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1.
Biochem Biophys Res Commun ; 449(3): 284-8, 2014 Jul 04.
Artículo en Inglés | MEDLINE | ID: mdl-24857983

RESUMEN

Binding of the utmost N-terminus of essential myosin light chains (ELC) to actin slows down myosin motor function. In this study, we investigated the binding constants of two different human cardiac ELC isoforms with actin. We employed circular dichroism (CD) and surface plasmon resonance (SPR) spectroscopy to determine structural properties and protein-protein interaction of recombinant human atrial and ventricular ELC (hALC-1 and hVLC-1, respectively) with α-actin as well as α-actin with alanin-mutated ELC binding site (α-actin(ala3)) as control. CD spectroscopy showed similar secondary structure of both hALC-1 and hVLC-1 with high degree of α-helicity. SPR spectroscopy revealed that the affinity of hALC-1 to α-actin (KD=575 nM) was significantly (p<0.01) lower compared with the affinity of hVLC-1 to α-actin (KD=186 nM). The reduced affinity of hALC-1 to α-actin was mainly due to a significantly (p<0.01) lower association rate (kon: 1,018 M(-1)s(-1)) compared with kon of the hVLC-1/α-actin complex interaction (2,908 M(-1)s(-1)). Hence, differential expression of ELC isoforms could modulate muscle contractile activity via distinct α-actin interactions.


Asunto(s)
Actinas/metabolismo , Miosinas Atriales/metabolismo , Cadenas Ligeras de Miosina/metabolismo , Miosinas Ventriculares/metabolismo , Actinas/química , Actinas/genética , Miosinas Atriales/química , Miosinas Atriales/genética , Dicroismo Circular , Humanos , Contracción Miocárdica , Cadenas Ligeras de Miosina/química , Cadenas Ligeras de Miosina/genética , Unión Proteica , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Resonancia por Plasmón de Superficie , Miosinas Ventriculares/química , Miosinas Ventriculares/genética
2.
Cardiovasc Res ; 90(3): 513-20, 2011 Jun 01.
Artículo en Inglés | MEDLINE | ID: mdl-21262909

RESUMEN

AIMS: In this paper, we tested the hypothesis that different binding affinities of the C-terminus of human cardiac alkali (essential) myosin light chain (A1) isoforms to the IQ1 motif of the myosin lever arm provide a molecular basis for distinct sarcomeric sorting and inotropic activity. METHODS AND RESULTS: We employed circular dichroism and surface plasmon resonance spectroscopy to investigate structural properties, secondary structures, and protein-protein interactions of a recombinant head-rod fragments of rat cardiac ß-myosin heavy chain aa664-915 with alanine-mutated IQ2 domain (rß-MYH(664-915)IQ(ala4)) and A1 isoforms [human atrial (hALC1) and human ventricular (hVLC-1) light chains]. Double epitope-tagging competition was used to monitor the intracellular localization of exogenously introduced hALC-1 and hVLC-1 constructs in neonatal rat cardiomyocytes. Contractile functions of A1 isoforms were investigated by monitoring shortening and intracellular-free Ca(2+) (Fura-2) of adult rat cardiomyocytes infected with adenoviral (Ad) vectors using hALC-1 or ß-galactosidase as expression cassettes. hALC-1 bound more strongly (greater than three-fold lower K(D)) to rß-MYH(664-915) than did hVLC-1. Sorting specificity of A1 isoforms to sarcomeres of cardiomyocytes rose in the order hVLC-1 to hALC-1. Replacement of endogenous VLC-1 by hALC-1 in adult rat cardiomyocytes increased contractility while the systolic Ca(2+) signal remained unchanged. CONCLUSION: Intense myosin binding of hALC-1 provides a mechanism for preferential sarcomeric sorting and Ca(2+)-independent positive inotropic activity.


Asunto(s)
Miosinas Cardíacas/química , Miosinas Cardíacas/metabolismo , Cadenas Ligeras de Miosina/química , Cadenas Ligeras de Miosina/metabolismo , Sustitución de Aminoácidos , Animales , Animales Recién Nacidos , Miosinas Atriales/química , Miosinas Atriales/genética , Miosinas Atriales/metabolismo , Secuencia de Bases , Señalización del Calcio/fisiología , Miosinas Cardíacas/genética , Dicroismo Circular , Cartilla de ADN/genética , Humanos , Técnicas In Vitro , Masculino , Mutagénesis Sitio-Dirigida , Contracción Miocárdica/fisiología , Miocitos Cardíacos/metabolismo , Cadenas Ligeras de Miosina/genética , Dominios y Motivos de Interacción de Proteínas , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Estructura Secundaria de Proteína , Ratas , Ratas Wistar , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Sarcómeros/metabolismo , Resonancia por Plasmón de Superficie , Transfección , Miosinas Ventriculares/química , Miosinas Ventriculares/genética , Miosinas Ventriculares/metabolismo
3.
Development ; 130(24): 6121-9, 2003 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-14573521

RESUMEN

The embryonic vertebrate heart is composed of two major chambers, a ventricle and an atrium, each of which has a characteristic size, shape and functional capacity that contributes to efficient circulation. Chamber-specific gene expression programs are likely to regulate key aspects of chamber formation. Here, we demonstrate that epigenetic factors also have a significant influence on chamber morphogenesis. Specifically, we show that an atrium-specific contractility defect has a profound impact on ventricular development. We find that the zebrafish locus weak atrium encodes an atrium-specific myosin heavy chain that is required for atrial myofibrillar organization and contraction. Despite their atrial defects, weak atrium mutants can maintain circulation through ventricular contraction. However, the weak atrium mutant ventricle becomes unusually compact, exhibiting a thickened myocardial wall, a narrow lumen and changes in myocardial gene expression. As weak atrium/atrial myosin heavy chain is expressed only in the atrium, the ventricular phenotypes in weak atrium mutants represent a secondary response to atrial dysfunction. Thus, not only is cardiac form essential for cardiac function, but there also exists a reciprocal relationship in which function can influence form. These findings are relevant to our understanding of congenital defects in cardiac chamber morphogenesis.


