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Induced pluripotent stem cell technology and inherited arrhythmia syndromes

Published:August 17, 2017DOI:https://doi.org/10.1016/j.hrthm.2017.08.013
      Inherited arrhythmia syndromes, including familial long QT syndrome, catecholaminergic polymorphic ventricular tachycardia, and Brugada syndrome, can cause life-threatening arrhythmias and are responsible for a significant proportion of sudden deaths in the young. Identification of genetic mutations and pathophysiological changes that underlie disease development can inform clinical practice and guide novel drug development. However, disease mechanisms in a large number of patients remain elusive and pharmacologic treatment is suboptimal, so many patients rely on implantable cardioverter–defibrillator therapy. Induced pluripotent stem cell models of disease facilitate analysis of disease mechanisms in patient-specific cardiomyocytes, overcoming limitations of animal models and human tissue restrictions. This review outlines how studies using induced pluripotent stem cell–derived cardiomyocytes are contributing to our understanding of the mechanisms that underpin disease pathogenesis and their potential to facilitate new pharmacologic therapies and personalized medicine.

      Keywords

      Abbreviations:

      AP (action potential), ARVC (arrhythmogenic right ventricular cardiomyopathy), BrS (Brugada syndrome), CPVT (catecholaminergic polymorphic ventricular tachycardia), CRISPR (clustered regularly interspaced short palindromic repeats), iPSC (induced pluripotent stem cell), iPSC-CM (induced pluripotent stem cell–derived cardiomyocyte), LQTS (long QT syndrome)

      Introduction

      Inherited arrhythmia syndromes, including long QT syndrome (LQTS), catecholaminergic polymorphic ventricular tachycardia (CPVT), and Brugada syndrome (BrS), present with heterogeneous clinical profiles that range from palpitations to syncope, arrhythmias, and sudden cardiac death. Cumulatively they are responsible for a significant proportion of sudden cardiac deaths in the young.
      • Bagnall R.D.
      • Weintraub R.G.
      • Ingles J.
      • et al.
      A prospective study of sudden cardiac death among children and young adults.
      Despite progress in our understanding of the mechanisms that underlie inherited arrhythmia syndromes, therapies focus on addressing symptoms and complications. Many patients require implantable cardioverter–defibrillator therapy to prevent sudden cardiac death.
      Animal studies have made substantial contributions to our understanding of disease but are limited by species differences.
      • Yang X.
      • Pabon L.
      • Murry C.E.
      Engineering adolescence: maturation of human pluripotent stem cell-derived cardiomyocytes.
      For example, the antiaddiction drug ibogaine can significantly prolong the QT in humans despite its ability to reduce the action potential (AP) duration in guinea pig cardiomyocytes.
      • Rubi L.
      • Eckert D.
      • Boehm S.
      • Hilber K.
      • Koenig X.
      Anti-addiction drug ibogaine prolongs the action potential in human induced pluripotent stem cell-derived cardiomyocytes.
      Assessment of human cardiac tissue is the gold standard; however, adequate samples of heart tissue are difficult to obtain. Induced pluripotent stem cells (iPSCs) provide an alternative source of species- and patient-specific cardiomyocytes.
      Many studies have shown that induced pluripotent stem cell–derived cardiomyocytes (iPSC-CMs) can effectively recapitulate inherited arrhythmia syndromes at the cellular level. Multiple techniques, including calcium imaging and AP analysis, are used to identify electrophysiological abnormalities. The introduction of clustered regularly interspaced short palindromic repeats (CRISPR), a highly efficient and relatively simple to use gene-editing platform, has facilitated the development of more advanced applications of iPSC-CMs.
      • Zhang F.
      • Wen Y.
      • Guo X.
      CRISPR/Cas9 for genome editing: progress, implications and challenges.
      CRISPR can be used to alter gene expression and introduce and correct genetic variation. Despite considerable expense, the use of iPSC-CMs in the pharmacologic industry is rapidly expanding, and drug toxicity risk can be determined in patient-derived iPSC-CMs.
      • Liang P.
      • Lan F.
      • Lee A.S.
      • et al.
      Drug screening using a library of human induced pluripotent stem cell-derived cardiomyocytes reveals disease-specific patterns of cardiotoxicity.
      • Matsa E.
      • Rajamohan D.
      • Dick E.
      • Young L.
      • Mellor I.
      • Staniforth A.
      • Denning C.
      Drug evaluation in cardiomyocytes derived from human induced pluripotent stem cells carrying a LQTS type 2 mutation.
      • Penttinen K.
      • Swan H.
      • Vanninen S.
      • Paavola J.
      • Lahtinen A.M.
      • Kontula K.
      • Aalto-Setala K.
      Antiarrhythmic effects of dantrolene in patients with CPVT and replication of the responses using iPSC models.
      iPSC-CMs have the potential to facilitate personalized medicine with the aim of targeting the most effective therapy for the patient.
      • Burridge P.W.
      • Li Y.F.
      • Matsa E.
      • et al.
      Human induced pluripotent stem cell-derived cardiomyocytes recapitulate the predilection of breast cancer patients to doxorubicin-induced cardiotoxicity.
      However, iPSC technology has limitations that need to be overcome before its true potential can be realized. This review highlights what we have learned from iPSC technology and its clinical role in the inherited arrhythmia syndromes.

       Basics of iPSC technology

       Development of iPSC technology

      iPSCs are embryonic stemlike cells that self-renew and possess the ability to differentiate into cells of all 3 embryonic germ layers.
      • Takahashi K.
      • Tanabe K.
      • Ohnuki M.
      • Narita M.
      • Ichisaka T.
      • Tomoda K.
      • Yamanaka S.
      Induction of pluripotent stem cells from adult human fibroblasts by defined factors.
      iPSCs have been generated from many somatic cells including peripheral blood mononuclear cells and fibroblasts through introduction of the Yamanaka pluripotency factors (OCT-4, SOX-2, KLF-4, and C-MYC).
      • Ross S.B.
      • Fraser S.T.
      • Nowak N.
      • Semsarian C.
      Generation of induced pluripotent stem cells from a hypertrophic cardiomyopathy patient with the pathogenic variant p.Val698Ala in beta-myosin heavy chain gene.
      iPSC and CRISPR gene-editing technologies can be combined to create patient-specific isogenic controls,
      • Wang Y.
      • Liang P.
      • Lan F.
      • et al.
      Genome editing of isogenic human induced pluripotent stem cells recapitulates long QT phenotype for drug testing.
      in which an individual’s genome can be studied with and without the presence of specific genetic variation (Figure 1). In the future, iPSC and CRISPR technologies may be used in the clinical cardiac setting with the ultimate goal of curing disease. In some fields of medicine, gene-editing technology is rapidly moving into the clinic.
      • Cyranoski D.
      CRISPR gene-editing tested in a person for the first time.
      Figure thumbnail gr1
      Figure 1Generation of iPSC-CMs from patients with inherited arrhythmia syndromes. CRISPR = clustered regularly interspaced short palindromic repeats; iPSC = induced pluripotent stem cell; iPSC-CM = induced pluripotent stem cell–derived cardiomyocyte.

