Background/Purpose:
Dilated cardiomyopathy (DCM) is characterized by left ventricular dilation and contractile dysfunction. The molecular basis of most DCM cases remains unknown. Nevertheless, 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 analyze 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 and healthy controls. Whole exome sequencing of iPSC of the 4 family members 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 iPSC were shown to harbour a heterozygous FLNc variant, which is located in a highly inter-species conserved, intrinsically disordered 82aa insertion of domain 20. iPSC of all family members were generated and subsequently differentiated into functional ventricular 3‑month‑old iPSC-CM. In order to generate isogenic controls, we performed CRISPR Cas9 genome editing to rescue the identified FLNc variant in both, father and daughter DCM iPSC-CM. Since FLNC acts as a scaffolding and sarcomere stabilization protein, we analyzed the sarcomeric regularity by staining the M-line and Z-disc with antibodies against titin and α-actinin and found a significantly dysregulated sarcomeric structure of the diseased iPSC‑CM from the father and the daughter compared to healthy wild type cardiomyocytes. Furthermore, sarcomeric regularity was completely restored in the isogenic controls to wild type level. To analyze whether dysregulated sarcomeric structure is accompanied by cardiac physiological dysfunction, Fura-2 AM epifluorescence Ca²⁺ measurements with Caffein applications and molecular analysis were performed focusing on the iPSC-CM and isogenic controls of the diseased daughter. We found a reduced, but not significant Caffeine-induced relaxation time, which might be explained by elevated SERCA activity in DCM-iPSC-CM compared to unrelated control CM. That is in line with an enhanced SERCA gene and protein expression in DCM-iPSC-CM. Both, enhanced SERCA expression and activity were completely restored in the isogenic control lines showing a direct association between the FLNc mutation and SERCA function. However, SR Ca²⁺ content, Ca²⁺ transient amplitude and relaxation time (80%) were not changed between analysed groups. To analyze the functional role of the FLNc variant in a 3D cellular environment, engineered heart muscles (EHM) with iPSC-CM of all family members and isogenic controls were generated. EHM composed of diseased iPSC-CM (father and daughter) showed significantly decreased contraction capacity, which was rescued in isogenic control EHM to wild type level.