Eberl, H. | bioRxiv Science

bioRxiv2025

Enhanced iPSC-Cardiomyocyte Maturation via Combined 3D-Culture and Metabolic Cues

Human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CM) have become an invaluable tool for disease modelling and drug testing. However, while many etiologies of heart failure involve defects in excitation-contraction coupling, mitochondrial energetics or both, iPSC-CM are limited by the developmental immaturity of these processes. Here, we report a combinatorial strategy to enhance the maturation of human iPSC-CM by integrating three-dimensional (3D) spheroid culture conditions with a defined hormone- and fatty acid-enriched maturation medium (MM). A comprehensive analysis of structural, electrophysiological and Ca2+ handling parameters was performed to evaluate cellular and functional maturation. The iPSC-CM generated under these conditions (3D_MM) exhibit many phenotypic characteristics that resemble those of isolated adult human CM, including (i) a rod-shaped morphology, (ii) cardiac ultrastructural features such as aligned myofilaments, unidirectional organized sarcomeres, and the presence of transverse (t)-tubules, (iii) refined action potential (AP) parameters and Ca2+ handling, and (iv) {beta}-adrenergic responsiveness and a positive force-frequency relationship. Compared with long-term (LT) monolayer cultures of 90 days or the individual cues (3D or MM alone), the 3D_MM protocol achieves mostly superior or at the least non-inferior maturation effects. This systematic investigation further demonstrates that while 3D culturing or MM alone improved specific aspects of maturation, only their synergistic combination produced a comprehensive enhancement of key CM processes, such as excitation-contraction coupling and mitochondrial energetics.

cell biology↗

bioRxiv2025

RBM20-variants induce distinct calcium handling and metabolic phenotypes in patient-specific stem cell models of dilated and non-compaction cardiomyopathy

Background and aimMutations in the splice regulator RBM20 account for [~]3 % of genetic cardiomyopathies. In particular, the highly conserved RS domain is a hotspot for disease-associated mutations. Previously, mutations at same amino acid position 634 in the hotspot RS-domain were found to cause dilated cardiomyopathy (DCM) with left ventricular non-compaction (R634L) or without (R634W), but the pathophysiological mechanisms that govern the heterogeneity in phenotype presentation remained unknown. Here, we identify the molecular events caused by the distinct RBM20 mutations from DCM and left-ventricular non-compaction (LVNC) using patient-specific stem cell models. MethodsWe generated induced pluripotent stem cell-derived cardiomyocytes (iPSC-CM) of one LVNC- and two DCM-patients harboring the RBM20-mutations R634L (LVNC) or R634W (DCM). We investigated alternative splicing activity, RBM20 localization, sarcomeric regularity, cAMP level, kinase-specific phosphorylation of key Ca2+ handling enzymes, physiological cardiac functions as Ca2+ homeostasis, and metabolic activity on a patient-specific cardiomyocyte level. Force generation was analyzed in patient-specific engineered myocardial tissues. Isogenic rescue and mutation insertion lines were generated by CRISPR/Cas9 technology to analyze the direct impact of the RBM20 mutations on the cardiac phenotype. ResultsWe observed common splicing aberrations for LVNC- and DCM-CM in TTN and RYR2, RBM20 cytoplasmatic accumulation and irregular sarcomeric structure. LVNC-CM harboring the RBM20-p.R634L variant show distinct molecular, cellular and functional impairments that manifest in CAMK2D, TRDN and IMMT mis-splicing. Splicing defects in LVNC-CM correlate with elevated systolic Ca2+ and faster Ca2+ kinetics with elevated cAMP levels and PLN-hyperphosphorylation. An increased metabolic activity and mitochondrial membrane potential support the hyperactive LVNC-CM. By contrast, DCM-CM (RBM20-p.R634W) distinctly present with decreased systolic Ca2+ and increased SR Ca2+leak but unchanged Ca2+ kinetics and metabolic activity. Both mutations lead to severely reduced force of contraction in engineered myocardium. CRISPR/Cas9 gene-edited isogenic control lines of both described RBM20 mutations in LVNC and DCM demonstrated the causative nature of the two mutations and their diverging effects. Further, L-type Ca2+ channel blockade by verapamil ameliorates the Ca2+ cycling and leakage phenotypes in LVNC- and DCM-CM. ConclusionWe show the first comparative iPSC-model of splice-defect-associated RBM20-dependent LVNC-p.R634L and DCM-p.R634W. We found shared and variant-specific phenotypes on a patient-specific level. Our data demonstrate that the different RBM20 mutations manifest in distinct molecular aberrations in alternative splicing and RBM20 cytoplasmic accumulation that convey various physiological impairments in structure, Ca2+ handling, metabolism and contractile force.

molecular biology↗