Cardiomyocyte BRAF and type 1 RAF inhibitors promote cardiomyocyte and cardiac hypertrophy in mice in vivo

Figures

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  Figure 1.. BRAF is up-regulated in human failing hearts. (A–C) Expression of ARAF, BRAF and RAF1 in human heart samples showed increased expression of BRAF and RAF1 in failing hearts compared with controls (unpaired two-tailed t-tests). (A) mRNA expression was assessed by qPCR. (B and C) Proteins were analysed by immunoblotting. Blots were reprobed for GAPDH. Densitometric analysis of the blots (C) is in (B). Positions of relative molecular mass markers (kDa) are on the right of the immunoblots. Data were normalised to GAPDH and expressed relative to the mean of the controls. (D) Data were mined from an RNASeq database of patients with dilated cardiomyopathy (n = 97) and normal controls (n = 108). Data for individual samples are shown with adjusted P values.

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  Figure 2.. Activated cardiomyocyte BRAF induces changes in cardiac gene expression in mouse hearts in vivo. (A) Study protocol. Mice heterozygous for floxed BRAFV600E and hemizygous for Myh6-directed tamoxifen-inducible Cre were treated with 40 mg/kg (i.p.) tamoxifen (Tam) or vehicle (Veh). Echocardiograms (ECHO) were taken at baseline (BL) then at 10 d or 6 weeks after tamoxifen treatment. (B) Confirmation of recombination for BRAFV600E knock-in was based on introduction of an Xba1 restriction site in the mRNA following recombination. cDNA was prepared from RNA extracted from heart, liver or kidney, 24 h or 10 d after treatment with tamoxifen or vehicle. cDNA was PCR amplified and subject to Xba1 digestion, resulting in appearance of additional bands (arrows) in hearts (not liver or kidney) following treatment with tamoxifen, but not vehicle. (C) qPCR analysis of mRNA expression of RAF isoforms in samples of mouse hearts treated with vehicle or tamoxifen for 24 h (upper panel) or 10 d (lower panel). (D and E) Immunoblotting of RAF isoforms or Gapdh, in addition to phosphorylated (phospho-) or total MKK1/2, ERK1/2, p90RSK, Akt and p70S6K in samples of mouse hearts treated with vehicle or…

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  Figure 3.. Activated cardiomyocyte BRAF promotes cardiac hypertrophy in mouse hearts in vivo. Mice heterozygous for floxed BRAFV600E and hemizygous for Myh6-directed tamoxifen-inducible Cre (BRAFV600E/WT/CreMCM/−) were treated with a single dose of 40 mg/kg (i.p.) tamoxifen or vehicle, and maintained for 10 d (A–C) or 6 weeks (D–G). Cardiac function and dimensions were measured by echocardiography. (A and D) Representative M-mode images from short-axis views for individual mice before and after treatment. (B and E) Quantification of echocardiograms represented in panels A and D, respectively. LV, left ventricle; AW, anterior wall; PW, posterior wall; ID, internal diameter; d, diastole; s, systole. C and G, Sections of BRAFV600E/WT/CreMCM/− mouse hearts stained with haemotoxylin and eosin (H&E) or Masson's Trichrome were analysed. Representative images are shown with quantification provided below (C) or in the right panel (G). (F) B-mode images of long-axis views of the heart were used for speckle-tracking strain analysis. Representative images are in the upper panels with cardiac function data in the lower panels. EDV, end diastolic volume; ESV, end systolic volume; EDLVM, end diastolic…

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  Figure 4.. Activation of RAF and ERK1/2 signalling in cardiomyocytes by the type 1 RAF kinase inhibitor, SB590885. (A–D) Superdex 200 FPLC separation of RAF complexes in unstimulated neonatal rat cardiomyocytes. Fractions were immunoblotted with antibodies to ARAF, or BRAF and RAF1. Representative blots of all column fractions (A) and comparing expression in fractions across different blots (B) are shown. (C) Column calibration with relative molecular mass (RMM) standards. (D) Elution profiles of ARAF, BRAF and RAF1. Results are means ± SEM (n = 3). (E) Immunoblots of extracts from neonatal rat cardiomyocytes exposed to 0.1 µM SB590885 or 0.1 µM endothelin-1 (ET-1) for the times indicated. Total extracts were immunoblotted for phosphorylated BRAF(S445) and RAF1(S338) (upper blot) or the total proteins (lower blot). Representative blots of four independent cardiomyocyte preparations are shown. (F) RAF kinase activities in neonatal rat cardiomyocytes exposed to the indicated concentrations of SB590885 or 0.1 µM ET-1 for the times shown. Activities were assayed using GST-MKK1 following immunoprecipitation (IP) of RAF1 or BRAF. Phosphorylation of GST-MKK1 was detected with antibodies to…

