HCM and DCM are the most frequently encountered genetic CMPs in daily clinical practice, therefore, unsurprisingly, they have been the most studied iPSC-CM-based models. In a recent report, Eschenhagen et al[19] comparatively analyzed 38 original papers that reported the characteristics of iPSC-CMs obtained from patients with HCM/DCM or generated from iPSC lines in which a HCM or DCM mutation had been genetically introduced[19]. In summary, compared with their respective controls, the main features exhibited by HCM iPSC-CMs were the following: larger cell size, increased nuclear localization of nuclear factor of activated T cells (NFAT, a transcription factor) and increased MYH7 (or MYH7/MYH6 ratio) expression[20-24]. The most constant aberration identified in DCM lines was reduced peak force development[25], the molecular mechanisms of which are discussed, and explain the main clinical presentation of the disease. As for similarities between the two considered diseases, three anomalies were the most documented: sarcomere disarray, increased NPPA/NPPB gene expression, and arrhythmic behavior[20,24,26,27].

Although not constantly associated with alterations in contractile force or kinetics, abnormal calcium handling appears to be a key pathological mechanism observed in iPSC-CM models of HCM[20,21,24,28]. Valuable insights related to the molecular mechanisms of HCM pathogenesis have been provided by Seeger et al[29] in a model of patient-derived iPSC-CMs harboring a premature stop codon in the MYBPC3 gene. When compared with the isogenic mutation-corrected iPSC lines, in addition to aberrant calcium signaling, patient-derived iPSC-CMs displayed molecular dysregulations without haploinsufficiency of the MYBPC3 protein. This observation could challenge the existing dogma of haploinsufficiency as the underlying mechanisms for HCM caused by MYBPC3 premature termination codon mutations. The specific molecular signature included dysregulation of genes involved in calcium handling (ATP2A2, ATP2B2, and CASQ1), cardiac hypertrophy (GP130, JAK2, RRAS, MEK1, TWEAKR, and NPPB), stress response (HSPB1, HSPB6, HSPB7, IGF1, and IGF2), and structural organization of sarcomeres and mechanosensors (CSRP3 and TCAP).

Disturbed calcium signaling has been shown to be a central pathological mechanism of diastolic dysfunction in familial HCM lines with variations in the MYH7, MYBPC3, and TNNT2 genes[30]. Using comprehensive functional imaging analysis (i.e. calcium imaging and traction force microscopy), Wu et al[30] revealed that diastolic Ca²⁺ overload and increased myofilament Ca²⁺ sensitivity contribute to diastolic dysfunction and demonstrated for the first time that patient-specific iPSC-CMs can recapitulate diastolic dysfunction characteristics at the single-cell level. Furthermore, calcium homeostasis was restored by Ca²⁺ and late Na⁺ blockers (verapamil, dilitiazem, ranolazine, and electlazine), which was reflected in diastolic function improvement in HCM iPSC-CMs.

More recently, it has been shown that molecular signaling differs within HCM iPSC-CMs with diverse gene mutations. Isogenic models of HCM revealed differential phenotypes and mechanism-driven possible therapeutic targets in MYH7 and ACTC1 cell lines, respectively[31]. In spite of sharing key disease hallmarks, such as intracellular calcium overload and calcium transient arrhythmias, which were common in both models, modifications in contractility were entirely divergent, namely decreased contractility of MYH7 cells and gain of hypercontractility of ACTC1 cells. Notably, the expression of Ca²⁺-binding proteins and hypertrophy-associated transcription factor activation also showed opposing behavior. Accordingly, compared with their respective controls, MYH7-R453C mutants were characterized by upregulation of CASQ2, CALM1 and CAMK2D along with MEF and NFAT nuclear translocation prompted by IRF8 downregulation. Reversed expression patterns of those genes were found in ACTC1-E99K iPSC-CMs. The study offered clinically-relevant data, given that the arrhythmogenic phenotype was rescued in both models following treatment with a mixture of dantrolene and ranolazine (a ryanodine receptor antagonist that inhibits sarcoplasmic Ca²⁺ release into the cytosol and a late sodium current blocker promoting intracellular Ca²⁺ efflux). In addition, the enhanced contractility displayed in ACTC1 iPSC-CMs was rescued by mavacamten (a selective allosteric inhibitor of cardiac myosin ATPase). Of note, an earlier study reported differences in the phenotypic features of iPSC-CMs obtained from a family with ACTC1-E99K mutation. These differences varied depending on the source subject, highlighting the value of isogenic iPSC-CMs in genotype-phenotype correlations. Thus, while arrhythmogenesis was manifested in all ACTC1-E99K iPSC-CM lines, it was more common in cells obtained from the father, and less apparent in those derived from the two sons. This suggests that, in addition to the causative mutation, other factors (either genetic or epigenetic) might contribute to the disease phenotype[32].

