J B Reisqs

A Moreau

Y Sleiman

M Boutjdir

P. Chevalier Sylvain Richard

P Chevalier
email: philippe.chevalier@chu-lyon.fr

Arrhythmogenic cardiomyopathy as a myogenic disease: highlights from cardiomyocytes derived from human induced pluripotent stem cells

Keywords: arrhythmogenic cardiomyopathy, transdifferentiation, personalized medicine, electrophysiology

  L'archive ouverte pluridisciplinaire

Introduction 1.Clinical presentation

Arrhythmogenic Cardiomyopathy (ACM) is a rare genetic disease predisposing to a high risk for ventricular arrhythmias and heart failure [START_REF] Basso | Arrhythmogenic right ventricular cardiomyopathy[END_REF][START_REF] Bueno-Beti | Histopathological features and protein markers of arrhythmogenic cardiomyopathy[END_REF]. Its prevalence is estimated between 1/1,000 and 1/5,000. The early clinical symptoms appear in young adults [START_REF] Thiene | Right ventricular cardiomyopathy and sudden death in young people[END_REF][START_REF] Roudijk | Clinical characteristics and follow-up of pediatric-onset arrhythmogenic right ventricular cardiomyopathy[END_REF]. Historically, ACM has been described as affecting predominantly the right ventricle but this concept has evolved [START_REF] Marcus | Right ventricular dysplasia: A report of 24 adult cases[END_REF]. Recent studies showed that ACM can manifest early with a biventricular pattern or even as an isolated left ventricular dysfunction [START_REF] Sen-Chowdhry | Arrhythmogenic cardiomyopathy: Etiology, diagnosis, and treatment[END_REF][START_REF] Pinamonti | Arrhythmogenic right ventricular cardiomyopathy: From genetics to diagnostic and therapeutic challenges[END_REF][START_REF] Westphal | Myocarditis or inherited disease? -the multifaceted presentation of arrhythmogenic cardiomyopathy[END_REF]. Four main disease stages are usually described. The first one is concealed with a risk of sudden death without structural abnormalities. During the second phase, structural changes appear gradually. The third and fourth phases are characterized by single and/or biventricular failure [START_REF] Thiene | Arrhythmogenic right ventricular cardiomyopathy/dysplasia[END_REF][START_REF] Asimaki | Pathogenesis of arrhythmogenic cardiomyopathy[END_REF].

The main feature of ACM is a loss of myocardium with fibro-fatty replacement. This phenomenon creates a conduction block causing asymmetric electrical conduction in the form of a loop through which the same electrical activity can propagate again and re-excite the tissue [START_REF] Farré | Philippe coumel: A founding father of modern arrhythmology[END_REF]. This process promotes electrical instability, thereby causing impaired ventricular mechanical function, potentially leading to sudden cardiac death [START_REF] Corrado | Arrhythmogenic cardiomyopathy[END_REF]. The various forms of arrhythmias include palpitations, premature ventricular beat, bundle branch block, ventricular tachycardia, and ventricular fibrillation [START_REF] Austin | Molecular mechanisms of arrhythmogenic cardiomyopathy[END_REF]. On the ECG, ACM can manifest with a large QRS duration (>110 m), Twave inversion, or also with the presence of an epsilon wave [START_REF] Delmar | The cardiac desmosome and arrhythmogenic cardiomyopathies: From gene to disease[END_REF][START_REF] Kalantarian | Long-term electrocardiographic and echocardiographic progression of arrhythmogenic right ventricular cardiomyopathy and their correlation with ventricular tachyarrhythmias[END_REF]. Structural heart remodeling manifests as fibrosis, fatty infiltration, and aneurysm, leading to ventricular dilation and decreased heart contraction [START_REF] Kohela | Fibro-fatty remodelling in arrhythmogenic cardiomyopathy[END_REF].

ACM is classified as an intercellular junction pathology [START_REF] Basso | Arrhythmogenic right ventricular cardiomyopathy[END_REF][START_REF] Marcus | Genetics of arrhythmogenic right ventricular cardiomyopathy: A practical guide for physicians[END_REF]. In 50% of cases, ACM patients harbor a variant in genes coding for desmosomal proteins, including plakophilin-2 (PKP2, the most affected gene), desmoplakin (DSP), desmoglein-2 (DSG2), junction plakoglobin (JUP) and desmocollin-2 (DSC2). Alternatively, variants in genes coding for non-desmosomal proteins such as the ryanodine receptor 2 (RYR2), transforming growth factor reduced β-3 (TGFβ3), transmembrane protein 43 (TMEM43), desmin (DES), titin (TTN), phospholamban (PLN), lamin A/C (LMNA) and sodium channel (SCN5A) proteins have also been described [START_REF] Quarta | Familial evaluation in arrhythmogenic right ventricular cardiomyopathy: Impact of genetics and revised task force criteria[END_REF][START_REF] Groeneweg | Arrhythmogenic cardiomyopathy: Diagnosis, genetic background, and risk management[END_REF][START_REF] Lazzarini | The ARVD/C genetic variants database: 2014 update[END_REF].

Desmosomes

Desmosomes are structures located at the intercalated discs in the myocardium and they are responsible for intercellular adhesion [START_REF] Garrod | Desmosome structure, composition and function[END_REF][START_REF] Nielsen | The intercalated disc: A unique organelle for electromechanical synchrony in cardiomyocytes[END_REF]. This desmosomal protein complex consequently ensures solid intercellular junctions and notably explains why these structures are mainly found in stretched tissues such as the skin or the heart [START_REF] Holthöfer | Structure and function of desmosomes[END_REF][START_REF] Najor | Desmosomes in human disease[END_REF]. Desmosomes were thought to have a specific physical role in intercellular adhesion, linking the intracellular cytoskeleton to the extracellular cadherins domain. The hypothesis regarding the pathophysiology of ACM implies the destabilization of the desmosome structure, due to variants in desmosomal protein, and may weaken the right myocardium stretch resistance [START_REF] Delmar | The cardiac desmosome and arrhythmogenic cardiomyopathies: From gene to disease[END_REF]. This process could lead to myocyte death and replacement with fibrofatty tissue due to the limited heart regeneration potential. Such a fibrofatty replacement, associated with inflammatory mechanisms, could provide an arrhythmogenic substrate [START_REF] Smith | Desmoplakin cardiomyopathy, a fibrotic and inflammatory form of cardiomyopathy distinct from typical dilated or arrhythmogenic right ventricular cardiomyopathy[END_REF]. Several studies have used cells or animal models to decipher the mechanisms involved. However, the precise clinical and biological features of ACM remain to be elucidated. This review recapitulates the knowledge about ACM and the recent contributions of human-induced pluripotent stem cells (hiPSC) for both a better understanding of the disease and the comprehensive development of precision therapy.

