**3.2 Dilated cardiomyopathy (DCM)**

DCM is a myocardial disease characterized by ventricular chamber enlargement and systolic dysfunction and progressive heart failure without significant change in ventricular wall thickness. Mutations in >30 genes encoding proteins of cytoskeleton, sarcomere, and nuclear lamina are found in 30–35% of DCM patients [87]. DCM patients with mutations in *RBM20*, encoding RNA binding motif protein 20 (RBM20), have an early onset of disease phenotype [88]. Isolated CMs from DCM patients carrying mutation in RBM20 displayed elongated and thinner sarcomere structure [88], and such disorganized sarcomeric structure phenotypes were recapitulated in DCM hiPSC-CMs carrying mutation in RBM20 [89, 90]. RBM20 is the main regulator of the heart-specific titin splicing, and N2BA isoform is predominantly expressed in CMs from DCM patient carrying mutation in the *RBM20* gene [91]. In vitro model of RBM20 hiPSC-CMs successfully mirrored the altered titin

**71**

*Modelling of Genetic Cardiac Diseases*

*DOI: http://dx.doi.org/10.5772/intechopen.84965*

isoform expression (titin isoform switch) [89, 90]. Furthermore, RBM20 hiPSC-CMs showed delayed Ca2+ extrusion and reuptake and more Ca2+ being released during each ECC, which resulted into deficient muscle contraction, the hallmark of cardiac dysfunction of DCM patients [89, 90]. In addition, a three-dimensional engineered heart muscle generated from RBM20 hiPSC-CMs showed an impaired force of contraction, and passive stress was decreased in response to stepwise increase in strain, suggesting higher viscoelasticity caused by mutation in *RBM20* [89]. Besides HCM, mutation in cTnT also caused DCM and resulted in shifts in Ca2+ sensitivity and force of contraction [92]. Sun and co-workers generated iPSCs from DCM patients carrying R173W mutation in cTnT and reported that DCM hiPSC-CMs exhibited altered Ca2+ handling, decreased contractility, and abnormal sarcomeric α–actinin distribution [93]. DCM patients with lamin A/C (LMNA) mutations show a highly variable phenotype. Cardiac biopsies from DCM patients harboring LMNA mutations exhibit reduced LMNA in nuclei with nuclear membrane damage such as focal disruption and nuclear pore clustering [94]. Nonsense mutation (R225X) in exon 4 of the LMNA gene causing DCM was associated with accelerated nuclear senescence and apoptosis of DCM hiPSC-CMs under electrical stimulation [95]. In another in vitro modeling of DCM, harboring A285V mutation in desmin (*DES*) using hiPSC-CMs displayed the pathogenic phenotypes of DCM such as diffuse abnormal DES aggregation, poor co-localization of DES with cTnT, and Z-disk streaming with accumulation of granulofilamentous materials or pleomorphic dense structures adjacent to the Z-disk or between the myofibrils [96]. DCM patients harboring R14del mutation in phospholamban (PLN) result in ventricular dilation, contractile dysfunction, and episodic ventricular arrhythmias [97]. Similarly, hiPSC-CMs carrying R14del mutation in PLN induced the Ca2+ handling abnormalities, irregular electrical activity, and abnormal intracellular distribution of PLN in DCM hiPSC-CMs [98]. These PLN R14del-associated disease phenotypes were mitigated upon correction of PLN R14del mutation by transcription activator-like effector nuclease (TALENs) gene editing technique [98]. Furthermore, genetic correction of PLN R14del mutation by TALENs improved the force development and restored the contractile function in threedimensional human engineered cardiac tissue derived from R14del-iPSCs [99].

**3.3 Arrhythmogenic right ventricular cardiomyopathy (ARVC)**

ARVC is rare genetic cardiac disease with the prevalence ranging from 1:000 to 1:5000 worldwide. The histopathological hallmark of ARVC is the substitution of the cardiac myocytes with fibro-fatty deposits, particularly within the free wall of the right ventricle. The consequent results from the disruption of normal myocardial architecture can lead to right ventricular dysfunction, life-threatening arrhythmias, and SCD [100]. ARVC is caused by mutations in genes encoding desmosomal proteins such as plakoglobin (JUP), desmoplakin (DSP), plakophilin-2 (PKP2), desmoglein-2 (DSG2), and desmocollin-2 (DSC2) [100]. Similar to immunohistological results from the biopsy sample from ARVC patients [101], ARVC hiPSC-CMs harboring a plakophilin 2 (PKP2) gene mutation mimicked the reduced *PKP2* immunosignal [102, 103]. In addition, clusters of lipid droplets accumulating within the cytoplasm were identified in ARVC-hiPSC-CMs associated with structural distortion of desmosomes [103]**.** Another study showed that induction of adult-like metabolic energetics from an embryonic/glycolytic state and abnormal peroxisome proliferator-activated receptor gamma (PPARγ) activation underlie the pathogenesis of ARVC [104]. It has been observed that male ARVC patients develop earlier and more severe phenotype than female ARVC patients [105]. To understand whether sex hormones in serum may contribute to the major arrhythmic cardiovascular events in ARVC, Akdis and co-workers combined a clinical study and in vitro

