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• W2510124332 abstract "•Cardiomyocytes and fibroblast-like cells can be purified separately from EBs•BRAF-mutant cardiomyocytes display hypertrophy and intrinsic Ca2+-handling defects•BRAF-mutant fibroblast-like cells influence cardiomyocyte hypertrophy through TGFβ•The hypertrophic phenotype can be rescued by TGFβ or RAS/MAPK inhibition Germline mutations in BRAF cause cardio-facio-cutaneous syndrome (CFCS), whereby 40% of patients develop hypertrophic cardiomyopathy (HCM). As the role of the RAS/MAPK pathway in HCM pathogenesis is unclear, we generated a human induced pluripotent stem cell (hiPSC) model for CFCS from three patients with activating BRAF mutations. By cell sorting for SIRPα and CD90, we generated a method to examine hiPSC-derived cell type-specific phenotypes and cellular interactions underpinning HCM. BRAF-mutant SIRPα+/CD90− cardiomyocytes displayed cellular hypertrophy, pro-hypertrophic gene expression, and intrinsic calcium-handling defects. BRAF-mutant SIRPα−/CD90+ cells, which were fibroblast-like, exhibited a pro-fibrotic phenotype and partially modulated cardiomyocyte hypertrophy through transforming growth factor β (TGFβ) paracrine signaling. Inhibition of TGFβ or RAS/MAPK signaling rescued the hypertrophic phenotype. Thus, cell autonomous and non-autonomous defects underlie HCM due to BRAF mutations. TGFβ inhibition may be a useful therapeutic option for patients with HCM due to RASopathies or other etiologies. Germline mutations in BRAF cause cardio-facio-cutaneous syndrome (CFCS), whereby 40% of patients develop hypertrophic cardiomyopathy (HCM). As the role of the RAS/MAPK pathway in HCM pathogenesis is unclear, we generated a human induced pluripotent stem cell (hiPSC) model for CFCS from three patients with activating BRAF mutations. By cell sorting for SIRPα and CD90, we generated a method to examine hiPSC-derived cell type-specific phenotypes and cellular interactions underpinning HCM. BRAF-mutant SIRPα+/CD90− cardiomyocytes displayed cellular hypertrophy, pro-hypertrophic gene expression, and intrinsic calcium-handling defects. BRAF-mutant SIRPα−/CD90+ cells, which were fibroblast-like, exhibited a pro-fibrotic phenotype and partially modulated cardiomyocyte hypertrophy through transforming growth factor β (TGFβ) paracrine signaling. Inhibition of TGFβ or RAS/MAPK signaling rescued the hypertrophic phenotype. Thus, cell autonomous and non-autonomous defects underlie HCM due to BRAF mutations. TGFβ inhibition may be a useful therapeutic option for patients with HCM due to RASopathies or other etiologies. The RASopathies are developmental disorders caused by mutations in the RAS/MAPK pathway, characterized by pleomorphic developmental defects including facial dysmorphism, short stature, neurocognitive delay, and cardiac defects. One of the commonest cardiac manifestations is hypertrophic cardiomyopathy (HCM) (Tartaglia and Gelb, 2010Tartaglia M. Gelb B.D. Disorders of dysregulated signal traffic through the RAS-MAPK pathway: phenotypic spectrum and molecular mechanisms.Ann. N. Y. Acad. Sci. 2010; 1214: 99-121Crossref PubMed Scopus (154) Google Scholar). HCM is defined as thickening of the myocardium that occurs in the absence of an underlying insult, usually resulting from mutations in various genes encoding sarcomeric components. Histologically, there is cardiomyocyte (CM) enlargement and increased fibrosis. HCM is molecularly characterized by upregulation of a fetal gene program including increased expression of atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP), often with dysregulated calcium handling (Konno et al., 2010Konno T. Chang S. Seidman J.G. Seidman C.E. Genetics of hypertrophic cardiomyopathy.Curr. Opin. Cardiol. 