Established molecular mechanisms in ACM

Various studies have focused on disturbances of intracellular pathways described for ACM related to desmosome destabilization [START_REF] Austin | Molecular mechanisms of arrhythmogenic cardiomyopathy[END_REF]. Desmosome alteration is one of the leading hypotheses of cardiomyocyte replacement in ACM. Indeed, the Wnt/β-catenin and Hippo pathways are altered because desmosomal variants can lead to fibro-fatty replacement [START_REF] Chen | The hippo pathway is activated and is a causal mechanism for adipogenesis in arrhythmogenic cardiomyopathy[END_REF]. Studies also highlighted an increase in PPARγ expression in human cardiomyocytes from ACM patients [START_REF] Djouadi | A potential link between peroxisome proliferator-activated receptor signalling and the pathogenesis of arrhythmogenic right ventricular cardiomyopathy[END_REF]. PPARγ gene is a known master regulator of adipogenesis [START_REF] Rosen | C/EBPalpha induces adipogenesis through PPARgamma: A unified pathway[END_REF][START_REF] Mal | Role of peroxisome proliferator-activated receptor gamma (PPARγ) in different disease States: Recent updates[END_REF].

The Wnt pathway

The canonical Wnt signaling pathway regulates developmental processes during embryogenesis and is involved in the maintenance of adult tissue homeostasis [START_REF] Logan | The Wnt signaling pathway in development and disease[END_REF][START_REF] Balatskyi | WNT/β-catenin pathway is a key regulator of cardiac function and energetic metabolism[END_REF]. This signaling pathway is associated with cell differentiation, polarization, and migration during development [START_REF] Steinhart | Wnt signaling in development and tissue homeostasis[END_REF]. The canonical Wnt pathway plays also a pivotal role in adult cardiac remodeling by reactivation of the developmental program to maintain contractile function in the left ventricle [START_REF] Bergmann | WNT signaling in adult cardiac hypertrophy and remodeling: Lessons learned from cardiac development[END_REF]. Canonical Wnt pathway activation inhibits the degradation of cytoplasmic β-catenin by the proteasome [START_REF] Lustig | The Wnt signaling pathway and its role in tumor development[END_REF][START_REF] Debruine | Assembly and architecture of the Wnt/βcatenin signalosome at the membrane[END_REF]. The β-catenin can thus translocate into the nucleus and interact with the T-cell factor/Lymphoid-enhancer binding factor (Tcf/Lef) to activate the canonical Wnt signaling pathway (Figure 1) [START_REF] Godsel | Desmoplakin assembly dynamics in four dimensions: Multiple phases differentially regulated by intermediate filaments and actin[END_REF][START_REF] Nusse | Wnt/β-Catenin signaling, disease, and emerging therapeutic modalities[END_REF]. This pathway favors cell proliferation and regulates cell fate specification including cardiomyocyte differentiation [START_REF] Rim | The Wnt pathway: From signaling mechanisms to synthetic modulators[END_REF].

In the ACM condition, an inhibition of the canonical Wnt pathway has been described in DSP-deficient mice and DSP-knockdown HL-1 cells leading to a morphological change of cardiomyocytes into adipocytes with lipid accumulation [START_REF] Garcia-Gras | Suppression of canonical Wnt/beta-catenin signaling by nuclear plakoglobin recapitulates phenotype of arrhythmogenic right ventricular cardiomyopathy[END_REF]. More recently, a study showed an alteration of canonical Wnt signaling in DSP-deficient zebrafish models [START_REF] Giuliodori | Loss of cardiac Wnt/β-catenin signalling in desmoplakin-deficient AC8 zebrafish models is rescuable by genetic and pharmacological intervention[END_REF]. Variants in genes coding for desmosomal proteins can induce a global desmosomal destabilization and lead to the cytoplasmic release of proteins usually retained at the plasma membrane. Using mice overexpressing the junction plakoglobin, Lombardi and coworkers further confirmed the ability of JUP to be translocated into the nucleus [START_REF] Lombardi | Nuclear plakoglobin is essential for differentiation of cardiac progenitor cells to adipocytes in arrhythmogenic right ventricular cardiomyopathy[END_REF]. The junction plakoglobin, also called γ-catenin competes with β-catenin which can also be found in both the cytoplasm and the nucleus. The nuclear relocalization of junctional plakoglobin prevents the interaction between β-catenin and Tcf/Lef and consequently affects the canonical Wnt pathway (Figure 1) [START_REF] Lombardi | Nuclear plakoglobin is essential for differentiation of cardiac progenitor cells to adipocytes in arrhythmogenic right ventricular cardiomyopathy[END_REF]. In cardiomyocytes, the suppression of Wnt signaling mainly promotes adipogenesis, thus potentially supporting lipid accumulation in the heart of ACM patients and the hypothesis of the transdifferentiation of cardiomyocytes into adipocytes. A relationship has been established in cellular models between the Wnt pathway and PPARγ expression [START_REF] Liu | Functional interaction between peroxisome proliferator-activated receptor gamma and beta-catenin[END_REF][START_REF] Parrotta | Deciphering the role of Wnt and rho signaling pathway in iPSC-derived ARVC cardiomyocytes by in silico mathematical modeling[END_REF]. PPARγ is the master regulator for adipocyte differentiation, lipogenesis, and adipocyte survival [START_REF] Cristancho | Forming functional fat: A growing understanding of adipocyte differentiation[END_REF][START_REF] Lefterova | PPARγ and the global map of adipogenesis and beyond[END_REF]. PPARγ is suspected to promote adipogenesis switch in ACM. In a study using DSP knockdown mice, Garcia-Gras et al. demonstrated a link between PPARγ overexpression and Wnt signaling suppression (Figure 1) [START_REF] Garcia-Gras | Suppression of canonical Wnt/beta-catenin signaling by nuclear plakoglobin recapitulates phenotype of arrhythmogenic right ventricular cardiomyopathy[END_REF]. This pathological mechanism may thus underlie the fibrofatty replacement characteristics of ACM.

