Generation of patient-specific induced pluripotent stem cell-derived cardiomyocytes as a cellular model of arrhythmogenic right ventricular cardiomyopathy.

AIMS: 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. METHODS AND 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). CONCLUSION: Patient-specific iPSC-derived cardiomyocytes display key features of ARVC, including reduced cell surface localization of desmosomal proteins and a more adipogenic phenotype.

Clinical applications of patient-specific induced pluripotent stem cells in cardiovascular medicine.

The emergence of induced pluripotent stem cell (iPSC) technology has had a great impact on the field of medicine ever since the ground-breaking discovery in 2006 that overexpression of four specific transcription factors was able to turn back the developmental clock of somatic cells into an embryonic-like state. The resulting iPSCs carry the developmental potential of human embryonic stem cells (hESC) without the embryo and have been heralded as a powerful tool to study development and disease. This technology has made it possible for the first time for researchers to transform end-differentiated cells from a particular individual into another cell type that remains specific to that individual, paving the way for novel methods of in vitro disease modelling and therapeutic applications. This paper reviews some of the key areas in cardiovascular medicine in which iPSC technology has been applied and discusses the future directions and ongoing challenges ahead in this exciting field.

Pin1 facilitates the phosphorylation-dependent ubiquitination of SF-1 to regulate gonadotropin beta-subunit gene transcription.

Pin1 is a peptidyl-prolyl cis-trans isomerase which catalyzes the isomerization of phosphorylated Ser/Thr-Pro peptide bonds. Pin1 knockout mice have marked abnormalities in their reproductive development and function. However, the molecular mechanisms underlying their reproductive defects are poorly understood. Herein, we demonstrate that Pin1 is required for both basal and GnRH-induced gonadotropin beta-subunit gene transcription, through interactions with the transcription factors SF-1, Pitx1, and Egr-1. Pin1 activates transcription of the gonadotropin beta-subunit genes synergistically with these transcription factors, either by modulating their stability or by increasing their protein-protein interactions. Notably, we provide evidence that Pin1 is required for the Ser203 phosphorylation-dependent ubiquitination of SF-1, which facilitates SF-1-Pitx1 interactions and therefore results in an enhancement of SF-1 transcriptional activity. Furthermore, we demonstrate that in gonadotrope cells, sufficient levels of activated Pin1 are maintained through transcriptional and posttranslational regulation by GnRH-induced signaling cascades. Our results suggest that Pin1 functions as a novel player in GnRH-induced signal pathways and is involved in gonadotropin beta-subunit gene transcription by modulating the activity of various specific transcription factors.