Understanding arrhythmogenic right ventricular cardiomyopathy: Use of patient‐specific induced pluripotent stem cell models

Arrhythmogenic right ventricular cardiomyopathy (ARVC), an inherited heart muscle disease, is associated with a high risk of arrhythmias and sudden cardiac death [1]. It is characterized primarily by fibrofatty replacement of the right ventricular myocardium, which is often accompanied by subsequent progression to the left ventricle [2], [3]. ARVC is genetically determined by autosomal dominant inheritance, although recessive subtypes have been observed [4]. Mutations in genes coding for desmosomal proteins, including desmoplakin, plakoglobin, plakophilin 2 (PKP 2), desmoglein 2, and desmocollin 2, have been identified in a significant number of patients [5]. The clinical presentation of ARVC varies widely; diagnosis is often complex and involves structural, histological, electrocardiographic, arrhythmic, and genetic tests for the disease [6]. Diagnosis is particularly difficult during the early phases of the disease when structural changes are not well-developed, leading to under-diagnosis. However, in this usually asymptomatic phase, some individuals may be at risk of sudden cardiac death. As such, it is important to increase the understanding of this complex condition through more patient-centered approaches, which may help with both diagnosis and management. Animal models have provided insight into the underlying mechanisms and pathophysiology of ARVC [4]. However, differences in functional and conduction properties between animal and human cardiomyocytes limit the general applicability of these results. The advent of induced pluripotent stem cell (iPSC) technology has greatly changed the landscape [7]. iPSCs are pluripotent stem cells derived from somatic cells through the induction of gene expression; these cells can then be reprogrammed to differentiate into other mature cell lines. In 2006, Yamanaka et al. pioneered this technology in mice followed by in human cells [7]. Since then, human iPSCs have been shown to be capable of differentiating into cardiomyocytes with cardiac-specific molecular, structural, and functional properties, indicating their great potential for the development of in vitro models of genetic cardiomyopathies [8]. Models patient-specific iPSCs have been used to study various inherited cardiac disorders. iPSC models were developed for the examination of long QT syndrome and have shed light on the mechanism of arrhythmogenicity in these cells (such as early-after depolarizations and triggered arrhythmias) and were used to evaluate the effectiveness of current and new agents to treat the disease phenotype [9]. For hypertrophic cardiomyopathy, iPSC models have increased the understanding of the molecular pathways of hypertrophy and arrhythmogenesis involved in abnormal calcium regulation and homeostasis within the cardiac sacromere [10]. Similar models have shed light on the pathogenesis and treatment of other cardiac disorders such as familial dilated cardiomyopathy [11] and catecholaminergic polymorphic ventricular tachycardia [12]. Thus, using patient-specific iPSCs models for studying ARVC has immense potential benefits, allowing for an increased understanding of the pathogenesis, risk stratification, and diagnosis of ARVC patients, as well as exploration of potential therapeutic options. However, stem cell models must accurately reflect the mutations and characteristics of the disease. In a histopathological study involving myocardial biopsy samples acquired from patients with ARVC, Asimaki et al. found a marked reduction in immunoreactive signals for plakoglobin, but normal levels of the non-desmosomal adhesion molecule N-cadherin in ARVC patients [13]. The authors suggested that this is a potentially useful new diagnostic test with a sensitivity and specificity of 91% and 82%, respectively. The test is limited because it requires myocardial biopsy, an invasive procedure with associated risks. Therefore, a less invasive but accurate and specific test may be more acceptable to patients and have a greater clinical impact. The first in vitro cellular model of ARVC by using patient-specific iPSC-derived cardiomyocytes was described by Ma et al. in 2012. They produced functional iPSC-derived cardiomyocytes by retroviral reprogramming of dermal fibroblasts taken from a male with clinical features of ARVC harboring a PKP2 gene mutation [14]. The results provided novel insights into the disease. First, reduced gene expression of desmosomal proteins (PKP2 and plakoglobin) was observed compared to controls, with lower immunofluorescence signals for