Abstract 172 Cardiology: Signaling Mechanisms and Cardiovascular Function Platform, Sunday, 5/2

Sudden death due to hypertrophic cardiomyopathy appears to occur in knockout mice lacking the atrial natriuretic peptide (ANP) receptor. Furthermore, in uncomplicated hypertrophy, ANP is expressed in the ventricle despite normal circulating levels. These data suggest that autocrine/paracrine anti-hypertrophic ANP actions may be important. We have previously shown that ANP causes rapid and specific activation of the mitogen activated protein kinase, ERK, in neonatal rat ventricular myocytes (NRVMC) and that this effect can be mimicked using cGMP analogs ( Pediatr Res 41:24A, 1997). To determine if ANP treatment inhibits the hypertrophic effects of the α-adrenergic agonist phenylephrine (PE) and if this effect requires ERK activation, we measured cell cross sectional area (CSA) using computer-assisted planimetry in serum-starved NRVMC transfected with pCMBβ-Gal and then treated for 48 hours with ANP (1µM), the MEK inhibitor PD098059 (10µM), and PE(1µM). Three separate identical experiments were performed in duplicate and at least 90 cells were measured in each group. PE caused an 80±5 % increase over basal CSA. ANP inhibited the PE-induced CSA increase by 50% and ANP's inhibitory effect was prevented by PD098059. These data indicate that ERK activation is required for ANP's anti-hypertrophic effect. To determine the level at which the ANP signal intersects with the ERK cascade, we studied ANP effects on MEK and Raf. Western immunoblots of whole cell lysates of ANP-treated (100nM) NRVMC probed with specific phospho-MEK antibodies demonstrated MEK phosporylation. In vitro kinase assays with immunoprecipitated MEK showed a nearly 4-fold increase in MEK activity. ANP-induced ERK phosphorylation was inhibited with PD098059. Western immunoblots of NRVMC probed with specific antibodies confirmed the presence of A-Raf, Raf-1, but not B-Raf. In vitro kinase assays of immunoprecipitated A-Raf or Raf-1 demonstrated an 8-fold increase of Raf activity in response to TPA, but ANP treatment had no effect. These data indicate that the ANP signal intersects with the ERK cascade at the level of MEK. Since both PE and ANP can activate ERK, we investigated the possibility that PE and ANP had differential effects on ERK nuclear translocation. Using immunoflourescent confocal microscopy we observed that ANP but no PE-treatment of NRVMC caused translocation of ERK from the cytoplasm to the nucleus suggesting that nuclear localization of ERK may be essential for its anti-hypertrophic action. Modulating the molecular pathways responsible for ANP's anti-hypertrophic effects may provide novel therapeutic strategies for the treatment of maladaptive hypertrophy.