       Cardiac differentiation of iPSCs

      iPSCs can be differentiated into iPSC-CMs through chemical manipulation of cardiac developmental pathways (Figure 1).
      • Burridge P.W.
      • Matsa E.
      • Shukla P.
      • et al.
      Chemically defined generation of human cardiomyocytes.
      iPSC-CMs are identified according to the presence of cardiac markers, and the ability to develop cardiac APs and display appropriate drug responses.
      • Itzhaki I.
      • Maizels L.
      • Huber I.
      • et al.
      Modelling LQTS with induced pluripotent stem cells.
      To create models that most accurately reflect disease pathology, iPSC-CMs can be purified and enriched for subtypes, including atrial-, nodal-, and ventricular-like iPSC-CMs.
      • Burridge P.W.
      • Matsa E.
      • Shukla P.
      • et al.
      Chemically defined generation of human cardiomyocytes.
      Although the majority of iPSC-CMs are ventricular-like and can be identified by MLC2v expression and specific AP characteristics, different protocols produce heterogeneous populations of iPSC-CMs, which can introduce variability in cell analysis.
      • Zhang Q.
      • Jiang J.
      • Han P.
      • et al.
      Direct differentiation of atrial and ventricular myocytes from human embryonic stem cells by alternating retinoid signals.
      Cell characteristics can be subtype specific.
      • Chen Z.
      • Xian W.
      • Bellin M.
      • et al.
      Subtype-specific promoter-driven action potential imaging for precise disease modelling and drug testing in hiPSC-derived cardiomyocytes.
      A number of assays can be performed in iPSC-CMs, including analysis of ion channels, calcium handling, APs, and gene expression (Figure 2).
      Figure thumbnail gr2
      Figure 2Assessment of cellular phenotype in induced pluripotent stem cell–derived cardiomyocytes (iPSC-CMs) from patients with inherited arrhythmia syndromes.

       iPSC-CM models of the inherited arrhythmia syndromes

       iPSC-CM models of LQTS

      Congenital LQTS, characterized by prolongation of ventricular repolarization, has a prevalence of up to 1 in 2,000 individuals and is primarily considered to be a monogenic disease with autosomal dominant inheritance.
      • Hofman N.
      • Tan H.L.
      • Alders M.
      • Kolder I.
      • de Haij S.
      • Mannens M.M.
      • Lombardi M.P.
      • Dit Deprez R.H.
      • van Langen I.
      • Wilde A.A.
      Yield of molecular and clinical testing for arrhythmia syndromes.
      Importantly, genetic testing can inform clinical practice and in LQTS has a yield of 60%–75%. The majority of patients present with LQTS types 1–3 (LQT1–3). LQT1 patients are likely to experience exercise-related symptoms and carry mutations in potassium channel subfamily Q member 1 (KCNQ1), which together with KCNE1 encodes the slowly activating delayed rectifier potassium channel. Patients with LQT2 most commonly have events triggered by sudden adrenergic stimuli, such as emotional stress, and carry mutations in potassium channel subfamily H member 2 (KCNH2), which encodes the alpha-subunit of the rapidly activating delayed rectifier potassium channel. Mutations in delayed rectifier potassium channels can prolong the QT interval by reducing the repolarizing current. Patients with LQT3 often experience symptoms at low heart rates (eg, during sleep) and carry mutations in SCN5A, which encodes the pore-forming subunit cardiac sodium channels. Gain-of-function SCN5A mutations are thought to cause LQT3 by increasing the late sodium depolarizing current. Despite differences in clinical presentation and underlying genetic etiology, the majority of LQTS subtypes are primarily treated with beta-blockers, most commonly nadolol and propranolol.
      Since the first iPSC-CM model of LQTS was reported in 2010,
      • Moretti A.
      • Bellin M.
      • Welling A.
      • et al.
      Patient-specific induced pluripotent stem-cell models for LQTS.
      numerous studies have shown that iPSC-CMs demonstrate key features of LQTS, including AP prolongation and early afterdepolarizations (Table 1). Consistent with the genetic etiologies that underlie LQTS, iPSC-CMs display abnormal expression of specific disease-related ion channels.
      • Itzhaki I.
      • Maizels L.
      • Huber I.
      • et al.
      Modelling LQTS with induced pluripotent stem cells.
      • Moretti A.
      • Bellin M.
      • Welling A.
      • et al.
      Patient-specific induced pluripotent stem-cell models for LQTS.
      • Ma D.R.
      • Wei H.M.
      • Lu J.
      • Huang D.
      • Liu Z.F.
      • Loh L.J.
      • Islam O.
      • Liew R.
      • Shim W.
      • Cook S.A.
      Characterization of a novel KCNQ1 mutation for type 1 LQTS and assessment of the therapeutic potential of a novel IKs activator using patient-specific induced pluripotent stem cell-derived cardiomyocytes.
      • Caballero R.
      • Utrilla R.G.
      • Amoros I.
      • et al.
      Tbx20 controls the expression of the KCNH2 gene and of hERG channels.
      Table 1Cellular disease phenotypes present in iPSC-CMs derived from patients with inherited arrhythmia syndromes
      DiseasePhenotype characteristicsDisease genesReference
      LQT1Prolongation of APD, increased susceptibility to EADs and arrhythmias, reduced IKs activationKCNQ1
      • Wang Y.
      • Liang P.
      • Lan F.
      • et al.
      Genome editing of isogenic human induced pluripotent stem cells recapitulates long QT phenotype for drug testing.
      • Moretti A.
      • Bellin M.
      • Welling A.
      • et al.
      Patient-specific induced pluripotent stem-cell models for LQTS.
      • Ma D.R.
      • Wei H.M.
      • Lu J.
      • Huang D.
      • Liu Z.F.
      • Loh L.J.
      • Islam O.
      • Liew R.
      • Shim W.
      • Cook S.A.
      Characterization of a novel KCNQ1 mutation for type 1 LQTS and assessment of the therapeutic potential of a novel IKs activator using patient-specific induced pluripotent stem cell-derived cardiomyocytes.
      • Sala L.
      • Yu Z.
      • Ward-van Oostwaard D.
      • van Veldhoven J.P.
      • Moretti A.
      • Laugwitz K.L.
      • Mummery C.L.
      • AP I.J.
      • Bellin M.
      A new hERG allosteric modulator rescues genetic and drug-induced long-QT syndrome phenotypes in cardiomyocytes from isogenic pairs of patient induced pluripotent stem cells.
      LQT2Prolongation of APD, increased susceptibility to EADs and triggered beats, reduced IKr densityKCNH2
      • Matsa E.
      • Rajamohan D.
      • Dick E.
      • Young L.
      • Mellor I.
      • Staniforth A.
      • Denning C.
      Drug evaluation in cardiomyocytes derived from human induced pluripotent stem cells carrying a LQTS type 2 mutation.
      • Wang Y.
      • Liang P.
      • Lan F.
      • et al.
      Genome editing of isogenic human induced pluripotent stem cells recapitulates long QT phenotype for drug testing.
      • Itzhaki I.
      • Maizels L.
      • Huber I.
      • et al.
      Modelling LQTS with induced pluripotent stem cells.
      • Caballero R.
      • Utrilla R.G.
      • Amoros I.
      • et al.
      Tbx20 controls the expression of the KCNH2 gene and of hERG channels.
      • Lahti A.L.
      • Kujala V.J.
      • Chapman H.
      • et al.
      Model for long QT syndrome type 2 using human iPS cells demonstrates arrhythmogenic characteristics in cell culture.
      • Sala L.
      • Yu Z.
      • Ward-van Oostwaard D.
      • van Veldhoven J.P.
      • Moretti A.
      • Laugwitz K.L.
      • Mummery C.L.
      • AP I.J.
      • Bellin M.
      A new hERG allosteric modulator rescues genetic and drug-induced long-QT syndrome phenotypes in cardiomyocytes from isogenic pairs of patient induced pluripotent stem cells.
      • Liu Q.N.
      • Trudeau M.C.
      Eag domains regulate LQT mutant hERG channels in human induced pluripotent stem cell-derived cardiomyocytes.
      LQT3Prolongation of APD, increased susceptibility to EADs and recovery from sodium channel inactivationSCN5A
      • Malan D.
      • Zhang M.
      • Stallmeyer B.
      • Muller J.
      • Fleischmann B.K.
      • Schulze-Bahr E.
      • Sasse P.
      • Greber B.
      Human iPS cell model of type 3 LQTS recapitulates drug-based phenotype correction.
      • Ma D.
      • Wei H.
      • Zhao Y.
      • Lu J.
      • Li G.
      • Sahib N.B.
      • Tan T.H.
      • Wong K.Y.
      • Shim W.
      • Wong P.
      • Cook S.A.
      • Liew R.
      Modeling type 3 LQTS with cardiomyocytes derived from patient-specific induced pluripotent stem cells.
      LQT15Prolongation of APD, impaired inactivation of l-type Ca2+ channels, reduced beating rateCALM1, CALM2
      • Rocchetti M.
      • Sala L.
      • Dreizehnter L.
      • et al.
      Elucidating arrhythmogenic mechanisms of long-QT syndrome CALM1-F142L mutation in patient-specific induced pluripotent stem cell-derived cardiomyocytes.
      • Yamamoto Y.
      • Makiyama T.
      • Harita T.
      • et al.
      Allele-specific ablation rescues electrophysiological abnormalities in a human iPS cell model of long-QT syndrome with a CALM2 mutation.
      • Limpitikul W.B.
      • Dick I.E.
      • Tester D.J.
      • et al.
      A precision medicine approach to the rescue of function on malignant calmodulinopathic long-QT syndrome.
      CPVT1Increased susceptibility to DADs and spontaneous sarcoplasmic reticulum Ca2+ release, abnormal Ca2+ transients, reduction in RYR2 complexesRYR2
      • Preininger M.K.
      • Jha R.
      • Maxwell J.T.
      • et al.
      A human pluripotent stem cell model of CPVT recapitulates patient-specific drug responses.
      • Fatima A.
      • Xu G.
      • Shao K.
      • et al.
      In vitro modeling of ryanodine receptor 2 dysfunction using human induced pluripotent stem cells.
      • Novak A.
      • Barad L.
      • Lorber A.
      • et al.
      Functional abnormalities in iPSC-derived cardiomyocytes generated from CPVT1 and CPVT2 patients carrying ryanodine or calsequestrin mutations.
      • Paavola J.
      • Vaananen H.
      • Larsson K.
      • Penttinen K.
      • Toivonen L.
      • Kontula K.
      • Laine M.
      • Aalto-Setala K.
      • Swan H.
      • Viitasalo M.
      Slowed depolarization and irregular repolarization in CPVT.
      • Sasaki K.
      • Makiyama T.
      • Yoshida Y.
      • et al.
      Patient-specific human induced pluripotent stem cell model assessed with electrical pacing validates S107 as a potential therapeutic agent for CPVT.
      • Jung C.B.
      • Moretti A.
      • Mederos y Schnitzler M.
      • et al.
      Dantrolene rescues arrhythmogenic RYR2 defect in a patient-specific stem cell model of CPVT.
      CPVT2Increased intracellular Ca2+ concentration, susceptibility to DADs, oscillatory prepotentials and diastolic Ca2+ leakCASQ2
      • Novak A.
      • Barad L.
      • Lorber A.
      • et al.
      Functional abnormalities in iPSC-derived cardiomyocytes generated from CPVT1 and CPVT2 patients carrying ryanodine or calsequestrin mutations.
      • Maizels L.
      • Huber I.
      • Arbel G.
      • Tijsen A.J.
      • Gepstein A.
      • Khoury A.
      • Gepstein L.
      Patient-Specific Drug Screening Using a Human Induced Pluripotent Stem Cell Model of CPVT Type 2.
      • Novak A.
      • Barad L.
      • Zeevi-Levin N.
      • Shick R.
      • Shtrichman R.
      • Lorber A.
      • Itskovitz-Eldor J.
      • Binah O.
      Cardiomyocytes generated from CPVTD307H patients are arrhythmogenic in response to beta-adrenergic stimulation.
      BrSIncreased beating interval variation and abnormal Ca2+ transients, reduced sodium current and maximal upstroke velocity of action potentialSCN5A
      • Liang P.
      • Sallam K.
      • Wu H.
      • et al.
      Patient-specific and genome-edited induced pluripotent stem cell-derived cardiomyocytes elucidate single-cell phenotype of Brugada syndrome.
      • Veerman C.C.
      • Mengarelli I.
      • Guan K.
      • et al.
      hiPSC-derived cardiomyocytes from Brugada Syndrome patients without identified mutations do not exhibit clear cellular electrophysiological abnormalities.
      • Cerrone M.
      • Lin X.
      • Zhang M.
      • et al.
      Missense mutations in plakophilin-2 cause sodium current deficit and associate with a Brugada syndrome phenotype.
      APD = action potential duration; BrS = Brugada syndrome; CPVT1, 2 = catecholaminergic polymorphic ventricular tachycardia type 1 or 2; DAD = delayed afterdepolarization; EAD = early afterdepolarization; IPSC-CM = induced pluripotent stem cell–derived cardiomyocyte; LQT1, 2, 3, 15 = long QT syndrome type 1, 2, 3, or 15.

