Abstract 19583: Autonomous and Non-Autonomous Defects Underlie Hypertrophic Cardiomyopathy in BRAF-Mutant hiPSC-Derived Cardiomyocytes

Hypertrophic cardiomyopathy (HCM) is a pathological disorder predominantly due to mutations in sarcomeric components. Germline mutations in BRAF cause a developmental syndrome called cardio-facio-cutaneous syndrome (CFCS), in which 40% of patients also develop HCM. Since the role of the RAS/MAPK pathway in HCM is still unclear, we generated a human induced pluripotent stem cell model for CFCS from three patients with activating BRAF T599R or Q257R mutations. In order to examine hiPSC-derived cell-type specific phenotypes and cellular interactions underpinning HCM, we generated a method to purify cardiomyocytes and non-cardiomyocytes simultaneously by cell sorting based on SIRPα and CD90 expression. Purified BRAF-mutant SIRPα+/CD90- cells were >95% cardiomyocytes, displayed cellular hypertrophy with pro-hypertrophic gene expression, and dysregulation of the RAS/MAPK pathway. BRAF-mutant cardiomyocytes also displayed intrinsic calcium handling defects, including increased calcium transient irregularity and increased stored calcium within the sarcoplasmic reticulum. In addition, purified BRAF-mutant SIRPα-/CD90+ cells, which were fibroblast-like, displayed activation of the RAS/MAPK pathway and exhibited a pro-fibrotic phenotype. Cross-culture studies revealed that BRAF-mutant fibroblast-like cells critically modulate cardiomyocyte hypertrophy through TGFβ paracrine signaling, as TGFβ inhibition prevented cardiomyocyte hypertrophy induced by BRAF-mutant fibroblast-like cells. Additionally, inhibition of RAS/MAPK signaling was capable of rescuing BRAF-mutant cardiomyocyte hypertrophy and cardiomyocyte-intrinsic calcium handling abnormalities. Thus, we show for the first time that cell autonomous and non-autonomous defects underlie RASopathy-associated HCM. As fibroblast activation has been documented previously in sarcomeric HCM, our findings suggest that cardiac fibroblasts may contribute to pathologic hypertrophy in addition to causing fibrosis in primary forms of HCM. TGFβ inhibition may be a useful therapeutic option for patients with HCM due to RASopathies or other etiologies

Autonomous and Non-autonomous Defects Underlie Hypertrophic Cardiomyopathy in BRAF-Mutant hiPSC-Derived Cardiomyocytes

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

Identification and Purification of Human Induced Pluripotent Stem Cell-Derived Atrial-Like Cardiomyocytes Based on Sarcolipin Expression

<div><p>The use of human stem cell-derived cardiomyocytes to study atrial biology and disease has been restricted by the lack of a reliable method for stem cell-derived atrial cell labeling and purification. The goal of this study was to generate an atrial-specific reporter construct to identify and purify human stem cell-derived atrial-like cardiomyocytes. We have created a bacterial artificial chromosome (BAC) reporter construct in which fluorescence is driven by expression of the atrial-specific gene sarcolipin (<i>SLN</i>). When purified using flow cytometry, cells with high fluorescence specifically express atrial genes and display functional calcium handling and electrophysiological properties consistent with atrial cardiomyocytes. Our data indicate that <i>SLN</i> can be used as a marker to successfully monitor and isolate hiPSC-derived atrial-like cardiomyocytes. These purified cells may find many applications, including in the study of atrial-specific pathologies and chamber-specific lineage development.</p></div

BAC transgene and expression in hiPSC-derived cardiomyocytes.

<p>(a) Schematic of recombineered BAC containing a tdTomato-<i>Rex1</i>-Neo^R cassette. (b) tdTomato fluorescence in differentiating EBs appeared at Day 10 of differentiation and increased over time, lasting up to 60 days. Scale bars, 1000 µm. (c) qPCR of differentiating EBs showing timeline of expression of <i>cTNT, SLN,</i> and <i>ANP</i> consistent with the appearance of red fluorescence. (d) Dissociated red⁺ cells were positive for cTNT by immunofluorescence. Scale bars, 100 µm.</p

Purification and characterization of red^high and red^low cells.

<p>(a) Cell sorting strategy to purify red^high and red^low cardiomyocytes. Cells were first gated on the total cardiomyocyte population, marked as SIRPα⁺/CD90⁻, and then purified based on high or low levels of red fluorescence. (b) qPCR for relevant atrial (<i>SLN, ANP)</i> and ventricular (<i>MYL2, HRT2)</i> genes in purified red^high (n = 3) and red^low (n = 3) populations. (**p<0.01, ***p<0.001, ****p<0.0001). (c) Immunofluorescence for MLC2v showing exclusive expression in red^low population. Scale bars, 200 µm. (d) Quantification of fraction red^low (n = 143) and red^high (n = 514) cells expressing MLC2v protein from (c).</p

Red^high cells display electrophysiological properties similar to atrial cardiomyocytes.

<p>(a) Representative triggered action potential traces recorded from red^high and red^low cells. (b) Quantification of APD₅₀ and APD₉₀ for red^high (n = 15) and red^low (n = 10) cells (***p<0.001, *p<0.05). (c–e) Expression of depolarization-activated potassium currents. (c) Upper panel: representative instantaneous outward currents. Lower panel: representative sustained potassium currents. (d) Comparison of peak instantaneous outward currents between red^high (n = 7) and red^low (n = 8) cells. (e) Comparison of I–V curves of sustained potassium currents between red^high and red^low cells (***p<0.001). (f) qPCR for gene expression of the I_Kur subunit <i>KCNA5</i> and I_KAch subunit <i>KCNJ3</i>, revealing increased expression in red^high cells (*p<0.05, ****p<0.0001). All genes normalized to expression of <i>GAPDH</i> and relative to gene expression in EBs.</p

Red^high cells possess functional Ca²⁺ handling properties similar to atrial cardiomyocytes.

<p>(a) Representative spontaneous calcium transients recorded from red^high and red^low cells. (b) Quantification of calcium transient decay and interval properties in red^high (n = 13) and red^low (n = 13) cells (**p<0.01). (c) Quantification of beating rate in spontaneously beating red^high (n = 22) and red^low (n = 23) cells (****p<0.0001).</p