2010; 25: 205-209Crossref PubMed Scopus (101) Google Scholar). About 70% of patients with HCM develop obstruction, while other complications include arrhythmias, heart failure, and sudden cardiac death (Harris et al., 2006Harris K.M. Spirito P. Maron M.S. Zenovich A.G. Formisano F. Lesser J.R. Mackey-Bojack S. Manning W.J. Udelson J.E. Maron B.J. Prevalence, clinical profile, and significance of left ventricular remodeling in the end-stage phase of hypertrophic cardiomyopathy.Circulation. 2006; 114: 216-225Crossref PubMed Scopus (497) Google Scholar). The role of RAS/MAPK signaling in cardiac hypertrophy remains unclear. Cardiac-restricted overexpression of Mek1 caused compensated hypertrophy with increased cardiac function (Bueno et al., 2000Bueno O.F. De Windt L.J. Tymitz K.M. Witt S.A. Kimball T.R. Klevitsky R. Hewett T.E. Jones S.P. Lefer D.J. Peng C.F. et al.The MEK1-ERK1/2 signaling pathway promotes compensated cardiac hypertrophy in transgenic mice.EMBO J. 2000; 19: 6341-6350Crossref PubMed Scopus (645) Google Scholar). In addition, Erk1−/−Erk2+/− mice were not protected from pressure overload or agonist stimulation (Purcell et al., 2007Purcell N.H. Wilkins B.J. York A. Saba-El-Leil M.K. Meloche S. Robbins J. Molkentin J.D. Genetic inhibition of cardiac ERK1/2 promotes stress-induced apoptosis and heart failure but has no effect on hypertrophy in vivo.Proc. Natl. Acad. Sci. USA. 2007; 104: 14074-14079Crossref PubMed Scopus (190) Google Scholar). However, oncogenic Hras over-expression led to pathological hypertrophy with fibrosis and calcium-handling defects (Hunter et al., 1995Hunter J.J. Tanaka N. Rockman H.A. Ross Jr., J. Chien K.R. Ventricular expression of a MLC-2v-ras fusion gene induces cardiac hypertrophy and selective diastolic dysfunction in transgenic mice.J. Biol. Chem. 1995; 270: 23173-23178Crossref PubMed Scopus (272) Google Scholar, Zheng et al., 2004Zheng M. Dilly K. Dos Santos Cruz J. Li M. Gu Y. Ursitti J.A. Chen J. Ross Jr., J. Chien K.R. Lederer J.W. et al.Sarcoplasmic reticulum calcium defect in Ras-induced hypertrophic cardiomyopathy heart.Am. J. Physiol. Heart Circ. Physiol. 2004; 286: H424-H433Crossref PubMed Scopus (72) Google Scholar), and dominant-negative Raf1 overexpression prevented pressure overload-induced cardiac hypertrophy (Harris et al., 2004Harris I.S. Zhang S. Treskov I. Kovacs A. Weinheimer C. Muslin A.J. Raf-1 kinase is required for cardiac hypertrophy and cardiomyocyte survival in response to pressure overload.Circulation. 2004; 110: 718-723Crossref PubMed Scopus (143) Google Scholar). Some suggest that the pathological effects of RAS and RAF signaling may not be exclusively mediated by downstream MAPK signaling but rather by cross-activation of other pathways (Heineke and Molkentin, 2006Heineke J. Molkentin J.D. Regulation of cardiac hypertrophy by intracellular signalling pathways.Nat. Rev. Mol. Cell Biol. 2006; 7: 589-600Crossref PubMed Scopus (1503) Google Scholar). Mice expressing the RASopathy Raf1L613V allele developed HCM, rescued by MEK inhibition (Wu et al., 2011Wu X. Simpson J. Hong J.H. Kim K.H. Thavarajah N.K. Backx P.H. Neel B.G. Araki T. MEK-ERK pathway modulation ameliorates disease phenotypes in a mouse model of Noonan syndrome associated with the Raf1(L613V) mutation.J. Clin. Invest. 