The contractility consequences seem to be dependent on the specific DNA change rather than the affected gene. In contrast to the hypo-contractile phenotype described in MYH7-R453C mutants[31], MYH7-R403Q, MYH7-V606, and MYH7-R719 iPSC-CMs exhibited hyperdynamic contraction resulting from an increased proportion of myosin molecules in a disordered relaxed state conformation[33]. Imbalance of myosin configurations (i.e. super and disordered relaxed states) led to the destabilization of interacting-heads motif interactions that were followed, in addition to increased contractility, by impaired relaxation, hypertrophic remodeling, excessive energy consumption, and metabolic stress. Mavacamten treatment restored the physiological super relaxed state/disordered relaxed state ratio and relieved downstream functional abnormalities, suggesting that chronic dysregulation of myosin conformations is a central mechanism of HCM.

Other reports have used iPSCs to decode genetic DCM physiopathology. The most frequently mutated gene in DCM is TTN, with truncating variants explaining up to 30% of familial cases[34,35]. Other genes involved in DCM etiology are shown in Table 1. The most commonly encountered genes responsible for DCM are LMNA, DES, MYH7, MYH6, SCN5A, MYBPC3, and TNNT2, although at a lower prevalence than TTN[36]. Patient-derived iPSC-CMs have characteristics consistent with DCM, including sarcomere disorganization, reduced contractile function, and altered calcium handling[25,37-39]. Hinson et al[38] investigated the disease phenotypes of iPSCs engineered from DCM subjects with either truncating or missense mutations in TTN, and showing sarcomere insufficiency, impaired responses to mechanical and βadrenergic stress, and attenuated growth factor and cell signaling activation.

In a series of studies that focused on TNNT2-R173W missense mutation, iPSCCMs lines generated from affected family members exhibited a compromised ability to regulate calcium flux, reduced contraction force, and heterogeneous myofilament organization that were exacerbated by βadrenergic stimulation[25]. In-depth analysis uncovered the epigenetic activation of phosphodiesterase genes PDE2A and PDE3A as the underlying mechanism responsible for the defective βadrenergic signaling and contractile dysfunction[40]. The latest data indicates that TNNT2-R173W hampers molecular interactions between troponin and tropomyosin and restricts the binding of PKA to local sarcomere microdomains, resulting in diminished troponin phospho-rylation and misalignment of sarcomeric proteins[41]. An R173W variant also altered the interaction between sarcomere microdomain and cytoskeleton filaments via MYH7 and AMPK, with consequent disturbance of sarcomere protein alignment and impaired contractility. In terms of phenotype rescue, AMPK activation by small molecules, such as A-769662, improved sarcomere-cytoskeleton attachment and partially recovered sarcomere protein misalignment and subsequent impaired contractility in mutated iPSC-CMs[41].

