Generation of patient-specific induced pluripotent stem cell-derived cardiomyocytes as a cellular model of arrhythmogenic right ventricular cardiomyopathy.
OBJECTIVE
Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a primary heart muscle disorder associated with sudden cardiac death. Its pathophysiology is still poorly understood. We aimed to produce an in vitro cellular model of ARVC using patient-specific induced pluripotent stem cell (iPSC)-derived cardiomyocytes and determine whether the model could recapitulate key features of the disease phenotype.


RESULTS
Dermal fibroblasts were obtained from a 30-year-old man with a clinical diagnosis of ARVC, harbouring a plakophilin 2 (PKP2) gene mutation. Four stable iPSC lines were generated using retroviral reprogramming, and functional cardiomyocytes were derived. Gene expression levels of desmosomal proteins (PKP2 and plakoglobin) in cardiomyocytes from ARVC-iPSCs were significantly lower compared with cardiomyocytes from control iPSCs (P< 0.01); there were no significant differences in the expression of desmoplakin, N-cadherin, and connexin 43 between the two groups. Cardiomyocytes derived from ARVC-iPSCs exhibited markedly reduced immunofluorescence signals when stained for PKP2 and plakoglobin, but similar levels of staining for desmoplakin, N-cadherin, and connexin 43 compared with control cardiomyocytes. Transmission electron microscopy showed that ARVC-iPSC cardiomyocytes were larger and contained darker lipid droplets compared with control cardiomyocytes. After 2 weeks of cell exposure to adiopgenic differentiation medium, ARVC-iPSC cardiomyocytes were found to contain a significantly greater amount of lipid, calculated using Oil Red O staining, compared with controls (734 ± 35.6 vs. 8.1 ± 0.49 a.u., respectively; n = 7, P = 0.001).


CONCLUSIONS
Patient-specific iPSC-derived cardiomyocytes display key features of ARVC, including reduced cell surface localization of desmosomal proteins and a more adipogenic phenotype.
Research and Development Unit (RDU), National Heart Centre Singapore, 17 Third Hospital Avenue, Singapore 168752, Singapore.
Dongrui
D
Ma
Heming
H
Wei
Jun
J
Lu
Shuswen
S
Ho
Guangqing
G
Zhang
Xiaoming
X
Sun
Yingzi
Y
Oh
Suat Hoon
SH
Tan
Mah Lee
ML
Ng
Winston
W
Shim
Philip
P
Wong
Reginald
R
Liew
D000328:Adult; D019571:Arrhythmogenic Right Ventricular Dysplasia / Q000235:genetics / Q000378:metabolism / Q000473:pathology*; D002120:Calcium Channel Agonists / Q000494:pharmacology; D003896:Desmosomes / Q000378:metabolism; D005347:Fibroblasts / Q000473:pathology; D005455:Fluorescent Antibody Technique; D020869:Gene Expression Profiling; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000473:pathology*; D008297:Male; D008955:Models, Cardiovascular; D009154:Mutation; D009200:Myocardial Contraction / Q000187:drug effects; D032383:Myocytes, Cardiac / Q000378:metabolism / Q000473:pathology* / Q000648:ultrastructure; D010641:Phenotype; D051191:Plakophilins / Q000235:genetics / Q000378:metabolism; D051185:gamma Catenin / Q000378:metabolism
D002120:Calcium Channel Agonists; C495337:PKP2 protein, human; D051191:Plakophilins; D051185:gamma Catenin