Abstract 416: B-Raf Loss Suppresses Extracellular-Regulated Kinase Activation and Cardiomyocyte Proliferation

Abstract

Objectives: The postnatal heart does not retain the proliferative capacity it had during fetal life. Unlike large mammals, murine cardiomyocytes (CM) continue to divide into the first week of life before terminal differentiation and binucleation. We hypothesized that B-Raf regulates ERK activation in newborn CM and that loss of B-Raf suppresses cyclin levels and reduced proliferation. To test this, we determined whether loss of B-Raf disrupts the ERK (extracellular-regulated kinase) cascade and impairs CM growth. Methods: CM specific knockout (KO) of B-Raf was generated using CRE/lox (floxed B-Raf x αMHC CRE) resulting in a truncated, unstable B-Raf and a null phenotype. KO mice (α-MHC-CRE / B-Raf lox/lox ) were compared to CRE negative / B-Raf lox/lox mice (wild type; WT). Hearts from 3d and 8d old pups were harvested for molecular analysis of B-Raf signaling and cell cycle markers. Hearts from 3d old pups were harvested and CMs isolated for culture using a trypsin/DNAse digestion. The cells were treated with Isoproterenol (Iso;10uM), forskolin (20uM), and IGF-1 (1ng/ml) for 15 min to determine if the loss of B-Raf results in reduced activation of ERK. Results: Heart weight to body weight (HW/BW) ratio was less in 3d KO versus 3d WT (n=50, p<0.05). HW/BW ratio became greater in 8d KO; there was no difference in 3d and 8d HW/BW in WT animals. Baseline B-Raf and phosphorylated ERK levels were reduced in KO hearts (*p<0.05). Cell cycle inhibitors p21 and p53 were increased in 3d KO hearts with decreased levels of all cyclins (p<0.05). In 8d KO hearts, increased p21, p27, and p53 expression was accompanied with increased cyclin levels (p<0.05). In vitro ERK activation was blunted in KO CMs by forskolin and Iso compared to IGF-1. Conclusions: ERK activation was suppressed in KO hearts resulting in smaller newborn hearts but which exceeded normal HW/BW by 8d. This is may represent premature hypertrophy as the proliferative period of CM development had ended. Cell cycle analysis supports reduced CM mitotis among 8d CM. Such early disturbances in normal CM growth may increase susceptibility for reduced cardiac function in the face of increased postnatal load stress.