#### *Modelling of Genetic Cardiac Diseases DOI: http://dx.doi.org/10.5772/intechopen.84965*

*Visions of Cardiomyocyte - Fundamental Concepts of Heart Life and Disease*

current densities, reduced transient outward K+

mitochondrial membrane potential [86].

**3.2 Dilated cardiomyopathy (DCM)**

elevation of β-myosin/α-myosin ratio, and calcineurin activation [75]. Furthermore, isolated CMs from HCM patients displayed the prolonged APDs, increased 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

DCM is a myocardial disease characterized by ventricular chamber enlargement and systolic dysfunction and progressive heart failure without significant change in ventricular wall thickness. Mutations in >30 genes encoding proteins of cytoskeleton, sarcomere, and nuclear lamina are found in 30–35% of DCM patients [87]. DCM patients with mutations in *RBM20*, encoding RNA binding motif protein 20 (RBM20), have an early onset of disease phenotype [88]. Isolated CMs from DCM patients carrying mutation in RBM20 displayed elongated and thinner sarcomere structure [88], and such disorganized sarcomeric structure phenotypes were recapitulated in DCM hiPSC-CMs carrying mutation in RBM20 [89, 90]. RBM20 is the main regulator of the heart-specific titin splicing, and N2BA isoform is predominantly expressed in CMs from DCM patient carrying mutation in the *RBM20* gene [91]. In vitro model of RBM20 hiPSC-CMs successfully mirrored the altered titin

current densities, abnormal Ca2+

**70**

isoform expression (titin isoform switch) [89, 90]. Furthermore, RBM20 hiPSC-CMs showed delayed Ca2+ extrusion and reuptake and more Ca2+ being released during each ECC, which resulted into deficient muscle contraction, the hallmark of cardiac dysfunction of DCM patients [89, 90]. In addition, a three-dimensional engineered heart muscle generated from RBM20 hiPSC-CMs showed an impaired force of contraction, and passive stress was decreased in response to stepwise increase in strain, suggesting higher viscoelasticity caused by mutation in *RBM20* [89]. Besides HCM, mutation in cTnT also caused DCM and resulted in shifts in Ca2+ sensitivity and force of contraction [92]. Sun and co-workers generated iPSCs from DCM patients carrying R173W mutation in cTnT and reported that DCM hiPSC-CMs exhibited altered Ca2+ handling, decreased contractility, and abnormal sarcomeric α–actinin distribution [93]. DCM patients with lamin A/C (LMNA) mutations show a highly variable phenotype. Cardiac biopsies from DCM patients harboring LMNA mutations exhibit reduced LMNA in nuclei with nuclear membrane damage such as focal disruption and nuclear pore clustering [94]. Nonsense mutation (R225X) in exon 4 of the LMNA gene causing DCM was associated with accelerated nuclear senescence and apoptosis of DCM hiPSC-CMs under electrical stimulation [95]. In another in vitro modeling of DCM, harboring A285V mutation in desmin (*DES*) using hiPSC-CMs displayed the pathogenic phenotypes of DCM such as diffuse abnormal DES aggregation, poor co-localization of DES with cTnT, and Z-disk streaming with accumulation of granulofilamentous materials or pleomorphic dense structures adjacent to the Z-disk or between the myofibrils [96]. DCM patients harboring R14del mutation in phospholamban (PLN) result in ventricular dilation, contractile dysfunction, and episodic ventricular arrhythmias [97]. Similarly, hiPSC-CMs carrying R14del mutation in PLN induced the Ca2+ handling abnormalities, irregular electrical activity, and abnormal intracellular distribution of PLN in DCM hiPSC-CMs [98]. These PLN R14del-associated disease phenotypes were mitigated upon correction of PLN R14del mutation by transcription activator-like effector nuclease (TALENs) gene editing technique [98]. Furthermore, genetic correction of PLN R14del mutation by TALENs improved the force development and restored the contractile function in threedimensional human engineered cardiac tissue derived from R14del-iPSCs [99].
