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Using hiPSC-CMs to understand mechanisms driving cardiomyocyte maturation and hypertrophic cardiomyopathy

In this thesis, I integrated experimental and computational approaches to investigate the epigenetic, transcriptional, and metabolic mechanisms underlying cardiomyocyte maturation and hypertrophic cardiomyopathy (HCM) using human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). In Chapter 3, I examined how hormonal and metabolic maturation-inducing factors (MIF) reprogram immature hiPSC-CMs toward a mature phenotype. Chromatin accessibility profiling revealed a global reduction in regulatory element accessibility during maturation, particularly at transcription start sites, alongside increased accessibility at distal regulatory regions at later stages, consistent with specialized cell fate programming. Transcriptomic and proteomic analyses demonstrated upregulation of metabolic and structural maturation markers. MIF treatment suppressed mTORC1-ULK1 signaling and was accompanied by increased expression of autophagy-related components. Using these mature hiPSC-CMs to model doxorubicin-induced cardiotoxicity, acute doxorubicin exposure revealed chromatin accessibility changes associated with extracellular matrix organization and electrophysiological regulation. In Chapter 4, I investigated epigenetic and metabolic remodeling driven by TNNT2 variants associated with HCM (I79N^+/- and R278C^+/-). Both variants exhibited reduced mitochondrial respiration; however, the R278C^+/-variant showed improved metabolic capacity following maturation, consistent with a milder phenotype, whereas the I79N^+/- variant maintained impaired metabolic activity. Widespread chromatin accessibility changes were observed in mature HCM cardiomyocytes compared with isogenic controls, with minimal changes during early differentiation. Transcriptomic analyses revealed variant-specific stress responses, altered extracellular matrix regulation, and dysregulation of sarcomere gene expression. Integration of multi-omics datasets demonstrated limited concordance between chromatin accessibility and transcriptional changes at the mature disease stage, suggesting post-transcriptional regulatory mechanisms. Pharmacological inhibition of mTORC1 with rapamycin in the more severe I79N^+/- variant reduced pathogenic protein signatures. In Chapter 5, I explored stage-specific modulation of mTORC1 signaling during cardiomyocyte differentiation. Early inhibition of mTORC1 impaired cardiomyocyte differentiation, whereas inhibition at the cardiac progenitor stage enhanced expression of key maturation markers and reduced fibroblast-associated protein expression, identifying a developmental window in which mTORC1 modulation supports cardiomyocyte maturation. Collectively, these findings provide molecular and metabolic insight into cardiomyocyte development, disease modeling, and therapeutic intervention strategies.