The Hippo-Yap pathway

The Hippo-YAP pathway was discovered in 1995 using Drosophila genetic screening to isolate new genes involved in the regulation of cell proliferation, survival, and differentiation [START_REF] Ma | The hippo pathway: Biology and pathophysiology[END_REF]. This pathway involves a cascade of protein kinases. In the control condition, the kinases of Hippo are inactivated, YAP and TAZ are hypo-phosphorylated, then translocated into the nucleus to bind to DNA [START_REF] Yu | Hippo pathway in organ size control, tissue homeostasis, and cancer[END_REF]. Once YAP is in the nucleus, the YAP pathway is activated, promoting cell proliferation and resistance to apoptosis (Figure 1). However, when Hippo is activated by neurofibromin-2, which is a multifunctional protein involved in cell-cell and cell-matrix adhesions, mammalian STE20-like protein kinase 1 (MST1) and MST2 phosphorylate and activate the kinases large tumor suppressor homologs (LATS1 and LATS2). The MST and LATS are central kinases of the Hippo pathway. The LATS phosphorylates YAP and TAZ, inducing their degradation in the cytoplasm [START_REF] Heallen | Hippo signaling impedes adult heart regeneration[END_REF]. The YAP pathway is inactive, which inhibits gene expression (Figure 1). ACM patients' samples, mouse models, and PKP2 knockdown in HL-1 cells have demonstrated an aberrant activation of the Hippo signaling pathway leading to cytoplasmic retention of YAP [START_REF] Chen | The hippo pathway is activated and is a causal mechanism for adipogenesis in arrhythmogenic cardiomyopathy[END_REF]. The cytoplasmic-retained YAP can interact with βcatenin and prevents its nuclear translocation into the nucleus (Figure 1). This further suppresses the Wnt signaling pathway leading to either the death of cardiomyocytes or their adipogenic trans-differentiation [START_REF] Imajo | A molecular mechanism that links Hippo signalling to the inhibition of Wnt/β-catenin signalling[END_REF][START_REF] Hu | Hippo activation in arrhythmogenic cardiomyopathy[END_REF]. Despite its role, the Hippo-Yap pathway remains poorly studied, which warrants further investigations to elucidate the exact role of this pathway in ACM. This pathology is a pathology relying on cardiomyocytes connection and the activation/inhibition of Hippo-YAP is regulated by cell adhesion [START_REF] Nishioka | The Hippo signaling pathway components Lats and Yap pattern Tead4 activity to distinguish mouse trophectoderm from inner cell mass[END_REF][START_REF] Piquer-Gil | Non coding RNAs as regulators of wnt/β-catenin and hippo pathways in arrhythmogenic cardiomyopathy[END_REF].

Cell types involved in fibro-adipogenesis

Several hypotheses have emerged to explain the origin of fibro-adipose replacement in the ventricles. Early hypotheses focused on adult cardiac stem cells as a source [START_REF] Stadiotti | Arrhythmogenic cardiomyopathy: The guilty party in adipogenesis[END_REF]. Cardiac progenitor cells express desmosomal proteins. Studies in mice have shown the involvement of cells expressing the multipotent marker Isl-1 as a source of adipogenesis [START_REF] Cohen | Wnt/beta-catenin signaling promotes expansion of Isl-1-positive cardiac progenitor cells through regulation of FGF signaling[END_REF][START_REF] Lombardi | Genetic fate mapping identifies second heart field progenitor cells as a source of adipocytes in arrhythmogenic right ventricular cardiomyopathy[END_REF]. This hypothesis has been supported by the coexpression of Isl-1 markers and adipogenic transcription factors in the heart of ACM patients [START_REF] Lombardi | Genetic fate mapping identifies second heart field progenitor cells as a source of adipocytes in arrhythmogenic right ventricular cardiomyopathy[END_REF]. The c-kit/Sca1 cellular progenitors have also been proposed as precursors of adipocytes [START_REF] Lombardi | Nuclear plakoglobin is essential for differentiation of cardiac progenitor cells to adipocytes in arrhythmogenic right ventricular cardiomyopathy[END_REF]. The c-kit and Sca1 markers are recognized markers of pluripotent stem cells in the hematopoietic system. Histological studies of transgenic mice over-expressing JUP showed an increase in the number of adipocytes and fibrosis in the heart [START_REF] Lombardi | Nuclear plakoglobin is essential for differentiation of cardiac progenitor cells to adipocytes in arrhythmogenic right ventricular cardiomyopathy[END_REF]. However, their low number suggests that only a small proportion of adipocytes originate from these cells [START_REF] Sommariva | Cardiac mesenchymal stromal cells are a source of adipocytes in arrhythmogenic cardiomyopathy[END_REF].

Other studies have looked at the involvement of cardiac pluripotent cells such as cardiac mesenchymal stromal cells [START_REF] Lombardi | Cardiac fibro-adipocyte progenitors express desmosome proteins and preferentially differentiate to adipocytes upon deletion of the desmoplakin gene[END_REF][START_REF] Sommariva | Cardiac mesenchymal stromal cells are a source of adipocytes in arrhythmogenic cardiomyopathy[END_REF]. These cells originate from the epicardium and participate in structural maintenance of the heart. They are pluripotent and involved in cardiac remodeling in pathological conditions. Their contribution to ACM has been demonstrated from studies of patient biopsies, from which these mesenchymal cells have been isolated and recultured. Under these conditions, these cells differentiated into adipocytes. [START_REF] Lombardi | Cardiac fibro-adipocyte progenitors express desmosome proteins and preferentially differentiate to adipocytes upon deletion of the desmoplakin gene[END_REF]. Another potential adipogenic source from pluripotent cells has been found in cardiac fibro-adipose progenitors. This previous study has shown that a mutation in a gene coding for a desmosomal protein can differentiate these cells into adipocytes. The authors estimated that 40% of adipocytes in the hearts of patients with AC originated from these progenitors [START_REF] Lombardi | Cardiac fibro-adipocyte progenitors express desmosome proteins and preferentially differentiate to adipocytes upon deletion of the desmoplakin gene[END_REF].