2011; 121: 1009-1025Crossref PubMed Scopus (156) Google Scholar). However, mice with activating BRAF mutations did not exhibit pathological cardiac remodeling (Andreadi et al., 2012Andreadi C. Cheung L.K. Giblett S. Patel B. Jin H. Mercer K. Kamata T. Lee P. Williams A. McMahon M. et al.The intermediate-activity (L597V)BRAF mutant acts as an epistatic modifier of oncogenic RAS by enhancing signaling through the RAF/MEK/ERK pathway.Genes Dev. 2012; 26: 1945-1958Crossref PubMed Scopus (48) Google Scholar, Urosevic et al., 2011Urosevic J. Sauzeau V. Soto-Montenegro M.L. Reig S. Desco M. Wright E.M. Canamero M. Mulero F. Ortega S. Bustelo X.R. et al.Constitutive activation of B-Raf in the mouse germ line provides a model for human cardio-facio-cutaneous syndrome.Proc. Natl. Acad. Sci. USA. 2011; 108: 5015-5020Crossref PubMed Scopus (53) Google Scholar). Thus, the pathogenesis of HCM in cardio-facio-cutaneous syndrome (CFCS), whereby 75% of cases have germline BRAF mutations (Rodriguez-Viciana et al., 2006Rodriguez-Viciana P. Tetsu O. Tidyman W.E. Estep A.L. Conger B.A. Cruz M.S. McCormick F. Rauen K.A. Germline mutations in genes within the MAPK pathway cause cardio-facio-cutaneous syndrome.Science. 2006; 311: 1287-1290Crossref PubMed Scopus (462) Google Scholar) and 40% develop HCM (Armour and Allanson, 2008Armour C.M. Allanson J.E. Further delineation of cardio-facio-cutaneous syndrome: clinical features of 38 individuals with proven mutations.J. Med. Genet. 2008; 45: 249-254Crossref PubMed Scopus (85) Google Scholar), is unclear. To study this, we generated an hiPSC model of CFCS and developed a method to examine hiPSC-derived cell type-specific phenotypes and cellular interactions underpinning HCM by cell sorting based on SIRPα and CD90 expression. We show that purified CMs derived from hiPSCs harboring the CFC-causing T599R or Q257R BRAF mutations display cellular hypertrophy and intrinsic calcium-handling defects. In addition, fibroblast-like cells (FLCs) derived from BRAF-mutant hiPSCs exhibit pro-fibrotic behavior and modulate the hypertrophic phenotype through paracrine transforming growth factor β (TGFβ) signaling. Both TGFβ and RAS/MAPK inhibition rescue the hypertrophic phenotype. hiPSCs were generated from dermal fibroblasts from three unrelated patients with CFCS. Patient 1 (BRAF1) harbored the known BRAF T599R mutation (Yoon et al., 2007Yoon G. Rosenberg J. Blaser S. Rauen K.A. Neurological complications of cardio-facio-cutaneous syndrome.Dev. Med. Child Neurol. 2007; 49: 894-899Crossref PubMed Scopus (94) Google Scholar), which alters the activation segment of the kinase domain and is kinase activating (Wan et al., 2004Wan P.T. Garnett M.J. Roe S.M. Lee S. Niculescu-Duvaz D. Good V.M. Jones C.M. Marshall C.J. Springer C.J. Barford D. et al.Mechanism of activation of the RAF-ERK signaling pathway by oncogenic mutations of B-RAF.Cell. 2004; 116: 855-867Abstract Full Text Full Text PDF PubMed Scopus (2239) Google Scholar). Patients 2 and 3 (BRAF2, BRAF3) harbored BRAF Q257R mutations, the commonest CFCS mutation, which alters the cysteine-rich domain of the conserved region 1 (CR1) and is also kinase activating (Rodriguez-Viciana et al., 2006Rodriguez-Viciana P. Tetsu O. Tidyman W.E. Estep A.L. Conger B.A. Cruz M.S. McCormick F. Rauen K.A. Germline mutations in genes within the MAPK pathway cause cardio-facio-cutaneous syndrome.Science. 2006; 311: 1287-1290Crossref PubMed Scopus (462) Google Scholar). Patients 1 and 2 displayed HCM while patient 3 did not. Wild-type (WT) hiPSC lines generated from three unrelated healthy individuals served as controls. DNA sequencing confirmed the heterozygous BRAF mutations in mutant hiPSC lines (Figure 1A ). Mutant lines had normal karyotypes, and pluripotency was confirmed with immunofluorescence, gene expression, and tri-lineage differentiation assays (Figure S1). hiPSCs were differentiated along a cardiogenic lineage as 3D embryoid bodies (EBs) using a modification of an established protocol (Yang et al., 2008Yang L. Soonpaa M.H. Adler E.D. Roepke T.K. Kattman S.J. Kennedy M. Henckaerts E. Bonham K. Abbott G.W. Linden R.M. et al.Human cardiovascular progenitor cells develop from a KDR+ embryonic-stem-cell-derived population.Nature. 