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  Figure 5.. SB590885 promotes immediate early gene expression and hypertrophy in cardiomyocytes. (A and B) Rat neonatal cardiomyocytes were exposed to the indicated concentrations of SB590885 for the times shown. Cytosolic and nuclear-enriched fractions were immunoblotted for phosphorylated (phospho-) or total ERK1/2. Representative blots are shown (A) with quantification for 0.1 µM SB590885 (B). Results are means ± SEM (n = 3). P values are relative to 0 min (one-way ANOVA with Holm–Sidak's post-test). Positions of relative molecular mass markers (kDa) are on the right of the immunoblots. (C) Rat neonatal cardiomyocytes were untreated or exposed to 0.1 µM SB590885 (1 h) with/without 2 µM PD184352. mRNA expression was measured by quantitative qPCR. Individual data points are shown with means ± SEM (n = 4). P values are for PD184352/SB590885 vs SB590885 (one-way ANOVA with Holm–Sidak's post-test). (D) Cardiomyocytes were incubated with 1 µM SB590885, 1 µM encorafenib or under control conditions (24 h) and immunostained for troponin T. Images (left panels) are representative of 6 (SB590885, controls) or 4 (encorafenib) independent myocyte preparations with analysis of the surface area of…

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  Figure 6.. SB590885 and encorafenib promote cardiac hypertrophy in vivo. C57Bl/6J mice were treated with vehicle, 0.5 mg/kg/d SB590885 (A–D), or 3 mg/kg/d encorafenib (E–H). Cardiac function and dimensions were measured by echocardiography. (A and E) M-mode images from short-axis views of the heart were analysed to provide data for cardiac dimensions. Representative images are in the upper panels with analysis of echocardiograms shown below. LV, left ventricle; AW, anterior wall; PW, posterior wall; ID, internal diameter. Diastolic measurements are shown with all data provided in Supplementary Table S10. (B and F) B mode images from mice at 3 d (upper panels) or 7 d (lower panels) were subject to strain analysis using speckle-tracking to gain an estimate of left ventricular mass. EDLVM, end diastolic left ventricular mass. Additional data are provided in Supplementary Table S9. (C and G) Haemotoxylin (H&E) and eosin staining of mouse heart sections. Representative images are on the left with quantification of cardiomyocyte size from H&E-stained sections on the right. Individual data points are shown with means ± SEM. Statistical analysis used a two-tailed paired t-test. (D and H) RNA was…

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  Figure 7.. Effects of SB590885 on hypertension-induced cardiac hypertrophy in mice in vivo. C57Bl/6J mice were treated with vehicle, 0.8 mg/kg/d angiotensin II (AngII) or AngII with 0.5 mg/kg/d SB590885 for 7 d. (A) Cardiac function and dimensions were measured by echocardiography with speckle-tracking analysis of B-mode long axis images. (B–D) RNA was isolated from hearts of mice treated with vehicle, AngII or AngII with SB590885, and mRNA expression assessed by qPCR. Data are individual points with means ± SEM. Statistical analysis: one-way ANOVA with Holm–Sidak's post-test.

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  Figure 8.. Schematic representation of the conclusions from this study. BRAF is up-regulated in human failing hearts, although the cells involved remain to be defined. In cardiomyocytes, BRAF and RAF1 exist in preformed complexes. Activation of cardiomyocyte BRAF by knock-in of the V600E mutation or with Type 1 RAF kinase ‘inhibitors' (e.g. SB590885, encorafenib) results in activation of MKK1/2 (mitogen-activated protein kinase kinases 1 and 2) and ERK1/2 (extracellular signal-regulated kinases 1 and 2). This leads to phosphorylation and activation of a range of substrates to modify activities of functional proteins in the cell, along with transcription and translation factors that promote expression of immediate early genes with stimulation of downstream gene expression, resulting in cardiomyocyte hypertrophy.