Most studies of ACM mention the phenomenon of cardiomyocytes transdifferentiation as a source for fibro-adipose replacement (d'Amati et al., 2000;[START_REF] Fujita | Markedly increased intracellular lipid droplets and disruption of intercellular junctions in biopsied myocardium from a patient with arrhythmogenic right ventricular cardiomyopathy[END_REF]. D'Amati and collaborators were the first to report this phenomenon through histological, immunochemical, and ultrastructure analyzes in human cardiac samples (d'Amati et al., 2000). They reported positive labeling for vimentin, a protein expressed in adipocytes, in some cardiomyocytes. These cells would therefore be a transition cell type between cardiomyocytes and adipocytes. In addition, a second study reported this phenomenon of transdifferentiation where the myocardial cells had strong similarities with an adipocyte. Analysis of this group of cells reveals a polymorphic nuclear change, perinuclear vacuolation, and finally an accumulation of lipid droplets [START_REF] Fujita | Markedly increased intracellular lipid droplets and disruption of intercellular junctions in biopsied myocardium from a patient with arrhythmogenic right ventricular cardiomyopathy[END_REF]. This phenomenon is supported by the idea that desmosome destabilization leads to the translocation of JUP from the membrane to the nucleus, thus inhibiting the action of β-catenin. The cellular source of fibro-adipose replacement in the ACM is shown in Figure 2.

Alterations of myocytes' electrical activity as a critical pathogenic mechanism

Electrical remodeling, due to the destabilization of desmosomes, is a hallmark of ACM [START_REF] Rizzo | Intercalated disc abnormalities, reduced Na(+) current density, and conduction slowing in desmoglein-2 mutant mice prior to cardiomyopathic changes[END_REF][START_REF] Stevens | Humanized dsp ACM mouse model displays stress-induced cardiac electrical and structural phenotypes[END_REF]. Desmosomes are physically close to gap junctions, belonging to the same macromolecular complex [START_REF] Sato | Interactions between ankyrin-G, Plakophilin-2, and Connexin43 at the cardiac intercalated disc[END_REF]Zhang J et al., 2021). Connexins are transmembrane proteins forming gap junctions enabling intercellular communication [START_REF] Desplantez | Gap junction channels and cardiac impulse propagation[END_REF][START_REF] Dhein | Remodeling of cardiac gap junctional cell-cell coupling[END_REF]. In the diseased myocardium, alterations of gap junction organization and connexin expression are often observed [START_REF] Severs | Remodelling of gap junctions and connexin expression in diseased myocardium[END_REF][START_REF] Chevalier | Short QT interval as a harbinger of an arrhythmogenic cardiomyopathy[END_REF]. Studies in patients harboring a variant in JUP or DSC2 highlighted abnormal connexin expression [START_REF] Gehmlich | Mechanistic insights into arrhythmogenic right ventricular cardiomyopathy caused by desmocollin-2 mutations[END_REF]. Similar findings have been observed in-vitro using RNA silencing technology to decrease the expression of PKP2 in neonatal rat ventricular myocytes. The loss of PKP2 expression leads to a drastic loss of Connexin43 [START_REF] Oxford | Connexin43 remodeling caused by inhibition of plakophilin-2 expression in cardiac cells[END_REF]. Alterations in intercellular coupling via gap junctions are thus expected to settle a strong pro-arrhythmogenic substrate.

The desmosome/connexin macro-molecular complex also involves Na v1.5 voltage-gated sodium channels [START_REF] Sato | Loss of plakophilin-2 expression leads to decreased sodium current and slower conduction velocity in cultured cardiac myocytes[END_REF]. In this study of neonatal rat ventricular myocytes, the knock-down of PKP2, using shRNA, revealed a loss of Na v 1.5 and gap junctions, suggesting a link between desmosomes, gap junctions, and Na v 1.5 at the intercalated disc. Variants in desmosomal proteins may also cause a drastic reduction in Na v 1.5 sodium current (I Na) densities [START_REF] Cerrone | Desmosomes and the sodium channel complex: Implications for arrhythmogenic cardiomyopathy and brugada syndrome[END_REF]. The use of a transgenic DSG2 mouse model revealed that the reduction in INa density occurs before cardiomyocyte necrosis or fibrosis [START_REF] Rizzo | Intercalated disc abnormalities, reduced Na(+) current density, and conduction slowing in desmoglein-2 mutant mice prior to cardiomyopathic changes[END_REF]. Altogether, these results suggest that reduced I Na densities due to variants in desmosomal proteins can establish an arrhythmogenic substrate and explain the conduction disturbances and arrhythmias seen early in ACM patients [START_REF] Zaklyazminskaya | The role of mutations in the SCN5A gene in cardiomyopathies[END_REF]. Moreover, I Na reduction may be causal in the pathology rather than resulting from pathological phenotypic remodeling. This channel not only forms an ion pore but also plays a role in a functional adhesion/excitability complex with mechanical junctions. Depending on the protein interaction affected, a variant in the gene coding for Nav1.5 (SCN5A) may thereby cause a mixed electrical and structural phenotype in ACM [START_REF] Te Riele | Multilevel analyses of SCN5A mutations in arrhythmogenic right ventricular dysplasia/cardiomyopathy suggest non-canonical mechanisms for disease pathogenesis[END_REF].

The study of a cardiomyocyte-specific tamoxifen-induced PKP2 knockout mouse model demonstrated the reduced expression of genes controlling intracellular calcium, such as the Ryanodine Receptor 2 (RYR2) and the voltage-gated calcium channel (CACNA1C) gene [START_REF] Cerrone | Plakophilin-2 is required for transcription of genes that control calcium cycling and cardiac rhythm[END_REF]. Variants in genes regulating calcium handling proteins were found in a cohort of patients diagnosed with ACM, particularly in genes encoding the RYR2 and phospholamban (PLN) [START_REF] Tiso | Identification of mutations in the cardiac ryanodine receptor gene in families affected with arrhythmogenic right ventricular cardiomyopathy type 2 (ARVD2)[END_REF][START_REF] Van Der Zwaag | Phospholamban R14del mutation in patients diagnosed with dilated cardiomyopathy or arrhythmogenic right ventricular cardiomyopathy: Evidence supporting the concept of arrhythmogenic cardiomyopathy[END_REF]. Like for sodium channels, the loss of desmosomal genes provokes alterations in calcium handling, contributing to the development of arrhythmogenic events in ACM [START_REF] Vallverdú-Prats | Alterations in calcium handling are a common feature in an arrhythmogenic cardiomyopathy cell model triggered by desmosome genes loss[END_REF]. These findings indicate that cardiomyocytes not only undergo morphological and structural remodeling but also electrophysiological remodeling which contributes to the development of the pathology.

