RESUMEN
In order to study the effects of Hepatocyte Growth Factor (HGF) in the heart, two transgenic mice were developed, one carrying a bidirectional HGF-TetO-GFP responder construct and the other carrying a α-MHC-tTA transactivator construct. Crosses were carried out between heterozygotes, so that litters contained bitransgenic α-MHC-tTA/HGF-TetO-GFP+, thus expressing HGF and GFP exclusively in the heart and only in the absence of Doxycycline. Our data show that the expression of HGF was indeed restricted to the heart and that the expression was limited to the timeframe of the absence of Doxycycline. Surprisingly the expression was variable even between bitransgenic littermates. In the setting of a model of ischemia-reperfusion, the expression of HGF ameliorates cardiac functionality, enhances proliferation and diminishes the scarred area, proving that this is a good model to study the beneficial influences and functional roles of HGF in the heart.
Asunto(s)
Doxiciclina/farmacología , Corazón/fisiopatología , Factor de Crecimiento de Hepatocito/metabolismo , Animales , Western Blotting , Línea Celular , Proliferación Celular , Colágeno/metabolismo , Cruzamientos Genéticos , Medios de Cultivo Condicionados/metabolismo , Perros , Ecocardiografía , Femenino , Regulación de la Expresión Génica , Proteínas Fluorescentes Verdes/metabolismo , Corazón/efectos de los fármacos , Factor de Crecimiento de Hepatocito/genética , Heterocigoto , Inmunohistoquímica , Ratones , Ratones Transgénicos , Modelos Animales , Daño por Reperfusión Miocárdica/tratamiento farmacológico , Daño por Reperfusión Miocárdica/fisiopatología , Embarazo , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
AIMS: The ErbB-neuregulin-1ß1 (Nrg1ß1) pathway is required for cardiac development and exerts chronic effects on the postnatal adult heart. Long-term application of Nrg1ß1 results in hypertrophy and protection against oxidative stress and cytotoxic agents. We performed experiments with acute Nrg1ß1 treatment to find evidence for a further protective role due to rapid modulation of adult cardiomyocyte function. METHODS AND RESULTS: In confocal fluorimetric measurements, Nrg1ß1 induced a calcium-independent increase in nitric oxide (NO) production in isolated adult rat ventricular myocytes (ARVCMs) that was blocked by the phosphoinositide-3-kinase (PI3K) inhibitor Wortmannin. Western blot analysis showed enhancement of endothelial nitric oxide synthase phosphorylation in Nrg1ß1-treated ARVCMs, which was attenuated by Wortmannin. Nrg1ß1 induced a significant increase in calcium transient amplitude (indo-1 ratiometric measurement) and accelerated the recovery of cytosolic calcium in the sarcoplasmic reticulum without affecting whole-cell L-type calcium current. Wortmannin or the protein kinase G inhibiting peptide (DT-2) abolished the increase in calcium transient amplitude and the acceleration of calcium recovery induced by Nrg1ß1 treatment. Immunofluorescence analysis revealed that Nrg1ß1 treatment increased phospholamban phosphorylation, and the effect was blocked by PI3K and protein kinase G inhibition. Caffeine-releasable sarcoplasmic reticulum calcium content was also higher during Nrg1ß1 administration. CONCLUSION: Rapid activation of PI3K, endothelial nitric oxide synthase and protein kinase G and a consequent improvement in diastolic calcium can be added to established Nrg1 protective roles.