**2.6 LQT type 8 (LQT8) or Timothy syndrome (TS)**

2.6 LQT type 8 (LQT8) or Timothy syndrome (TS) is a very rare genetic cardiac disease which results from mutation in *CACNA1C* gene encoding Ca2+ channel (CaV1.2). LQT8 is the most severe type of LQTS, which is characterized by markedly prolonged QT interval, severe ventricular arrhythmia, and multiorgan dysfunction [66]. hiPSC-CMs derived from TS patients recapitulated the disease phenotypes, but roscovitine rescued those abnormalities such as altered Ca2+ channel inactivation, prolonged APD, higher incidences of arrhythmias, and abnormal Ca2+ handling [67].

### **2.7 Short QT (SQT)**

SQT is a rare inherited cardiac disease characterized by QT internal shortening, which is in contrast to QT prolongation observed in LQTS. SQT is associated with mutations in genes associated with K<sup>+</sup> channel or Ca2+ channels [68]. The prevalence of SQT is between 0.02–0.1% and 0.05% in adults and children, respectively [69]. Recently El-Battrawy and co-workers had generated hiPSCs from SQT type 1 patients carrying a mutation (N588K) in *KCNH2*, and hiPSC-CMs mimicked the clinical phenotype of SQT by showing a shortened APD as a result of increased IKr current densities [70]. In addition, SQT hiPSC-CMs exhibited abnormal Ca2+ transients and rhythmic activities, which are enhanced by carbachol, but quinidine alleviated those carbachol-induced arrhythmias and prolonged the APD [70].

## **3. Cardiomyopathy phenotypes in hiPSC-CMs**

Cardiomyopathies are diseases of cardiac muscle and associated with structural and/or functional abnormalities. The most common genetic cardiomyopathies are hypertrophic cardiomyopathy (HCM), dilated cardiomyopathy (DCM), and arrhythmogenic right ventricular cardiomyopathy/dysplasia (ARVC/D). These genetic cardiomyopathies have been also extensively studied using hiPSC-CMs [71, 72].

#### **3.1 Hypertrophic cardiomyopathy (HCM)**

HCM is one of the most common genetic cardiac diseases with an estimate prevalence of 1 in 500. HCM is characterized by unexplained symmetrical or asymmetrical left ventricular hypertrophy. Mutations in sarcomeric proteins account for ~60% of all HCM cases including mutation in β-myosin heavy chain (MYH7), cardiac myosinbinding protein C (MYBPC3), cardiac troponin I (cTnI), cardiac troponin T (cTnT), and tropomyosin (TPM1) [73]. Hypertrophy of myocytes and disarray of sarcomere are the histological hallmarks of HCM seen in cardiac biopsies from HCM patients [74], and these histological phenotypes are also observed in hiPSC-CM model of HCM [25, 75–77]. In addition, HCM hiPSC-CMs also demonstrated other hallmarks of HCM such as nuclear translocation of nuclear factor of activated T cells (NFAT) [75–77],

elevation of β-myosin/α-myosin ratio, and calcineurin activation [75]. Furthermore, isolated CMs from HCM patients displayed the prolonged APDs, increased Ca2+ current densities, reduced transient outward K+ current densities, abnormal Ca2+ handling, and increased frequency of arrhythmias [21]. These electrophysiological and Ca2+ transient irregularity phenotypes have been faithfully recapitulated in HCM hiPSC-CMs [25, 75, 76, 78]. When HCM tissues carrying a mutation in *MYBPC3* gene were compared with donor heart sample, no specific truncated MyBP-C peptides were detected, but the overall level of MyBP-C in myofibrils was significantly reduced [79]. Similar haploinsufficiency results were also shown in HCM hiPSC-CMs with mutation in *MYBPC3* gene [25, 80], and gene replacement in HCM hiPSC-CMs partially improves the haploinsufficiency and reduces cellular hypertrophy [80]. Similar to higher myofilament Ca2+ sensitivity observed in isolated cardiac biopsies from HCM with E99K mutation in cardiac actin [81], in vitro model of HCM hiPSC-CMs carrying E99K mutation in cardiac actin demonstrated significantly stronger contraction and increased arrhythmogenic events [82] Furthermore, a study in HCM mice harboring I79N mutation in cTnT resulted in increased cardiac contractility, altered Ca2+ transients, and remodeling of action potential [83]. These phenotypes were faithfully recapitulated by HCM hiPSC-CMs carrying the same I79N mutation in cTnT [84]. These hypercontractility and increased arrhythmogenicity phenotypes were reversed in HCM hiPSC-CMs when the E99K mutation in cardiac actin [82] and I79N mutation in cTnT [84] were corrected using CRISPR/Cas9 gene editing technique. Recently, we have shown that HCM hiPSC-CMs carrying *TPM1-Asp175Asn* mutation exhibited VT type of arrhythmias [78], and this observation is in line with earlier clinical observation of HCM patients with *TPM1-Asp175Asn* mutation being at increased risk of fatal arrhythmias [85]. Currently, there is no specific pharmacological therapy for HCM patients, and drugs are prescribed mainly based on symptoms and personal history. However, drug therapy has also resulted in poor outcomes in HCM patients [12]. We reported the similar poor antiarrhythmic efficiency of β-blocker in preventing lethal arrhythmias in HCM hiPSC-CMs [78]. In another HCM report, several environmental factors were investigated with hiPSC-CMs to study their effect on disease progression [77]. They found that endothelin (ET)-1 was able to induce HCM phenotypes such as cellular hypertrophy and myofibrillar disarray in hiPSC-CMs, which are inhibited by ET receptor type A blocker [77]. HCM patients exhibited defects in mitochondrial functions and ultrastructure and abnormal energy metabolism [74]. These structural and functional phenotypes were recapitulated in hiPSC-CMs carrying m.2336 T > C mutation in mitochondrial genome causing HCM [86]. They reported that HCM hiPSC-CMs expressed reduced levels of mitochondrial proteins, ATP/ADP ratio, and mitochondrial membrane potential [86].
