Modeling structural and functional deficiencies ofRBM20familial dilated cardiomyopathy using human induced pluripotent stem cells

Abstract

Dilated cardiomyopathy (DCM) is a leading cause of heart failure. In families with autosomal-dominant DCM, heterozygous missense mutations were identified in RNA-binding motif protein 20 (RBM20), a spliceosome protein induced during early cardiogenesis. Dermal fibroblasts from two unrelated patients harboring an RBM20 R636S missense mutation were reprogrammed to human induced pluripotent stem cells (hiPSCs) and differentiated to beating cardiomyocytes (CMs). Stage-specific transcriptome profiling identified differentially expressed genes ranging from angiogenesis regulator to embryonic heart transcription factor as initial molecular aberrations. Furthermore, gene expression analysis for RBM20-dependent splice variants affected sarcomeric (TTN and LDB3) and calcium (Ca(2+)) handling (CAMK2D and CACNA1C) genes. Indeed, RBM20 hiPSC-CMs exhibited increased sarcomeric length (RBM20: 1.747 ± 0.238 µm versus control: 1.404 ± 0.194 µm; P < 0.0001) and decreased sarcomeric width (RBM20: 0.791 ± 0.609 µm versus control: 0.943 ± 0.166 µm; P < 0.0001). Additionally, CMs showed defective Ca(2+) handling machinery with prolonged Ca(2+) levels in the cytoplasm as measured by greater area under the curve (RBM20: 814.718 ± 94.343 AU versus control: 206.941 ± 22.417 AU; P < 0.05) and higher Ca(2+) spike amplitude (RBM20: 35.281 ± 4.060 AU versus control:18.484 ± 1.518 AU; P < 0.05). β-adrenergic stress induced with 10 µm norepinephrine demonstrated increased susceptibility to sarcomeric disorganization (RBM20: 86 ± 10.5% versus control: 40 ± 7%; P < 0.001). This study features the first hiPSC model of RBM20 familial DCM. By monitoring human cardiac disease according to stage-specific cardiogenesis, this study demonstrates RBM20 familial DCM is a developmental disorder initiated by molecular defects that pattern maladaptive cellular mechanisms of pathological cardiac remodeling. Indeed, hiPSC-CMs recapitulate RBM20 familial DCM phenotype in a dish and establish a tool to dissect disease-relevant defects in RBM20 splicing as a global regulator of heart function.

A BAG3 chaperone complex maintains cardiomyocyte function during proteotoxic stress

“…Brackets indicate significant differences (P < 0.05) by 1-way ANOVA with Bonferroni's test for multiple comparisons. insight.jci.org https://doi.org/10.1172/jci.insight.94623 disease phenotype after repetitive dosing of norepinephrine (37,38). However, BAG3-mutant lines displayed a positive chronotropic response to stimulation of both α-and β-adrenergic receptors by phenylephrine and isoproterenol, respectively (Supplemental Figure 8).…”

Pharmacological Modulation of Calcium Homeostasis in Familial Dilated Cardiomyopathy: An In Vitro Analysis From an RBM20 Patient‐Derived iPSC Model

“…In vitro stress testing of hiPSC‐CMs with β‐adrenergic agonist, NE, has led to increased cardiac contractility by the G‐protein coupled second messenger system . Familial DCM has been associated with increased intracellular Ca 2+ overload as well as an increased susceptibility to chronotropic stress from NE . To modulate this Ca 2+ overload and minimize chronotropic stress response, we utilized pretreatment with carvedilol, β‐blocker, and verapamil, L‐type Ca 2+ channel blocker ( Figure a ).…”

“…Disruption of normal splicing therefore results in anomalous isoforms of a whole network of cardiac proteins and prevents the heart from effectively responding to both extrinsic and intrinsic stressors . Recent reports have shown that RBM20 familial DCM hiPSC‐CMs have an increased susceptibility to β‐adrenergic stimulation and exhibit stress‐induced phenotypic change in sarcomeric structure …”

Dilated Cardiomyopathy

“…iPSC-derived cardiomyocytes with the RBM20 R636S mutation develop a gene expression and splicing profile consistent with cardiomyopathy affecting not only TTN but also the CAMK2D and CACNA1C genes 112 . Sarcomeres within these RBM20 mutant lines were thinner, similar to what was described for TTN mutant iPSC-cardiomyocytes 68 .…”