Abstract 185: Genome Editing of Isogenic Human Induced Pluripotent Stem Cells Allows for Functional and Transcriptomic Insights Into Hereditary Dilated Cardiomyopathy Caused by Phospholamban Mutations

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Dilated cardiomyopathy (DCM) can be caused by genetic mutations in numerous cardiac proteins, including phospholamban (PLN). PLN mutations are quite rare and obtaining patient samples for mechanistic insights can be challenging. We used genome editing with CRISPR/Cas9 to successfully insert R14del and R9C PLN mutations into a human induced pluripotent stem cell (hiPSC) line from an individual with no cardiovascular disease. HiPSC-cardiomyocytes (hiPSC-CMs) with the inserted R14del PLN mutation recapitulate the phenotype observed in patient-derived R14del hiPSC-CMs, characterized by abnormal intracellular calcium cycling and arrhythmogenicity. Insertion of R9C PLN results in hiPSC-CMs displaying an abnormal response to β-agonists, defective calcium handling, and a hypertrophic phenotype. In human engineered cardiac tissues (hECTs) created from hiPSC-CMs in a 3D matrix, R14del results in a progressive worsening of developed force and R9C PLN demonstrates an abnormal lusitropic response following β-adrenergic stimulation. Further, transcriptional profiling using RNAseq suggests a role for lipid metabolism in R14del. DNA methylation studies showed that differentially expressed genes were enriched for lipoprotein metabolism and chylomicron-mediated lipid transport pathways in R14del PLN. This was also confirmed with the observation of lipid deposition in R14del hECTs and human myocardial tissues from explanted hearts of affected patients. Furthermore, small RNAseq identified 2 miRNAs that were differentially regulated in R14del hiPSC-CMs (miR-449c-5p and miR-483-3p). For R9C PLN, RNAseq suggests that the mutation results in profibrotic signaling, activation of autophagy, and an altered metabolic state. Our findings demonstrate that gene editing of hiPSCs can be used to successfully create models and delineate molecular mechanisms of human PLN mutations associated with DCM.

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