Highlights from hiPSC-CMs

Animal and cell models have considerably contributed to highlighting morphological and electrophysiological remodeling. However, these models do not always reproduce all features of human pathology because of different limitations. It is difficult to consider the successful development of new treatments using only those models. The technology developed by Pr. S. Yamanaka, allows the reprogramming of adult mouse or human fibroblasts into pluripotent stem cells using four transcription factors (KLF4, OCT3/4, SOX2, and C-Myc) [START_REF] Takahashi | Induction of pluripotent stem cells from adult human fibroblasts by defined factors[END_REF][START_REF] Yamanaka | Induced pluripotent stem cells: Past, present, and future[END_REF]. The newly obtained pluripotent stem cells are named induced pluripotent stem cells (iPSC). The human iPSCs (hiPSC) express embryonic factors such as TRA1-60 or SSEA-1, maintain their pluripotency, and demonstrate high self-renewal capabilities. The method has rapidly been expanded and hiPSC can now be obtained from several original tissue types including blood or urine [START_REF] Hou | Pluripotent stem cells induced from mouse somatic cells by small-molecule compounds[END_REF][START_REF] Moreau | Induced pluripotent stem-cellderived cardiomyocytes: Cardiac applications, opportunities, and challenges[END_REF][START_REF] Shi | Induced pluripotent stem cell technology: A decade of progress[END_REF].

The capacity to differentiate hiPSC into spontaneously beating cardiomyocytes was a major advance in the understanding of cardiac pathologies [START_REF] Burridge | Production of de novo cardiomyocytes: Human pluripotent stem cell differentiation and direct reprogramming[END_REF]. Indeed, hiPSCderived cardiomyocytes (hiPSC-CM) are essential in the study of cardiomyopathies, channelopathies, and molecule screening during drug development or safety studies [START_REF] Moretti | Patient-specific induced pluripotent stem-cell models for long-QT syndrome[END_REF][START_REF] Sun | Patientspecific induced pluripotent stem cells as a model for familial dilated cardiomyopathy[END_REF][START_REF] Moreau | Induced pluripotent stem-cellderived cardiomyocytes: Cardiac applications, opportunities, and challenges[END_REF][START_REF] Sleiman | Modeling polymorphic ventricular tachycardia at rest using patient-specific induced pluripotent stem cell-derived cardiomyocytes[END_REF][START_REF] Poulin | iPSC-derived cardiomyocytes from patients with myotonic dystrophy type 1 have abnormal ion channel functions and slower conduction velocities[END_REF][START_REF] Benichou | Enhanced delivery of ligand-conjugated antisense oligonucleotides (C16-HA-ASO) targeting dystrophia myotonica protein kinase transcripts for the treatment of myotonic dystrophy type 1[END_REF][START_REF] Jauvin | Generation of four myotonic dystrophy type 1 patient iPSC lines (CBRCULi002-A, CBRCULi003-A, CBRCULi004-A, CBRCULi005-A) and a control (CBRCULi001-A) derived from lymphoblastoids cell lines[END_REF] At the early stage of culture (20 days), hiPSC-CMs exhibit immature morphological characteristics. Analysis by Transmission Electronic Microscopy (TEM) and immunofluorescence revealed a poorly organized contractile machinery, with a low number of myofibrils, which lacked alignment, immature Z-band, and T-tubule. However, with prolonged time in culture (360 days), cells become larger and more elongated, with increases in the density and alignment of myofibrils. Interestingly, the number of MLC2v-positive cells increases in the late stage of culture, indicating maturing of ventricular-type hiPSC-CM [START_REF] Kamakura | Ultrastructural maturation of human-induced pluripotent stem cell-derived cardiomyocytes in a long-term culture[END_REF][START_REF] Lundy | Structural and functional maturation of cardiomyocytes derived from human pluripotent stem cells[END_REF]. To go further, the use of molecules and new culture media makes it possible to obtain more mature cardiomyocytes [START_REF] Feyen | Metabolic maturation media improve physiological function of human iPSCderived cardiomyocytes[END_REF][START_REF] Chirico | Small molecule-mediated rapid maturation of human induced pluripotent stem cell-derived cardiomyocytes[END_REF]. On this topic, targeting signaling pathways such as PPARα, Pitx2 or the metabolic switch from glucose to fatty acid makes it possible to work with cardiomyocytes presenting a more mature morphology and phenotype at an earlier stage. [START_REF] Song | LEFTY-PITX2 signaling pathway is critical for generation of mature and ventricular cardiac organoids in human pluripotent stem cell-derived cardiac mesoderm cells[END_REF][START_REF] Bissoli | Induced pluripotent stem cell-based models: Are we ready for that heart in a dish? Front[END_REF].