2008; 453: 524-528Crossref PubMed Scopus (1154) Google Scholar) (Figure 1B). In addition to three WT hiPSC lines, two clones from each mutant hiPSC line were used in subsequent experiments (Table S1). Spontaneous beating was observed between days 10 and 12 of differentiation. To detect potential molecular defects intrinsic to CMs and non-CMs, we purified these populations separately using flow cytometry based on their expression of SIRPα, a CM marker (Dubois et al., 2011Dubois N.C. Craft A.M. Sharma P. Elliott D.A. Stanley E.G. Elefanty A.G. Gramolini A. Keller G. SIRPA is a specific cell-surface marker for isolating cardiomyocytes derived from human pluripotent stem cells.Nat. Biotechnol. 2011; 29: 1011-1018Crossref PubMed Scopus (405) Google Scholar), and CD90, a marker that labels the majority of non-CMs derived from PSCs (Dubois et al., 2011Dubois N.C. Craft A.M. Sharma P. Elliott D.A. Stanley E.G. Elefanty A.G. Gramolini A. Keller G. SIRPA is a specific cell-surface marker for isolating cardiomyocytes derived from human pluripotent stem cells.Nat. Biotechnol. 2011; 29: 1011-1018Crossref PubMed Scopus (405) Google Scholar, Kisselbach et al., 2009Kisselbach L. Merges M. Bossie A. Boyd A. CD90 Expression on human primary cells and elimination of contaminating fibroblasts from cell cultures.Cytotechnology. 2009; 59: 31-44Crossref PubMed Scopus (90) Google Scholar). SIRPα+/CD90− cells were >95% CMs based on cardiac troponin T (cTNT; TNNT2) expression (Figures 1C and 1D). Expression of the cardiac-specific genes ANP, MYH6, and TNNT2 was restricted to the SIRPα+ cells. Re-cultured SIRPα+/CD90− cells formed synchronous monolayers that beat spontaneously and homogeneously expressed cTNT. While differentiation efficiency was variable among lines, no reproducible difference was observed between WT and BRAF-mutant lines (Figure S2). To determine whether BRAF-mutant CMs displayed hypertrophy, we dissociated whole EBs and measured the cellular area of re-plated single cTNT+ cells. Across lines, BRAF-mutant CMs were on average more than three times larger than WT CMs (p < 0.0001) (Figure 2A ). Using flow cytometry to analyze forward scatter (FSC) as a surrogate of 3D cellular size, BRAF-mutant CMs were also larger than WT CMs (Figure S3). Increased sarcomeric organization, a hallmark of cardiac hypertrophy (Aoki et al., 2000Aoki H. Sadoshima J. Izumo S. Myosin light chain kinase mediates sarcomere organization during cardiac hypertrophy in vitro.Nat. Med. 2000; 6: 183-188Crossref PubMed Scopus (123) Google Scholar) demonstrated by a striated cTNT staining pattern, was apparent in BRAF-mutant CMs (58%) compared with WT (16%; p < 0.0001). The most pronounced sarcomere organization was observed in severely hypertrophied BRAF-mutant CMs (Figure 2B). Upregulation of a fetal gene program, classically observed in HCM (Kuwahara et al., 2003Kuwahara K. Saito Y. Takano M. Arai Y. Yasuno S. Nakagawa Y. Takahashi N. Adachi Y. Takemura G. Horie M. et al.NRSF regulates the fetal cardiac gene program and maintains normal cardiac structure and function.EMBO J. 2003; 22: 6310-6321Crossref PubMed Scopus (176) Google Scholar), was also noted in purified BRAF-mutant CMs, which displayed >5-fold and >6-fold increased expression of ANP and BNP, respectively, compared with WT (p < 0.0001), as well as a >8-fold increased β-myosin heavy chain (MYH7) to α-myosin heavy chain (MYH6) ratio (p = 0.078) (Figure 2C). Dysregulation of calcium (Ca2+) handling has been observed in many HCM models (Molkentin, 2004Molkentin J.D. Calcineurin-NFAT signaling regulates the cardiac hypertrophic response in coordination with the MAPKs.Cardiovasc. Res. 2004; 63: 467-475Crossref PubMed Scopus (372) Google Scholar), including an hiPSC model of familial HCM (Lan et al., 2013Lan F. Lee A.S. Liang P. Sanchez-Freire V. Nguyen P.K. Wang L. Han L. Yen M. Wang Y. Sun N. et al.Abnormal calcium handling