The phenotypes and the causal disease mechanisms linked to LMNA variants have also been intensively studied. In iPSC-CMs with either a nonsense or missense mutation in LMNA, Siu et al[39] noticed enhanced nuclear senescence and augmented electrical stress-induced apoptosis that was significantly attenuated by pharmacological inhibition of the ERK1/2 pathway with the MEK1/2 inhibitors U0126 and selumetinib. Other investigators analyzed iPSC-CMs from three patients with distinctive LMNA mutations (R225X, Q354, and T518fs)[42]. Although all three types of diseased cells recapitulated the pathophysiological hallmarks of LMNA-related DCM, the positive effect of ataluren treatment on the expression of full-length Lamin A/C, nuclear blebbing, apoptosis, and contractility was detected only in the LMNA-R225X mutant, suggesting that the effect might be codon selective[42]. Other traits expressed by LMNA-mutated iPSC-CMs were bradyarrhythmia, beat rate variability, abnormal calcium handling, stress hypersensitivity, disorganized sarcomeres, and increased cell size[43,44]. The incriminating mechanisms were aberrant activation of PDGF-signaling, which was rescued by pharmacological and molecular inhibition of PDGF receptor B[43], or epigenetic inhibition of SCN5A that was rescued by SCN5A overexpression[45]. Mutations in other DCM-related genes such as DES[37], MYH7[46], DMD[47,48], PLN[26], or even compound mutations have been modeled[49].

Characterization of iPSC-CMs from family members with LVNC who carried a nonsense variant in cardiac transcription factor TBX20, compared with iPSCCMs from unaffected relatives, revealed abnormal activation of transforming growth factor-β signaling leading to decreased cell proliferation capability. Moreover, inhibition of the transforming growth factor-β signaling pathway and correction of TBX20 alteration by CRISPR-Cas9 technology, successfully rectified the pathological phenotype[50].

Impairments in contractility, calcium handling, and metabolic activity have been nominated as key features in another model of iPSC-CMs generated from patients with delayed-onset LVNC caused by a missense mutation in the GATA4 gene[51].

In an iPSC model with a TPM1-R178H mutation, it was shown that mislocalization of tropomyosin 1 was a central pathological change, triggering disturbance of the sarcomere structure and impaired calcium handling. Comprehensive analysis found involvement of complex molecular pathways centered on downregulation of the expression of numerous genes controlling heart development and positive regulation of cellular processes, including transcription factors (GATA4, GATA6) and sarcomeric proteins (MYBPC3, MYH6, TTN, TNNI3, TNNT2), thus linking sarcomeric dysfunction to LVNC[52].

ACM is another inherited CMP studied in iPSC models. In a series of reports published in 2013, CMs engineered from subjects having mutations in the PKP2 gene efficiently recapitulated key disease features, including reduced cell surface localization of desmosomal proteins with altered desmosomal structure and a more adipogenic phenotype[53]. These phenotypical changes were accompanied by upregulation of the pro-adipogenic transcription factor peroxisome proliferator-activated receptor (PPAR)-γ and enhanced activation of respective signaling pathways[54,55]. Furthermore, lipid droplets accumulation was prevented by admini-stration of a specific inhibitor of glycogen synthase kinase 3β (6-bromoindirubin-3'-oxime)[55]. Subsequent work revealed novel mechanistic insights in ACM pathogenesis or confirmed those already cited. Wen et al[56] reported that coactivation of normal PPAR-α and abnormal PPAR-γ pathways in ACM iPSC-CMs triggered markedly increased lipogenesis, apoptosis, Na⁺ channel downregulation and defective intracellular calcium handling[56]. In another iPSC-based model it was found that RhoA/ROCK signaling at the intercalated disc was essential for cardiomyocyte homeostasis[57]. Using patient-derived iPSC-CMs with impaired cell-cell adhesion due to a PKP2 frameshift mutation, or disturbed RhoA signaling caused by a nonsense MYH10 mutation, Dorn et al[57] elegantly demonstrated that cardiomyocyte identity was lost following disruption of the RhoA/MRTF/SRF-signaling circuit. RhoA recruitment to cell-cell junctions was abridged in diseased cells, prompting increased levels of cytosolic G-actin and successive cytoplasmic sequestration of transcription factors such as MRTF that are involved in myocyte identity, preventing their entry into the nucleus. Finally, when exposed to an adipogenic environment, the mutated cells were poised to switch to a brown/beige adipocyte lineage, providing a possible molecular explanation of the clinical phenotype observed in ACM. Interestingly, a recent study reported lipid accumulation, increased pleomorphism, irregular Z-bands, and increased L-type calcium currents in iPSC-CMs carrying a novel frameshift mutation (L5218fs) in the OBSCN gene[58]. The phenotypic alterations were accompanied by activation of adipocytokines and PPAR signaling pathways, diminished expression of the mutant protein and its anchor protein Ank1.5, in addition to downregulation of other desmosomal coding genes (PKP2, JUP, DSP)[58].

