Background: Dilated cardiomyopathy (DCM) is characterized by left ventricular dilation and contractile dysfunction. Nearly 35% of all cases have a family history, linked to mutations in more than 30 gene loci mainly encoding cytoskeleton, sarcomere, splicing factors and mitochondrial proteins. The aim of this study was to analyse the underlying genetic and molecular causes in a family of hereditary DCM by using patient-specific induced pluripotent stem cell cardiomyocytes (iPSC-CM).

Methods and Results: For this purpose a 4-member family was recruited containing 2 patients (father and daughter) with severe DCM followed by heart transplantation. Whole exome sequencing pointed towards a new genetic variant in the filamin c (FLNc) gene coding for the structural protein FLNC, an actin-crosslinking protein within the sarcomere of striated muscle. Diseased iPSCs were shown to harbour a heterozygous FLNc variant, which is located in a highly inter-species conserved 82aa insertion domain. iPSCs of all family members were generated and differentiated into functional ventricular 3-month-old iPSC-CMs. All iPSC-CMs expressed general cardiac markers, such as α and β myosin heavy chain (α-/β-MHC), α‑actinin and cTNT, whereas α-MHC expression was downregulated in diseased iPSC-CMs compared to control pointing to stress-induced cardiac dysfunction. Although, FLNc expression itself was not changed between DCM and healthy control iPSC-CMs, FLNc interaction partners XIN and myopodin were increased expressed in all DCM iPSC-CMs. Since FLNc acts as a scaffolding and stabilization protein, we analysed the sarcomeric regularity by staining the M-line/Z-band with antibodies against titin/α-actinin and found a dysregulated sarcomeric structure of the diseased iPSC‑CMs. Furthermore, DCM iPSC-CMs of the father showed a significantly decreased cell size compared to control CMs. We performed CRISPR Cas9 genome editing to rescue the identified FLNc variant in DCM iPSC-CMs and demonstrated a restored sarcomeric regularity as well as cell size in all analysed isogenic iPSC-CMs. To analyse whether dysregulated sarcomeric structure is accompanied by cardiac physiological dysfunction, Ca²⁺ measurements were performed. We found significantly reduced diastolic [Ca²⁺] and fastened Ca²⁺ elimination time in diseased DCM-CMs compared to healthy family controls, which is in line with increased CAMKIIδ-dependent phosphorylation of PLN-Thr17 in DCM-CMs versus control. Evaluation of SR Ca²⁺ handling by Caffeine application showed a significantly reduced Caffeine-induced relaxation time, which might be explained by increased NCX expression in DCM-CMs compared to control-CMs. However, SR Ca²⁺ content and SERCA activity were not changed. Engineered heart muscles (EHM) from DCM-CMs were generated and underlined the DCM phenotype by significantly impaired force generation in DCM-EHM. Future studies of isogenic rescue iPSC-CMs and EHMs will give further insights into the contribution of this newly identified FLNc variant to the development of DCM.

Conclusion: Using a ps-iPSC-CM model of a 4-member family with two severe DCM patients, we identified a new disease-causing heterozygous mutation in the FLNc gene that is directly linked to DCM. CRISPR Cas9 repair of the FLNc mutation in the diseased iPSC-CMs underscored the role of FLNc mutations in cardiomyopathies as some cellular phenotypes were rescued for DCM-CMs. This makes FLNc a new therapeutic target for DCM on a personalized level.