RESUMEN
Nkx2-5 gene mutations cause cardiac abnormalities, including deficits of function in the atrioventricular conduction system (AVCS). In the chick, Nkx2-5 is elevated in Purkinje fiber AVCS cells relative to working cardiomyocytes. Here, we show that Nkx2-5 expression rises to a peak as Purkinje fibers progressively differentiate. To disrupt this pattern, we overexpressed Nkx2-5 from embryonic day 10, as Purkinje fibers are recruited within developing chick hearts. Overexpression of Nkx2-5 caused inhibition of slow tonic myosin heavy chain protein (sMHC), a late Purkinje fiber marker but did not affect Cx40 levels. Working cardiomyocytes overexpressing Nkx2-5 in these hearts ectopically up-regulated Cx40 but not sMHC. Isolated embryonic cardiomyocytes overexpressing Nkx2-5 also displayed increased Cx40 and suppressed sMHC. By contrast, overexpression of a human NKX2-5 mutant did not effect these markers in vivo or in vitro, suggesting one possible mechanism for clinical phenotypes. We conclude that a prerequisite for normal Purkinje fiber maturation is precise regulation of Nkx2-5 levels.
Asunto(s)
Proteínas Aviares/biosíntesis , Diferenciación Celular/fisiología , Regulación del Desarrollo de la Expresión Génica/fisiología , Proteínas de Homeodominio/biosíntesis , Ramos Subendocárdicos/citología , Factores de Transcripción/biosíntesis , Adenoviridae , Animales , Proteínas Aviares/genética , Biomarcadores , Núcleo Celular/metabolismo , Embrión de Pollo , Conexinas/metabolismo , Vectores Genéticos , Proteínas de Homeodominio/genética , Miocitos Cardíacos/metabolismo , Cadenas Pesadas de Miosina/biosíntesis , Cadenas Pesadas de Miosina/genética , Ramos Subendocárdicos/metabolismo , Factores de Transcripción/genética , Proteína alfa-5 de Unión ComunicanteRESUMEN
Functional mitochondria require up to 1000 proteins to function properly, with 99% synthesized as precursors in the cytoplasm and transported into the mitochondria with the aid of cytosolic chaperones and mitochondrial translocators (import components). Proteins to be imported are chaperoned to the mitochondria by the cytosolic heat shock protein (cHSP70) and are immediately pursued by Translocators of the Outer Membrane (TOMs), followed by transient interactions of the unfolded proteins with Translocators of the Inner Membrane (TIMs). In the present study, we describe a human gene, TOMM70A, orthologous to the yeast Tom70 import component. TOMM70A is ubiquitously expressed in human tissues, maps on chromosome 3q13.1-q13.2 and consists of 12 coding exons spanning over 37 kb. TOMM70A localizes in the mitochondria of COS-7 cells, and in organello import assays confirmed its presence in the Outer Mitochondrial membrane (OM) of rat liver mitochondria. TOMM70A could play a significant role in the import of nuclear-encoded mitochondrial proteins with internal targeting sites such as ADP/ATP carriers and the uncoupling proteins.
Asunto(s)
Membranas Intracelulares/metabolismo , Proteínas de la Membrana , Proteínas de Transporte de Membrana/biosíntesis , Proteínas de Transporte de Membrana/genética , Mitocondrias Hepáticas/metabolismo , Secuencia de Aminoácidos , Animales , Transporte Biológico , Células COS , Núcleo Celular/metabolismo , Cromosomas Humanos Par 3 , Proteínas Fúngicas , Humanos , Células Híbridas , Hibridación Fluorescente in Situ , Proteínas de Transporte de Membrana Mitocondrial , Proteínas del Complejo de Importación de Proteínas Precursoras Mitocondriales , Proteínas Mitocondriales , Modelos Genéticos , Datos de Secuencia Molecular , Unión Proteica , Transporte de Proteínas , Proteínas , Ratas , Homología de Secuencia de Aminoácido , Distribución TisularRESUMEN
Nkx2.5 and serum response factor (SRF) are critically important transcription factors in cardiac morphogenesis. They are also widely expressed in adult cardiomyocytes, but there is little data to indicate their possible role in adult cardiac cells. In this paper we demonstrate that the interaction of Nkx2.5 and SRF in cardiac-specific gene regulation is different between neonatal and adult cardiomyocytes. Our experimental model utilizes transient transfection and adenovirus mediated gene transfer of the proximal promoter fragment of the cardiac isoform of the sodium-calcium exchanger gene (NCX1). This promoter construct (NCX184) contains a single Nkx2.5-response element (NKE) and a single serum response element (CArG). In rat neonatal cardiomyocytes NCX184 activity is substantially induced with Nkx2.5 or SRF and additively with both. Mutagenesis of these NKE and CArG elements demonstrated the specificity of the interactions, which was confirmed with gel retardation analysis of cardiac ventricular tissue. In contrast, in adult cardiomyocytes, co-infection of Nkx2.5 and SRF adenovirus vectors showed Nkx2.5 induction but SRF did not have additive effects on NCX1 promoter regulation. As opposed to NCX1, the proximal atrial natriuretic factor (ANF) promoter was regulated identically in response to SRF and Nkx2.5 in both adult and neonatal cardiomyocytes. These results show that Nkx2.5-SRF interactions are capable of producing different transcriptional responses in adult versus neonatal cardiomyocytes, implying important differences in NCX1 promoter tertiary complex formation dependent on developmental stage.