IPSC-CMs generated from an ACM patient with a DSG2 mutation exhibited complex ion channel dysfunctions and abnormal cellular electrophysiology as well as increased sensitivity to adrenergic stimulation, indicating involvement of ion channel dysfunctions in arrhythmogenesis, independent of structural abnormalities[59]. Subsequent work conducted by the same group established for the first time that enhanced NDPK-B expression, via activating SK4 channels, contributed to arrhythmogenesis in DSG2-related ACM, suggesting that NDPK-B could be a specific therapeutic target in selected patients[60].

Ng et al[61] reported for the first time that some desmoplakin missense variants, such as DSP-R451G, are functionally equivalent to truncating alleles by promoting pathological vulnerability to calpain proteolysis and subsequent desmoplakin insufficiency.

There are few data related to restrictive cardiomyopathy modeling by iPSC. CMs harboring homozygous DES-Y122H mutation were reported to display abnormal desmin cytoplasmic aggregates responsible for the pathological phenotype[62].

A second category of genetic cardiac conditions extensively modeled using iPSC technology is represented by CNPs, for which electrophysiology studies exposed alterations of action potential, field potential, or Ca²⁺ transients in engineered CMs. LQTS, in particular the first three types (LQTS1, LQTS2 and LQTS3), benefit from the most well-characterized iPSC-CMs models. Types LQTS that differ according to the underlying channel or gene mutation are shown in Table 1.

