts showed marked increases in diastolic and peak systolic intracellular Ca2+ ([Ca2+]i), and prolonged systolic rise and diastolic fall of [Ca2+]i. Perfused Tnnt2R141W/+ hearts had slower intrinsic rates in sinus rhythm and reduced peak heart rates in response to isoproterenol. Tnnt2R141W/+ hearts exhibited a reduction in phosphorylated phospholamban relative to wildtype mice. However, crossing Tnnt2R141W/+ mice with phospholamban knockout (Pln-/-) mice, which exhibit increased Ca2+ transients and contractility, had no effect on the DCM phenotype. We conclude that the Tnnt2 R141W mutation causes a Ca2+ desensitization and mice adapt by increasing Ca2+-transient amplitudes, which impairs Ca2+ handling dynamics, metabolism and responses to β-adrenergic activation

Representative APs recorded from wildtype and <i>Tnnt2</i>^R141W/+ hearts.

<p><b>(A)</b> Spontaneously beating <i>Tnnt2</i>^R141W/+ hearts had an intrinsically lower heart rate. <b>(B)</b> <i>Tnnt2</i>^R141W/+ hearts exhibited relatively small increases in heart rate at 1 and 10 μM concentrations.</p

Troponin I expression.

<p>Immunoblot and densitometry of phosphorylated troponin I (P-TnI) and total troponin I (TnI) in 8-week-old wildtype (WT) and <i>Tnnt2</i>^R141W/+ hearts. <i>n</i> = 3 for wildtype and <i>n</i> = 3 for <i>Tnnt2</i>^R141W/+.</p

Representative Ca²⁺ transients from wildtype (WT) and <i>Tnnt2</i>^R141W/+ hearts.

<p><b>(A)</b> Langendorff perfused hearts were loaded with Rhod-2/AM and imaged on a 16x16 element photodiode array to map Ca²⁺ transients (CaT). Spontaneously beating, <i>Tnnt2</i>^R141W/+ hearts exhibited a markedly slower intrinsic sinus rhythm heart rate (71±12 bpm, top right traces) compared to wildtype hearts (343±52 bpm top left traces; <i>n</i> = 6 each, <i>P</i><0.03). Calibration of Rhod-2 through measurements of F_max (maximum Rhod-2 fluorescence when saturated with Ca²⁺) and F_min (minimum Rhod-2 fluorescence when all Ca²⁺ is washed out and chelated with EGTA) detected increases in diastolic and peak systolic cytosolic Ca²⁺ in <i>Tnnt2</i>^R141W/+ compared to wildtype hearts (lower left panel). <i>Tnnt2</i>^R141W/+ hearts had a longer rise time of [Ca²⁺]_i, (WT = 14.3±2.09 ms, <i>n</i> = 4 and <i>Tnnt2</i>^R141W/+ = 17.20±0.67, <i>n</i> = 6; <i>P</i><0.05), and a longer time to recover to diastolic[Ca²⁺]_i. Lower right traces show the superposition of normalized CaTs from a wildtype and a <i>Tnnt2</i>^R141W/+ heart recorded from the center of the left ventricles, when both hearts were paced at the same cycle length (350 ms). The superposition of CaTs exposes marked differences in Ca²⁺ dynamics associated with Ca²⁺ desensitization in <i>Tnnt2</i>^R141W/+ hearts. <b>(B)</b> <i>Tnnt2</i>^R141W/+ hearts exhibited lower intrinsic heart rates and lower peak heart rates in response to isoproterenol at 1 and 10 μM concentrations, compared to wildtype, <i>P</i><0.001.</p

Expression of Ca²⁺ handling proteins.

<p>Immunoblots and densitometry analysis of total cardiac protein extracts from wildtype (WT) and <i>Tnnt2</i>^R141W/+hearts at ages 2 and 8 weeks. CASQ2, calsequestrin 2; NCX1, sodium/calcium exchanger; P-PLN, Ser¹⁶ phosphorylated phospholamban; SERCA2, sarcoplasmic reticulum cardiac Ca²⁺ ATPase 2; T-PLN, total phospholamban. *, <i>P</i><0.01 versus wildtype.</p

Ca²⁺ sensitivity and maximum force generation in skinned papillary muscle fibers.

<p>Ca²⁺ sensitivity and maximum force generation in skinned papillary muscle fibers.</p

Primer sequences used for real-time quantitative PCR (QPCR).

<p>Primer sequences used for real-time quantitative PCR (QPCR).</p

Generation of <i>Tnnt2</i>^R141W/+ mice.

<p><b>(A)</b> Homologous recombination strategy used to introduce the DCM Arg141Trp (R141W) mutation into exon 9 of the murine <i>Tnnt2</i> gene in ES cells. The resultant mice were mated with EIIa-Cre recombinase mice to remove the floxed <i>neo/zeo</i> cassette. <b>(B)</b> ES clones having undergone successful homologous recombination with the R141W targeting construct were identified by long range PCR using primer pairs F1 and R1 at the 5’ end, and F2 and R2 at the 3’ end. Positions of the primers are shown in panel A. Clones 1, 2, 4, and 5 showed appropriate homologous recombination at both the 5’ and the 3’ ends. <b>(C)</b> Expression of mutant <i>Tnnt2</i> mRNA in <i>Tnnt2</i>^R141W/+ mice was confirmed by reverse transcription-PCR and sequencing of total cardiac RNA. WT, wildtype.</p

Pacing kinetics.

<p>AP durations were shorter in <i>Tnnt2</i>^R141W/+ relative to wildtype hearts at all pacing cycle lengths. <i>n</i> = 4 for WT, <i>n</i> = 3 for all <i>Tnnt2</i>^R141W/+ because not all <i>Tnnt2</i>^R141W/+ hearts could be paced at faster rates or shorter cycle lengths. *, <i>P</i><0.001 vs. wildtype.</p