Abstract
Dilated cardiomyopathy (DCM) is a highly heterogeneous trait with sarcomeric gene mutations predominating. The cause of a substantial percentage of DCMs remains unknown, and no gene-specific therapy is available. On the basis of resequencing of 513 DCM cases and 1,150 matched controls from various cohorts of distinct ancestry, we discovered rare, functional RAF1 mutations in 3 of the cohorts (South Indian, North Indian and Japanese). The prevalence of RAF1 mutations was ∼9% in childhood-onset DCM cases in these three cohorts. Biochemical studies showed that DCM-associated RAF1 mutants had altered kinase activity, resulting in largely unaltered ERK activation but in AKT that was hyperactivated in a BRAF-dependent manner. Constitutive expression of these mutants in zebrafish embryos resulted in a heart failure phenotype with AKT hyperactivation that was rescued by treatment with rapamycin. These findings provide new mechanistic insights and potential therapeutic targets for RAF1-associated DCM and further expand the clinical spectrum of RAF1-related human disorders.
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Acknowledgements
The authors thank A. Mir and C. Evan for their technical support in the zebrafish studies, P. Poulikakos (Icahn School of Medicine at Mount Sinai) and M. Baccarini (Max F. Perutz Laboratories, University of Vienna) for the Braf and Raf1 knockout MEFs, and P. Vaideeswar for the initial collection of samples from cardiomyopathy cases. This work was supported in part by grants from the National Heart, Lung, and Blood Institute to B.D.G. (HL071207) and D.L. (HL097357) and by grants from Telethon-Italy to M.T. (GGP10020 and GGP13107). J.J.E. was a research fellow supported by the Sarnoff Cardiovascular Research Foundation. M.A.R. is a postdoctoral fellow of the American Heart Association (Great Rivers Affiliate). K.T. was supported by a Network project grant (CARDIOMED-BSC0122) from the Council of Scientific and Industrial Research (CSIR) of the government of India.
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P.S.D. conceived the project, with input from B.D.G. P.S.D., M.A.R., U.M., S.K., D.S.R., G.L., A.B., M.K., A.R., E.F., M.T., T.Y., M.F., T. Nishizawa, T. Nakanishi, P.G., B.R., R.M. and K.T. collected and sequenced the various cardiomyopathy cases and controls. P.S.D., M.A.R., J.J.E. and I.R. performed the functional studies, with the help of J.S., S.P. and S.M.-N. J.R., R.J.H., D.L., K.C.S., K.T. and B.D.G. provided reagents for the study. P.S.D. drafted the manuscript, with input from B.D.G.
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B.D.G. and P.S.D. are named inventors on a patent application filed with the US Patent and Trademark Office involving RAF1 mutations and dilated cardiomyopathy.
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Supplementary Figure 2 DCM-associated mutant RAF1 proteins hyperactivate AKT but not ERK1/2.
Representative immunoblot with total lysates from HEK293 cells expressing wild-type RAF1 (WT), DCM-associated mutant RAF1 proteins (Pro332Ala, Leu603Pro, His626Arg, Thr641Met and Arg254fs) and HCM-associated mutant RAF1 proteins (Ser257Leu and Leu613Val) that were stimulated with EGF for 15 min. The blot was probed with anti-phospho-ERK1/2, anti-ERK1/2, anti-phospho-AKT, anti-AKT and anti-RAF1 antibodies. Expression levels were normalized to respective total protein levels and are expressed as relative expression compared to the levels in the cells overexpressing wild-type RAF1. GAPDH levels were used as a loading control. Data are the means ± s.d. from two independent experiments assayed in duplicate. *P < 0.05, **P < 0.01, ***P < 0.001 compared to wild type.
Supplementary Figure 3 DCM-associated RAF1 mutants activate the AKT-mTOR pathway as indicated by tuberin phosphorylation.
Representative immunoblot with total lysates from HEK293 cells expressing wild-type RAF1 (WT), DCM-associated mutant RAF1 proteins (Pro332Ala, Leu603Pro, His626Arg, Thr641Met and Arg254fs) and HCM-associated mutant RAF1 proteins (Ser257Leu and Leu613Val) that were stimulated with EGF for 15 min. The blot was probed with anti-phospho-TSC2, anti-RAF1 and anti-GAPDH antibodies. Expression levels were normalized to RAF1 levels and are expressed as relative expression compared to levels in cells expressing wild-type RAF1. GAPDH levels were used as a loading control. Data are the means ± s.d. from two independent experiments assayed in duplicate. **P < 0.01 compared to wild type.
Supplementary Figure 4 DCM-associated RAF1 mutants show impaired Erk1/2 activation in Raf1–/– MEFs.
Immunoblot with total lysates from MEFs from Raf1 knockout mice (Raf1–/–) expressing wild-type RAF1 (WT) and two representative DCM mutants (Pro332Ala and Leu603Pro) that were stimulated with EGF for 0, 5, 15, 30, 45 and 60 min and probed with anti-phospho-Erk1/2, anti-Erk1/2 and anti-RAF1 antibodies. Expression levels were normalized to respective total protein levels and are expressed as relative expression compared to levels in cells expressing wild-type RAF1 at the indicated time points. β-actin levels were used as a loading control.
Supplementary Figure 5 DCM-associated RAF1 mutants fail to activate Erk1/2 in Braf–/– MEFs.
Immunoblot with total lysates from MEFs from Braf knockout mice (Braf –/–) expressing wild-type RAF1 (WT) and two representative DCM mutants (Pro332Ala and Leu603Pro) that were stimulated with EGF for 0, 5, 15 and 30 min and probed with anti-phospho-Erk1/2, anti-Erk2 and anti-RAF1 antibodies. Expression levels were normalized to respective total protein levels and are expressed as relative expression compared to levels in cells expressing wild-type RAF1 at the indicated time points. β-actin levels were used as a loading control.
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Dhandapany, P., Razzaque, M., Muthusami, U. et al. RAF1 mutations in childhood-onset dilated cardiomyopathy. Nat Genet 46, 635–639 (2014). https://doi.org/10.1038/ng.2963
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DOI: https://doi.org/10.1038/ng.2963
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