RESUMEN

Duchenne muscular dystrophy (DMD) associated cardiomyopathy remains incurable. Connexin 43 (Cx43) is upregulated and remodeled in the hearts of mdx mice, a mouse model of DMD. Hearts from Wild Type, mdx, and mdx:Cx43(+/-) mice were studied before (4-6 months) and after (10-15 months) the onset of cardiomyopathy to assess the impact of decreasing Cx43 levels on cardiac pathology in dystrophic mice. Increased connexin 43 protein levels in mdx hearts were not observed in mdx:Cx43(+/-) hearts. Cx43 remodeling in mdx hearts was attenuated in mdx:Cx43(+/-) hearts. At time-point 4-6 months, isolated cardiomyocytes from mdx hearts displayed enhanced ethidium bromide uptake, augmented intracellular calcium signals and increased production of reactive oxygen species. These pathological features were improved in mdx:Cx43(+/-) cardiomyocytes. Isoproterenol-challenged mdx:Cx43(+/-) mice did not show arrhythmias or acute lethality observed in mdx mice. Likewise, isoproterenol-challenged mdx:Cx43(+/-) isolated hearts were also protected from arrhythmogenesis. At time-point 10-15 months, mdx:Cx43(+/-) mice showed decreased cardiac fibrosis and improved ventricular function, relative to mdx mice. These results suggest that normalization of connexin 43 protein levels in mdx mice reduces overall cardiac pathology.

Asunto(s)

Calcio/metabolismo , Cardiomiopatías/metabolismo , Conexina 43/metabolismo , Distrofia Muscular de Duchenne/metabolismo , Animales , Cardiomiopatías/patología , Modelos Animales de Enfermedad , Femenino , Masculino , Ratones Transgénicos , Distrofia Muscular de Duchenne/patología , Miocardio/patología , Miocitos Cardíacos/patología

RESUMEN

Aims: Duchenne muscular dystrophy (DMD) is an inherited devastating muscle disease with severe and often lethal cardiac complications. Emerging evidence suggests that the evolution of the pathology in DMD is accompanied by the accumulation of mitochondria with defective structure and function. Here, we investigate whether defects in the housekeeping autophagic pathway contribute to mitochondrial and metabolic dysfunctions in dystrophic cardiomyopathy. Methods and results: We employed various biochemical and imaging techniques to assess mitochondrial structure and function as well as to evaluate autophagy, and specific mitochondrial autophagy (mitophagy), in hearts of mdx mice, an animal model of DMD. Our results indicate substantial structural damage of mitochondria and a significant decrease in ATP production in hearts of mdx animals, which developed cardiomyopathy. In these hearts, we also detected enhanced autophagy but paradoxically, mitophagy appeared to be suppressed. In addition, we found decreased levels of several proteins involved in the PINK1/PARKIN mitophagy pathway as well as an insignificant amount of PARKIN protein phosphorylation at the S65 residue upon induction of mitophagy. Conclusions: Our results suggest faulty mitophagy in dystrophic hearts due to defects in the PINK1/PARKIN pathway.

Asunto(s)

Autofagia , Cardiomiopatías/enzimología , Mitocondrias Cardíacas/enzimología , Mitofagia , Distrofia Muscular de Duchenne/complicaciones , Miocitos Cardíacos/enzimología , Proteínas Quinasas/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Cardiomiopatías/etiología , Cardiomiopatías/genética , Cardiomiopatías/patología , Senescencia Celular , Modelos Animales de Enfermedad , Ratones Endogámicos mdx , Proteínas Asociadas a Microtúbulos/metabolismo , Mitocondrias Cardíacas/ultraestructura , Distrofia Muscular de Duchenne/enzimología , Distrofia Muscular de Duchenne/genética , Miocitos Cardíacos/ultraestructura , Fosforilación , Transducción de Señal

RESUMEN

Cardiac troponin C (cTnC) is a key effector in cardiac muscle excitation-contraction coupling as the Ca2+ sensing subunit responsible for controlling contraction. In this study, we generated several FRET sensors for divalent cations based on cTnC flanked by a donor fluorescent protein (CFP) and an acceptor fluorescent protein (YFP). The sensors report Ca2+ and Mg2+ binding, and relay global structural information about the structural relationship between cTnC's N- and C-domains. The sensors were first characterized using end point titrations to decipher the response to Ca2+ binding in the presence or absence of Mg2+. The sensor that exhibited the largest responses in end point titrations, CTV-TnC, (Cerulean, TnC, and Venus) was characterized more extensively. Most of the divalent cation-dependent FRET signal originates from the high affinity C-terminal EF hands. CTV-TnC reconstitutes into skinned fiber preparations indicating proper assembly of troponin complex, with only ~0.2 pCa unit rightward shift of Ca2+-sensitive force development compared to WT-cTnC. Affinity of CTV-TnC for divalent cations is in agreement with known values for WT-cTnC. Analytical ultracentrifugation indicates that CTV-TnC undergoes compaction as divalent cations bind. C-terminal sites induce ion-specific (Ca2+ versus Mg2+) conformational changes in cTnC. Our data also provide support for the presence of additional, non-EF-hand sites on cTnC for Mg2+ binding. In conclusion, we successfully generated a novel FRET-Ca2+ sensor based on full length cTnC with a variety of cellular applications. Our sensor reveals global structural information about cTnC upon divalent cation binding.