The first model of an arrhythmic syndrome using patient-specific iPSC-CMs was reported in 2010 by Moretti et al[63], who generated iPSCs from two affected members of a family with LQTS1 caused by a missense mutation (R190Q) in the KCNQ1 gene. Mutant iPSC-CMs effectively reproduced the relevant features of the disease, namely prolongation of the action potential duration into atrial-like and ventricular-like cells, and increased occurrence of arrhythmic events when exposed to β-adrenergic agonists. Voltage clamp analysis revealed a substantial 70% to 80% reduction in the slowly activating delayed rectifier potassium currents (IKs) of LQTS1-iPSC-derived ventricular CMs due to a decreased number of functional KCNQ1 channels in the sarcolemma compared with the healthy counterpart. Beta-blocker treatment of LQTS1 CMs had a protective effect against catecholamine-induced arrhythmia. Similar findings have been reported in subsequent models of iPSC-CMs from LQTS1 with KCNQ1 missense or frameshift mutations[64,65]. Additionally, Wang et al[66] identified abnormalities in Ca²⁺ handling linked to three distinct KCNQ1 variants (R190Q, G269S, and G345E). CMs derived from all three edited iPSC lines displayed a characteristic LQTS phenotype and significant prolongation of the action potential duration compared with isogenic controls, which were corrected by treatment with L-type calcium channel antagonists. Similar results have been reported recently following an increase in the number of iPSC-CMs models of autosomal dominant, recessive, and compound heterozygous LQTS1[67-73], including analysis of models derived from specific populations[67,72]. A plethora of LQTS2 iPSC-CMs models developed from patients harboring missense mutations in KCNH2 have reproducibly shown prolonged action potential, increased arrhythmogenicity, and reduced rapidly activating delayed rectifier potassium current (IKr), compared with healthy control lines. The first analyzes of iPSC-based LQTS2 models were published in 2011 by two independent groups[74,75]. Itzhaki et al[74] generated iPSC lines from a patient with a KCNH2-A614V variant. As expected, the derived-CMs exhibited substantial prolongation of the action potential, diminished IKr, early after depolarizations (EADs), and triggered arrhythmias. Specific pharmacological inhibition of IKr worsened the cellular phenotype, while administration of other pharmacological agents such as Ca²⁺ channel blockers, or KATP-channel openers alleviated the pathological features[74]. The in vitro model developed by the second group effectively replicated the variation in clinical phenotypes of two family members carrying the same KCNH2-A561V mutation. Although the action potential duration was increased in the iPSC-CMs derived from both the clinically symptomatic patient and the clinically asymptomatic mother, an increased sensitivity (appearance of EADs) to stress (i.e. β-adrenoreceptor stimulation) was detected only in the symptomatic patient-derived cells[75]. By comparing the electrophysiological properties of spontaneously beating CMs produced from LQTS2 cases and controls, it was suggested that cell-to-cell contacts in the syncytium result in compensatory mechanisms with a tendency to protect the repolarization system from major aberrations of physiological parameters. Although a considerable signal difference was detected between LQTS2 and control iPSC-CMs on single-cell patch-clamp recordings (a 66% increase in action potential in LQTS2 cells), the differences were more modest (10%-20%) when using a microelectrode array technique on cell aggregates, similar to the surface electrocardiogram in respective patients[76]. In later studies, the diseased phenotype was rescued either by genetic correction of the KCNH2 mutation[77], or by allele-specific ribonucleic acid (RNA) interference, which selectively destroyed the mutant mRNA while leaving the wild-type mRNA undamaged[78]. Various pharmacological agents were also shown to correct the electrophysiological anomalies[79-81], although for some molecules the effect was mutation-specific[82]. An interesting observation was noted by Spencer et al[83], who established that in iPSC-CMs with a KCNH2-A422T mutation, the action potentials and intracellular calcium transients were prolonged in parallel. Furthermore, exposure to a Ca²⁺ antagonist such as nifedipine, abbreviated the action potentials despite the IKr deficit.

Although abnormal calcium handling is common in both LQTS1 and LQTS2, there are major differences in this regard. Joutsijoki et al[84] used an innovative approach to differentiate the Ca²⁺ transient statistics between these two LQTS-mutated iPSC-CMs. By combining machine learning and iPSC technology, the authors analyzed 90 LQTS1 transient signals from two cell lines and 138 LQTS2 signals from four cell lines, resulting in classification accuracies of up to 100%. The findings support the hypothesis that Ca²⁺ transients are disease-specific or even mutation-specific.

Patient-specific iPSC-CMs models harboring gain-of-function mutations in the SCN5A gene efficaciously summarized LQTS3 pathognomonic electrophysiological traits, such as abnormal sodium currents and prolonged APD[85-88]. A study by Malan at al[89] complemented prior findings by also showing a high incidence of EADs, a recognized trigger mechanism for arrhythmia, in disease cells. Treatment with mexiletine, specific sodium channel inhibitors, reduced action and field potential durations in LQT3 iPSC-CMs and alleviated EADs in a dose-dependent manner. Other types of LQTS, including LQTS7[90], LQTS8[91], LQTS14, and LQTS15[92-94] have been successfully investigated with iPSC technology.

