Yuta Yamamoto

All Publications

• Electrophysiological Analysis of hiPSC-Derived Cardiomyocytes Using a Patch-Clamp Technique. Methods in molecular biology (Clifton, N.J.) Yamamoto, Y., Hirose, S., Wuriyanghai, Y., Yoshinaga, D., Makiyama, T. 2021; 2320: 121-133

  Abstract

  Electrophysiological analysis of human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) using a patch-clamp technique enables the most precise evaluation of electrophysiological properties in single cells. Compared to multielectrode array (MEA) and membrane voltage imaging, patch-clamp recordings offer quantitative measurements of action potentials, and the relevant ionic currents which are essential for the research of disease modeling of inherited arrhythmias, safety pharmacology, and drug discovery using hiPSC-CMs. In this chapter, we describe the detail flow of patch-clamp recordings in hiPSC-CMs.

  View details for DOI 10.1007/978-1-0716-1484-6_13

  View details for PubMedID 34302654

• Allele-specific ablation rescues electrophysiological abnormalities in a human iPS cell model of long-QT syndrome with a CALM2 mutation HUMAN MOLECULAR GENETICS Yamamoto, Y., Makiyama, T., Harita, T., Sasaki, K., Wuriyanghai, Y., Hayano, M., Nishiuchi, S., Kohjitani, H., Hirose, S., Chen, J., Yokoi, F., Ishikawa, T., Ohno, S., Chonabayashi, K., Motomura, H., Yoshida, Y., Horie, M., Makita, N., Kimura, T. 2017; 26 (9): 1670-1677

  Abstract

  Calmodulin is a ubiquitous Ca2+ sensor molecule encoded by three distinct calmodulin genes, CALM1-3. Recently, mutations in CALM1-3 have been reported to be associated with severe early-onset long-QT syndrome (LQTS). However, the underlying mechanism through which heterozygous calmodulin mutations lead to severe LQTS remains unknown, particularly in human cardiomyocytes. We aimed to establish an LQTS disease model associated with a CALM2 mutation (LQT15) using human induced pluripotent stem cells (hiPSCs) and to assess mutant allele-specific ablation by genome editing for the treatment of LQT15. We generated LQT15-hiPSCs from a 12-year-old boy with LQTS carrying a CALM2-N98S mutation and differentiated these hiPSCs into cardiomyocytes (LQT15-hiPSC-CMs). Action potentials (APs) and L-type Ca2+ channel (LTCC) currents in hiPSC-CMs were analyzed by the patch-clamp technique and compared with those of healthy controls. Furthermore, we performed mutant allele-specific knockout using a CRISPR-Cas9 system and analyzed electrophysiological properties. Electrophysiological analyses revealed that LQT15-hiPSC-CMs exhibited significantly lower beating rates, prolonged AP durations, and impaired inactivation of LTCC currents compared with control cells, consistent with clinical phenotypes. Notably, ablation of the mutant allele rescued the electrophysiological abnormalities of LQT15-hiPSC-CMs, indicating that the mutant allele caused dominant-negative suppression of LTCC inactivation, resulting in prolonged AP duration. We successfully recapitulated the disease phenotypes of LQT15 and revealed that inactivation of LTCC currents was impaired in CALM2-N98S hiPSC model. Additionally, allele-specific ablation using the latest genome-editing technology provided important insights into a promising therapeutic approach for inherited cardiac diseases.

  View details for DOI 10.1093/hmg/ddx073

  View details for Web of Science ID 000400912200007

  View details for PubMedID 28335032

• Effects of a Sustained-Release Form of Isosorbide Dinitrate on Left Atrial Pressure in Dogs with Experimentally Induced Mitral Valve Regurgitation JOURNAL OF VETERINARY INTERNAL MEDICINE Yamamoto, Y., Suzuki, S., Hamabe, L., Aytemiz, D., Huai-Che, H., Kim, S., Yoshiyuki, R., Fukayama, T., Fukushima, R., Tanaka, R. 2013; 27 (6): 1421-1426