The functional and electrophysiological features of hiPSC-CMs have been documented. Electrophysiological investigations revealed a heterogeneous population of cells characterized by nodal-, atrial-or ventricular-like action potentials (AP) [START_REF] Zhang | Functional cardiomyocytes derived from human induced pluripotent stem cells[END_REF][START_REF] Zhao | A platform for generation of chamber-specific cardiac tissues and disease modeling[END_REF]. Several ionic currents have been described in iPSC-CMs, reflecting notably the presence and function of the major ionic channels underlying an action potential. These currents include the inward sodium and calcium (L-and T-type) currents, the transient outward potassium, and the rapid and slow delayed rectifier potassium currents [START_REF] Honda | Electrophysiological characterization of cardiomyocytes derived from human induced pluripotent stem cells[END_REF][START_REF] Zhao | Ion Channel expression and characterization in human induced pluripotent stem cell-derived cardiomyocytes[END_REF]. In atrial and ventricular hiPSC-CMs, the expression of a hyperpolarization-activated cyclic nucleotide-gated (HCN) channel may contribute to spontaneous electrical activity. The density of the inward rectifier potassium current (I K1 ) was lower than in native human ventricular cardiomyocytes [START_REF] Ma | High purity human-induced pluripotent stem cell-derived cardiomyocytes: Electrophysiological properties of action potentials and ionic currents[END_REF]. Despite evidence for incomplete electrophysiological maturity, the hiPSC-CMs revealed the presence of a functional excitation-contraction coupling close to native cardiomyocytes [START_REF] Itzhaki | Calcium handling in human induced pluripotent stem cell derived cardiomyocytes[END_REF]. The hiPSC-CMs, therefore, provide a great opportunity for studying cardiac pathology, due to their morphological and electrophysiological phenotypes close to native human cardiomyocytes and for drug screening [START_REF] Doss | Current challenges of iPSC-based disease modeling and therapeutic implications[END_REF]. The AP profile of hiPSC-CMs and consequently the activity of individual membrane currents during the AP, differs from that of native human cardiomyocytes, largely due to the almost negligible expression of IK1 [START_REF] Hoekstra | Induced pluripotent stem cell derived cardiomyocytes as models for cardiac arrhythmias[END_REF]. To overcome this problem, an artificial method consists of artificially injecting the I K1 current into the cells to make them a more reliable model for investigating mechanisms underlying cardiac arrhythmias [START_REF] Meijer Van Putten | Ion channelopathies in human induced pluripotent stem cell derived cardiomyocytes: A dynamic clamp study with virtual Ik1[END_REF]. Improving the maturity of hiPSC-CM to be as close as possible to native cardiomyocytes remains a challenge that could be met in part thanks to the development of 3D culture approaches [START_REF] Mannhardt | Human engineered heart tissue: Analysis of contractile force[END_REF]. 3D bioprinting makes it possible to build organoids with a structure closer to native tissue. This technique has shown better cardiomyocyte morphology as well as improved electrophysiological function [START_REF] Maiullari | A multi-cellular 3D bioprinting approach for vascularized heart tissue engineering based on HUVECs and iPSC-derived cardiomyocytes[END_REF][START_REF] Kupfer | In situ expansion, differentiation, and electromechanical coupling of human cardiac muscle in a 3D bioprinted, chambered organoid[END_REF]. Another method to develop a more mature model of hiPSC cardiomyocytes is the use of an extracellular matrix (ECM). Studies have shown that ECM improves electrical propagation velocity as well as action potential upstroke velocity, hiPSC-CM hypertrophy, and increased expression of SCN5A, Kir2.1, Cx43 and cardiac troponin I [START_REF] Herron | Extracellular matrix-mediated maturation of human pluripotent stem cell-derived cardiac monolayer structure and electrophysiological function[END_REF]. The use of human ECM could encounter hiPSC-CM immaturity issues for optimal pre-clinical drug discoveries [START_REF] Block | Human perinatal stem cell derived extracellular matrix enables rapid maturation of hiPSC-CM structural and functional phenotypes[END_REF]. Finally, another study showed that the use of a cardioid platform allows a better understanding of the stages of cardiomyogenesis and improves the organization of hiPSC-CMs [START_REF] Hofbauer | Cardioids reveal self-organizing principles of human cardiogenesis[END_REF].

