RESUMEN
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.
Asunto(s)
Insuficiencia Cardíaca , Miocitos Cardíacos , Animales , Muerte Súbita Cardíaca , Insuficiencia Cardíaca/genética , Insuficiencia Cardíaca/metabolismo , Masculino , Ratones , Ratones Noqueados , Miocitos Cardíacos/metabolismo , Oxidación-Reducción , Volumen SistólicoRESUMEN
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 ß-adrenergic activation.
Asunto(s)
Calcio/metabolismo , Cardiomiopatía Dilatada/genética , Cardiomiopatía Dilatada/metabolismo , Corazón/fisiopatología , Mutación Puntual , Troponina T/genética , Animales , Cardiomiopatía Dilatada/fisiopatología , Femenino , Técnicas de Sustitución del Gen , Marcación de Gen , Humanos , Masculino , Ratones , Ratones Transgénicos , Contracción MiocárdicaRESUMEN
DJ-1, a protein that promotes the action of multiple anti-apoptotic/pro-survival pathways, is expressed prominently in human reactive astrocytes and in many human cancers. Glioblastomas (GBMs) are the most common adult primary brain tumor, and most show either abnormalities in p53 or epidermal growth factor receptor (EGFR) amplification, but not both. In this retrospective study of 40 surgically resected GBMs, we compared the immunohistochemical intensity of DJ-1 expression (based on blinded scoring by independent examiners) to these and other molecular factors associated with GBM oncogenesis. We report here that: (i) most of the GBMs that we studied expressed DJ-1 protein at significant levels, and typically in a cytoplasmic, non-nuclear fashion; (ii) DJ-1 staining intensity varied directly with strong nuclear p53 expression (assessed by immunostaining); and (iii) DJ-1 staining intensity varied inversely with EGFR amplification (assessed by fluorescent in situ hybridization). Since the anti-apoptotic/pro-survival actions of DJ-1 have been clearly linked in in vitro systems to p53 and receptor tyrosine kinase (i.e. EGFR) pathways that are hypothesized to be critical to GBM genesis, these observations indicate that DJ-1 expression may play a role in the biology of some types of GBMs. Therefore, given the new associations presented here between DJ-1, p53 and EGFR amplification in GBMs, future investigations of these tumors should include an analysis of DJ-1 to determine whether its expression pattern is important for tumor progression, prognosis and responsiveness to therapy.
Asunto(s)
Neoplasias Encefálicas/metabolismo , Receptores ErbB/biosíntesis , Glioblastoma/metabolismo , Proteínas Oncogénicas/biosíntesis , Proteína p53 Supresora de Tumor/biosíntesis , Adulto , Anciano , Anciano de 80 o más Años , Neoplasias Encefálicas/genética , Receptores ErbB/genética , Femenino , Amplificación de Genes , Expresión Génica , Perfilación de la Expresión Génica , Glioblastoma/genética , Humanos , Inmunohistoquímica , Hibridación Fluorescente in Situ , Péptidos y Proteínas de Señalización Intracelular , Masculino , Persona de Mediana Edad , Proteína Desglicasa DJ-1 , Estudios RetrospectivosRESUMEN
Ventricular tachyarrhythmias are the main cause of sudden death in patients after myocardial infarction. Here we show that transplantation of embryonic cardiomyocytes (eCMs) in myocardial infarcts protects against the induction of ventricular tachycardia (VT) in mice. Engraftment of eCMs, but not skeletal myoblasts (SMs), bone marrow cells or cardiac myofibroblasts, markedly decreased the incidence of VT induced by in vivo pacing. eCM engraftment results in improved electrical coupling between the surrounding myocardium and the infarct region, and Ca2+ signals from engrafted eCMs expressing a genetically encoded Ca2+ indicator could be entrained during sinoatrial cardiac activation in vivo. eCM grafts also increased conduction velocity and decreased the incidence of conduction block within the infarct. VT protection is critically dependent on expression of the gap-junction protein connexin 43 (Cx43; also known as Gja1): SMs genetically engineered to express Cx43 conferred a similar protection to that of eCMs against induced VT. Thus, engraftment of Cx43-expressing myocytes has the potential to reduce life-threatening post-infarct arrhythmias through the augmentation of intercellular coupling, suggesting autologous strategies for cardiac cell-based therapy.
Asunto(s)
Arritmias Cardíacas/complicaciones , Arritmias Cardíacas/prevención & control , Conexina 43/metabolismo , Infarto del Miocardio/complicaciones , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/trasplante , Animales , Arritmias Cardíacas/patología , Arritmias Cardíacas/fisiopatología , Conexina 43/genética , Embrión de Mamíferos/citología , Corazón/fisiología , Corazón/fisiopatología , Humanos , Ratones , Ratones Transgénicos , Infarto del Miocardio/patología , Infarto del Miocardio/fisiopatología , Miocardio/citología , Miocardio/patología , PerfusiónRESUMEN
Sympathetic activity and spatial dispersion of repolarization (DOR) have been implicated as mechanisms that promote arrhythmia vulnerability; yet there are no direct measurements of the effects of autonomic nerve stimulation on DOR. Rabbit hearts were perfused in a Langendorff apparatus with full sympathetic and parasympathetic innervation and were optically mapped to measure action potential durations and DOR (apex-base) over the left ventricles. DOR was measured under sinus rhythm, during bilateral sympathetic nerve stimulation (SNS) and right and/or left vagus nerve stimulation and was compared with DOR during isoproterenol (100 nmol/L) or acetylcholine (1 micromol/L) infusion. In sinus rhythm, repolarization started at the apex and systematically progressed toward the base. SNS (10 to 15 Hz) increased DOR by 29% (from Deltaaction potential duration=17+/-0.7 to -22+/-1.6 ms, n=6) and reversed DOR as the direction of repolarization from apex-->base in sinus rhythm shifted to base-->apex in 5 to 15 seconds after SNS. DOR flipped back to its sinus rhythm DOR pattern 115+/-15 seconds after the interruption of SNS. During right or left vagus nerve stimulation, there was no change in the direction of DOR, but bilateral vagus nerve stimulation increased and reversed DOR to base-->apex direction. Infusion of isoproterenol or acetylcholine increased DOR but did not alter the direction of repolarization sequences. These findings demonstrate that bilateral autonomic activity (SNS or vagus nerve stimulation) cause reversible shifts of apex-base DOR and that the spatial heterogeneities of autonomic effects on the ventricles are most likely attributable to a greater innervation at the base than the apex of the heart.