  Abstract

  The effects of isosorbide dinitrate (ISDN) have not been sufficiently investigated in conscious dogs with mitral valve regurgitation (MR).The objective was to investigate the effects of a sustained-release form of ISDN (sr-ISDN) on hemodynamics and the autonomic nervous system in dogs with MR.Six healthy Beagles weighing 11.2 ± 2.2 kg (2 years of age; 2 males and 4 females) were used.Experimental, crossover, and interventional study. Dogs with experimentally induced MR were administered placebo, 2, 5, and 10 mg/kg sr-ISDN PO on separate days with a 7-day washout period between randomized dosings. Left atrial pressure (LAP) had been recorded continuously from 30 minutes before administration of sr-ISDN to 12 hours after administration.LAP was significantly decreased after administration in the 5 and 10 mg/kg groups. Significant decrease was observed at 3 and 4 hours after administration in the 5 mg/kg group. In the 10 mg/kg group, significant decrease was observed at 2, 3, 4, 5, 6, 7, 10, and 11 hours after administration. The lowest value was observed at 4 hours after administration in the 5 and 10 mg/kg groups (20.9 ± 4.2 to 15.9 ± 3.9 mmHg, P < .01, and 21.3 ± 4.0 to 13.6 ± 4.2 mmHg, P < .001).Sustained-release form of ISDN showed significant decrease of LAP in the 5 mg/kg and 10 mg/kg groups, and duration of effect was dose related.

  View details for DOI 10.1111/jvim.12184

  View details for Web of Science ID 000326972600017

  View details for PubMedID 24010635

• Improved Cardiac Performance and Decreased Arrhythmia in Hypertrophic Cardiomyopathy With Non-β-Blocking R-Enantiomer Carvedilol. Circulation Seo, K., Yamamoto, Y., Kirillova, A., Kawana, M., Yadav, S., Huang, Y., Wang, Q., Lane, K. V., Pruitt, B. L., Perez, M. V., Bernstein, D., Wu, J. C., Wheeler, M. T., Parikh, V. N., Ashley, E. A. 2023

  Abstract

  Hypercontractility and arrhythmia are key pathophysiologic features of hypertrophic cardiomyopathy (HCM), the most common inherited heart disease. β-Adrenergic receptor antagonists (β-blockers) are the first-line therapy for HCM. However, β-blockers commonly selected for this disease are often poorly tolerated in patients, where heart-rate reduction and noncardiac effects can lead to reduced cardiac output and fatigue. Mavacamten, myosin ATPase inhibitor recently approved by the US Food and Drug Administration, has demonstrated the ability to ameliorate hypercontractility without lowering heart rate, but its benefits are so far limited to patients with left ventricular (LV) outflow tract obstruction, and its effect on arrhythmia is unknown.We screened 21 β-blockers for their impact on myocyte contractility and evaluated the antiarrhythmic properties of the most promising drug in a ventricular myocyte arrhythmia model. We then examined its in vivo effect on LV function by hemodynamic pressure-volume loop analysis. The efficacy of the drug was tested in vitro and in vivo compared with current therapeutic options (metoprolol, verapamil, and mavacamten) for HCM in an established mouse model of HCM (Myh6R403Q/+ [myosin heavy chain 6]) and iPSC cardiomyocytes derived from patients with HCM (MYH7R403Q/+) [myosin heavy chain 7]).We identified that carvedilol, a β-blocker not commonly used in HCM, suppresses contractile function and arrhythmia by inhibiting RyR2 (ryanodine receptor type 2). Unlike metoprolol (a β1-blocker), carvedilol markedly reduced LV contractility through RyR2 inhibition, while maintaining stroke volume through α1-adrenergic receptor inhibition in vivo. Clinically available carvedilol is a racemic mixture, and the R-enantiomer, devoid of β-blocking effect, retains the ability to inhibit both α1-receptor and RyR2, thereby suppressing contractile function and arrhythmias without lowering heart rate and cardiac output. In Myh6R403Q/+ mice, R-carvedilol normalized hyperdynamic contraction, suppressed arrhythmia, and increased cardiac output better than metoprolol, verapamil, and mavacamten. The ability of R-carvedilol to suppress contractile function was well retained in MYH7R403Q/+ induced pluripotent stem cell cardiomyocytes.R-enantiomer carvedilol attenuates hyperdynamic contraction, suppresses arrhythmia, and at the same time, improves cardiac output without lowering heart rate by dual blockade of α1-adrenergic receptor and RyR2 in mouse and human models of HCM. This combination of therapeutic effects is unique among current therapeutic options for HCM and may particularly benefit patients without LV outflow tract obstruction.