       Genetics and molecular mechanisms that underlie LQTS

      Functional studies using iPSC-CMs can provide evidence regarding the pathogenicity of gene mutations. Identification of a clinically meaningful mutation relies on meeting classification criteria with determinants of pathogenicity, including rarity, previous association with disease, and segregation and experimental data.
      • Burns C.
      • Ingles J.
      • Davis A.M.
      • Connell V.
      • Gray B.
      • Hunt L.
      • McGaughran J.
      • Semsarian C.
      Clinical and genetic features of Australian families with long QT syndrome.
      iPSC-CM models have been used to better understand the pathogenic nature of mutations identified in families with LQTS.
      • Caballero R.
      • Utrilla R.G.
      • Amoros I.
      • et al.
      Tbx20 controls the expression of the KCNH2 gene and of hERG channels.
      • Terrenoire C.
      • Wang K.
      • Tung K.W.
      • et al.
      Induced pluripotent stem cells used to reveal drug actions in a long QT syndrome family with complex genetics.
      With the introduction of CRISPR, the number of studies that use iPSC-CMs and gene editing to delineate disease-causing mutations from background noise is likely to increase substantially.
      Patient-derived iPSC-CMs can also be used to identify the pathophysiological changes that link gene mutations to disease phenotypes. Analysis of patient-derived iPSC-CMs revealed that heterozygous mutations in calmodulin (CALM1-3) cause LQT15 through dominant negative mutation mechanisms.
      • Rocchetti M.
      • Sala L.
      • Dreizehnter L.
      • et al.
      Elucidating arrhythmogenic mechanisms of long-QT syndrome CALM1-F142L mutation in patient-specific induced pluripotent stem cell-derived cardiomyocytes.
      • Yamamoto Y.
      • Makiyama T.
      • Harita T.
      • et al.
      Allele-specific ablation rescues electrophysiological abnormalities in a human iPS cell model of long-QT syndrome with a CALM2 mutation.
      • Limpitikul W.B.
      • Dick I.E.
      • Tester D.J.
      • et al.
      A precision medicine approach to the rescue of function on malignant calmodulinopathic long-QT syndrome.
      This discovery explains why mutations in just 1 of 6 identical calmodulin alleles cause such a severe LQTS phenotype and may facilitate development of new gene therapies.
      • Yamamoto Y.
      • Makiyama T.
      • Harita T.
      • et al.
      Allele-specific ablation rescues electrophysiological abnormalities in a human iPS cell model of long-QT syndrome with a CALM2 mutation.
      • Limpitikul W.B.
      • Dick I.E.
      • Tester D.J.
      • et al.
      A precision medicine approach to the rescue of function on malignant calmodulinopathic long-QT syndrome.
      Future exploration of mutation mechanisms with iPSC-CMs may help us understand why specific mutations, or mutations in the same genes, can cause distinct disease phenotypes, best illustrated by gain-of-function SCN5A mutations, which cause LQT3, whereas loss-of-function SCN5A mutations result in BrS.
      Multiple studies have used iPSC-CMs to explore the physiological changes that occur in LQTS. Moretti et al
      • Moretti A.
      • Bellin M.
      • Welling A.
      • et al.
      Patient-specific induced pluripotent stem-cell models for LQTS.
      used iPSC-CMs derived from an LQT1 patient with the KCNQ1 mutation p.R190Q to show that the primary change underlying the LQT1 phenotype was a reduction in the slow outward potassium current. Studies have also identified increased recovery from inactivation of depolarizing sodium channels in iPSC-CMs derived from LQT3 patients with mutations in SCN5A.
      • Terrenoire C.
      • Wang K.
      • Tung K.W.
      • et al.
      Induced pluripotent stem cells used to reveal drug actions in a long QT syndrome family with complex genetics.
      • Malan D.
      • Zhang M.
      • Stallmeyer B.
      • Muller J.
      • Fleischmann B.K.
      • Schulze-Bahr E.
      • Sasse P.
      • Greber B.
      Human iPS cell model of type 3 LQTS recapitulates drug-based phenotype correction.
      In these cases, effective treatment with mexiletine further supports the role of inappropriate sodium channel activity in pathological AP prolongation.
      The rise in genetic testing has resulted in increased identification of asymptomatic mutation carriers, which highlights the issue of incomplete penetrance. Analysis of iPSC-CMs from asymptomatic mutation carriers may help us to understand the variable disease penetrance seen in LQTS. Lahti et al
      • Lahti A.L.
      • Kujala V.J.
      • Chapman H.
      • et al.
      Model for long QT syndrome type 2 using human iPS cells demonstrates arrhythmogenic characteristics in cell culture.
      generated iPSC-CMs from an asymptomatic carrier of LQT2 Finnish founder mutation KCNH2 p.R176W with a significant family history of sudden cardiac death. Despite the asymptomatic nature of the patient, iPSC-CMs displayed significant AP prolongation and potassium current reduction in comparison to controls. These findings suggest that asymptomatic mutation carriers have increased susceptibility to developing LQTS and that multiple genetic or environmental factors may be required for full disease onset.

       Pharmacologic treatment of LQTS

      iPSC-CMs derived from LQTS patients show promise for enhanced pharmacologic development and safety testing (Table 2). Importantly, patient and patient-derived iPSC-CM drug responses appear to be consistent.
      • Matsa E.
      • Rajamohan D.
      • Dick E.
      • Young L.
      • Mellor I.
      • Staniforth A.
      • Denning C.
      Drug evaluation in cardiomyocytes derived from human induced pluripotent stem cells carrying a LQTS type 2 mutation.
      • Terrenoire C.
      • Wang K.
      • Tung K.W.
      • et al.
      Induced pluripotent stem cells used to reveal drug actions in a long QT syndrome family with complex genetics.
      Patient-derived iPSC-CMs demonstrate responses to adrenergic stress, such as QT prolongation and early afterdepolarizations, which are effectively treated with beta-blocker therapy.
      • Moretti A.
      • Bellin M.
      • Welling A.
      • et al.
      Patient-specific induced pluripotent stem-cell models for LQTS.
      Table 2Cellular responses to drug treatment in iPSC-CMs derived from patients with inherited arrhythmia syndromes
      DiseaseCompoundsDrug classOutcomeReference
      LQT1IsoproterenolBeta-adrenergic agonistIncreased APD and arrhythmia susceptibility
      • Moretti A.
      • Bellin M.
      • Welling A.
      • et al.
      Patient-specific induced pluripotent stem-cell models for LQTS.
      PropranololBeta-blockerDecreased proarrhythmic effects after isoproterenol treatment
      • Moretti A.
      • Bellin M.
      • Welling A.
      • et al.
      Patient-specific induced pluripotent stem-cell models for LQTS.
      ML277IKs activatorIncreased activation of IKs, reduction in APD
      • Ma D.R.
      • Wei H.M.
      • Lu J.
      • Huang D.
      • Liu Z.F.
      • Loh L.J.
      • Islam O.
      • Liew R.
      • Shim W.
      • Cook S.A.
      Characterization of a novel KCNQ1 mutation for type 1 LQTS and assessment of the therapeutic potential of a novel IKs activator using patient-specific induced pluripotent stem cell-derived cardiomyocytes.
