RBM20 variants disrupt Ca²⁺ handling and metabolism in dilated and non-compaction cardiomyopathy stem cell models

Mutations in the splice-regulator RBM20 cause heart failure with reduced ejection fraction (HFrEF), typically manifesting as dilated cardiomyopathy (DCM). Mutations at position 634 in the RS-domain cause DCM with (R634L) or without (R634W) left ventricular non-compaction (LVNC). However, the mechanisms underlying phenotype variability and personalized therapy beyond HFrEF remain unclear. We generated induced pluripotent stem cell-derived cardiomyocytes (iPSC-CM), 3D-cardiospheres and engineered myocardial tissues from patients with RBM20 mutations R634L (LVNC) or R634W (DCM). Using CRISPR/Cas9, we created isogenic rescue and mutation-insertion lines, identifying RBM20 mis-localization, splicing errors in TTN and RYR2, and sarcomere irregularities in both. DCM-CM showed increased resting Ca²⁺ leak and reduced Ca²⁺ transient amplitude, typical of HFrEF, and spatial disorganization of sarcoplasmic reticulum and mitochondria. In contrast, LVNC-CM exhibited elevated Ca²⁺ transient amplitude with faster kinetics, driven by elevated cAMP and mis-spliced, hyperactive CAMK2D, leading to PLN-hyperphosphorylation and increased metabolic respiration. Further, LVNC showed desmosomal derangement potentially from mis-splicing of Junction plakoglobin and reduced 3D cardiosphere compaction. Despite distinct mechanisms, contractile force was reduced in both. Isogenic controls confirm mutation causality. Drug intervention with verapamil partially improved selected abnormal Ca²⁺ handling and contractile phenotypes in LVNC- and DCM-CM, whereas the CAMK2D inhibitor AIP improved systolic Ca²⁺ handling predominantly in LVNC-CM. In conclusion, different amino acid substitutions at the same RBM20-residue induce opposing Ca²⁺-handling and structural phenotypes. While DCM features impaired Ca²⁺ handling, LVNC shows defective cell-cell coupling and activated Ca²⁺ handling and metabolism, yet insufficient to compensate for organ-level dysfunction. This supports personalized pharmacological therapies in early HF, and potential CRISPR/Cas9 repair for RBM20 cardiomyopathy.