Introduction: Arrhythmogenic right ventricular cardiomyopathy (ARVC) is an inherited disease of the heart characterized by ventricular arrhythmias, cardiomyocyte (CM) degeneration, and a fibro-fatty replacement of right ventricular myocardium. However, the link between ARVC and lipotoxicity is still undefined. Aim of the study was to analyze the role of cardiac lipotoxicity in triggering arrhythmic events using ARVC patient-specific induced pluripotent stem cell (iPSC)-derived CMs.

Methods and results: Patient-specific iPSCs were generated from peripheral blood samples obtained from healthy control and a 61-year-old patient, harboring a PKP2 heterozygous mutation with a clinical diagnosis of ARVC (cardiac imaging confirmed and T-wave inversion in ECG), as well as from his 32-year-old son who carries the same mutation without clinical findings of ARVC. Reprogramming factors (OCT4, SOX2, KLF4, and c-MYC) were introduced by infecting peripheral blood mononuclear cells (PBMCs) with non-integrating SeV vectors to create iPSCs. After reprogramming, the karyotype showed genomic integrity in all iPSC lines and the PKP2 mutation was confirmed in PKP2-iPSC using Sanger sequencing. CMs derived from PKP2-iPSCs (5 weeks old) exhibited significantly reduced protein levels of the desmosomal protein plakophilin-2 (0.31 ± 0.04 and 0.19±0.1 versus 1.0 ± 0.24, respectively; p < 0.01) and increased lipogenic transcription factors (PPARγ) (2.7 ± 0.89 and 5.06±1.37 versus 1.0 ± 0.28, respectively; p < 0.001)  when compared with control CMs. In addition, desmosomal abnormalities in the PKP2-iPSC-CMs were detected in both transmission electron microscopy (TEM) as well as PKP2 and plakoglobin immunofluorescence staining. Further, TEM showed that 5 weeks old PKP2-iPSC-derived CMs showed lipid droplet accumulation and typical sarcomeric protein derangement compared with control CMs. CMs derived from ARVC-iPSCs exhibited markedly abnormal frequency of intracellular calcium ([Ca²⁺]_i) transients compared with control CMs (0.5 ± 0.2 and 0.4 ± 0.13 versus 1.0 ± 0.10 respectively; p < 0.001). Exposing PKP2-iPSC-derived CMs (2 weeks old) to high fatty acids medium (10 µg/mL insulin, 0.2 mM FAs [palmitic acid conjugated to BSA], 10 mmol/L glucose, 10 nmol/L endothelin-1 and 1 µmol/L cortisol in DMEM) for one week resulted in increased lipid accumulation (65.5 ± 8.4% and 40.8 ± 6.3% versus 10.7 ± 2.80% respectively; p < 0.001), degeneration of CMs and loss of the regular [Ca2+]_i transient (72±7, 43±5 versus 10.2±6.3; p<0.001) indicating an early trigger to develop an ARVC phenotype in 3 weeks old PKP2-iPSC-derived CMs compared to control iPSC-derived CMs.

Conclusion:  From these results, we conclude that exposure of patient-specific PKP2 iPSC-derived CM to lipotoxic stimuli facilitates the development of an ARVC phenotype in vitro.