properties underlie familial hypertrophic cardiomyopathy pathology in patient-specific induced pluripotent stem cells.Cell Stem Cell. 2013; 12: 101-113Abstract Full Text Full Text PDF PubMed Scopus (452) Google Scholar). In purified BRAF-mutant CMs, phospholamban (PLN) expression was significantly decreased (p = 0.016) (Figure 2C) with a trend toward increased SERCA2a expression (data not shown). To assess whether BRAF-mutant CMs had impaired Ca2+ handling, we analyzed Ca2+ transients in paced cells. Compared with WT, BRAF-mutant CMs displayed a significantly increased frequency of irregular transients (28% versus 6%, p < 0.0001), defined as an extra Ca2+ transient peak during the decay of the previous transient, indicating unstable Ca2+ release from the sarcoplasmic reticulum (SR). A small percentage of BRAF-mutant CMs also displayed discordance between the timing of electrical stimulation and resulting Ca2+ transient, further indicating underlying instability (Figure 3A ). Consistent with increased Ca2+ release observed in mouse hearts overexpressing SERCA2a (Baker et al., 1998Baker D.L. Hashimoto K. Grupp I.L. Ji Y. Reed T. Loukianov E. Grupp G. Bhagwhat A. Hoit B. Walsh R. et al.Targeted overexpression of the sarcoplasmic reticulum Ca2+-ATPase increases cardiac contractility in transgenic mouse hearts.Circ. Res. 1998; 83: 1205-1214Crossref PubMed Scopus (179) Google Scholar), BRAF-mutant CMs also displayed increased Ca2+ release from the SR, as reflected by increased transient amplitude compared with WT (3.6 a.u. versus 2.6 a.u., p < 0.0001) (Figure 3B). Exposure to the ryanodine receptor activator caffeine revealed that BRAF-mutant CMs contained increased stored Ca2+ compared with WT (6.2 a.u. versus 4.5 a.u., p < 0.001) (Figure 3C), similar to observations during early cardiac hypertrophy (Delbridge et al., 1997Delbridge L.M. Satoh H. Yuan W. Bassani J.W. Qi M. Ginsburg K.S. Samarel A.M. Bers D.M. Cardiac myocyte volume, Ca2+ fluxes, and sarcoplasmic reticulum loading in pressure-overload hypertrophy.Am. J. Physiol. 1997; 272: H2425-H2435PubMed Google Scholar, Sipido et al., 2000Sipido K.R. Volders P.G. de Groot S.H. Verdonck F. Van de Werf F. Wellens H.J. Vos M.A. Enhanced Ca(2+) release and Na/Ca exchange activity in hypertrophied canine ventricular myocytes: potential link between contractile adaptation and arrhythmogenesis.Circulation. 2000; 102: 2137-2144Crossref PubMed Scopus (238) Google Scholar). Our results demonstrate that BRAF-mutant CMs possess intrinsic defects in Ca2+ handling, representative of early-stage HCM. To assess RAS/MAPK pathway activation, we analyzed ERK activation basally and after stimulation with epidermal growth factor (EGF) or angiotensin II (AngII). ERK activation was sustained over time in BRAF-mutant hiPSCs compared with WT (Figure S5A). In contrast, ERK activation was not increased in BRAF-mutant CMs compared with WT CMs (Figure 4A ). The contribution of non-CMs as crucial to the hypertrophic response has been increasingly recognized (Fujiu and Nagai, 2014Fujiu K. Nagai R. Fibroblast-mediated pathways in cardiac hypertrophy.J. Mol. Cell Cardiol. 2014; 70C: 64-73Abstract Full Text Full Text PDF Scopus (58) Google Scholar). To address whether BRAF-mutant non-CMs display activation of the RAS/MAPK pathway, we purified the SIRPα−/CD90+ population (Figure 1C). CD90 labeled the majority of non-CM cells across hiPSC lines (Figure S2C). To investigate the specific cell type to which the CD90+ cells are most closely related, we performed gene-expression profiling. CD90+ cells did not express markers of stem cells (REX1) or endothelial cells (CD31, CDH5), but robustly expressed three fibroblast markers (VIM, COL1A2, and DDR2), which were also expressed by human fetal SIRPα−/CD90+ cells (Figure S4A). Within cardiac