CPVT comprises two main subtypes, CPTV1 (caused by mutations in the RYR2 gene) and CPTV2 (determined by mutations in the CASQ2 gene). Both genes are key regulators of CM calcium homeostasis, and dysfunction of either gene prompts abnormal intracellular Ca²⁺ handling and signaling, and increased arrhythmogenicity under adrenergic stimulation. To date, numerous CPVT models have been developed using the iPSC platform, successfully recapitulating the arrhythmogenic phenotype seen in patients[95-104]. In a study published in 2011, Fatima et al[95] analyzed the functional properties of iPSC-CMs from healthy donors and a patient with CPVT1 carrying a novel heterozygous autosomal dominant mutation (RYR2-F2483I). Compared with healthy CMs, the mutated cells displayed arrhythmias and delayed afterdepolarizations (DADs), higher amplitudes and longer duration of spontaneous Ca²⁺ release events in the basal state, as revealed by patch-clamp recordings and calcium imaging studies. Additionally, in CPVT-iPSC-CMs the Ca²⁺-induced Ca²⁺-release events continued after repolarization and were eliminated by increasing cytosolic cAMP levels with forskolin. In another CPVT1 model of iPSC-CMs harboring the RYR2-M4109R mutation, intracellular electrophysiological recordings evidenced increased development of DADs in CPVT-iPSCs-CMs compared with healthy CMs, which were further exacerbated by β-adrenergic stimulation; and, as opposed to previous findings, by forskolin. In contrast, thapsigargin (a specific inhibitor of the sarcoplasmic reticulum calcium ATPase pump) eradicated all afterdepolarizations in those cells, indicating that internal Ca²⁺ stores play a central role in the pathogenesis of DADs. Indeed, laser-confocal Ca²⁺ imaging revealed extensive whole-cell Ca²⁺ anomalies (such as frequent local and large-storage Ca²⁺-release events, broad and double-humped transients, and triggered activity) that were aggravated under catecholaminergic stress and alleviated by beta-blockers. Also, RYR2-M4109R mutations significantly reduced the threshold for store-overload-induced Ca²⁺ release[96]. Dantrolene[97], flecainide[105], and S107 (an RYR stabilizer[104]) were other pharmacological agents shown to ameliorate the disease phenotypes.

More recently, the combined iPSC and CRISPR/Cas9 gene editing technics were used to validate preliminary data and, more importantly, to gain further insight into dysfunction produced by variations in RYR2 gene. Wei et al[106], introduced a point mutation into wild-type RYR2 iPSCs by CRISPR/Cas9 gene editing. Similar to CMs generated from CPVT1 patient harboring F2483I-RyR2 mutation[101], edited iPSC-CMs carrying the same CPVT1-associated variant showed abnormal intracellular Ca²⁺ handling, including longer and wandering Ca²⁺ sparks, elevated diastolic Ca²⁺ leaks, reduced sarcoplasmic reticulum (SR) Ca²⁺ content, and increased susceptibility to arrhythmias caused by isoproterenol[106], suggesting that F2483I-RyR2 mutation produces leaky RyR2 channels. The same approach was used by Zhang et al[107] to assess aberrant Ca²⁺ signaling and pharmacological sensitivity to three distinct CPVT1-associated mutations. While all three diseased iPSC-CM lines exhibited some abnormalities in calcium handling (i.e. irregular, long-lasting, spatially wandering Ca²⁺ sparks and aberrant Ca²⁺ releases), enhanced SR Ca²⁺ leaks and diminished SR Ca²⁺ contents were seen only in cells with Q4201R and F2483I, but not R420Q. Moreover, fractional Ca²⁺ release and calcium-induced calcium release gain were higher in Q4201R than in R420Q and F2483I iPSC-CMs, emphasizing that Ca²⁺ signaling abnormalities may vary depending on the mutation site. Several potential therapeutic interventions, including flecainide, dantrolene, and JTV519 (a Ca²⁺-dependent blocker of SERCA) were tested, indicating that drug sensitivities may also be mutation dependent. Using a wide-ranging methodology integrating optogenetics, tissue engineering, lab-on-a-chip technology, gene editing, and iPSC technology, Park et al[108] identified calmodulin-dependent protein kinase II activation as a key molecular pathway underlying exercise-triggered arrhythmia in CPVT patients, suggesting that its inhibition might be an effective therapeutic strategy in selected cases.

