Analysis of splice-defect associated cardiac diseases using a patient-specific iPSC-cardiomyocyte system

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The induced pluripotent stem cell (iPSCs) based cardiomyocyte system (iPSC-CMs) is a powerful tool to serve as a human in vitro model for cardiac diseases. Dilated cardiomyopathy (DCM) and left ventricular non-compaction cardiomyopathy (LVNC) are diseases resulting in reduced and thin (DCM) or trabeculated (LVNC) muscle tissue. This project aims to analyze the influence of genetic mutations on aberrant splicing events leading to the development of DCM and LVNC. For this purpose, two families were recruited: 1) A LVNC family with a missense mutation in a conserved region of the splicing factor RNA binding motif protein 20 (RBM20) (p.R634L). 2) A DCM family with a missense mutation at the same amino acid position in RBM20 (p.R634W). Patient-specific and healthy control iPSCs and iPSC-CMs were successfully established for all family members of LVNC and DCM. RBM20 is a splice factor with highest expression in the heart. Therefore, selected splice targets of RBM20 were analyzed and compared between control-, LVNC- and DCM-CMs. Common splice defects were detected for the titin (TTN) isoform switch and a 24 bp insertion in the gene ryanodine receptor 2 (RYR2). Differential missplicing was observed for the Ca2+ handling genes triadin (TRDN) (exon 9) and the Ca2+/calmodulin-dependent protein kinase 2 δ (CAMK2δ) (exon 14) in LVNC-CMs, and the structural gene Lim-domain binding protein 3 (LDB3) (exon 5) for DCM-CMs. To test cellular and physiological outcomes in the iPSC-CMs, analyses of the sarcomeric regularity, Ca2+ cycling as well as beating rate and regularity were performed. Intriguingly, both LVNC- and DCM-CMs showed impaired sarcomere regularity, which appears to be a common characteristic among RBM20-based cardiomyopathies. Correct Ca2+ cycling is a crucial process in cardiomyocytes since it couples the electrical excitation with mechanical contraction. LVNC-CMs presented with fastened Ca2+ cycling and an inadequate response to β-adrenergic stimulation with Isoprenaline (Iso). In contrast, DCM-CMs exhibited normal Ca2+ cycling kinetics and reaction to Iso similar to control-CMs but showed a significantly increased Ca2+ leakage and decreased amounts of diastolic and systolic Ca2+ levels. Intriguingly, the electrophysiological assessment of beating rate, field potential duration and beating regularity did not reveal any significant deviations between LVNC-, DCM- and control-CMs. Isogenic controls of LVNC- and DCM-iPSCs were generated in order to directly link molecular and physiological aberrations to the RBM20 mutation. The gene editing of RBM20 into wildtype was successful using the CRISPR/Cas9 technology and generated isogenic rescue (res) lines of LVNC- and DCM-iPSCs, termed resLVNC and resDCM, by using the CRISPR/Cas9 technology. The assessment of these isogenic iPSC-CMs demonstrated a regular sarcomeric structure, which was accompanied by a rescue of the TTN missplicing in both resLVNC- and resDCM-CMs. Furthermore, the differential Ca2+ pathologies observed in LVNC-CMs (fastened Ca2+ cycling, impaired reaction to Iso stimulation) and DCM-CMs (increased Ca2+ leakage) was abolished in their respective rescue counterparts underscoring the RBM20 mutations as a major driver for the sarcomeric irregularity and the differential pathological Ca2+ phenotypes. Concurrent to the rescue of Ca2+ cycling, splicing of TRDN and CAMK2δ was restored in resLVNC-CMs. Further molecular investigations demonstrated CAMK2δ-dependent hyperphosphorylation of the specific target Phospholamban (PLN) in LVNC-CMs, which possibly contributes to the fastened Ca2+ kinetics and is caused by CAMK2δ missplicing exclusively observed in LVNC-CMs. The treatment of LVNC-CMs with the Ca2+ channel blocker Verapamil (30 nM) showed beneficial effects on the Ca2+ cycling identifying Verapamil as a promising candidate for medical treatment of RBM20-mutation based LVNC. RBM20-dependent missplicing of CAMK2δ focusses on exon 14, which codes for a nuclear localization sequence (NLS), however, altered distribution patterns of CAMK2δ were not detected in this work. Next generation sequencing (NGS) identified novel RBM20-dependent splice targets, which could be verified for exon 7/8 of K+ voltage-gated channel subfamily H member 2 (KCNH2), exon 13 of Ca2+ voltage-gated channel subunit alpha1 C (CACNA1C), exon 14 of adenylate cyclase 6 (ADCY6) and exon 4 of bone morphogenic protein 7 (BMP7). Investigations regarding developmental defects of RBM20 in LVNC-CMs demonstrated that RBM20 is expressed early during cardiogenesis. In addition, LVNC-CMs with the RBM20 mutation p.R634L showed retained cell growth and proliferation rates. Single-cell sequencing (SCS) unveiled upregulation of atrial and fetal marker genes in LVNC-CMs underscoring an impaired and delayed development. Taken together, these results demonstrate that two distinct missense mutations at the same amino acid position p.634 of RBM20 can degenerate into two distinct cardiomyopathies. RBM20-dependent missplicing differs depending on the mutation present, which causes differential pathologies demonstrated in this work by different Ca2+ handling pathologies in LVNC- and DCM-CMs. Novel splice targets were discovered, which expands the scope of RBM20 and opens a new avenue for further disease mechanisms conveyed by these genes. In the future, this stem cell platform, which comprises healthy control-, patient- and isogenic control-iPSC-CMs, will be used to screen for patient-specific drug applications and to elucidate further RBM20-based disease mechanisms.