RESUMEN
Since the discovery of Xin repeat-containing proteins in 1996, the importance of Xin proteins in muscle development, function, regeneration, and disease has been continuously implicated. Most Xin proteins are localized to myotendinous junctions of the skeletal muscle and also to intercalated discs (ICDs) of the heart. The Xin gene is only found in vertebrates, which are characterized by a true chambered heart. This suggests that the evolutionary origin of the Xin gene may have played a key role in vertebrate origins. Diverse vertebrates including mammals possess two paralogous genes, Xinα (or Xirp1) and Xinß (or Xirp2), and this review focuses on the role of their encoded proteins in cardiac muscles. Complete loss of mouse Xinß (mXinß) results in the failure of forming ICD, severe growth retardation, and early postnatal lethality. Deletion of mouse Xinα (mXinα) leads to late-onset cardiomyopathy with conduction defects. Molecular studies have identified three classes of mXinα-interacting proteins: catenins, actin regulators/modulators, and ion-channel subunits. Thus, mXinα acts as a scaffolding protein modulating the N-cadherin-mediated adhesion and ion-channel surface expression. Xin expression is significantly upregulated in early stages of stressed hearts, whereas Xin expression is downregulated in failing hearts from various human cardiomyopathies. Thus, mutations in these Xin loci may lead to diverse cardiomyopathies and heart failure.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Regulación de la Expresión Génica , Proteínas con Dominio LIM/metabolismo , Miocardio/metabolismo , Proteínas Nucleares/metabolismo , Actinas/metabolismo , Animales , Cardiomiopatías/metabolismo , Cortactina/metabolismo , Proteínas del Citoesqueleto , Citoesqueleto/metabolismo , Regulación hacia Abajo , Corazón/fisiología , Insuficiencia Cardíaca/metabolismo , Humanos , Ratones , Mutación , Canales de Potasio/metabolismo , Estructura Terciaria de Proteína , Factores de Tiempo , beta Catenina/metabolismoRESUMEN
The Xin repeat-containing proteins, Xinα (Xirp1) and Xinß (Xirp2), localize to the intercalated discs (ICDs) of mammalian hearts. Mouse Xinα (mXinα) directly interacts with ß-catenin and actin filaments, potentially coupling the N-cadherin/ß-catenin complexes to the underlying actin cytoskeleton and modulating ICD integrity and function. Supporting this possibility, mXinα-null hearts develop ICD structural defects and cardiomyopathy with conduction defects. However, the underlying mechanisms leading to these defects remain unclear. Here, we showed that mXinα also interacted with p120-catenin and cortactin. Different from the ß-catenin binding domain, there existed multiple p120-catenin binding sites on mXinα, while only the extreme N-terminus of mXinα containing a SH3-binding motif could interact with cortactin. In mouse heart, a significant fraction of cortactin was co-localized with N-cadherin to ICDs, whereas in mXinα-null heart, this fraction of cortactin was drastically reduced. Therefore, mXinα may modulate ICD integrity and function through its interactions with catenins and cortactin. Analyses of the in vivo consequence of p120-catenin and mXinα interaction revealed that force-expressed mXinα or its fragments significantly suppressed the p120-catenin-induced branching phenotypes. It is known that p120-catenin directly regulates Rho GTPases, leading to the branching phenotype. Thus, mXinα may sequester the p120-catenin from inhibiting RhoA activity and/or from activating Rac1 activity.
