Various studies have focused on disturbances of intracellular pathways described for ACM related to desmosome destabilization (Austin et al., 2019). 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 (Chen et al., 2014). Studies also highlighted an increase in PPARγ expression in human cardiomyocytes from ACM patients (Djouadi et al., 2009). PPARγ gene is a known master regulator of adipogenesis (Rosen et al., 2002; Mal et al., 2021).

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) (Takahashi et al., 2007; Yamanaka, 2012). 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 (Hou et al., 2013; Moreau et al., 2017; Shi et al., 2017).

The capacity to differentiate hiPSC into spontaneously beating cardiomyocytes was a major advance in the understanding of cardiac pathologies (Burridge et al., 2012). Indeed, hiPSC- derived cardiomyocytes (hiPSC-CM) are essential in the study of cardiomyopathies, channelopathies, and molecule screening during drug development or safety studies (Moretti et al., 2010; Sun et al., 2012; Moreau et al., 2017; Sleiman et al., 2020; Poulin et al., 2021; Ait Benichou et al., 2022; Jauvin et al., 2023).

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 (Kamakura et al., 2013; Lundy et al., 2013). To go further, the use of molecules and new culture media makes it possible to obtain more mature cardiomyocytes (Feyen et al., 2020; Chirico et al., 2022). 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. (Song et al., 2021; Bissoli et al., 2023).

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) (Zhang et al., 2009; Zhao et al., 2019). 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 (Honda et al., 2011; Zhao et al., 2018). 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 (Ma et al., 2011). Despite evidence for incomplete electrophysiological maturity, the hiPSC-CMs revealed the presence of a functional excitation-contraction coupling close to native cardiomyocytes (Itzhaki et al., 2011). 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 (Doss and Sachinidis, 2019). 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 I_K1 (Hoekstra et al., 2012). 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 (Meijer van Putten et al., 2015). 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 (Mannhardt et al., 2016). 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 (Maiullari et al., 2018; Kupfer et al., 2020). 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 (Herron et al., 2016). The use of human ECM could encounter hiPSC-CM immaturity issues for optimal pre-clinical drug discoveries (Block et al., 2020). 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 (Hofbauer et al., 2021).

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) (Ma et al., 2013). TEM also showed a widened and distorted desmosome in ACM condition, which is one of the characteristics of the weakening desmosomal complex (Figure 3B) (Caspi et al., 2013). 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) (Kim et al., 2013). 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 (Wen et al., 2015). 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) (El-Battrawy et al., 2018). 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 (Gusev et al., 2020). 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 (El-Battrawy et al., 2018). 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 (Khudiakov et al., 2020). 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) (Khudiakov et al., 2020). 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 (Hawthorne et al., 2021). 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 (Inoue et al., 2022).

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 (Moreau et al., 2021). 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 (Chevalier et al., 2021; Moreau et al., 2021). 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) (Chelko et al., 2019). 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 (Reisqs et al., 2022). A very recent publication demonstrates the beneficial effect of spironolactone, an anti-diuretic, to prevent the onset of arrhythmias (Reisqs et al., 2023). 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 (Feaster et al., 2022). 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 (Ten Tusscher and Panfilov, 2007). 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 (Blom et al., 2019). 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 (Kohela et al., 2021; Zhang K et al., 2021). 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 (Moreau et al., 2021). These suggestions link to a clinical study where patients presented electrical disturbances in absence of structural modifications in the ventricle (Gomes et al., 2012).

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 (Ermakov and Scheinman, 2015). 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 (Moreau et al., 2021). 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 (Shiba et al., 2021). This cellular model can also be used to discover new therapeutic pathways like NF-κB or using the anti-diuretic (Figure 4) (Chelko et al., 2019; Reisqs et al., 2023).

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 (Vera et al., 2022; Loiben et al., 2023). 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 (Moreau et al., 2021). 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 (Bliley et al., 2021). 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 (Caspi et al., 2013; Ma et al., 2013; Reisqs et al., 2022). 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 (Zhang et al., 2019).

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.