B‐Raf is Required for Isoproterenol‐Induced Hypertrophy
Authors: Rees Katherina , Takahashi Maho , Stork Philip J , Thornburg Kent L , Chattergoon Natasha N 
Published: November 2023

Objectives

B‐Raf, an upstream regulator of ERK, is responsive to cAMP stimulation. Isoproterenol (ISO) augments cAMP production via β‐adrenergic receptor stimulation. Cardiomyocyte (CM)‐specific loss of B‐Raf leads to early reduction in CM volume fraction and increased cardiac mass compared to controls. We hypothesized that CM‐specific loss of B‐Raf protects the heart against pathophysiological hypertrophy in adult mice.

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). Mice were randomized into ISO (60mg/kg/day) or vehicle (1X PBS) treatments. Following baseline echocardiographs at 12 weeks old, mini‐osmotic pumps were implanted subcutaneously, under sterile conditions, for continuous administration of ISO or vehicle. Echocardiographs were repeated 1 week after pump implantation. Hearts were harvested for molecular analysis of B‐Raf signaling, β‐adrenergic receptors (β1AR, β2AR), and calcium handling genes (NCX, Serca2a, RyR2, PMCA, PLB). Gene expression was quantified using qPCR and analyzed with ANOVA. Significance was set at p<0.05.

Results

KO hearts had larger diastolic endocardial and epicardial areas and lengths, end‐systolic volume (ESV) and end‐diastolic volume (EDV), indicating a dilated ventricle compared to WT. ISO increased cardiac dimensions measured by echocardiography in WT mice but not in KO mice. Total cardiac mass increased in both WT and KO with ISO but with a greater increase in LV mass in KO hearts. At baseline, KO mice had a lower heart rate (HR), but a larger ejection fraction (EF), stroke volume (SV) and cardiac output (CO) compared to WT. ISO treatment increased CO in both groups primarily by elevating HR. ISO treated WT mice had worsening cardiac function with reduced EF and increased EDV and ESV. mRNA levels for Col1, Col3, ANP genes were increased in ISO treated WT animals but with lower expression of ERK1. ISO increased mTOR expression in KO mice compared to baseline but not compared to WT ISO. ISO treatment decreased calcium handling gene expression in WT hearts but increased expression in KO hearts. WT and KO mice had similar levels of β‐adrenergic receptor expression.

Conclusions

WT mice respond to ISO as expected by showing signs of early heart failure. WT ISO mice had hypertrophy of the ventricles, increased ANP and collagen gene expression, reduced expression of calcium handling genes, and evidence of worsening cardiac function (EF, EDV, ESV). These changes were not seen in KO mice. KO mice had a dilated ventricle at baseline but paradoxically better initial cardiac performance. ISO increased hypertrophic signals (ANP and mTOR) as well as calcium handling genes in KO hearts but did not depress function in KO hearts. We conclude that loss of B‐Raf is protective against severe ISO‐induced hypertrophy and functional suppression.

Support or Funding Information

This study was supported by NIH‐NHLBI K01HL118677 and the MJ Murdock Charitable Trust.