The hiPSCs model constitutes a revolutionizing method to obtain spontaneously contracting cardiomyocytes, which opens many avenues for studying cardiac pathologies. In 2013, Ma and co-authors were the first to differentiate hiPSC into cardiomyocytes using dermal fibroblasts from an ACM patient. Since, several studies using ACM-derived hiPSC-CMs showed that this cell type can recapitulate the key features of clinical pathology. Experiences by TEM and Oil Red O staining revealed clusters of lipid droplets in ACM hiPSC-CMs (Figure 3A) [START_REF] Ma | Generation of patientspecific induced pluripotent stem cell-derived cardiomyocytes as a cellular model of arrhythmogenic right ventricular cardiomyopathy[END_REF]. TEM also showed a widened and distorted desmosome in ACM condition, which is one of the characteristics of the weakening desmosomal complex (Figure 3B) [START_REF] Caspi | Modeling of arrhythmogenic right ventricular cardiomyopathy with human induced pluripotent stem cells[END_REF]. More specifically, using polymerase chain reaction (PCR) and immunostaining, these studies showed a significant decrease in the expression of PKP2, JUP, and connexin43. Kim and collaborators observed a nuclear localization of JUP and very low β-catenin activity, which has been previously described to induce adipogenic switch (Figure 3C) [START_REF] Kim | Studying arrhythmogenic right ventricular dysplasia with patient-specific iPSCs[END_REF]. Interestingly, these studies have shown an important role of PPARγ in the appearance of an ACM phenotype. The activation of this gene by rosiglitazone and indomethacin demonstrated an exaggerated lipogenesis and increased apoptosis in ACM hiPSC-CMs whereas the blockade of PPARγ rescued all ACM phenotypes [START_REF] Wen | Maturation-based model of arrhythmogenic right ventricular dysplasia using patient-specific induced pluripotent stem cells[END_REF]. Besides morphological changes, other studies on hiPSC also revealed electrophysiological remodeling in ACM. The patch-clamp technique demonstrated a decrease in the amplitude and the maximal upstroke velocity of action potential in comparison with control hiPSC-CMs. These reductions involved a decrease in the peak I Na in ACM hiPSC-CMs (Figure 3D) [START_REF] El-Battrawy | Electrical dysfunctions in human-induced pluripotent stem cell-derived cardiomyocytes from a patient with an arrhythmogenic right ventricular cardiomyopathy[END_REF]. Surprisingly, despite an increase in the rapid delayed rectifying potassium current (I Kr ), there was no difference in the action potential duration (APD). This could also rely on hiPSC-CMs on their developmental stage or/and heterogeneity in their stage of differentiation (35 days) at the time of investigations. These ion channel problems were also found in another hiPSC line carrying a variant in the DSP gene [START_REF] Gusev | Impact of the DSP-H1684R genetic variant on ion channels activity in iPSCderived cardiomyocytes[END_REF]. The hiPSC-CMs from an ACM patient were also more sensitive to adrenergic stimulation than control cells. Isoprenaline shortened APD in ACM hiPSC-CM and epinephrine unleashed more arrhythmogenic events early after depolarization (EAD)-like or delayed after depolarization (DAD)-like [START_REF] El-Battrawy | Electrical dysfunctions in human-induced pluripotent stem cell-derived cardiomyocytes from a patient with an arrhythmogenic right ventricular cardiomyopathy[END_REF]. One publication demonstrated the link between I Na and Wnt/B-catenin activity in ACM hiPSC-CMs with a variant in the gene encoding PKP2 [START_REF] Khudiakov | Sodium current abnormalities and deregulation of Wnt/β-catenin signaling in iPSC-derived cardiomyocytes generated from patient with arrhythmogenic cardiomyopathy harboring compound genetic variants in plakophilin 2 gene[END_REF]. To recapitulate, the PKP2 variant induced a significant reduction of Wnt activity and I Na density, which was restored by the inhibition of Glycogen synthase kinase-3 beta (GSK3β) [START_REF] Khudiakov | Sodium current abnormalities and deregulation of Wnt/β-catenin signaling in iPSC-derived cardiomyocytes generated from patient with arrhythmogenic cardiomyopathy harboring compound genetic variants in plakophilin 2 gene[END_REF]. A new DSG2 variant in hiPSC-CM demonstrated an increased pro-inflammatory cytokine expression, accompanied by a shortened APD and a calcium transient decay reduced [START_REF] Hawthorne | Altered electrical, biomolecular, and immunologic phenotypes in a novel patientderived stem cell model of desmoglein-2 mutant ARVC[END_REF]. Collectively, the aberrant cytoskeletal organization, cytokine expression, electrophysiology, and calcium handling disturbance found in ACM-hiPSC-CMs could explain the arrhythmogenic mechanisms of the disease in ACM patients [START_REF] Inoue | Modeling reduced contractility and impaired desmosome assembly due to plakophilin-2 deficiency using isogenic iPS cell-derived cardiomyocytes[END_REF]. hiPSCs are also used to explain therapies in patients. A study demonstrated the beneficial effect of the combination of sotalol and flecainide in ACM patients, using this cellular tool [START_REF] Moreau | Deciphering DSC2 arrhythmogenic cardiomyopathy electrical instability: From ion channels to ECG and tailored drug therapy[END_REF]. Indeed, the sotalol resulted in the normalization of APD by blocking potassium currents. Flecainide had the effect of decreasing cellular excitability by decreasing the number of aberrant calcium sparks. Moreover, this publication has shed light on an early repolarization disorder in some ACM patients [START_REF] Chevalier | Short QT interval as a harbinger of an arrhythmogenic cardiomyopathy[END_REF][START_REF] Moreau | Deciphering DSC2 arrhythmogenic cardiomyopathy electrical instability: From ion channels to ECG and tailored drug therapy[END_REF]. Other studies use hiPSCs to demonstrate signaling pathway involvement in disease spread. A study shows the role of Nuclear Factor-κB (NF-κB) signaling in the inflammation of hiPSC harboring a variant in the PKP2 gene (c.2013delC) [START_REF] Chelko | Therapeutic modulation of the immune response in arrhythmogenic cardiomyopathy[END_REF]. This study defines inflammatory signaling which is activated in ACM and drives key features of the disease. Targeting inflammatory pathways may be an effective new mechanism-based therapy for ACM. Another study defines the role of PPARγ signaling in electrophysiological and calcium disturbances in ACM hiPSC-CMs and the inhibition of this pathway can rescue these troubles [START_REF] Reisqs | The PPARγ pathway determines electrophysiological remodelling and arrhythmia risks in DSC2 arrhythmogenic cardiomyopathy[END_REF]. A very recent publication demonstrates the beneficial effect of spironolactone, an anti-diuretic, to prevent the onset of arrhythmias [START_REF] Reisqs | Spironolactone as a potential new treatment to prevent arrhythmias in arrhythmogenic cardiomyopathy cell model[END_REF]. All the discoveries and advances obtained by hiPSCs are summarized in Table 1.

The hiPSC-CMs system offers unique access to study cardiac cellular functions and signaling pathways directly related to a given patient with ACM, which is not possible to reproduce the complete genetic background of a given patient in any animal model. Features found in non-hiPSC-CM and hiPSC-CM models are summarized in Table 2. The hiPSC-CMs allow a more cell-centered approach and have brought to light the greater importance of the myogenic origin of arrhythmias. The hiPSC-CMs form a monolayer or an organoid which also enables a multicellular approach to contractile function [START_REF] Feaster | Evaluation of cardiac contractility modulation therapy in 2D human stem cell-derived cardiomyocytes[END_REF]. Recent studies conducted on ACM-hiPSC-CM reproduced hallmarks of ACM, such as contractile apparatus defects accompanied by fibro-fatty and lipid accumulation. The electrical modifications of ACM-hiPSC-CM are likely to create conduction blocks facilitating reentry arrhythmias [START_REF] Ten Tusscher | Influence of diffuse fibrosis on wave propagation in human ventricular tissue[END_REF]. The hiPSC-CMs are therefore a suitable approach for comprehensive studies of ACM and use as a valuable model for therapeutic investigations. Moreover, a study reported that two patients diagnosed with ACM presented ventricular fibrillation without structural abnormalities [START_REF] Blom | Late evolution of arrhythmogenic cardiomyopathy in patients with initial presentation as idiopathic ventricular fibrillation[END_REF]. Interestingly, studies with ACM-hiPSC-CMs demonstrated electrical and calcium disturbances supporting the idea of direct involvement of cardiomyocytes in the genesis of arrhythmias and not only following structural and morphological remodeling [START_REF] Kohela | Epicardial differentiation drives fibro-fatty remodeling in arrhythmogenic cardiomyopathy[END_REF][START_REF] Zhang | Plakophilin-2 truncating variants impair cardiac contractility by disrupting sarcomere stability and organization[END_REF]. Arrhythmias would result not only from fibro-fatty replacement disturbing electrical wave propagation in the heart but also directly from a profound modification of the cell's electrical profile. Electrical disturbances would thus constitute a predominant mechanism to underly the early rhythm disturbances in ACM [START_REF] Moreau | Deciphering DSC2 arrhythmogenic cardiomyopathy electrical instability: From ion channels to ECG and tailored drug therapy[END_REF]. These suggestions link to a clinical study where patients presented electrical disturbances in absence of structural modifications in the ventricle [START_REF] Gomes | Electrophysiological abnormalities precede overt structural changes in arrhythmogenic right ventricular cardiomyopathy due to mutations in desmoplakin-A combined murine and human study[END_REF].