      LQT2E-4031, cisaprideK+ channel antagonistsProlonged APD, increased number and complexity of EADs
      • Matsa E.
      • Rajamohan D.
      • Dick E.
      • Young L.
      • Mellor I.
      • Staniforth A.
      • Denning C.
      Drug evaluation in cardiomyocytes derived from human induced pluripotent stem cells carrying a LQTS type 2 mutation.
      • Itzhaki I.
      • Maizels L.
      • Huber I.
      • et al.
      Modelling LQTS with induced pluripotent stem cells.
      NifedipineCa2+ channel antagonistReduced APD, eliminated EADs and triggered beats
      • Itzhaki I.
      • Maizels L.
      • Huber I.
      • et al.
      Modelling LQTS with induced pluripotent stem cells.
      Pinacidil, NicorandilK+ channel agonistsReduced APD, eliminated EADs and triggered beats
      • Matsa E.
      • Rajamohan D.
      • Dick E.
      • Young L.
      • Mellor I.
      • Staniforth A.
      • Denning C.
      Drug evaluation in cardiomyocytes derived from human induced pluripotent stem cells carrying a LQTS type 2 mutation.
      • Itzhaki I.
      • Maizels L.
      • Huber I.
      • et al.
      Modelling LQTS with induced pluripotent stem cells.
      RanolazineLate Na+ channel agonistNo effect on APD, reduced number of EADs and triggered beats
      • Itzhaki I.
      • Maizels L.
      • Huber I.
      • et al.
      Modelling LQTS with induced pluripotent stem cells.
      IsoprenalineBeta-adrenergic agonistReduced APD, increased susceptibility to EADs
      • Matsa E.
      • Rajamohan D.
      • Dick E.
      • Young L.
      • Mellor I.
      • Staniforth A.
      • Denning C.
      Drug evaluation in cardiomyocytes derived from human induced pluripotent stem cells carrying a LQTS type 2 mutation.
      LUF7346Allosteric modulator of hERGReduced APD and EAD development
      • Sala L.
      • Yu Z.
      • Ward-van Oostwaard D.
      • van Veldhoven J.P.
      • Moretti A.
      • Laugwitz K.L.
      • Mummery C.L.
      • AP I.J.
      • Bellin M.
      A new hERG allosteric modulator rescues genetic and drug-induced long-QT syndrome phenotypes in cardiomyocytes from isogenic pairs of patient induced pluripotent stem cells.
      LQT3Mexiletine, ranolazine, phenytoinNa+ channel antagonistsReduced APD, eliminated EADs
      • Malan D.
      • Zhang M.
      • Stallmeyer B.
      • Muller J.
      • Fleischmann B.K.
      • Schulze-Bahr E.
      • Sasse P.
      • Greber B.
      Human iPS cell model of type 3 LQTS recapitulates drug-based phenotype correction.
      • Ma D.
      • Wei H.
      • Zhao Y.
      • Lu J.
      • Li G.
      • Sahib N.B.
      • Tan T.H.
      • Wong K.Y.
      • Shim W.
      • Wong P.
      • Cook S.A.
      • Liew R.
      Modeling type 3 LQTS with cardiomyocytes derived from patient-specific induced pluripotent stem cells.
      LQT15Verapamil, amlodipineCa2+ channel antagonistsReduced QTc
      • Rocchetti M.
      • Sala L.
      • Dreizehnter L.
      • et al.
      Elucidating arrhythmogenic mechanisms of long-QT syndrome CALM1-F142L mutation in patient-specific induced pluripotent stem cell-derived cardiomyocytes.
      CPVT1IsoproterenolBeta-adrenergic agonistNegative chronotropic response, increased arrhythmias, DADs, Ca2+ sparks, and diastolic intracellular Ca2+ concentration
      • Preininger M.K.
      • Jha R.
      • Maxwell J.T.
      • et al.
      A human pluripotent stem cell model of CPVT recapitulates patient-specific drug responses.
      • Fatima A.
      • Xu G.
      • Shao K.
      • et al.
      In vitro modeling of ryanodine receptor 2 dysfunction using human induced pluripotent stem cells.
      • Novak A.
      • Barad L.
      • Lorber A.
      • et al.
      Functional abnormalities in iPSC-derived cardiomyocytes generated from CPVT1 and CPVT2 patients carrying ryanodine or calsequestrin mutations.
      DantroleneRYR2 antagonistReduced Ca2+ sparks and beat irregularity with beta-adrenergic stimulation
      • Jung C.B.
      • Moretti A.
      • Mederos y Schnitzler M.
      • et al.
      Dantrolene rescues arrhythmogenic RYR2 defect in a patient-specific stem cell model of CPVT.
      NadololBeta-blockerNegligible reduction in Ca2+ waves and sparks
      • Paavola J.
      • Vaananen H.
      • Larsson K.
      • Penttinen K.
      • Toivonen L.
      • Kontula K.
      • Laine M.
      • Aalto-Setala K.
      • Swan H.
      • Viitasalo M.
      Slowed depolarization and irregular repolarization in CPVT.
      FlecainideNa+ channel antagonistReduced frequency and duration of Ca2+ waves and sparks
      • Paavola J.
      • Vaananen H.
      • Larsson K.
      • Penttinen K.
      • Toivonen L.
      • Kontula K.
      • Laine M.
      • Aalto-Setala K.
      • Swan H.
      • Viitasalo M.
      Slowed depolarization and irregular repolarization in CPVT.
      Abbreviations as in Table 1.
      Patient-derived iPSC-CMs have been used to assess the potential of different drug classes, including calcium and late sodium channel antagonists and potassium channel agonists, to reduce AP duration and ameliorate the LQTS phenotype (Table 2). Manipulation of hERG to increase the delayed rectifying potassium current has demonstrated efficacy in congenital and drug-induced forms of LQTS and is being extensively investigated.
      • Sala L.
      • Yu Z.
      • Ward-van Oostwaard D.
      • van Veldhoven J.P.
      • Moretti A.
      • Laugwitz K.L.
      • Mummery C.L.
      • AP I.J.
      • Bellin M.
      A new hERG allosteric modulator rescues genetic and drug-induced long-QT syndrome phenotypes in cardiomyocytes from isogenic pairs of patient induced pluripotent stem cells.
      Significant work is still required to move these therapeutic approaches into the clinic, specifically to determine how to identify optimal drug concentrations and prevent the development of a short QT interval. However, these early studies with patient-derived iPSC-CMs are facilitating the exploration of new, much needed drugs for LQTS.
      Patient-derived iPSC-CMs provide an unprecedented opportunity to explore individual responses to drug therapies. Consistent with clinical observations, iPSC-CMs derived from LQT3 patients are more effectively treated with different sodium channel inhibitors than with beta-blockers.
      • Malan D.
      • Zhang M.
      • Stallmeyer B.
      • Muller J.
      • Fleischmann B.K.
      • Schulze-Bahr E.
      • Sasse P.
      • Greber B.
      Human iPS cell model of type 3 LQTS recapitulates drug-based phenotype correction.
      Additionally, using iPSC-CMs from an LQT3 patient with suboptimal drug therapy, Terrenoire et al
      • Terrenoire C.
      • Wang K.
      • Tung K.W.
      • et al.
      Induced pluripotent stem cells used to reveal drug actions in a long QT syndrome family with complex genetics.
      found that a combination of cardiac pacing and mexiletine treatment was more beneficial than addition of a second sodium channel inhibitor. Importantly, these in vitro findings are consistent with best clinical treatment of the patient to date. Overall, these studies support preclinical assessment of drug efficacy with patient-derived iPSC-CMs for the development of personalized treatment strategies.