  View details for DOI 10.1161/CIRCULATIONAHA.123.065017

  View details for PubMedID 37850394

• Novel Calmodulin Variant p.E46K Associated With Severe Catecholaminergic Polymorphic Ventricular Tachycardia Produces Robust Arrhythmogenicity in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes. Circulation. Arrhythmia and electrophysiology Gao, J., Makiyama, T., Yamamoto, Y., Kobayashi, T., Aoki, H., Maurissen, T. L., Wuriyanghai, Y., Kashiwa, A., Imamura, T., Aizawa, T., Huang, H., Kohjitani, H., Nishikawa, M., Chonabayashi, K., Fukuyama, M., Manabe, H., Nakau, K., Wada, T., Kato, K., Toyoda, F., Yoshida, Y., Makita, N., Woltjen, K., Ohno, S., Kurebayashi, N., Murayama, T., Sakurai, T., Horie, M., Kimura, T. 2023: e011387

  Abstract

  BACKGROUND: CaM (calmodulin) is a ubiquitously expressed, multifunctional Ca2+ sensor protein that regulates numerous proteins. Recently, CaM missense variants have been identified in patients with malignant inherited arrhythmias, such as long QT syndrome and catecholaminergic polymorphic ventricular tachycardia (CPVT). However, the exact mechanism of CaM-related CPVT in human cardiomyocytes remains unclear. In this study, we sought to investigate the arrhythmogenic mechanism of CPVT caused by a novel variant using human induced pluripotent stem cell (iPSC) models and biochemical assays.METHODS: We generated iPSCs from a patient with CPVT bearing CALM2 p.E46K. As comparisons, we used 2 control lines including an isogenic line, and another iPSC line from an patient with long QT syndrome bearing CALM2 p.N98S (also reported in CPVT). Electrophysiological properties were investigated using iPSC-cardiomyocytes. We further examined the cardiac RyR2 (ryanodine receptor) and Ca2+ affinities of CaM using recombinant proteins.RESULTS: We identified a novel de novo heterozygous variant, CALM2 p.E46K, in 2 unrelated patients with CPVT accompanied by neurodevelopmental disorders. The E46K-cardiomyocytes exhibited more frequent abnormal electrical excitations and Ca2+ waves than the other lines in association with increased Ca2+ leakage from the sarcoplasmic reticulum via RyR2. Furthermore, the [3H]ryanodine binding assay revealed that E46K-CaM facilitated RyR2 function especially by activating at low [Ca2+] levels. The real-time CaM-RyR2 binding analysis demonstrated that E46K-CaM had a 10-fold increased RyR2 binding affinity compared with wild-type CaM which may account for the dominant effect of the mutant CaM. Additionally, the E46K-CaM did not affect CaM-Ca2+ binding or L-type calcium channel function. Finally, antiarrhythmic agents, nadolol and flecainide, suppressed abnormal Ca2+ waves in E46K-cardiomyocytes.CONCLUSIONS: We, for the first time, established a CaM-related CPVT iPSC-CM model which recapitulated severe arrhythmogenic features resulting from E46K-CaM dominantly binding and facilitating RyR2. In addition, the findings in iPSC-based drug testing will contribute to precision medicine.

  View details for DOI 10.1161/CIRCEP.122.011387

  View details for PubMedID 36866681

• Disrupted CaV1.2 selectivity causes overlapping long QT and Brugada syndrome phenotypes in the CACNA1C-E1115K iPS cell model. Heart rhythm Kashiwa, A., Makiyama, T., Kohjitani, H., Maurissen, T. L., Ishikawa, T., Yamamoto, Y., Wuriyanghai, Y., Gao, J., Huang, H., Imamura, T., Aizawa, T., Nishikawa, M., Chonabayashi, K., Mishima, H., Ohno, S., Toyoda, F., Sato, S., Yoshiura, K., Takahashi, K., Yoshida, Y., Woltjen, K., Horie, M., Makita, N., Kimura, T. 2023; 20 (1): 89-99

  Abstract

  BACKGROUND: A missense mutation in the alpha1c subunit of voltage-gated L-type Ca2+ channel-coding CACNA1C-E1115K, located in the Ca2+ selectivity site, causes a variety of arrhythmogenic phenotypes.OBJECTIVE: We aimed to investigate the electrophysiological features and pathophysiological mechanisms of CACNA1C-E1115K in patient-specific induced pluripotent stem cell (iPSC)-derived cardiomyocytes (CMs).METHODS: We generated iPSCs from a patient carrying heterozygous CACNA1C-E1115K with overlapping phenotypes of long QT syndrome, Brugada syndrome, and mild cardiac dysfunction. Electrophysiological properties were investigated using iPSC-CMs. We used iPSCs from a healthy individual and an isogenic iPSC line corrected using CRISPR-Cas9-mediated gene editing as controls. A mathematical E1115K-CM model was developed using a human ventricular cell model.RESULTS: Patch-clamp analysis revealed that E1115K-iPSC-CMs exhibited reduced peak Ca2+ current density and impaired Ca2+ selectivity with an increased permeability to monovalent cations. Consequently, E1115K-iPSC-CMs showed decreased action potential plateau amplitude, longer action potential duration (APD), and a higher frequency of early afterdepolarization compared with controls. In optical recordings examining the antiarrhythmic drug effect, late Na+ channel current (INaL) inhibitors (mexiletine and GS-458967) shortened APDs specifically in E1115K-iPSC-CMs. The AP-clamp using a voltage command obtained from E1115K-iPSC-CMs with lower action potential plateau amplitude and longer APD confirmed the upregulation of INaL. An in silico study recapitulated the invitro electrophysiological properties.CONCLUSION: Our iPSC-based analysis in CACNA1C-E1115K with disrupted CaV1.2 selectivity demonstrated that the aberrant currents through the mutant channels carried by monovalent cations resulted in specific action potential changes, which increased endogenous INaL, thereby synergistically contributing to the arrhythmogenic phenotype.

