B-RAF and its novel negative regulator reticulocalbin 1 (RCN1) modulates cardiomyocyte hypertrophy.
OBJECTIVE
Activation of the kinase RAF and its downstream targets leads to cardiomyocyte hypertrophy. It has been hypothesized that B-RAF might be the main activator of MEK in various cell types. Therefore, the aim of this study was to investigate the role of B-RAF and its modulating factors in cardiomyocyte hypertrophy.


RESULTS
Neonatal rat cardiomyocytes were pre-treated with and without the specific B-RAF inhibitor SB590885 and then stimulated with phenylephrine to induce hypertrophy. Inhibition of B-RAF completely impeded the hypertrophic response and led to a significant reduction of MEK1/2 phosphorylation. By applying a eukaryotic cDNA expression screen, based on a dual-luciferase reporter assay for B-RAF activity measurement, we identified RCN1 as a new negative modulator of B-RAF activity. Adenovirus-mediated overexpression of reticulocalbin 1 (RCN1) completely impeded phenylephrine-induced hypertrophy and led to significantly reduced MEK1/2 phosphorylation. Conversely, adenoviral knockdown of RCN1 with a specific synthetic miRNA induced cardiomyocyte hypertrophy and significantly increased MEK1/2 phosphorylation.


CONCLUSIONS
In summary, our results show that the inhibition of B-RAF abolishes cardiomyocyte hypertrophy and we identified RCN1 as novel negative modulator of cardiomyocyte hypertrophy by inhibition of the mitogen-activated protein kinase signalling cascade. Our results show that B-RAF kinase activity is essential for cardiac hypertrophy and RCN1, its newly identified negative regulator, abolishes hypertrophic response of cardiomyocytes in vitro.
Department of Cardiology and Pulmonology, University Medical Centre Göttingen, Robert-Koch-Straße 40, 37075 Göttingen, Germany.
Nadine
N
Kramann
Gerd
G
Hasenfuß
Tim
T
Seidler
D000818:Animals; D002135:Calcium-Binding Proteins / Q000378:metabolism*; D006332:Cardiomegaly / Q000378:metabolism*; D002478:Cells, Cultured; D006801:Humans; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D032383:Myocytes, Cardiac / Q000378:metabolism*; D010656:Phenylephrine / Q000494:pharmacology; D010766:Phosphorylation; D048493:Proto-Oncogene Proteins B-raf / Q000378:metabolism*; D017208:Rats, Wistar; D015398:Signal Transduction / Q000502:physiology
D002135:Calcium-Binding Proteins; C078865:reticulocalbin; D010656:Phenylephrine; C482123:Braf protein, rat; D048493:Proto-Oncogene Proteins B-raf; D020928:Mitogen-Activated Protein Kinases
B-RAF; Cardiomyocyte hypertrophy; MAPK; RCN1; Reticulocalbin 1