       iPSC-CM models of CPVT

      CPVT is a severe arrhythmia syndrome with an estimated prevalence of 1 in 10,000 individuals.
      • Pflaumer A.
      • Davis A.M.
      Guidelines for the diagnosis and management of CPVT.
      Beta-blockers are the main form of treatment of CPVT but are ineffective in a large proportion of patients. CPVT is primarily caused by mutations in the cardiac ryanodine receptor (RYR2) and cardiac calsequestrin 2 (CASQ2).
      • Gray B.
      • Bagnall R.D.
      • Lam L.
      • Ingles J.
      • Turner C.
      • Haan E.
      • Davis A.
      • Yang P.C.
      • Clancy C.E.
      • Sy R.W.
      • Semsarian C.
      A novel heterozygous mutation in cardiac calsequestrin causes autosomal dominant CPVT.
      Mutations in RYR2 cause type 1 CPVT (CPVT1), which accounts for 50%–60% of CPVT cases. Mutations in CASQ2 cause type 2 CPVT (CPVT2), a more rare autosomal recessive form of CPVT. A small number of CASQ2 mutations that cause an autosomal dominant form of CPVT2 have been identified.
      • Gray B.
      • Bagnall R.D.
      • Lam L.
      • Ingles J.
      • Turner C.
      • Haan E.
      • Davis A.
      • Yang P.C.
      • Clancy C.E.
      • Sy R.W.
      • Semsarian C.
      A novel heterozygous mutation in cardiac calsequestrin causes autosomal dominant CPVT.
      RYR2 and CASQ2 proteins are major components of a complex that controls coordinated release of calcium from the sarcoplasmic reticulum during systole. Consistently, genetic etiologies and animal studies suggest that CPVT primarily results from abnormal storage and release of calcium from the sarcoplasmic reticulum.
      • Knollmann B.C.
      • Chopra N.
      • Hlaing T.
      • et al.
      Casq2 deletion causes sarcoplasmic reticulum volume increase, premature Ca2+ release, and CPVT.
      Analysis of iPSC-CM lines derived from CPVT patients has revealed a cellular phenotype of CPVT (Table 1). Consistent with the absence of a baseline arrhythmic phenotype in CPVT patients, the majority of CPVT iPSC-CMs require beta-adrenergic stimulation with drugs such as isoproterenol to induce arrhythmic features.
      • Preininger M.K.
      • Jha R.
      • Maxwell J.T.
      • et al.
      A human pluripotent stem cell model of CPVT recapitulates patient-specific drug responses.
      However, underlying abnormalities in calcium handling have been identified in CPVT1 iPSC-CMs at baseline.
      • Fatima A.
      • Xu G.
      • Shao K.
      • et al.
      In vitro modeling of ryanodine receptor 2 dysfunction using human induced pluripotent stem cells.
      Isoproterenol-treated iPSC-CMs from a CPVT1 patient displayed a negative chronotropic response with development of arrhythmias.
      • Fatima A.
      • Xu G.
      • Shao K.
      • et al.
      In vitro modeling of ryanodine receptor 2 dysfunction using human induced pluripotent stem cells.
      This abnormal response to beta-adrenergic stimulation is consistent with arrhythmia development in CPVT patients and iPSC-CMs derived from both CPVT1 and CPVT2 patients.
      • Novak A.
      • Barad L.
      • Lorber A.
      • et al.
      Functional abnormalities in iPSC-derived cardiomyocytes generated from CPVT1 and CPVT2 patients carrying ryanodine or calsequestrin mutations.

