Molecular Rewiring in TNNT2-Linked Hypertrophic Cardiomyopathy hiPSC-Derived Cardiomyocytes upon Metabolic Maturation

Abstract

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are widely used to model cardiac development and inherited cardiomyopathies, yet their immature metabolic state limits interpretation of disease-associated molecular programs. While multiple strategies promote structural and functional maturation, less is known about the molecular regulation of metabolic maturation as a distinct developmental transition. Here, we examine how metabolic maturation reshapes molecular and metabolic states in wild-type and TNNT2-linked hypertrophic cardiomyopathy (HCM) hiPSC-CMs. Using integrated transcriptomic, chromatin accessibility, proteomic, and metabolic profiling, we define the molecular trajectory associated with metabolic maturation in wild-type hiPSC-CMs, characterized by coordinated transcriptional and epigenetic remodeling, enhanced mitochondrial oxidative metabolism, and progressive suppression of mTORC1 signaling. In contrast, hiPSC-CMs carrying TNNT2 HCM variants (I79N+/- and R278C+/-) exhibit variant-specific deviations from this metabolic maturation trajectory. While early CM differentiation is largely preserved, metabolic maturation reveals defects in mitochondrial respiration and chromatin organization, with the more clinically severe I79N+/- variant showing sustained metabolic impairment. We further find that mTORC1 activity is temporally misregulated during metabolic maturation in HCM hiPSC-CMs, with reduced signaling at early stages and normalization at later time points. Pharmacological inhibition of mTORC1 with rapamycin partially improves disease-associated protein expression signatures in the I79N+/- variant, particularly when applied during early differentiation. Together, these findings demonstrate that TNNT2-linked HCM involves disrupted metabolic maturation programs coupled to altered gene regulatory states, highlighting metabolic maturation as a critical context for studying cardiomyopathy-associated molecular phenotypes in hiPSC-CMs.