Purpose:
Left ventricular non-compaction cardiomyopathy (LVNC) is a rare genetic cardiac disease and characterized by a trabeculated myocardium leading to cardiac dysfunction. LVNC is complex and incompletely understood, however dysfunctional myocytes and non-myocytes were shown to contribute to LVNC pathology. Previously, a mutation at position p.R634 in the hotspot RS-domain was found to cause LVNC (R634L). We aimed here to identify the molecular events caused by the distinct RBM20 mutation from LVNC patient using a patient-specific stem cell model and to analyze the contribution of cardiomyocytes and endothelial cells with regard to the cardiac LVNC phenotype in a patient-specific context.

Methods:
We generated induced pluripotent stem cell-derived cardiomyocytes (iPSC-CM) and iPSC-derived endothelial cells (iPSC-EC) from a LVNC patient with an RBM20 mutation p.R634L. We investigated RBM20-dependent alternative splicing activity, Ca²⁺ homeostasis with the Fluo-4-probe and the contribution of cardiomyocyte’s paracrine effects on endothelial cells by scratch assay. To investigate the impact of the genetic background, isogenic rescue lines were generated by CRISPR/Cas9 technology (rescue-CM: R634R).

Results:
The LVNC-iPSC-CM show missplicing of the RBM20 target genes Titin, RYR2, CAMK2d and TRDN compared to heathy- and rescue-iPSC-CM. In line with missplicing of the sarcomeric gene Titin, significant sarcomeric disarray was observed. In regard to the Ca²⁺ handling genes RYR2, CAMK2d and TRDN, the LVNC-iPSC-CM exhibited severely fastened Ca²⁺ kinetics and no response to β-adrenergic stimulation. In addition, LVNC-iPSC-CM showed elevated systolic Ca²⁺, whereas Ca²⁺ load and leakage remained inconspicuous. In line with the fastened Ca²⁺ kinetics, we observed a hyperphosphorylation of CAMK2d-dependent PLN_T17 site in LVNC-iPSC-CM and an overall downregulated expression of phospholamban. In contrast, SERCA2a was significantly downregulated on gene and protein level, suggesting a cardiac dysfunction. All of these pathological effects were reversed in the rescue-line underscoring this specific RBM20 mutation as the main causative driver. To further test the paracrine effects of CM on non-CM in LVNC pathology, LVNC- and rescue-iPSC-EC were generated and incubated either with supernatant of LVNC- or rescue-iPSC-CM. Using scratch assays, both LVNC-iPSC-EC and the rescue-iPSC-EC demonstrated a dysfunctional migration behavior if supernatant from diseased LVNC-iPSC-CM was added compared to supernatant from rescue-iPSC-CM. This suggests paracrine effects of LVNC cardiomyocyte’s on endothelial function.

Conclusion:
We show the first iPSC-model of splice-defect associated RBM20-dependent LVNC. In summary, our results suggest that the RBM20-R634L variant causes aberrant alternative splice events that lead to structural and calcium handling impairments and has a negative impact on the surrounding endothelial cells.