  View details for DOI 10.1016/j.hrthm.2022.08.021

  View details for PubMedID 36007726

• Gradient-based parameter optimization method to determine membrane ionic current composition in human induced pluripotent stem cell-derived cardiomyocytes SCIENTIFIC REPORTS Kohjitani, H., Koda, S., Himeno, Y., Makiyama, T., Yamamoto, Y., Yoshinaga, D., Wuriyanghai, Y., Kashiwa, A., Toyoda, F., Zhang, Y., Amano, A., Noma, A., Kimura, T. 2022; 12 (1): 19110

  Abstract

  Premature cardiac myocytes derived from human induced pluripotent stem cells (hiPSC-CMs) show heterogeneous action potentials (APs), probably due to different expression patterns of membrane ionic currents. We developed a method for determining expression patterns of functional channels in terms of whole-cell ionic conductance (Gx) using individual spontaneous AP configurations. It has been suggested that apparently identical AP configurations can be obtained using different sets of ionic currents in mathematical models of cardiac membrane excitation. If so, the inverse problem of Gx estimation might not be solved. We computationally tested the feasibility of the gradient-based optimization method. For a realistic examination, conventional 'cell-specific models' were prepared by superimposing the model output of AP on each experimental AP recorded by conventional manual adjustment of Gxs of the baseline model. Gxs of 4-6 major ionic currents of the 'cell-specific models' were randomized within a range of ± 5-15% and used as an initial parameter set for the gradient-based automatic Gxs recovery by decreasing the mean square error (MSE) between the target and model output. Plotting all data points of the MSE-Gx relationship during optimization revealed progressive convergence of the randomized population of Gxs to the original value of the cell-specific model with decreasing MSE. The absence of any other local minimum in the global search space was confirmed by mapping the MSE by randomizing Gxs over a range of 0.1-10 times the control. No additional local minimum MSE was obvious in the whole parameter space, in addition to the global minimum of MSE at the default model parameter.

  View details for DOI 10.1038/s41598-022-23398-0

  View details for Web of Science ID 000885173700057

  View details for PubMedID 36351955

  View details for PubMedCentralID PMC9646722

• Propranolol Attenuates Late Sodium Current in a Long QT Syndrome Type 3-Human Induced Pluripotent Stem Cell Model FRONTIERS IN CELL AND DEVELOPMENTAL BIOLOGY Hirose, S., Makiyama, T., Melgari, D., Yamamoto, Y., Wuriyanghai, Y., Yokoi, F., Nishiuchi, S., Harita, T., Hayano, M., Kohjitani, H., Gao, J., Kashiwa, A., Nishikawa, M., Wu, J., Yoshimoto, J., Chonabayashi, K., Ohno, S., Yoshida, Y., Horie, M., Kimura, T. 2020; 8: 761

