Genomweite Suche neuer Modulatoren der Signaltransduktion in kardialer Hypertrophie und Herzinsuffizienz

Abstract

Activated through cytokines, growth factors and hormones the MAPK signaling pathway regulates cell response to environmental cues. The signaling cascade is involved in fundamental cellular processes such as growth, proliferation, differentiation and survival. Therefore this pathway must be carefully controlled. Proteins of the MAPK signaling pathway have been reported to play a role in cancer, autoimmune disorders and heart failure. Constitutive activation of the MAPK signaling pathway leads to cardiac hypertrophy followed by heart failure. In this study B-Raf was identified to play an important role as regulator of cardiac hypertrophy. To determine this, hypertrophy had been induced in cultured neonatal rat cardiomyocytes by phenylephrine stimulation and their cell size was investigated planimetrically. The kinase activity of B-Raf was inhibited by the commercial available inhibitor SB590885. Neonatal rat cardiomyocytes stimulated with phenylephrine and simultaneously with the B-RAF Inhibitor SB590885 showed no evidence for cellular hypertrophy. Moreover, the inhibition of B-Raf led to significant reduction of Mek1/2 phosphorylation in both phenylephrine treated and untreated cardiomyocytes. This result revealed that B-Raf is the main RAF-Isoform responsible for signal transduction to Mek in neonatal rat cardiomyocytes and its primary function is to activate Mek, either directly or through its ability to recruit C-Raf. Signal transduction from B-RAF to Mek is an important key regulator of the MAPK signaling pathway in cardiomyocytes. Therefore, aim of this study was to identify new proteins which are able to modulate signal transduction from B-Raf to Mek. A high-throughput mammalian two hybrid screening method was established to identify cDNAs from a mouse embryo full length cDNA library which were able to affect the interaction between B-Raf and Mek1. Several cDNAs which were able to facilitate the B-Raf/Mek1 interaction were identified by luciferase reporter assay. In this study, Rcn1 was detected as inhibitor of the B-RAF/Mek1 interaction and was verified for its ability to modulate cardiac hypertrophy. The expression of Rcn1 is significantly upregulated at both, mRNA and protein level, in hypertrophied cardiac tissue of human and mouse. Rcn1 was able to completely abolish phenylephrine induced hypertrophy in neonatal rat cardiomyocytes in vitro and was also able to inhibit B-Raf and C-Raf kinase activity. Overexpression of Rcn1 prevents the neonatal rat cardiomyocytes to develop a hypertrophic growth of the cells, comparable to the treatment of the cells with the B-Raf Inhibitor SB590885. In addition, the overexpression of Rcn1 in neonatal rat cardiomyocytes led to dose dependent inhibition of Mek1/2 phosphorylation. Interestingly, in contrast, the knockdown of Rcn1 using specific miRNAs resulted in increased Mek1/2 phosphorylation in neonatal rat cardiomyocytes. This result was confirmed by Western-Blot and luciferase reporter assay. Moreover, knockdown of Rcn1 induced hypertrophic growth in cardiomyocytes.