B‐Raf is required for normal cardiomyocyte growth

Abstract

ObjectivesThe 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. This postnatal proliferation and maturation window offers the ability to study the mechanisms of CM terminal differentiation. We hypothesize that B‐Raf regulates ERK (extracellular‐regulated kinase) activation in newborn CM and that loss of B‐Raf suppresses cyclin levels and reduces CM proliferation. To test this, we determined whether the loss of B‐Raf disrupts the ERK cascade and impairs CM growth.MethodsCM 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 1d, 3d, 8d and 14d old pups were harvested for molecular analysis of B‐Raf signaling and cell cycle markers (p21, p27, p53, cyclins A1, B1, D1 and E). Mid‐wall sections of the heart were stained with Masson's trichrome to determine changes in tissue composition.ResultsHeart 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. 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 (Cyclin A1 and B1, p<0.05). In 8d KO hearts, increased p21, p27, and p53 expression was accompanied with decreased cyclin levels (p<0.05). In 14d KO hearts, gene expression of p21, p27, and p53 are increased (p<0.05), along with cyclins D1 and E. Tissue composition of cardiomyocytes decreased and collagen increased in 3d and 8d hearts (p<0.05).ConclusionsERK 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 mitosis among 8d CM. Trichrome analysis supports proliferation has ended prematurely by the reduction of the CM fraction. Such early disturbances in normal CM growth may increase susceptibility for reduced cardiac function in the face of increased postnatal load stress.