Asunto(s)
Uniones Adherentes/metabolismo , Cateninas/metabolismo , Forma de la Célula/fisiología , Cortactina/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas Nucleares/metabolismo , Citoesqueleto de Actina/metabolismo , Citoesqueleto de Actina/fisiología , Uniones Adherentes/fisiología , Animales , Sitios de Unión , Células CHO , Cadherinas/metabolismo , Adhesión Celular , Cricetinae , Proteínas del Citoesqueleto , ADN Complementario/genética , ADN Complementario/metabolismo , Corazón/fisiología , Ratones , Ratones Endogámicos C57BL , Microscopía Fluorescente , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/fisiología , Fenotipo , Plásmidos/genética , Plásmidos/metabolismo , Mapeo de Interacción de Proteínas , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transfección , Dominios Homologos src , Catenina deltaRESUMEN
The intercalated disc (ICD) is a unique structure to the heart and plays vital roles in communication and signaling among cardiomyocytes. ICDs are formed and matured during postnatal development through a profound redistribution of the intercellular junctions, as well as recruitment and assembly of more than 200 proteins at the termini of cardiomyocytes. The molecular mechanism underlying this process is not completely understood. The mouse orthologs (mXinα and mXinß) of human cardiomyopathy-associated (CMYA)/Xin actin-binding repeat-containing protein (XIRP) genes (CMYA1/XIRP1 and CMYA3/XIRP2, respectively) encode proteins localized to ICDs. Ablation of mXinα results in adult late-onset cardiomyopathy with conduction defects and up-regulation of mXinß. ICD structural defects are found in adult but not juvenile mXinα-null hearts. On the other hand, loss of mXinß leads to ICD defects at postnatal day 16.5, a developmental stage when the heart is forming ICDs, suggesting mXinß is required for ICD formation. Using quantitative Western blot, we showed in this study that mXinß but not mXinα was uniquely up-regulated during the redistribution of intercellular junction from the lateral membrane of cardiomyocytes to their termini. In the absence of mXinß, the intercellular junctions failed to be restricted to the termini of the cells, and the onset of such defect correlated with the peak expression of mXinß. Immunofluorescence staining and subcellular fractionation showed that mXinß preferentially associated with the forming ICDs, further suggesting that mXinß functioned locally to promote ICD maturation. In contrast, the spatiotemporal expression profile of mXinα and the lack of more severe ICD defects in mXinα-/-;mXinß-/- double knockout hearts than in mXinß-/- hearts suggested that mXinα was not essential for the postnatal formation of ICDs. A two-step model for the development of ICD is proposed where mXinß is essential for the redistribution of intercellular junction components from the lateral puncta to the cell termini.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Corazón/crecimiento & desarrollo , Proteínas con Dominio LIM/metabolismo , Miocardio/metabolismo , Proteínas Nucleares/metabolismo , Animales , Animales Recién Nacidos , Western Blotting , Proteínas del Citoesqueleto , Proteínas de Unión al ADN/genética , Femenino , Corazón/embriología , Inmunohistoquímica , Uniones Intercelulares/metabolismo , Proteínas con Dominio LIM/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microscopía Confocal , Miocardio/citología , Miocitos Cardíacos/metabolismo , Proteínas Nucleares/genéticaRESUMEN
Intercalated discs (ICDs) are cardiac-specific structures responsible for mechanical and electrical communication among adjacent cardiomyocytes and are implicated in signal transduction. The striated muscle-specific Xin repeat-containing proteins localize to ICDs and play critical roles in ICD formation and cardiac function. Knocking down the Xin gene in chicken embryos collapses the wall of developing heart chambers and leads to abnormal cardiac morphogenesis. In mammals, a pair of paralogous genes, Xinalpha and Xinbeta exist. Ablation of the mouse Xinalpha (mXinalpha) does not affect heart development. Instead, mXinalpha-deficient mice show adult late-onset cardiac hypertrophy and cardiomyopathy with conduction defects. The mXinalpha-deficient hearts up-regulate mouse Xinbeta (mXinbeta, suggesting a partial compensatory role of mXinbeta. Complete loss of mXinbeta however, leads to failure of forming ICD, mis-localization of mXinalpha, and early postnatal lethality. In this review, we will briefly discuss recent advances in the anatomy and function of ICDs. We will then review what we know about Xin repeat-containing proteins and how this protein family promotes ICD maturation and stability for normal cardiac function.