Background: Mutations in the splice factor RBM20 account for ~3% of genetic cardiomyopathies. Mutations in the hotspot RS-domain (p.633-637) were found to cause dilated cardiomyopathy or in a few cases left ventricular non-compaction cardiomyopathy (LVNC) (R634L), but the pathophysiological mechanisms that govern the heterogeneity in phenotype presentation remain unknown.

Purpose: We aimed here to investigate the molecular events caused by the distinct RBM20-p.R634L variant from a LVNC patient using a patient-specific induced pluripotent stem cell model (iPSC) and describe how these molecular act in molecular, cellular, and metabolic function of RBM20 to govern physiological impairments.

Methods: We generated iPSC-cardiomyocytes (iPSC-CM) of a LVNC patient harboring the RBM20-mutation R634L, and investigated RBM20-mediated alternative splicing, RBM20 localization, sarcomeric regularity, phosphorylation of Ca²⁺ players, and Ca²⁺ as well as mitochondrial function. To investigate the impact of the genetic background, isogenic rescue lines were generated by CRISPR/Cas9.

Results: We investigated the splicing pattern of the RBM20 mutation p.R634L in LVNC-iPSC-CM and observed isoform changes in titin (TTN), a 24bp-insertion in the gene ryanodine receptor 2 (RYR2), missplicing in the Ca²⁺ handling genes triadin (TRDN) and the Ca²⁺ handling gene CAMK2δ and the mitochondrial gene Mitochondrial Inner Membrane Protein (IMMT). As a possible physiological consequence, we observed severe RBM20-dependent sarcomeric irregularity at the Z-disc and M-line. The mis-spliced CAMK2δ leads to CAMK2δ-dependent hyperphosphorylation of its target PLN-Thr17 and subsequently to shortened Ca²⁺ elimination time and weakened response to β-adrenergic stimulation. The mitochondrial and metabolic profile of LVNC-CM revealed an increased mitochondrial membrane potential with a concomitant increase in respiratory oxygen consumption rate, maybe a consequence of the increased systolic Ca²⁺ content. In line with reports of other RBM20 mutations, confocal microscopy verifies that mutant RBM20 maintains very limited nuclear localization potential. Isogenic CRISPR/Cas9 repair of the RBM20 mutation (resLVNC) rescued the gene missplicing, sarcomeric regularity, Ca²⁺ handling aberrations and mitochondrial pathologies underscoring the causative nature of the mutation.

Conclusion: We show the first iPSC-model of splice-defect associated RBM20-dependent LVNC. In summary, our results suggest that the RBM20 mis-localization and molecular aberrations in alternative splicing convey various physiological impairments. Our results highlight a RBM20 mutation-dependent pathogenic mechanism in cardiac disease through splicing-dependent and -independent pathways.