       Molecular mechanisms that underlie CPVT

      The main mechanism of CPVT development is considered to be aberrant diastolic calcium release from the sarcoplasmic reticulum.
      • Lehnart S.E.
      • Wehrens X.H.
      • Laitinen P.J.
      • Reiken S.R.
      • Deng S.X.
      • Cheng Z.
      • Landry D.W.
      • Kontula K.
      • Swan H.
      • Marks A.R.
      Sudden death in familial polymorphic ventricular tachycardia associated with calcium release channel (ryanodine receptor) leak.
      • Paavola J.
      • Vaananen H.
      • Larsson K.
      • Penttinen K.
      • Toivonen L.
      • Kontula K.
      • Laine M.
      • Aalto-Setala K.
      • Swan H.
      • Viitasalo M.
      Slowed depolarization and irregular repolarization in CPVT.
      Analysis of iPSC-CMs derived from CPVT1 patients suggests that disease-causing RYR2 mutations are gain-of-function mutations that result in calcium leak due to increased open probability of RYR2.
      • Preininger M.K.
      • Jha R.
      • Maxwell J.T.
      • et al.
      A human pluripotent stem cell model of CPVT recapitulates patient-specific drug responses.
      • Fatima A.
      • Xu G.
      • Shao K.
      • et al.
      In vitro modeling of ryanodine receptor 2 dysfunction using human induced pluripotent stem cells.
      Calcium handling abnormalities identified in CPVT1 iPSC-CMs include reduced calcium transient amplitudes and sarcoplasmic reticulum calcium stores and increased RYR2-mediated SR calcium leak and fractional calcium release.
      • Preininger M.K.
      • Jha R.
      • Maxwell J.T.
      • et al.
      A human pluripotent stem cell model of CPVT recapitulates patient-specific drug responses.
      Evidence for a pathogenic increase in the open probability of RYR2 supports the use of compounds such as S107, which can increase binding of calstabin-2 to RYR2 promoting the closed RYR2 state.
      • Sasaki K.
      • Makiyama T.
      • Yoshida Y.
      • et al.
      Patient-specific human induced pluripotent stem cell model assessed with electrical pacing validates S107 as a potential therapeutic agent for CPVT.
      Dantrolene, which stabilizes the closed state of RYR2, has been shown to prevent arrhythmia development in iPSC-CMs derived from CPVT1 patients.
      • Jung C.B.
      • Moretti A.
      • Mederos y Schnitzler M.
      • et al.
      Dantrolene rescues arrhythmogenic RYR2 defect in a patient-specific stem cell model of CPVT.
      The efficacy of dantrolene, which increases interdomain interactions in RYR2, supports the domain unzipping mechanism, whereby the interactions between domains in RYR2 are weakened due to RYR2 mutations and result in an increased open probability.
      Patient-derived iPSC-CMs have been used to explore mechanistic differences between CPVT1 and CPVT2.
      • Novak A.
      • Barad L.
      • Lorber A.
      • et al.
      Functional abnormalities in iPSC-derived cardiomyocytes generated from CPVT1 and CPVT2 patients carrying ryanodine or calsequestrin mutations.
      Although both CPVT1 and CPVT2 iPSC-CMs displayed immature ultrastructures, reduced ryanodine receptor expression, and increased arrhythmia susceptibility after adrenergic stimulation, underlying differences were identified between the 2 forms of CPVT. CPVT1 and CPVT2 iPSC-CMs demonstrated disparities in the storage and stimulated release of calcium from the sarcoplasmic reticulum. Importantly, beta-adrenergic stimulation of both CPVT1 and CPVT2 iPSC-CMs with isoproterenol resulted in a marked increase in diastolic intracellular calcium concentration, a key feature of CPVT.

       Pharmacologic treatment of CPVT

      Current pharmacologic treatment of CPVT is suboptimal, and both patients and clinicians would benefit from preclinical assessment of patient-specific drug efficacy. Supporting the use of iPSC-CM pharmacology testing to guide patient therapy, multiple studies have shown that drug responses in iPSC-CMs correlate strongly with clinical responses in the CPVT patients from whom they were derived.
      • Penttinen K.
      • Swan H.
      • Vanninen S.
      • Paavola J.
      • Lahtinen A.M.
      • Kontula K.
      • Aalto-Setala K.
      Antiarrhythmic effects of dantrolene in patients with CPVT and replication of the responses using iPSC models.
      • Preininger M.K.
      • Jha R.
      • Maxwell J.T.
      • et al.
      A human pluripotent stem cell model of CPVT recapitulates patient-specific drug responses.
      • Maizels L.
      • Huber I.
      • Arbel G.
      • Tijsen A.J.
      • Gepstein A.
      • Khoury A.
      • Gepstein L.
      Patient-Specific Drug Screening Using a Human Induced Pluripotent Stem Cell Model of CPVT Type 2.
      Preininger et al
      • Preininger M.K.
      • Jha R.
      • Maxwell J.T.
      • et al.
      A human pluripotent stem cell model of CPVT recapitulates patient-specific drug responses.
      and Maizels et al
      • Maizels L.
      • Huber I.
      • Arbel G.
      • Tijsen A.J.
      • Gepstein A.
      • Khoury A.
      • Gepstein L.
      Patient-Specific Drug Screening Using a Human Induced Pluripotent Stem Cell Model of CPVT Type 2.
      evaluated drug responses in iPSC-CMs derived from CPVT1 and CPVT2 patients, respectively, whose arrhythmic events were not clinically treatable with widely used beta-blockers. Consistent with patient clinical responses, iPSC-CMs demonstrated only a minimal antiarrhythmic response to beta-blocker treatment. Alternatively, both patient and iPSC-CMs responded more effectively to flecainide therapy. Further comprehensive studies are required to move this technology into the clinic and to better understand how genetic variation can affect drug efficacy.
      Table 2 summarizes some of the drug compounds that have been evaluated with iPSC-CMs derived from CPVT1 patients. Recent iPSC-CM studies and a small clinical trial in CPVT1 patients have provided evidence supporting the use of drugs, such as dantrolene, that increase the closed state of RYR2.
      • Penttinen K.
      • Swan H.
      • Vanninen S.
      • Paavola J.
      • Lahtinen A.M.
      • Kontula K.
      • Aalto-Setala K.
      Antiarrhythmic effects of dantrolene in patients with CPVT and replication of the responses using iPSC models.
      However, the efficacy of dantrolene appears to be dependent on mutation location,
      • Penttinen K.
      • Swan H.
      • Vanninen S.
      • Paavola J.
      • Lahtinen A.M.
      • Kontula K.
      • Aalto-Setala K.
      Antiarrhythmic effects of dantrolene in patients with CPVT and replication of the responses using iPSC models.
      which suggests that optimal treatment of CPVT1 may require different pathways to be targeted even among individuals with mutations in the same genes. Patient and iPSC-CMs revealed consistent responses to dantrolene, supporting the ability of iPSC-CMs to accurately model personalized drug responses in the context of CPVT1.
      • Penttinen K.
      • Swan H.
      • Vanninen S.
      • Paavola J.
      • Lahtinen A.M.
      • Kontula K.
      • Aalto-Setala K.
      Antiarrhythmic effects of dantrolene in patients with CPVT and replication of the responses using iPSC models.