tissue, fibroblasts exclusively express DDR2 (Camelliti et al., 2005Camelliti P. Borg T.K. Kohl P. Structural and functional characterisation of cardiac fibroblasts.Cardiovasc. Res. 2005; 65: 40-51Crossref PubMed Scopus (700) Google Scholar). In addition, hiPSC-derived CD90+ cells displayed a spindle-shaped morphology similar to fibroblasts (Figure S4B). Thus, we henceforth refer to CD90+/SIRPα− cells as FLCs. BRAF-mutant FLCs displayed increased ERK activation compared with WT FLCs, with significantly larger activation in the basal state (1.8 a.u. versus 0.9 a.u., p = 0.0059) (Figure 4B). Importantly, activation of AKT was not observed in BRAF-mutant CMs or FLCs (Figures S5B and S5C), distinct from AKT/mTOR involvement in a related RASopathy syndrome due to PTPN11 mutations (Marin et al., 2011Marin T.M. Keith K. Davies B. Conner D.A. Guha P. Kalaitzidis D. Wu X. Lauriol J. Wang B. Bauer M. et al.Rapamycin reverses hypertrophic cardiomyopathy in a mouse model of LEOPARD syndrome-associated PTPN11 mutation.J. Clin. Invest. 2011; 121: 1026-1043Crossref PubMed Scopus (191) Google Scholar), but similar to findings in mice with mutations in RAF isoforms (Wu et al., 2011Wu X. Simpson J. Hong J.H. Kim K.H. Thavarajah N.K. Backx P.H. Neel B.G. Araki T. MEK-ERK pathway modulation ameliorates disease phenotypes in a mouse model of Noonan syndrome associated with the Raf1(L613V) mutation.J. Clin. Invest. 2011; 121: 1009-1025Crossref PubMed Scopus (156) Google Scholar). To investigate whether activated BRAF-mutant FLCs were modulating the CM hypertrophic phenotype, we co-cultured purified WT CMs along with purified WT or BRAF-mutant FLC populations (Figure 5A ). Strikingly, the cellular area of WT CMs doubled when co-cultured with BRAF-mutant FLCs compared with WT FLCs (p < 0.0001) (Figures 5B and 5C). Next, we examined whether BRAF-mutant FLCs displayed behavior similar to activated fibroblasts in the setting of fibrosis. Compared with WT, BRAF-mutant FLCs were hyperproliferative (15% versus 7%, p = 0.03) (Figure 5D). BRAF-mutant FLCs also expressed significantly increased levels of fibrosis-associated genes, including TGFβ1 (p = 0.002), periostin (POSTN) (p = 0.01), connective tissue growth factor (CTGF) (p = 0.002), and collagen type I (COL1A2) (p = 0.003), and a trend toward increased expression of endothelin-1 (ET-1) (p = 0.07) (Figure 5E). Thus, the BRAF-mutant FLCs displayed a pro-fibrotic phenotype with upregulation of several TGFβ pathway members, suggesting a role for TGFβ signaling in mediating the hypertrophic response. To determine whether the effect of the BRAF-mutant FLCs on the hypertrophic phenotype was mediated by a paracrine effect, we cultured purified WT and BRAF-mutant CMs with conditioned media from purified WT and BRAF-mutant FLCs (Figure 6A ). Remarkably, when WT CMs were exposed to BRAF-mutant FLC conditioned media, their cellular area nearly doubled (p < 0.0001) and was similar to that of BRAF-mutant CMs exposed to BRAF-mutant FLC conditioned media. Exposure to BRAF-mutant FLC conditioned media also resulted in significantly increased ANP expression in WT CMs (p = 0.03). When BRAF-mutant CMs were cultured with WT FLC conditioned media, their cellular area decreased by 30% (p = 0.008) (Figures 6B–6D). Thus, we concluded that BRAF-mutant FLCs were modulating CM hypertrophy through a paracrine mechanism. TGFβ has been implicated in signaling between fibroblasts and CMs, mediating hypertrophic growth (Gray et al., 1998Gray M.O. Long C.S. Kalinyak J.E. Li H.T. Karliner J.S. Angiotensin II stimulates cardiac myocyte hypertrophy via paracrine release of TGF-beta 1 and endothelin-1 from fibroblasts.Cardiovasc. Res. 1998; 40: 352-363Crossref PubMed Scopus (355) Google Scholar, Koitabashi et al., 2011Koitabashi N. Danner T. Zaiman A.L. Pinto Y.M. Rowell J. Mankowski J. Zhang D. Nakamura T. Takimoto E. Kass D.A. Pivotal role of cardiomyocyte TGF-beta signaling in the murine pathological response to sustained pressure overload.J. Clin. Invest. 