The hiPSC models open up immense prospects for progressing towards personalized medicine strategies for the diagnosis (functional and mechanistic at the cellular level) and the management of patients suffering from pathologies linked to variants. In ACM, one objective is to reduce the risk of arrhythmia for the patient. ICD placement and catheter ablation are frequently used in ACM patients. However, these methods do not prevent the occurrence of arrhythmias in these patients. That's why we use the hiPSC model to find therapies to prevent the onset of ventricular arrhythmias. Antiarrhythmic drugs are therefore used to reduce this risk [START_REF] Ermakov | Arrhythmogenic right ventricular cardiomyopathyantiarrhythmic therapy[END_REF]. However, studies on Anti-arrhythmic drugs (AAD) prescription for ACM patients are lacking, due to the rarity of cases and the complexity of this pathology limiting the information available to understand disease progression and improve the prevention and/or treatment of patients. In an ACM-hiPSC-CMs model, we demonstrated the potential beneficial effects of the combination of sotalol and flecainide, where the sotalol normalized APD while flecainide normalized calcium handling [START_REF] Moreau | Deciphering DSC2 arrhythmogenic cardiomyopathy electrical instability: From ion channels to ECG and tailored drug therapy[END_REF]. These results provided a mechanistic validation for the use of these drugs used sometimes empirically in patients with ACM. They also support the hiPSC-CMs system as a valuable tool to improve and rationalize patient treatments. Furthermore, the recapitulation and correction of this pathology using gene therapy in ACM-hiPSC-CM, support the development of personalized medicine and provide evidence for gene replacement therapy [START_REF] Shiba | Phenotypic recapitulation and correction of desmoglein-2-deficient cardiomyopathy using humaninduced pluripotent stem cell-derived cardiomyocytes[END_REF]. This cellular model can also be used to discover new therapeutic pathways like NF-κB or using the anti-diuretic (Figure 4) [START_REF] Chelko | Therapeutic modulation of the immune response in arrhythmogenic cardiomyopathy[END_REF][START_REF] Reisqs | Spironolactone as a potential new treatment to prevent arrhythmias in arrhythmogenic cardiomyopathy cell model[END_REF].

Most studies were carried out based on PKP2 variants, which could limit the relevance on a larger scale and for other variants. However, the emergence of new hiPSC cell lines bearing other variants will allow us to refine our findings [START_REF] Vera | Generation of two induced pluripotent stem cell lines carrying the phospholamban R14del mutation for modeling ARVD/C[END_REF][START_REF] Loiben | Generation of human iPSC line from an arrhythmogenic cardiomyopathy patient with a DSP protein-truncating variant[END_REF]. This could also help to elucidate a generalized pathological mechanism across all ACM genetic sources. In addition, these studies on hiPSC-CM were conducted between 30 and 60 days of culture. We have seen that a more prolonged culture (360 days) could improve cell maturity. However, we found the main highlights of the ACM at 30-60 days. In addition, we demonstrated a shortened APD in ACM-hiPSC-CM which shed light on the problem of QT duration in the ACM patient's ECG [START_REF] Moreau | Deciphering DSC2 arrhythmogenic cardiomyopathy electrical instability: From ion channels to ECG and tailored drug therapy[END_REF]. The use of hiPSC-CM at 30-60 days is, therefore, suitable to translate the phenotype in patients. To overcome these problems of immaturity, the emergence of novel 3D culture models or culture conditions will improve cardiomyocytes' phenotypes, and pathological modeling, and favor the development of new therapies [START_REF] Bliley | Dynamic loading of human engineered heart tissue enhances contractile function and drives a desmosome-linked disease phenotype[END_REF]. This latter publication describes impaired contractility associated with a reduction in desmosome number in a 3D model made of hiPSC-CM carrying a DSP variant. Since hiPSC-CMs are cultured in a Petri dish, the involvement of other cells, such as c-kits, SMCs, or FAPs, in fibro-adipose replacement is difficult to assess. However, this approach has an essential advantage for studying the mechanisms in pure myocytes. These limits interpretations at the cardiomyocytes' level but not in the context of tissue coherence integrating different cell types. These characteristics allowed the elucidation of potential transdifferentiation pathways in ACM-hiPSC-CM [START_REF] Caspi | Modeling of arrhythmogenic right ventricular cardiomyopathy with human induced pluripotent stem cells[END_REF][START_REF] Ma | Generation of patientspecific induced pluripotent stem cell-derived cardiomyocytes as a cellular model of arrhythmogenic right ventricular cardiomyopathy[END_REF][START_REF] Reisqs | The PPARγ pathway determines electrophysiological remodelling and arrhythmia risks in DSC2 arrhythmogenic cardiomyopathy[END_REF]. However, the co-culture of hiPSC-CM with cardiac fibroblasts from hiPSC could be useful to recreate a fibro-fatty infiltration in a dish to build a better model of ACM using hiPSC [START_REF] Zhang | Functional cardiac fibroblasts derived from human pluripotent stem cells via second heart field progenitors[END_REF].

Conclusion

This review summarizes the most advanced knowledge regarding the appropriate and relevant cellular model ACM pathological development. It further depicted several advances provided using hiPSC in studies of ACM. In addition to the fact that this model recapitulates the clinical hallmarks of the pathology, the results obtained using hiPSC, firstly, reinforce the previous cellular and animal models. Secondly, results obtained by hiPSC, are directly related to the patient. This model allows us to describe the pathology of the patient with the aim of more personalized medicine. This is of particular importance for cardiac diseases in general. Furthermore, hiPSC-CM is a cell-centered approach and deciphers the critical role of cardiomyocytes in the early manifestation and progression of ACM and serves as a unique patient-specific model for drug screening and hence therapy.

Funding

A CIFRE grant from SANOFI R&D supported this work for JR. Fond Marion Elisabeth Brancher supported the post-doctoral fellowship of AM.

Author contributions

Conceptualization: JR, AM; Writing-original draft preparation: JR, AM; Prepared the tables and figures: JR, YS; Writing-review and editing: MB, SR, and PC; All authors have read and agreed to the published version of the manuscript. All authors contributed to the article and approved the submitted version.

Conflict of interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.