Summary Familial hypertrophic cardiomyopathy (HCM) is a prevalent hereditary cardiac disorder linked to arrhythmia and sudden cardiac death. While the causes of HCM have been identified as genetic mutations in the cardiac sarcomere, the pathways by which sarcomeric mutations engender myocyte hypertrophy and electrophysiological abnormalities are not understood. To elucidate the mechanisms underlying HCM development, we generated patient-specific induced pluripotent stem cell cardiomyocytes (iPSC-CMs) from a ten-member family cohort carrying a hereditary HCM missense mutation (Arg663His) in the MYH7 gene. Diseased iPSC-CMs recapitulated numerous aspects of the HCM phenotype including cellular enlargement and contractile arrhythmia at the single-cell level. Calcium (Ca2+) imaging indicated dysregulation of Ca2+ cycling and elevation in intracellular Ca2+ ([Ca2+]i) are central mechanisms for disease pathogenesis. Pharmacological restoration of Ca2+ homeostasis prevented development of hypertrophy and electrophysiological irregularities. We anticipate that these findings will help elucidate the mechanisms underlying HCM development and identify novel therapies for the disease.

Background Cardiotoxicity is a leading cause for drug attrition during pharmaceutical development and has resulted in numerous preventable patient deaths. Incidents of adverse cardiac drug reactions are more common in patients with pre-existing heart disease than the general population. Here we generated a library of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) from patients with various hereditary cardiac disorders to model differences in cardiac drug toxicity susceptibility for patients of different genetic backgrounds. Methods and Results Action potential duration (APD) and drug-induced arrhythmia were measured at the single cell level in hiPSC-CMs derived from healthy subjects and patients with hereditary long QT syndrome (LQT), familial hypertrophic cardiomyopathy (HCM), and familial dilated cardiomyopathy (DCM). Disease phenotypes were verified in LQT, HCM, and DCM iPSC-CMs by immunostaining and single cell patch clamp. Human embryonic stem cell-derived cardiomyocytes (hESC-CMs) and the human ether-a-go-go-related gene (hERG) expressing human embryonic kidney (HEK293) cells were used as controls. Single cell PCR confirmed expression of all cardiac ion channels in patient-specific hiPSC-CMs as well as hESC-CMs, but not in HEK293 cells. Disease-specific hiPSC-CMs demonstrated increased susceptibility to known cardiotoxic drugs as measured by APD and quantification of drug-induced arrhythmias such as early after depolarizations (EADs) and delayed after depolarizations (DADs). Conclusions We have recapitulated drug-induced cardiotoxicity profiles for healthy subjects, LQT, HCM, and DCM patients at the single cell level for the first time. Our data indicate that healthy and diseased individuals exhibit different susceptibilities to cardiotoxic drugs and that use of disease-specific hiPSC-CMs may predict adverse drug responses more accurately than standard hERG test or healthy control hiPSC-CM/hESC-CM screening assays.

Background Drug-induced arrhythmia is the most common cause of drug development failure and withdrawal from market. This study tested whether human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) combined with a low-impedance microelectrode array (MEA) system could improve upon industry-standard, preclinical cardiotoxicity screening methods, identify the effects of well-characterized drugs, and elucidate underlying risk factors for drug-induced arrhythmia. Human iPSC-CMs may be advantageous over immortalized cell lines because they possess similar functional characteristics as primary human cardiomyocytes and can be generated in unlimited quantities. Methods and Results Pharmacological responses of beating embryoid bodies (EBs) exposed to a comprehensive panel of drugs at 65 to 95 days post-induction were determined. Responses of hiPSC-CMs to drugs were qualitatively and quantitatively consistent with the reported drug effects in literature. Torsadogenic hERG blockers such as sotalol and quinidine produced statistically and physiologically significant effects, consistent with patch-clamp studies on human embryonic stem cell-derived cardiomyocytes (hESC-CMs). False negative and false positive hERG blockers were identified accurately. Consistent with published studies using animal models, early afterdepolarizations (EADs) and ectopic beats were observed in 33% and 40% of embryoid bodies treated with sotalol and quinidine, respectively, compared to negligible EADs and ectopic beats in untreated controls. Conclusions We found that drug-induced arrhythmias can be recapitulated in hiPSC-CMs and documented with MEA. Our data indicate that the MEA/hiPSC-CM assay is a sensitive, robust, and efficient platform for testing drug effectiveness and for arrhythmia screening. We believe that this system holds great potential for reducing drug development costs and may provide significant advantages over current industry standard assays that use immortalized cell lines or animal models.

Background Brugada Syndrome is a disorder associated with characteristic ECG precordial ST elevation and predisposes afflicted patients to ventricular fibrillation and sudden cardiac death. Despite marked achievements in outlining the organ level pathophysiology of the disorder, the understanding of human cellular phenotype has lagged due to lack of adequate human cellular models of the disorder. Methods and Results We recruited two patients with Type 1 Brugada Syndrome (BrS) carrying two different SCN5A variants and two healthy controls. We generated induced pluripotent stem cells (iPSCs) from their skin fibroblasts by using integration-free Sendai virus. We utilized directed differentiation to create purified populations of iPSC-derived cardiomyocytes (iPSC-CMs). BrS iPSC-CMs showed reductions in inward Na+ current density and reduced maximal upstroke velocity of action potential compared to healthy control iPSC-CMs. Furthermore, BrS iPSC-CMs showed increased burden of triggered activity, abnormal Ca2+ transients, and beating interval variation. Correction of the causative variant by genome editing was performed and resultant iPSC-CMs showed resolution of triggered activity and abnormal Ca2+ transients. Gene expression profiling of iPSC-CMs showed clustering of BrS compared to controls. Furthermore, BrS iPSC-CM gene expression correlated with gene expression from BrS human cardiac tissue gene expression. Conclusions Patient-specific iPSC-CMs are able to recapitulate single cell phenotype features of BrS, including blunted inward sodium current, increased triggered activity and abnormal Ca2+ handling. This novel human cellular model creates future opportunities to further elucidate cellular disease mechanism and identify novel therapeutic targets.