2011; 121: 2301-2312Crossref PubMed Scopus (258) Google Scholar). Using an ELISA, we found that the amounts of active and total TGFβ protein secreted by BRAF-mutant FLCs were increased compared with those from WT cells. No difference in TGFβ1 gene expression or secreted TGFβ protein level was observed between BRAF-mutant and WT CMs (Figures S6A–S6C). To verify whether the paracrine effect of the BRAF-mutant FLCs was mediated by increased TGFβ secretion, we pre-incubated conditioned media with a pan-TGFβ neutralizing antibody (TGFβ-NA). TGFβ-NA-treated conditioned media from BRAF-mutant FLCs failed to produce a hypertrophic effect on WT or BRAF-mutant CMs (p = 0.01), and resulted in significantly decreased expression of BNP in BRAF-mutant CMs (p = 0.008) (Figures 6B, 6C, and 6E). Thus, we concluded that TGFβ signaling was necessary for the hypertrophy observed in the BRAF-mutant CMs. To determine whether TGFβ signaling was sufficient for the induction of CM hypertrophy, we incubated CMs with recombinant human TGFβ (rhTGFβ). Upon exposure to rhTGFβ, the cellular area of BRAF-mutant and WT CMs significantly increased (p < 0.0001 and p = 0.03, respectively) (Figure S6D). Exposure of purified CMs to TGFβ-NA demonstrated no significant change in their cellular area, further implicating the source of increased TGFβ as derived from BRAF-mutant FLCs rather than BRAF-mutant CMs. Together, our results reveal non-CM autonomous defects in BRAF-mutant FLCs, which contribute to the hypertrophic phenotype observed in BRAF-mutant CMs through increased TGFβ signaling. To test whether MEK inhibition could ameliorate the hypertrophic phenotype in hiPSC-derived CMs, we exposed a mixed population of CMs and non-CMs to the MEK inhibitor U0126. While U0126 treatment had no effect on WT CM cellular area, the cellular area of BRAF-mutant CMs was reduced by 36% (p < 0.0001) to a size not significantly different from WT (Figure 7A ), likely due to MEK inhibition affecting BRAF-mutant FLCs. In addition, treatment of purified BRAF-mutant CMs in the absence of FLCs with U0126 normalized their intrinsic Ca2+-handling defects, including decreasing the percentage of irregular transients and SR Ca2+ content to levels not significantly different from WT (Figures 7B and 7C). To document further that the CM hypertrophy was due to BRAF gain of function, we exposed purified WT and BRAF-mutant CMs to conditioned media from WT or BRAF-mutant FLCs that had or had not been treated with the BRAF inhibitor GDC-0879 (GDC). Both WT and BRAF-mutant CMs displayed significantly reduced cellular area after exposure to conditioned media taken from BRAF-mutant FLCs treated with GDC (p = 0.01 and p = 0.0006, respectively) (Figures S7A and S7B). To demonstrate whether activated BRAF was sufficient to induce CM hypertrophy, we overexpressed BRAF T599R cDNA in WT hiPSCs (WT-T599R). The presence of mutant cDNA was confirmed by Sanger sequencing, and increased expression of BRAF was confirmed by qPCR (Figure S7C). Compared with conditioned media from WT FLCs expressing only an empty vector, exposure to conditioned media from WT-T599R FLCs induced significantly increased cellular area (p = 0.02) and expression of ANP and BNP in WT CMs. Expression of those genes reached levels similar to those observed with BRAF-mutant CMs exposed to BRAF-mutant FLC conditioned media (Figures S7D–S7F). The largest enlargement in cellular area was observed in WT-T599R CMs exposed to WT-T599R FLC conditioned media (p < 0.0001). These data suggest that overactivation of BRAF and the RAS/MAPK pathway can engender CM