       iPSC-CM models of BrS

      BrS is characterized by ST-segment elevation and a significant risk of life-threatening ventricular fibrillation and sudden cardiac death.
      • Antzelevitch C.
      • Brugada P.
      • Borggrefe M.
      • et al.
      Brugada syndrome: report of the second consensus conference.
      BrS is increasingly being referred to as an overlap syndrome, following its identification in patients with features of other cardiomyopathies.
      • Corrado D.
      • Basso C.
      • Buja G.
      • Nava A.
      • Rossi L.
      • Thiene G.
      Right bundle branch block, right precordial ST-segment elevation, and sudden death in young people.
      Management of BrS patients is limited primarily to lifestyle modifications, with symptomatic patients requiring implantable cardioverter–defibrillator therapy. New treatment options, such as quinidine therapy and ablation, are being explored. BrS is considered a monogenic syndrome, although complex genetic mechanisms have been reported. The genetic yield in BrS is only 20%–25% (mainly SCN5A), with the majority of BrS cases remaining genetically elusive.
      Despite evidence that arrhythmic features of BrS result from tissue-level conduction abnormalities,
      • Yan G.X.
      • Antzelevitch C.
      Cellular basis for the Brugada syndrome and other mechanisms of arrhythmogenesis associated with ST-segment elevation.
      cellular features of BrS are identifiable in iPSC-CMs derived from patients who carry SCN5A mutations (Table 1). iPSC-CMs derived from BrS patients with SCN5A mutations display abnormal AP profiles and a reduction in sodium channel function with significantly slower depolarization, increased triggered activity, and calcium transient abnormalities.
      • Liang P.
      • Sallam K.
      • Wu H.
      • et al.
      Patient-specific and genome-edited induced pluripotent stem cell-derived cardiomyocytes elucidate single-cell phenotype of Brugada syndrome.
      Encouragingly, gene expression patterns in iPSC-CMs derived from 2 unrelated BrS patients correlated with gene expression patterns identified in cardiac tissue isolated from BrS patients.
      • Liang P.
      • Sallam K.
      • Wu H.
      • et al.
      Patient-specific and genome-edited induced pluripotent stem cell-derived cardiomyocytes elucidate single-cell phenotype of Brugada syndrome.
      The underlying etiology of BrS remains unclear and is likely a combination of multiple genetic factors interacting with a range of environmental influences. Veerman et al
      • Veerman C.C.
      • Mengarelli I.
      • Guan K.
      • et al.
      hiPSC-derived cardiomyocytes from Brugada Syndrome patients without identified mutations do not exhibit clear cellular electrophysiological abnormalities.
      found that a clear cellular phenotype of BrS was not identifiable in iPSC-CMs derived from genotype-negative BrS patients. This finding may support recent suggestions that BrS can present as an overlap syndrome resulting from a number of different underlying pathophysiological mechanisms.
      • Gray B.
      • Semsarian C.
      • Sy R.W.
      Brugada syndrome: a heterogeneous disease with a common ECG phenotype?.
      Furthermore, it suggests that the approximately 80% of patients with inconclusive genetic testing may not have underlying defects in ion channel function, and many oligogenic or nongenetic factors may contribute to BrS development. The BrS phenotype in these patients may be the result of a complex interaction of multiple cell types, genetics, and the environment, and therefore iPSC-CMs may be an unsuitable model. As the technology advances and limitations, primarily with respect to maturation, are overcome, a cellular disease phenotype may become identifiable.

       Genetic and molecular mechanisms that underlie BrS

      CRISPR gene editing has been used to correct SCN5A mutations in patient-derived iPSC-CMs, resulting in resolution of the BrS phenotype with respect to both AP and calcium handling abnormalities.
      • Liang P.
      • Sallam K.
      • Wu H.
      • et al.
      Patient-specific and genome-edited induced pluripotent stem cell-derived cardiomyocytes elucidate single-cell phenotype of Brugada syndrome.
      Additional analyses comparing the cellular phenotype of corrected iPSC-CMs to control iPSC-CMs would be of value in determining the contribution of genetic background to BrS. Such comparisons will be important because the genetic etiologies are not well understood, and inheritance patterns do not consistently support a monogenic disease.
      • Cho H.C.
      Modeling and genome-editing Brugada syndrome in a dish.
      iPSC-CMs have been used to assess whether mutations in plakophilin-2 (PKP2), a well-described cause of arrhythmogenic right ventricular cardiomyopathy (ARVC), can cause a BrS phenotype.
      • Cerrone M.
      • Lin X.
      • Zhang M.
      • et al.
      Missense mutations in plakophilin-2 cause sodium current deficit and associate with a Brugada syndrome phenotype.
      PKP2 is a component of the cardiac desmosome, which is important for intercellular communication and myocardium integrity. After screening of 200 otherwise genotype-negative BrS patients, 5 probands were found to carry missense mutations in PKP2. Assessment of iPSC-CMs derived from the 5 patients revealed findings consistent with previous BrS studies using animal models and cardiac cell lines. iPSC-CMs with mutant PKP2 had reduced sodium channel expression and current, thus supporting a role for PKP2 mutations in BrS. Further work is required to understand the role of PKP2 mutations in BrS and the underlying disease mechanisms that result in an overlap syndrome of both BrS and ARVC.
      As the number of iPSC-CM studies assessing BrS increases, we will likely see a larger contribution of this technology to our understanding of disease development. In a recent study, Liang et al
      • Liang P.
      • Sallam K.
      • Wu H.
      • et al.
      Patient-specific and genome-edited induced pluripotent stem cell-derived cardiomyocytes elucidate single-cell phenotype of Brugada syndrome.
      found that iPSC-CMs derived from BrS patients with SCN5A mutations have significantly reduced sodium channel function and expression at the cell membrane, which supports previous indications of reduced sodium channel expression in BrS. A reduction in maximum intracellular calcium concentration and rate of depolarization was also identified. Analysis also revealed a decrease in the expression of KCNJ2, which encodes Kir2.1 inwardly rectifying potassium channels and may represent a compensatory mechanism.