Asunto(s)
Función Atrial/fisiología , Miosinas Atriales/metabolismo , Atrios Cardíacos/embriología , Ventrículos Cardíacos/embriología , Contracción Miocárdica/fisiología , Cadenas Pesadas de Miosina/metabolismo , Proteínas de Pez Cebra/metabolismo , Pez Cebra/embriología , Animales , Miosinas Atriales/genética , Factor Natriurético Atrial/genética , Factor Natriurético Atrial/metabolismo , Epigénesis Genética , Corazón/fisiología , Atrios Cardíacos/metabolismo , Atrios Cardíacos/ultraestructura , Ventrículos Cardíacos/metabolismo , Ventrículos Cardíacos/ultraestructura , Morfogénesis , Mutación , Miocardio/citología , Miocardio/metabolismo , Cadenas Pesadas de Miosina/genética , Proteínas de Pez Cebra/genética
4.
Am J Physiol Heart Circ Physiol ; 284(3): H830-7, 2003 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-12424097

RESUMEN

Previous studies have shown that endurance exercise training increases myocardial contractility. We have previously described training-induced alterations in myocardial contractile function at the cellular level, including an increase in the Ca(2+) sensitivity of tension. To determine the molecular mechanism(s) of these changes, oligonucleotide microarrays were used to analyze the gene expression profile in ventricles from endurance-trained rats. We used an 11-wk treadmill training protocol that we have previously shown results in increased contractility in cardiac myocytes. After the training, the hearts were removed and RNA was isolated from the ventricles of nine trained and nine control rats. With the use of an Affymetrix Rat Genome U34A Array, we detected altered expression of 27 genes. Several genes previously found to have increased expression in hypertrophied myocardium, such as atrial natriuretic factor and skeletal alpha-actin, were decreased with training in this study. From the standpoint of altered contractile performance, the most significant finding was an increase in the expression of atrial myosin light chain 1 (aMLC-1) in the trained ventricular tissue. We confirmed microarray results for aMLC-1 using RT-PCR and also confirmed a training-induced increase in aMLC-1 protein using two-dimensional gel electrophoresis. aMLC-1 content has been previously shown to be increased in human cardiac hypertrophy and has been associated with increased Ca(2+) sensitivity of tension and increased power output. These results suggest that increased expression of aMLC-1 in response to training may be responsible, at least in part, for previously observed training-induced enhancement of contractile function.


Asunto(s)
Miosinas Atriales/metabolismo , Perfilación de la Expresión Génica , Ventrículos Cardíacos/metabolismo , Miocardio/metabolismo , Cadenas Ligeras de Miosina/metabolismo , Esfuerzo Físico/fisiología , Animales , Miosinas Atriales/análisis , Miosinas Atriales/genética , Factor Natriurético Atrial/análisis , Factor Natriurético Atrial/genética , Factor Natriurético Atrial/metabolismo , Electroforesis en Gel Bidimensional , Prueba de Esfuerzo , Femenino , Ventrículos Cardíacos/química , Miocardio/química , Cadenas Ligeras de Miosina/análisis , Cadenas Ligeras de Miosina/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , Condicionamiento Físico Animal , Ratas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa
5.
J Cell Biochem ; 86(3): 422-31, 2002.
Artículo en Inglés | MEDLINE | ID: mdl-12210749

RESUMEN

Expression of the human atrial myosin light chain 1 (hALC-1) in the cardiac ventricle in vivo as well as in primary cultivated adult cardiomyocytes caused a pronounced positive inotropic effect. Therefore, it is one of the most promising candidate gene to treat congestive heart failure (CHF). In this work, we investigated, whether hALC-1 expression also modifies the energetic state of cardiomyocytes. Primary cultivated neonatal rat hearts cells (NRHC) were infected with adenoviral vectors (Ad vectors) containing a hALC-1 cDNA (AdCMV.hALC-1) or a control Ad vector. Infection efficiency of NRHC reached 100% at 50 multiplicity of infection (MOI). Interestingly and in contrast to primary cultures of liver cells, there were no cytotoxic side effects or induction of apoptosis up to MOI 50 in Ad vector infected NRHC. NRHC expressed large amounts of hALC-1 upon infection with AdCMV.hALC-1 which could easily been detected by protein staining and Western blot analysis. Analysis of intracellular hALC-1 localization by double-labeling immunofluorescence of AdCMV.hALC-1 infected cardiomyocytes revealed the typical myofibrillar striation pattern, as well as co-localization of hALC-1 with myosin heavy chains. There was no difference in the oxygen consumption between controls and AdCMV.hALC-1 infected NRHC. These data suggest that first: adenoviral vectors could be used as a safe and effective tool for gene transfer to cardiomyocytes, and second: that a positive inotropic effect of hALC-1 is not associated with enhanced oxygen consumption.


Asunto(s)
Adenoviridae/genética , Miosinas Atriales/metabolismo , Corazón/fisiología , Miocardio/citología , Miocardio/metabolismo , Cadenas Ligeras de Miosina/metabolismo , Consumo de Oxígeno , Animales , Apoptosis , Miosinas Atriales/genética , Ciclo Celular , Células Cultivadas , Efecto Citopatogénico Viral , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Expresión Génica , Vectores Genéticos/genética , Corazón/virología , Humanos , Cadenas Ligeras de Miosina/genética , Ratas , Ratas Sprague-Dawley
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