Abstract 390: Ras-Association Factor 1 - A Novel Antihypertrophic Protein in the Myocardium
Cellular and transgenic studies demonstrate that the small GTP-binding protein Ras controls a number of key signaling pathways in cardiomyocyte hypertrophy (e.g. ERK, JNK and Akt). Increased expression of H-Ras has been observed in patients with cardiac hypertrophy. Notably, activating H-Ras mutations also cause Costello syndrome, in which patients have a propensity to hypertrophic cardiomyopathy and cancer. A novel effector of Ras, Ras-Association Factor-1 isoform A (RASSF1A), has recently been identified to have an anti-proliferative effect in cancer cells. RASSF1A is strongly expressed in rat neonatal cardiomyocytes and in the adult mouse heart. We investigated the role of RASSF1A in cardiac cellular hypertrophy using an adenoviral construct encoding human RASSF1A to over-express the protein in rat neonatal cardiomyocytes. The alpha-adrenergic receptor agonist phenylephrine was used to induce hypertrophy as it had previously been shown to activate Ras. Phenylephrine induced a 1.6 fold increase in cell area in control infected cells; however expression of RASSF1A reduced this increase in cell size by 92% (n=6, p<0.05). Likewise, increased ANP expression in response to phenylephrine was completely inhibited by over-expression of RASSF1A, increases in protein content and BNP expression were also reduced by 66%. To investigate whether RASSF1A influences Ras signaling the activation of downstream kinases by phenylephrine was monitored. RASSF1A significantly reduced activation of ERK by 50% (n=6, p<0.05) but did not alter the activation of JNK, p38 or Akt. Expression of RASSF1A also reduced the activation of ERK in response to other hypertrophic stimuli including endothelin-1 and isoprenaline. We examined whether RASSF1A altered ERK-activation through altering the activation of Ras. Using a Ras-GTP immunoprecipitation assay we showed that Ras activation in response to phenylephrine was unaltered in RASSF1A expressing cells, thus demonstrating that RASSF1A acts downstream of Ras in cardiac cells. In conclusion this data identifies RASSF1A as a novel and powerful anti-hypertrophic protein in the heart which functions in part through inhibition of the ERK pathway.