RBM20-variants induce distinct calcium handling and metabolic phenotypes in patient-specific stem cell models of dilated and non-compaction cardiomyopathy
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Background and aim
Mutations 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.
Methods
We 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 Ca 2+ handling enzymes, physiological cardiac functions as Ca 2+ 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.
Results
We 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 Ca 2+ and faster Ca 2+ 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 Ca 2+ and increased SR Ca 2+ leak but unchanged Ca 2+ 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 Ca 2+ channel blockade by verapamil ameliorates the Ca 2+ cycling and leakage phenotypes in LVNC- and DCM-CM.
Conclusion
We 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, Ca 2+ handling, metabolism and contractile force.