these proteins at the cell periphery. Second, after exposure of cardiomyocytes to adipogenic differentiation medium for 2 weeks, the investigators found greater amounts of lipids in ARVC cells compared to in control cells. This was confirmed by both Oil Red O staining for intracellular lipid droplets and qualitatively based on transmission electron microscopy. These findings indicate the abnormal trafficking or expression of desmosomal proteins in mutant cells and indicate an increased adipogenic potential in mutant cells, which may predispose patients to fibro-fatty changes observed in ARVC. Two other groups subsequently published further data on in vitro iPSCs models of ARVC that have improved our understanding of this disease. Kim et al. developed iPSC models using episomal methods from 2 ARVC patients, as well as with PKP2 mutations [15]. Detailed electrophysiological characterization of mutant and control cardiomyocytes and alteration of the external pathogenic conditions using hormones and small molecules resulted in accelerated pathogenesis of the adult-onset phenotype. Important changes in intracellular calcium handling in mutant iPSC-derived cardiomyocytes were also observed, as well as the induction of adult-like metabolism and abnormal peroxisome proliferator-activated receptor gamma (PPAR-γ) activation, which were important in the pathogenesis of ARVC. To explain the predominant pathological characteristics of ARVC in the right ventricle, the investigators increased the number of Islet 1-positive cardiac progenitor cells by approximately 4-fold in mutant PKP2 iPSCs, simulating natural right ventricle formation of the secondary heart field. Caspi et al. also modeled ARVC from 2 patients with PKP2 mutations [16]. Similarly, they found reduced PKP2 and plakoglobin expression as well as increased adipogenicity in ARVC cells, along with upregulation of PPAR-γ. Additionally, they found that the degree of adipogenicity was closely related to the extent of desmosomal abnormalities, suggesting “crosstalk” between desmosomal destruction and adipogenesis, and that ARVC cardiomyocytes were at an increased risk of apoptosis. Despite important advances in our understanding of inherited cardiovascular diseases based on patient-specific iPSC-models, the technology remains challenging and shows some limitations [17]. The currently used models predominantly involve single cells; thus, it is unclear whether these cells replicate more complex processes involving synchronized groups of beating cardiomyocytes at the tissue or organ level, rather than just at the cardiomyocyte level. The currently used models do not fully assess the pathophysiological changes that may affect cell–cell interactions, which may play a role in diseases such as ARVC. Models must be refined to include layers of synchronized functional cardiomyocytes and explore the intercellular interactions in mutant and control cells. Furthermore, ARVC and other cardiomyopathies such as dilated cardiomyopathy are predominantly adult-onset diseases. Because iPSC-derived cardiomyocytes are relatively immature cells, it is unclear whether the changes observed in these cells truly reflect all pathophysiological changes in adult cells. Several novel methods have recently been reported, and they may improve the maturation of iPSC-derived cardiomyocytes and thus help overcome this limitation. These techniques include prolonged in vitro cell culture [18], [19], bio-mimetic culturing using forced expression of Kir2.1 [20], and using a novel platform (consisting of biowires submitted to electrical stimulation) which combines 3-dimensional cell cultivation with electrical stimulation [21]. Finally, although ARVC iPSC-models described to date show adipogenic changes in mutant cells in vitro, the precise mechanisms and external factors responsible for these changes in ARVC patients in vivo remain unknown. Despite these challenges, the ability to use patient-specific iPSCs to model ARVC was shown to be useful in studies of the disease, providing vital knowledge regarding the pathogenesis of AVRC as well as providing opportunities to design and test novel diagnostic strategies and therapeutics. ARVC-iPSC models described to date involved patients with PKP2 gene mutations. Future studies examining ARVC patients with different mutations are necessary to determine whether there is a common pathophysiological abnormality in AVRC or varying mechanisms depending on the specific mutation. More detailed electrophysiological and functional studies are needed to elucidate the mechanisms underlying life-threatening cardiac arrhythmias observed in patients with ARVC. None.