Asunto(s)

Calcio/metabolismo , Cationes Bivalentes/metabolismo , Proteínas Luminiscentes/metabolismo , Troponina C/química , Troponina C/metabolismo , Técnicas Biosensibles/instrumentación , Cationes Bivalentes/química , Cristalografía por Rayos X , Humanos , Proteínas Luminiscentes/química , Magnesio , Modelos Moleculares , Unión Proteica , Estructura Secundaria de Proteína

RESUMEN

AIMS: Nicotinamide adenine dinucleotide oxidases (NOXs) are important contributors to cellular oxidative stress in the cardiovascular system. The NOX2 isoform is upregulated in numerous disorders, including dystrophic cardiomyopathy, where it drives the progression of the disease. However, mechanisms underlying NOX2 overexpression are still unknown. We investigated the role of microRNAs (miRs) in the regulation of NOX2 expression. METHODS AND RESULTS: Duchenne muscular dystrophy (DMD) was used as a model of cardiomyopathy. After screening with miRNA target prediction databases and following qRT-PCR analysis, we found drastic downregulation of miR-448-3p in hearts of mdx mice, an animal model of DMD. The downregulation correlated with overexpression of the Ncf1 gene, encoding the NOX2 regulatory subunit p47(phox). Specificity of Ncf1 targeting by miR-448-3p was validated by luciferase reporter assay. Silencing of miR-448-3p in wild-type mice had a dramatic effect on cellular and functional properties of cardiac muscle as assessed by western blotting, qRT-PCR, confocal imaging, echocardiography, and histology. Acute treatment of mice with LNA-miR-448 inhibitors led to increased Ncf1 expression, abnormally elevated reactive oxygen species (ROS) production and exacerbated Ca(2+) signalling in cardiomyocytes, reminiscent of features previously observed in dystrophic cardiac cells. In addition, chronic inhibition of miR-448-3p resulted in dilated cardiomyopathy and arrhythmia, hallmarks of dystrophic cardiomyopathy. CONCLUSIONS: Our studies suggest that downregulation of miR-448-3p leads to the increase in the expression of Ncf1 gene and p47(phox) protein, as well as to the substantial increase in NOX2-derived ROS production. Cellular oxidative stress subsequently triggers events that finally culminate in cardiac tissue damage and development of cardiomyopathy.

Asunto(s)

Cardiomiopatía Dilatada/enzimología , MicroARNs/metabolismo , Miocardio/enzimología , Estrés Oxidativo , Especies Reactivas de Oxígeno/metabolismo , Animales , Arritmias Cardíacas/genética , Arritmias Cardíacas/metabolismo , Señalización del Calcio , Cardiomiopatía Dilatada/genética , Cardiomiopatía Dilatada/patología , Modelos Animales de Enfermedad , Regulación Enzimológica de la Expresión Génica , Silenciador del Gen , Predisposición Genética a la Enfermedad , Células HEK293 , Humanos , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Ratones Endogámicos mdx , MicroARNs/genética , Distrofia Muscular de Duchenne/complicaciones , Distrofia Muscular de Duchenne/genética , Miocardio/patología , NADPH Oxidasa 2 , NADPH Oxidasas/genética , NADPH Oxidasas/metabolismo , Fenotipo , Factores de Tiempo , Transfección , Remodelación Ventricular

RESUMEN

Actomyosin kinetics in both skinned skeletal muscle fibers at maximum Ca2+-activation and unregulated in vitro motility assays are modulated by solvent microviscosity in a manner consistent with a diffusion limited process. Viscosity might also influence cardiac thin filament Ca2+-regulatory protein dynamics. In vitro motility assays were conducted using thin filaments reconstituted with recombinant human cardiac troponin and tropomyosin; solvent microviscosity was varied by addition of sucrose or glucose. At saturating Ca2+, filament sliding speed (s) was inversely proportional to viscosity. Ca2+-sensitivity (pCa50 ) of s decreased markedly with elevated viscosity (η/η0 ≥ ~1.3). For comparison with unloaded motility assays, steady-state isometric force (F) and kinetics of isometric tension redevelopment (kTR ) were measured in single, permeabilized porcine cardiomyocytes when viscosity surrounding the myofilaments was altered. Maximum Ca2+-activated F changed little for sucrose ≤ 0.3 M (η/η0 ~1.4) or glucose ≤ 0.875 M (η/η0 ~1.66), but decreased at higher concentrations. Sucrose (0.3 M) or glucose (0.875 M) decreased pCa50 for F. kTR at saturating Ca2+ decreased steeply and monotonically with increased viscosity but there was little effect on kTR at sub-maximum Ca2+. Modeling indicates that increased solutes affect dynamics of cardiac muscle Ca2+-regulatory proteins to a much greater extent than actomyosin cross-bridge cycling.

RESUMEN

Striated muscle contraction is regulated by dynamic and cooperative interactions among Ca(2+), troponin, and tropomyosin on the thin filament. While Ca(2+) regulation has been extensively studied, little is known about the dynamics of individual regulatory units and structural changes of individual tropomyosin molecules in relation to their mechanical properties, and how these factors are altered by cardiomyopathy mutations in the Ca(2+) regulatory proteins. In this hypothesis paper, we explore how various experimental and analytical approaches could broaden our understanding of the cooperative regulation of cardiac contraction in health and disease.