Background Human pluripotent stem cells (PSCs) play an important role in disease modeling and drug testing. However, the current methods are time consuming and lack an isogenic control. Objectives To establish an efficient technology to generate human PSCs-based disease models with isogenic control. Methods The ion channel genes KCNQ1 and KCNH2 with dominant negative mutations causing long QT syndrome (LQTS) type-1 and -2, respectively, were stably integrated into a safe harbor AAVS1 locus using zinc finger nuclease (ZFN) technology. Results Patch-clamp recording revealed that the edited induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) displayed characteristic LQTS phenotype and significant prolongation of the action-potential duration (APD) compared with the un-edited control cells. Finally, addition of nifedipine (L-type calcium channel blocker) or pinacidil (KATP-channel opener) shortened the APD of iPSC-CMs, confirming the validity of isogenic iPSC lines for drug testing in the future. Conclusions Our study demonstrates that PSC-based disease models can be rapidly generated by overexpression of dominant negative gene mutants.

Induced pluripotent stem cells (iPSCs) hold great hopes for therapeutic application in various diseases. While ongoing research is dedicated to achieving clinical translation of iPSCs, further understanding of the mechanisms that underlie complex pathogenic conditions is required. Compared to other classical models for studying diseases, iPSCs provide considerable advantages. A newly emerging application of iPSCs is in vitro disease modeling, which can significantly improve the never-ending search for new pharmacological cures. Here, we will discuss current efforts to create iPSC-dependent, patient-specific disease models. Furthermore, we will review the use of iPSCs for development and testing of new therapeutic agents, and the implications for high-throughput drug screening.

Background Human-induced pluripotent stem cells (iPSCs) are a potentially unlimited source for generation of cardiomyocytes (iPSC-CMs). However, current protocols for iPSC-CM derivation face a number of challenges, including variability in somatic cell sources and inconsistencies in cardiac differentiation efficiency. Objectives We aimed to assess the effect of epigenetic memory on differentiation and function of iPSC-CMs generated from somatic cell sources of cardiac versus non-cardiac origins. Methods Cardiac progenitor cells (CPCs) and skin fibroblasts from the same donors were reprogrammed into iPSCs and differentiated into iPSC-CMs via embryoid body and monolayer-based differentiation protocols. Results Differentiation efficiency was found to be higher in CPC-derived iPSC-CMs (CPC-iPSC-CMs) than in fibroblast-derived iPSC-CMs (Fib-iPSC-CMs). Gene expression analysis during cardiac differentiation demonstrated upregulation of cardiac transcription factors in CPC-iPSC-CMs, including NKX2-5, MESP1, ISL1, HAND2, MYOCD, MEF2C, and GATA4. Epigenetic assessment revealed higher methylation in the promoter region of NKX2-5 in Fib-iPSC-CMs compared to CPC-iPSC-CMs. Epigenetic differences were found to dissipate with increased cell passaging, and a battery of in vitro assays revealed no significant differences in their morphological and electrophysiological properties at early passage. Finally, cell delivery into small animal myocardial infarction (MI) model indicated that CPC-iPSC-CMs and Fib-iPSC-CMs possess comparable therapeutic capabilities in improving functional recovery in vivo. Conclusions This is the first study to compare differentiation of iPSC-CMs from human CPCs versus human fibroblasts from the same donors. We demonstrate that while epigenetic memory improves differentiation efficiency of cardiac versus non-cardiac somatic cell source in vitro, it does not contribute to improved functional outcome in vivo.

Rational Viral myocarditis is a life-threatening illness that may lead to heart failure or cardiac arrhythmias. A major causative agent for viral myocarditis is the B3 strain of coxsackievirus, a positive-sense RNA enterovirus. However, human cardiac tissues are difficult to procure in sufficient enough quantities for studying the mechanisms of cardiac-specific viral infection. Objective This study examined whether human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) could be used to model the pathogenic processes of coxsackievirus-induced viral myocarditis and to screen antiviral therapeutics for efficacy. Methods and Results Human iPSC-CMs were infected with a luciferase-expressing coxsackievirus B3 strain (CVB3-Luc). Brightfield microscopy, immunofluorescence, and calcium imaging were utilized to characterize virally-infected hiPSC-CMs for alterations in cellular morphology and calcium handling. Viral proliferation in hiPSC-CMs was quantified using bioluminescence imaging. Antiviral compounds including interferon beta 1 (IFNβ1), ribavirin, pyrrolidine dithiocarbamate, and fluoxetine were tested for their capacity to abrogate CVB3-Luc proliferation in hiPSC-CMs in vitro. The ability of these compounds to reduce CVB3-Luc proliferation in hiPSC-CMs was consistent with reported drug effects in previous studies. Mechanistic analyses via gene expression profiling of hiPSC-CMs infected with CVB3-Luc revealed an activation of viral RNA and protein clearance pathways after IFNβ1 treatment. Conclusions This study demonstrates that hiPSC-CMs express the coxsackievirus and adenovirus receptor, are susceptible to coxsackievirus infection, and can be used to predict antiviral drug efficacy. Our results suggest that the hiPSC-CM/CVB3-Luc assay is a sensitive platform that can screen novel antiviral therapeutics for their effectiveness in a high-throughput fashion.