Recent data indicate that RYR2 screening should not be indicated only in subjects with stress- or exercise-induced symptoms. Using patient-specific iPSC-CMs, it has been shown that RYR2-H29D variants elicit alteration of calcium homeostasis and molecular modifications such as aberrant SR Ca²⁺ leak under physiological pacing, pro-arrhythmic electrical phenotypes, impaired and asynchronous contractile properties, and aberrant RyR2 post-translational modifications that occur only at rest[109]. Furthermore, the authors hypothesized that the uncommon location of RYR2-H29D mutations outside the four hot-spot regions linked to CPVT1, might be responsible for the distinct phenotypic expression. iPSC-based platforms have also been used to explore functional abnormalities in CMs generated from CPVT2 patients[99,102,110,111]. Under beta-adrenergic stimulation, patient-derived iPSC-CMs carrying the CASQ2-D307H variant demonstrated disease-specific arrhythmogenic characteristics due to Ca²⁺-transient anomalies, afterdepolarization, reduced threshold for store overload-induced Ca²⁺-release, and upsurge of diastolic intracellular calcium concentration[99,102,110].

SQTS is a rare inheritable, autosomal dominant cardiac condition characterized by abnormally short QT intervals and an increased risk of atrial and ventricular tachyarrhythmias. The causal ion channel genes are shown in Table 2, variation in KCNH2 being the most frequently observed in genotyped cases[7,112]. The first SQTS model utilizing the iPSC platform was reported in 2018 by El-Battrawy et al[113]. The authors generated iPSC-CMs from a patient with a KCNH2-N588K mutation and two healthy control subjects. Mutated cardiac myocytes exhibited enhanced IKr density and shortened APD compared with control cells, along with abnormal calcium transients and arhythmic propensity. Carbachol, an acetylcholine receptor agonist, increased the occurrence of arrhythmic events in diseased iPSC-CMs, while quinidine, and not sotalol or metoprolol, prolonged the APD and alleviated carbachol-prompted arrhythmias. In subsequent studies, patient-specific and gene-corrected iPSC-CMs were used to elucidate the SQTS phenotype either at single-cell[114] or tissue level[115]. When compared with healthy control and gene-corrected CMs, KCNH2-T618I iPSC-CMs were shown to display shortened APDs and increased beat-beat interval variability. Although no significant differences in total KCNH2 expression in control, gene-corrected, and SQTS iPSC-CMs were seen, membrane expression of KCNH2 was approximately 8-fold higher in mutated iPSC-CMs than in isogenic cells, suggesting that the aforesaid variant results in enhanced membrane expression of KCNH2, which may contribute to the increased IKr density. Moreover, the phenotype was successfully rescued by BmKKx2, a short-peptide scorpion toxin acting as a selective IKr blocker[114]. Shinnawi et al[115] examined conduction and arrhythmogenesis in confluent 2-dimensional iPSC-derived cardiac cell monolayers generated from a symptomatic SQTS patient also with KCNH2-N588K mutation. SQTS-iPSC-CM monolayers were characterized by abnormal repolarization properties and induced sustained re-entrant arrhythmias, while retaining a normal conduction appearance.

BrS is another cardiac channelopathy that has been modeled using iPSC technology. Various genes encoding either sodium, potassium, or calcium channels have been linked to BrS[116]. Among them, the SCN5A gene was found to be most commonly mutated (Table 2). That gene encodes the alpha subunit of the main cardiac sodium channel (Nav1.5); loss of function variants result in reduced availability of functional Nav1.5 channels either through decreased plasma membrane channel expression or through altered channel gating properties[117]. iPSC-CMs generated from BrS patients were shown to reflect the pro-arrhythmic phenotype associated with the disease and caused by blunted inward sodium currents, increased triggered activity, and calcium transient abnormalities. Davis et al[118] were the first to describe the molecular mechanisms that underlie BrS by using patient-specific iPSC-CMs harboring SCN5A_1795insD mutation, which effectively recapped the INa peak reduction and persistent INa associated with overlapped LQTS3/BrS[118]. Another group investigated sodium currents, action potentials and calcium dynamics in iPSC-CMs derived from patients with type 1 BrS carrying two different SCN5A variants and in healthy control subjects[119]. Mutated cardiac cells showed reductions in inward sodium current density, reduced maximal upstroke velocity of the action potential (AP), increased burden of triggered activity, abnormal calcium transients, and beating interval variation compared with control iPSC-CMs from healthy controls. Further analysis revealed markedly reduced expression of KCNJ2, which encodes Kir2.1 inwardly rectifying potassium channel, an observation not previously described in BrS. Correction of the causative variant by CRISPR/Cas9-mediated genome editing prompted efficient resolution of triggered activity and abnormal Ca²⁺ transients.