hypertrophy. In this study we have shown that activating BRAF mutations leading to increased RAS/MAPK pathway signaling induce a hypertrophic phenotype in hiPSC-derived CMs. While BRAF-mutant CMs display intrinsic defects in Ca2+ handling, several aspects of their phenotype require paracrine TGFβ secretion by activated, pro-fibrotic BRAF-mutant FLCs. Inhibition of TGFβ or RAS/MAPK signaling rescues the hypertrophic phenotype. Interestingly, while patient 3 did not show clinical evidence of HCM, we detected underlying defects indistinguishable from those in patients 1 and 2, both diagnosed with HCM. Similar subclinical pathology has been demonstrated in other hiPSC models of HCM (Lan et al., 2013Lan F. Lee A.S. Liang P. Sanchez-Freire V. Nguyen P.K. Wang L. Han L. Yen M. Wang Y. Sun N. et al.Abnormal calcium handling properties underlie familial hypertrophic cardiomyopathy pathology in patient-specific induced pluripotent stem cells.Cell Stem Cell. 2013; 12: 101-113Abstract Full Text Full Text PDF PubMed Scopus (452) Google Scholar). However, it is also possible that more complex factors active in 3D multi-organ systems such as hemodynamic load, which are inadequately modeled using the 2D hiPSC system, may play a role in disease progression. We recently generated a 3D human engineered cardiac tissue (hECT) model, in which BRAF-mutant hECTs displayed increased twitch force and contraction and relaxation rates, and a lower excitation threshold compared with WT (Cashman et al., 2016Cashman T.J. Josowitz R. Johnson B.V. Gelb B.D. Costa K.D. Human engineered cardiac tissues created using induced pluripotent stem cells reveal functional characteristics of BRAF-mediated hypertrophic cardiomyopathy.PLoS One. 2016; 11: e0146697Crossref Scopus (53) Google Scholar). In the future, these hECT models may be helpful for investigating more complex factors such as tissue perfusion, flow dynamics, and mechanical stress, to enable higher-fidelity physiologic measurements of muscle function. Some aspects of the intrinsic CM phenotype we document have been associated with enhanced cellular maturation in culture, including organized sarcomeres and increased cellular area (Yang et al., 2014Yang X. Pabon L. Murry C.E. Engineering adolescence: maturation of human pluripotent stem cell-derived cardiomyocytes.Circ. Res. 2014; 114: 511-523Crossref PubMed Scopus (644) Google Scholar). However, matured stem cell-derived CMs develop into elongated rods with myofibrils arranged parallel to the long axis of the cell, and do not display the irregular, generalized increase in cellular area that we observed. In addition, immature derived CMs possess sophisticated excitation-contraction coupling and do not display increased Ca2+ transient amplitude, irregularity, or increased SR Ca2+ stores upon maturation (Lundy et al., 2013Lundy S.D. Zhu W.Z. Regnier M. Laflamme M.A. Structural and functional maturation of cardiomyocytes derived from human pluripotent stem cells.Stem Cells Dev. 2013; 22: 1991-2002Crossref PubMed Scopus (493) Google Scholar). Although genotype-specific influences on CM maturation may contribute to the CM phenotype, the HCM phenotype we observe in its totality cannot be attributed to them. In addition, the variations in cardiac differentiation efficiencies we document do not segregate WT an" @default.
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• W2510124332 date "2016-09-01" @default.
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• W2510124332 title "Autonomous and Non-autonomous Defects Underlie Hypertrophic Cardiomyopathy in BRAF-Mutant hiPSC-Derived Cardiomyocytes" @default.
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• W2510124332 doi "https://doi.org/10.1016/j.stemcr.2016.07.018" @default.
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