Electrophysiological and cellular analysis of filamin-C mutations causing cardiomyopathy using human iPSC-derived cardiomyocytes

Background: Arrhythmogenic Cardiomyopathy (AC) is a genetic cardiac disease resulting from different mutations within proteins constituting the intercalated disc, including desmosomal and nondesmosomal proteins. Recent studies have revealed that mutations in filamin-C (FLNC) may lead to AC. The arrhythmogenesis and electrophysiological effects of FLNC-related AC are incompletely understood. Therefore, the aim of this study is to assess the potential electrophysiological consequences of FLNC loss as occurs in AC in human induced pluripotent stem cell-derived cardiomyocytes (hiPSCCMs). Specifically, I aimed to characterise abnormal electrical activity and the expression and function of key proteins in cardiac electrical activity such as gap junction protein connexin 43 (Cx43).// Methods: hiPSC-CMs were differentiated and observed by immunofluorescence microscopy. Small interfering RNA (siRNA) transfection was utilised to knockdown the expression of FLNC in hiPSC-CMs. Protein analysis was performed using western blotting to confirm the knockdown efficiency. Electrophysiological properties were recorded using a multielectrode array and manual patch clamping. Optical recording of membrane potential and calcium activity from hiPSC-CMs were also carried out using parameter sensitive dyes.// Results: Silencing of FLNC led to markedly decreased immunofluorescence signals of FLNC, Cx43, desmoplakin, and junctional plakoglobin. No significant reductions were noted in the immunofluorescence signals of voltage-gated sodium channel (Nav1.5) and plakophilin-2 compared with control hiPSC-CMs. Western blotting showed the reduction of FLNC and Cx43 expression following silencing of FLNC. Knockdown of FLNC resulted in disturbances to the recorded action and field potential signals of hiPSC-CMs and arrhythmic likeevents. Transfected hiPSC-CMs with siRNA-FLNC were associated with prolongation of calcium transient durations, optical action potential duration, and action potentials measured with patch clamping.// Conclusion: The current findings indicated that loss of FLNC resulted in a complex arrhythmogenic phenotype in hiPSC-CM