Additional data were provided by the work of Ma et al[120], who reported that a repolarization deficit was involved in BrS. By comparing electrophysiological properties of iPSC-CMs generated from a patient carrying a compound SCN5A mutation (A226V and R1629X) and a healthy sibling control, they observed an over 75% loss of sodium current in diseased cells. The decline in INa was reflected by altered action potential morphology with reduced maximum upstroke velocity and action potential amplitude at normal 1.0 Hz pacing frequency. At slow a slow pacing 0.1 Hz pacing frequency, an increased phase-1 repolarization action potential pattern characterized by a marked reduction of action potential duration and increased resting membrane potential occurred in a fraction of BrS CMs. Furthermore, disparities in the levels of transient outward K⁺ currents (Ito) among the iPSC-CMs from either compound carriers or healthy controls were noticed, with 19% to 23% of the studied cells displaying high Ito densities. Importantly, in patient-derived iPSC-CMs, treatment with 4-Aminopyridine, an Ito blocker, completely reversed the increased phase-1 repolarization and restored the APD, indicating a coordinated role of INa and Ito in the arrhythmogenic mechanism of BrS. In-depth analysis of iPSC-CMs derived from two BrS subjects with an SCN5A-S1812X variant revealed reduced INa, amplified Ito, and increased ICaL window current probability along with conduction slowing, demonstrating coexistence of repolarization and depolarization impairment in diseased cells[121].

At present, it is widely acknowledged that patient-specific genetic background is a key determinant of the phenotypical manifestation of BrS, as was reported by a team of researchers from Spain and United Kingdom[122]. As expected, iPSC-CMs from a patient with a SCN5A variation recapitulated the loss of function of Nav1.5 associated with the syndrome. Also, a shift in both activation and inactivation voltage-dependence curves and faster recovery from inactivation were reported. Remarkably, conventional heterologous expression systems (i.e. immortalized HEK293 cells co-expressing wild-type and mutant channels) failed to exhibit pro-arrhythmic changes in channel function, showing only a reduction in sodium current density, highlighting once again the need to assess the pathophysiological mechanisms of sodium channel mutations in a cardiac- and patient-specific model.

IPSC technology has also been used to model BrS caused by mutations in genes other than SCN5A. Cerrone et al[123] were the first to describe the association of BrS and genetic variation in PKP2. They analyzed iPSC-CMs derived from five index cases carrying missense mutations in PKP2 and perceived reduced sodium channel expression and current. The phenotype was rescued by transfection of wild-type PKP2, demonstrating that not only loss of PKP2, but also single amino acid mutations, can interfere with INa.

In the vast majority of clinically-diagnosed BrS cases (85%), the genetic cause is not known despite extensive use of NGS[124]. Few research groups have used iPSC technology to uncover disease mechanisms at the cellular level in phenotype-positive genotype-negative patients[125,126]. Notably, no clear cellular electrophysiological differences between the iPSC-CMs obtained from BrS patients without identified pathogenic mutations and control-derived cells were seen. That finding indicated that alternative pathophysiological mechanisms may be involved in those specific cases, such as right ventricular fibrosis or diminished cardiomyocyte coupling through gap junctions. Last but not least, BrS may be a multifactorial disorder, caused by an interaction of common genetic variations and environmental factors[125].