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Miocitos Cardíacos/metabolismo , Proteínas Nucleares/metabolismo , Animales , Cateninas/metabolismo , Comunicación Celular , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Cardiopatías/etiología , Cardiopatías/metabolismo , Cardiopatías/patología , Humanos , Uniones Intercelulares/metabolismo , Canales Iónicos/metabolismo , Proteínas de la Membrana/metabolismo , Ratones , Modelos Cardiovasculares , Miocitos Cardíacos/ultraestructura , Proteínas Nucleares/química , Proteínas Nucleares/genética , Transducción de Señal , Proteínas Wnt/metabolismoRESUMEN
Mouse Xin-alpha (mXin-alpha) encodes a Xin repeat-containing, actin-binding protein localized to the intercalated disc (ICD). Ablation of mXin-alpha progressively leads to disrupted ICD structure, cardiac hypertrophy and cardiomyopathy with conduction defects during adulthood. Such conduction defects could be due to ICD structural defects and/or cell electrophysiological property changes. Here, we showed that despite the normal ICD structure, juvenile mXina-null cardiomyocytes (from 3~4-week-old mice) exhibited a significant reduction in the transient outward K+ current (ITO), similar to adult mutant cells. Juvenile but not adult mutant cardiomyocytes also had a significant reduction in the delayed rectifier K+ current. In contrast, the mutant adult ventricular myocytes had a significant reduction in the inward rectifier K+ current (IK1) on hyperpolarization. These together could account for the prolongation of action potential duration (APD) and the ease of developing early afterdepolarization observed in juvenile mXin-alpha-null cells. Interestingly, juvenile mXin-alpha-null cardiomyocytes had a notable decrease in the amplitude of intracellular Ca2+ transient and no change in the L-type Ca2+ current, suggesting that the prolonged APD did not promote an increase in intracellular Ca2+ for cardiac hypertrophy. Juvenile mXin-alpha-null ventricles had reduced levels of membrane-associated Kv channel interacting protein 2, an auxiliary subunit of ITO, and filamin, an actin cross-linking protein. We further showed that mXin-alpha interacted with both proteins, providing a novel mechanism for ITO surface expression.
Asunto(s)
Ventrículos Cardíacos/metabolismo , Proteínas Musculares/metabolismo , Animales , Secuencia de Bases , Células Cultivadas , Cartilla de ADN , Ventrículos Cardíacos/citología , Ventrículos Cardíacos/ultraestructura , Masculino , Ratones , Ratones Endogámicos C57BL , Microscopía Electrónica de Transmisión , Proteínas Musculares/fisiología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Técnicas del Sistema de Dos HíbridosRESUMEN
RATIONALE: The Xin repeat-containing proteins mXinalpha and mXinbeta localize to the intercalated disc of mouse heart and are implicated in cardiac development and function. The mXinalpha directly interacts with beta-catenin, p120-catenin, and actin filaments. Ablation of mXinalpha results in adult late-onset cardiomyopathy with conduction defects. An upregulation of the mXinbeta in mXinalpha-deficient hearts suggests a partial compensation. OBJECTIVE: The essential roles of mXinbeta in cardiac development and intercalated disc maturation were investigated. METHODS AND RESULTS: Ablation of mXinbeta led to abnormal heart shape, ventricular septal defects, severe growth retardation, and postnatal lethality with no upregulation of the mXinalpha. Postnatal upregulation of mXinbeta in wild-type hearts, as well as altered apoptosis and proliferation in mXinbeta-null hearts, suggests that mXinbeta is required for postnatal heart remodeling. The mXinbeta-null hearts exhibited a misorganized myocardium as detected by histological and electron microscopic studies and an impaired diastolic function, as suggested by echocardiography and a delay in switching off the slow skeletal troponin I. Loss of mXinbeta resulted in the failure of forming mature intercalated discs and the mislocalization of mXinalpha and N-cadherin. The mXinbeta-null hearts showed upregulation of active Stat3 (signal transducer and activator of transcription 3) and downregulation of the activities of Rac1, insulin-like growth factor 1 receptor, protein kinase B, and extracellular signal-regulated kinases 1 and 2. CONCLUSIONS: These findings identify not only an essential role of mXinbeta in the intercalated disc maturation but also mechanisms of mXinbeta modulating N-cadherin-mediated adhesion signaling and its crosstalk signaling for postnatal heart growth and animal survival.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Corazón/crecimiento & desarrollo , Corazón/fisiopatología , Proteínas Nucleares/metabolismo , Animales , Animales Recién Nacidos , Proliferación Celular , Supervivencia Celular , Proteínas del Citoesqueleto , Proteínas de Unión al ADN/deficiencia , Proteínas con Dominio LIM , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas Nucleares/deficienciaRESUMEN
