RESUMO
The aim of this study was to identify and functionally characterize cardiac subtypes during early stages of development. For this purpose, transgenic embryonic stem cells were generated using the alpha-myosin heavy chain promoter driving the expression of the enhanced green fluorescent protein (EGFP). EGFP-positive clusters of cells were first observed as early as 7 days of development, thus, even before the initiation of the contractile activity. Flow cytometry and single-cell fluorescence measurements evidenced large diversities of EGFP intensity. Patch-clamp experiments showed EGFP expression exclusively in pacemaker and atrial but not ventricular cells. The highest fluorescence intensities were detected in pacemaker-like cardiomyocytes. In accordance, multielectrode-array recordings of whole embryoid bodies confirmed that the pacemaker center coincided with strongly EGFP-positive areas. The cardiac subtypes displayed already at this early stage differential characteristics of electrical activity and ion channel expression. Thus, quantitation of the alpha-myosin heavy chain driven reporter gene expression allows identification and functional characterization of early cardiac subtypes.
Assuntos
Embrião de Mamíferos/citologia , Átrios do Coração/citologia , Sistema de Condução Cardíaco/citologia , Miócitos Cardíacos/citologia , Células-Tronco/citologia , Animais , Carbacol/farmacologia , Diferenciação Celular , Separação Celular , Células Clonais , Eletrofisiologia , Citometria de Fluxo , Fluorescência , Expressão Gênica , Proteínas de Fluorescência Verde/análise , Proteínas de Fluorescência Verde/genética , Átrios do Coração/embriologia , Sistema de Condução Cardíaco/embriologia , Camundongos , Camundongos Transgênicos , Microscopia Confocal , Agonistas Muscarínicos/farmacologia , Miócitos Cardíacos/classificação , Miócitos Cardíacos/metabolismo , Cadeias Pesadas de Miosina/genética , Regiões Promotoras Genéticas/genética , Células-Tronco/classificação , Células-Tronco/metabolismo , Transfecção , Miosinas Ventriculares/genéticaRESUMO
The binding of one ADP molecule at the catalytic site of the nucleotide depleted F1-ATPase results in a decrease in the initial rate of ATP hydrolysis. The addition of an equimolar amount of ATP to the nucleotide depleted F1-ATPase leads to the same effect, but, in this case, inhibition is time dependent. The half-time of this process is about 30 s, and the inhibition is correlated with Pi dissociation from the F1-ATPase catalytic site (uni-site catalysis). The F1-ATPase-ADP complex formed under uni-site catalysis conditions can be reactivated in two ways: (i) slow ATP-dependent ADP release from the catalytic site (tau 1/2 20 s) or (ii) binding of Pi in addition to MgADP and the formation of the triple F1-ATPase-MgADP-Pi complex. GTP and GDP are also capable of binding to the catalytic site, however, without changes in the kinetic properties of the F1-ATPase. It is proposed that ATP-dependent dissociation of the F1-ATPase-GDP complex occurs more rapidly, than that of the F1-ATPase-ADP complex.