       iPSC models of inherited cardiomyopathies

      iPSC-CMs derived from patients with inherited cardiomyopathies such as ARVC have been used to explore disease mechanisms and drug development.
      • Lan F.
      • Lee A.S.
      • Liang P.
      • et al.
      Abnormal calcium handling properties underlie familial hypertrophic cardiomyopathy pathology in patient-specific induced pluripotent stem cells.
      • Ma D.
      • Wei H.
      • Lu J.
      • et al.
      Generation of patient-specific induced pluripotent stem cell-derived cardiomyocytes as a cellular model of ARVC.
      • Tanaka A.
      • Yuasa S.
      • Mearini G.
      • et al.
      Endothelin-1 induces myofibrillar disarray and contractile vector variability in hypertrophic cardiomyopathy-induced pluripotent stem cell-derived cardiomyocytes.
      • Tse H.F.
      • Ho J.C.
      • Choi S.W.
      • et al.
      Patient-specific induced-pluripotent stem cells-derived cardiomyocytes recapitulate the pathogenic phenotypes of dilated cardiomyopathy due to a novel DES mutation identified by whole exome sequencing.
      • Ross S.B.
      • Fraser S.T.
      • Semsarian C.
      Induced pluripotent stem cells in the inherited cardiomyopathies: From disease mechanisms to novel therapies.
      Multiple studies have identified cellular characteristics of ARVC, including reduced PKP2 protein, increased lipid accumulation, and apoptosis in iPSC-CMs.
      • Akdis D.
      • Saguner A.M.
      • Shah K.
      • et al.
      Sex hormones affect outcome in ARVC/D: from a stem cell derived cardiomyocyte-based model to clinical biomarkers of disease outcome.
      • Caspi O.
      • Huber I.
      • Gepstein A.
      • Arbel G.
      • Maizels L.
      • Boulos M.
      • Gepstein L.
      Modeling of ARVC with human induced pluripotent stem cells.
      • Kim C.
      • Wong J.
      • Wen J.
      • et al.
      Studying ARVD with patient-specific iPSCs.
      • Te Riele A.S.
      • Agullo-Pascual E.
      • James C.A.
      • et al.
      Multilevel analyses of SCN5A mutations in ARVC/D suggest non-canonical mechanisms for disease pathogenesis.
      iPSC-CMs have been used to explore the genetics of ARVC, which currently has a genetic yield of 30%–50% and primarily involves genes of the cardiac desmosome.
      • Te Rijdt W.P.
      • Jongbloed J.D.
      • de Boer R.A.
      • Thiene G.
      • Basso C.
      • van den Berg M.P.
      • van Tintelen J.P.
      Clinical utility gene card for ARVC.
      The pathogenicity of mutant PKP2, the most common genetic cause of ARVC, has been demonstrated in iPSC-CMs by the loss of ARVC phenotype after transfection of wild-type PKP2 protein.
      • Cerrone M.
      • Lin X.
      • Zhang M.
      • et al.
      Missense mutations in plakophilin-2 cause sodium current deficit and associate with a Brugada syndrome phenotype.
      • Kim C.
      • Wong J.
      • Wen J.
      • et al.
      Studying ARVD with patient-specific iPSCs.
      Analysis of iPSC-CMs has also provided support for the pathogenicity of single amino acid substitutions in PKP2 in addition to the known loss-of-function mutation mechanism.
      • Cerrone M.
      • Lin X.
      • Zhang M.
      • et al.
      Missense mutations in plakophilin-2 cause sodium current deficit and associate with a Brugada syndrome phenotype.
      iPSC-CM studies have further demonstrated the importance of abnormal peroxisome proliferator-activated receptor-gamma activation in ARVC development
      • Caspi O.
      • Huber I.
      • Gepstein A.
      • Arbel G.
      • Maizels L.
      • Boulos M.
      • Gepstein L.
      Modeling of ARVC with human induced pluripotent stem cells.
      and testosterone in gender differences in ARVC whereby prevalence and severity of disease are increased in males.
      • Akdis D.
      • Saguner A.M.
      • Shah K.
      • et al.
      Sex hormones affect outcome in ARVC/D: from a stem cell derived cardiomyocyte-based model to clinical biomarkers of disease outcome.
      Overall, studies using iPSC-CMs to model ARVC and other inherited cardiomyopathies are significantly contributing to our understanding of the genetic and molecular mechanisms underlying disease development.

       Technical challenges and limitations of iPSC-CMs

      iPSC-CMs have significant limitations, in particular their immature fetal-like state, isolated culture environment, and challenges with reproducibility. At present, the fetal-like characteristics of iPSC-CMs limit their ability to model inherited arrhythmia syndromes, which generally have an adult onset. Immature iPSC-CMs have important differences, including a lack of T-tubules, abnormal morphology, altered gene expression, and reduced sarcomere organisation.
      • Yang X.
      • Pabon L.
      • Murry C.E.
      Engineering adolescence: maturation of human pluripotent stem cell-derived cardiomyocytes.
      Although the major cardiac channels, including those implicated in development of inherited arrhythmia syndromes, are present in iPSC-CMs, they have significantly altered expression and current amplitudes.
      • Bett G.C.
      • Kaplan A.D.
      • Lis A.
      • Cimato T.R.
      • Tzanakakis E.S.
      • Zhou Q.
      • Morales M.J.
      • Rasmusson R.L.
      Electronic “expression” of the inward rectifier in cardiocytes derived from human-induced pluripotent stem cells.
      Importantly, sodium channels contribute less to AP activity as iPSC-CMs have relatively depolarized diastolic membrane potentials.
      • Terrenoire C.
      • Wang K.
      • Tung K.W.
      • et al.
      Induced pluripotent stem cells used to reveal drug actions in a long QT syndrome family with complex genetics.
      Efforts are underway to increase iPSC-CM maturity, and a number of methodological factors, including time in culture, modulation of metabolism, electrical stimulation, and growth as microtissues, have shown improvement.
      • Kim C.
      • Wong J.
      • Wen J.
      • et al.
      Studying ARVD with patient-specific iPSCs.
      The in vitro nature of iPSC-CM models must also be taken into consideration as cardiac function is largely affected by systemic involvement and the heart is made up of a heterogeneous population of cells in addition to cardiomyocytes.

       What is on the iPSC-CM horizon?

      Although iPSC-CM models have and will continue to contribute to our understanding of inherited arrhythmia syndromes, the field likely will see an increase in culture of iPSC-CMs as cardiac microtissues.
      • Ma Z.
      • Koo S.
      • Finnegan M.A.
      • Loskill P.
      • Huebsch N.
      • Marks N.C.
      • Conklin B.R.
      • Grigoropoulos C.P.
      • Healy K.E.
      Three-dimensional filamentous human diseased cardiac tissue model.
      Cardiac microtissues have significant advantages that increase their physiological relevance, such as enhanced electrical coupling, force generation and maturation, and the ability to co-culture iPSC-CMs with multiple cell types.
      • Kurokawa Y.K.
      • George S.C.
      Tissue engineering the cardiac microenvironment: Multicellular microphysiological systems for drug screening.
      In comparison to adult cardiac tissues, iPSC-derived cardiac microtissues currently remain relatively immature, with reduced cardiac current amplitudes and contraction forces.
      • Ma Z.
      • Koo S.
      • Finnegan M.A.
      • Loskill P.
      • Huebsch N.
      • Marks N.C.
      • Conklin B.R.
      • Grigoropoulos C.P.
      • Healy K.E.
      Three-dimensional filamentous human diseased cardiac tissue model.
      At present, the use of cardiac microtissues for disease modeling and drug testing remains limited by significant time and cost restrictions.

      Conclusion

      Our understanding of the mechanisms that cause inherited arrhythmia syndromes has been limited by accessibility of human tissue and species discordance. Patient-derived iPSC-CMs have the potential to provide an unlimited source of patient-specific cardiomyocytes, and studies to date show that they already are contributing to many aspects of the inherited arrhythmia syndrome field. As cardiac microtissues become more time and cost efficient, we will likely see a shift from 2- to 3-dimensional iPSC-CM–derived models of disease. The insight gained from iPSC-CM studies will likely impact directly on the clinical practice of cardiac electrophysiologists, enhancing aspects of diagnosis and management of inherited arrhythmia patients, with more precise and targeted approaches (Figure 3). Specifically, patient-derived iPSC-CMs will likely be used to aid in the assessment of clinically actionable genetic mutations, to evaluate optimal treatment strategies with personalized preclinical drug testing, and to identify new drug compounds that facilitate better clinical outcomes for patients with inherited arrhythmia syndromes.
      Figure thumbnail gr3
      Figure 3Potential future implications of induced pluripotent stem cell-derived cardiomyocyte (iPSC-CM) technology for the clinical cardiac electrophysiologist.

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