The cardiac-specific -497 bp promoter of rat cardiac troponin T (cTnT) contains two similar modules, D and F, each of which possesses TCTG(G/C) direct repeats and A/T-rich sites. To identify cis-elements critical for cardiac specificity, a -249 bp promoter containing only module F and its site-directed mutations were used to generate transgenic mice. Transgene expression of the -249 bp promoter remained cardiac-specific, despite low and nonuniform expression. The nonuniform expression pattern of the transgene coincided with differential expression of HMGB1, which appeared to be the predominant form of HMGB family proteins in the heart. The HMGB1 binds to the A/T-rich/MEF2-like sites of the cTnT promoter, as determined by chromatin immunoprecipitation assays. Mice carrying the -249 bp promoter with point mutations disrupting the direct repeats expressed transgene at lower levels in the heart and ectopically in the brain. Ectopic expression of transgene was also observed in developing limbs and head. These results suggest an important role for the direct repeat in determining the cardiac specificity. Furthermore, mice carrying a mutant promoter simultaneously disrupting the direct repeats and overlapping GATA site failed to express the transgene in any tissues tested. Therefore, the direct repeat and overlapping GATA site are critical for the expression level and cardiac specificity. The F module controls one level of cardiac specificity. For a uniform and high level of cardiac-specific expression, the upstream element (-497 to -250 bp) is further required, possibly through the D enhancer module and the combination of Nkx2.5 and GATA sites.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica/genética , Corazón/embriología , Miocardio/metabolismo , Regiones Promotoras Genéticas/genética , Secuencias Repetitivas de Ácidos Nucleicos/genética , Troponina T/genética , Animales , Secuencia de Bases/genética , Tipificación del Cuerpo/genética , Señalización del Calcio/genética , Diferenciación Celular/genética , Elementos de Facilitación Genéticos/genética , Factores de Transcripción GATA/genética , Ratones , Ratones Transgénicos , Contracción Muscular/genética , Mutagénesis Sitio-Dirigida , Miocardio/ultraestructura , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/ultraestructura , Mutación Puntual/genética , Elementos Reguladores de la Transcripción/genética , Transcripción Genética/genética , Transgenes/genética , Troponina T/biosíntesisRESUMEN
Myosin Va, an actin-based motor protein that transports intracellular cargos, can bundle actin in vitro. Whether myosin Va regulates cellular actin dynamics or cell migration remains unclear. To address this, we compared Chinese Hamster Ovary (CHO) cells that stably express GFP fused to either full length mouse myosin Va (GFP-M5) or heavy meromyosin Va (GFP-M5Delta). GFP-M5 and GFP-M5Delta co-immunoprecipitate with CHO myosin Va and serve as overexpression of wild-type and dominant negative mutants of myosin Va. Compared to non-expressing control cells, GFP-M5-overexpressing cells have peripheral endocytic vesicles, spread slowly after plating, as well as produce robust interior actin stress fibers, myosin II bundles, and focal adhesions. However, these cells display normal cell migration and lamellipodial dynamics. In contrast, GFP-M5Delta-expressing cells have perinuclear endocytic vesicles, produce thin interior actin and myosin bundles and contain no interior focal adhesions. In addition, these cells spread rapidly, migrate slowly and display reduced lamellipodial dynamics. Similarly, neurite outgrowth is compromised in neurons cultured from transgenic Drosophila that express M5Delta-dsRed and in neurons cultured from Drosophila that produce a tailless version of endogenous myosin V. Together, these data suggest that myosin Va overexpression induces actin bundles in vivo whereas the tailless version fails to bundle actin and disrupts cell motility.