Modeling of Arrhythmogenic Right Ventricular Cardiomyopathy With Human Induced Pluripotent Stem Cells

Abstract

dilated and hypertrophic cardiomyopathies, 13,14 and cathecolaminergic polymorphic ventricular tachycardia. 15 In the current study, we aimed to use the emerging hiPSC technology to establish an in vitro model of ARVC. We Background-Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a primary heart muscle disorder resulting from desmosomal protein mutations. ARVC is characterized pathologically by fibrofatty infiltration and clinically by arrhythmias and sudden cardiac death. We aimed to establish a patient-/disease-specific human induced pluripotent stem cell (hiPSC) model of ARVC. Methods and Results-Dermal fibroblasts were obtained from 2 patients with ARVC with plakophilin-2 (PKP2) mutations, reprogrammed to generate hiPSCs, coaxed to differentiate into cardiomyocytes (CMs), and then compared with healthy control hiPSC-derived CMs (hiPSC-CMs). Real-time polymerase chain reaction showed a significant decrease in the expression of PKP2 in the ARVC-hiPSC-CMs. Immunostainings revealed reduced densities of PKP2, the associated desmosomal protein plakoglobin, and the gap-junction protein connexin-43. Electrophysiological assessment demonstrated prolonged field potential rise time in the ARVC-hiPSC-CMs. Transmission electron microscopy identified widened and distorted desmosomes in the ARVC-hiPSC-CMs. Clusters of lipid droplets were identified in the ARVC-CMs that displayed the more severe desmosomal pathology. This finding was associated with upregulation of the proadipogenic transcription factor peroxisome proliferator-activated receptor-γ. Exposure of the cells to apidogenic stimuli augmented desmosomal distortion and lipid accumulation. The latter phenomenon was prevented by application of a specific inhibitor of glycogen synthase kinase 3β (6-bromoindirubin-3'-oxime). Conclusions-This study highlights the unique potential of the hiPSC technology for modeling inherited cardiac disorders in general and ARVC specifically. The hiPSC-CMs were demonstrated to recapitulate the ARVC phenotype in the dish, provide mechanistic insights into early disease pathogenesis, and provide a unique platform for drug discovery and testing in this disorder.

Desmoglein 2–Dependent Arrhythmogenic Cardiomyopathy Is Caused by a Loss of Adhesive Function

“…The birth of viable animals with inconspicuous gross heart morphology provides evidence that Dsg2 is not essential for growth and function of the by guest on May 8, 2018 http://circgenetics.ahajournals.org/ Downloaded from heart during late embryogenesis and early postnatal life. This is remarkable because Dsg2 is by far the major Dsg isoform expressed in cardiomyocytes.…”

“…The situation in other AC types is less clear. Although some reports support an adhesion deficiency as evidenced by intercalated disc widening and dissociation in murine AC models, 31 in patient-derived iPSCs, 32 and in patients, 29 a recent publication portrays a more complex situation. 33 Depletion of either plakoglobin or plakophilin resulted in weakened cellcell adhesion as was observed by others before, 34,35 whereas mutant polypeptides did not (for contrasting results, see, however, Sato et al 34 ).…”

“…by guest on May 8, 2018 http://circgenetics.ahajournals.org/ Downloaded from acids consisting of the 29 amino acid signal peptide, the 25 amino acid prosequence, 24 amino acids of the extracellular domain EC1, and the 2 carboxyterminal amino acids histidine and leucine because of erroneous splicing of exon 3 onto exon 6. The processed carboxyterminal 26 amino acid peptide encompassing the W2 adhesion site, which is nonfunctional in Dsg2, 28 may still be secreted.…”

Inherited heart disease – what can we expect from the second decade of human iPS cell research?

“…Importantly, the human heart is composed not only of CMs but also vascular, smooth muscle and epicardial cells; to better mimic its function, we predict that 3D cardiac tissue structures will be widely implemented, especially where interactions between different cell types might underlie the disease. As an example, ARVC has been modelled in hiPSC‐CMs and these are the major cellular players in the cardiac dysfunction in this disease 30, 34, 73; however, the suspected contribution of epicardial cells to fibro‐fatty substitution and the role of inflammation could not so far be studied in two‐dimensional monotypic cultures. The expectation is that complex multicellar structures will be necessary to reflect fully the pathology of the condition.…”

Persistent Integration of Reprogramming Factors Impairs the In Vitro Cardiogenic Potential of Induced Pluripotent Stem Cells

“…This observation is contrary to what has been reported by multiple laboratories that used other integrating vectors to reprogram fibroblasts. [16][17][18] Additional studies are required to delineate whether the apparent reduced responsiveness of transgene-containing iPSC-derived cardiomyocytes to β-adrenergic stimulation is specific to the cell lines examined, is restricted to iPSC derived with the piggyBac system or is widely occurring but underappreciated in iPSC-derived cardiomyocytes.…”