  Abstract

  Long QT syndrome type 3 (LQT3) is caused by gain-of-function mutations in the SCN5A gene, which encodes the α subunit of the cardiac voltage-gated sodium channel. LQT3 patients present bradycardia and lethal arrhythmias during rest or sleep. Further, the efficacy of β-blockers, the drug used for their treatment, is uncertain. Recently, a large multicenter LQT3 cohort study demonstrated that β-blocker therapy reduced the risk of life-threatening cardiac events in female patients; however, the detailed mechanism of action remains unclear.This study aimed to establish LQT3-human induced pluripotent stem cells (hiPSCs) and to investigate the effect of propranolol in this model.An hiPSCs cell line was established from peripheral blood mononuclear cells of a boy with LQT3 carrying the SCN5A-N1774D mutation. He had suffered from repetitive torsades de pointes (TdPs) with QT prolongation since birth (QTc 680 ms), which were effectively treated with propranolol, as it suppressed lethal arrhythmias. Furthermore, hiPSCs were differentiated into cardiomyocytes (CMs), on which electrophysiological functional assays were performed using the patch-clamp method.N1774D-hiPSC-CMs exhibited significantly prolonged action potential durations (APDs) in comparison to those of the control cells (N1774D: 440 ± 37 ms vs. control: 272 ± 22 ms; at 1 Hz pacing; p < 0.01). Furthermore, N1774D-hiPSC-CMs presented gain-of-function features: a hyperpolarized shift of steady-state activation and increased late sodium current compared to those of the control cells. 5 μM propranolol shortened APDs and inhibited late sodium current in N1774D-hiPSC-CMs, but did not significantly affect in the control cells. In addition, even in the presence of intrapipette guanosine diphosphate βs (GDPβs), an inhibitor of G proteins, propranolol reduced late sodium current in N1774D cells. Therefore, these results suggested a unique inhibitory effect of propranolol on late sodium current unrelated to β-adrenergic receptor block in N1774D-hiPSC-CMs.We successfully recapitulated the clinical phenotype of LQT3 using patient-derived hiPSC-CMs and determined that the mechanism, by which propranolol inhibited the late sodium current, was independent of β-adrenergic receptor signaling pathway.

  View details for DOI 10.3389/fcell.2020.00761

  View details for Web of Science ID 000566255400001

  View details for PubMedID 32903469

  View details for PubMedCentralID PMC7438478

• Phenotype-Based High-Throughput Classification of Long QT Syndrome Subtypes Using Human Induced Pluripotent Stem Cells STEM CELL REPORTS Yoshinaga, D., Baba, S., Makiyama, T., Shibata, H., Hirata, T., Akagi, K., Matsuda, K., Kohjitani, I., Wuriyanghai, Y., Umeda, K., Yamamoto, Y., Conklin, B. R., Horie, M., Takita, J., Heike, T. 2019; 13 (2): 394-404

  Abstract

  For long QT syndrome (LQTS), recent progress in genome-sequencing technologies enabled the identification of rare genomic variants with diagnostic, prognostic, and therapeutic implications. However, pathogenic stratification of the identified variants remains challenging, especially in variants of uncertain significance. This study aimed to propose a phenotypic cell-based diagnostic assay for identifying LQTS to recognize pathogenic variants in a high-throughput manner suitable for screening. We investigated the response of LQT2-induced pluripotent stem cell (iPSC)-derived cardiomyocytes (iPSC-CMs) following IKr blockade using a multi-electrode array, finding that the response to IKr blockade was significantly smaller than in Control-iPSC-CMs. Furthermore, we found that LQT1-iPSC-CMs and LQT3-iPSC-CMs could be distinguished from Control-iPSC-CMs by IKs blockade and INa blockade, respectively. This strategy might be helpful in compensating for the shortcomings of genetic testing of LQTS patients.

  View details for DOI 10.1016/j.stemcr.2019.06.007

  View details for Web of Science ID 000481410000013

  View details for PubMedID 31378668

  View details for PubMedCentralID PMC6700479

• Clinical Manifestations and Long-Term Mortality in Lamin A/C Mutation Carriers From a Japanese Multicenter Registry CIRCULATION JOURNAL Nakajima, K., Aiba, T., Makiyama, T., Nishiuchi, S., Ohno, S., Kato, K., Yamamoto, Y., Doi, T., Shizuta, S., Onoue, K., Yagihara, N., Ishikawa, T., Watanabe, I., Kawakami, H., Oginosawa, Y., Murakoshi, N., Nogami, A., Aonuma, K., Saito, Y., Kimura, T., Yasuda, S., Makita, N., Shimizu, W., Horie, M., Kusano, K. 2018; 82 (11): 2707-+

  Abstract

  Mutation in the lamin A/C gene (LMNA) is associated with several cardiac phenotypes, such as cardiac conduction disorders (CCD), atrial arrhythmia (AA), malignant ventricular arrhythmia (MVA) and left ventricular dysfunction (LVD), leading to sudden cardiac death (SCD) and/or end-stage heart failure. We investigated how these phenotypes are associated with each other and which of them are most important for total mortality. Methods and Results: A multicenter registry included 110 LMNA mutation carriers (age, 43±15 years, male: 62%) from 60 families. After genetic diagnosis of LMNA mutation (missense: 27%, non-missense: 73%), patients or subjects were followed to evaluate the manifestations of their phenotypes and the risk of total mortality; 90 patients could be followed (median: 5 [0-35] years). Prevalence of the 4 clinical phenotypes was significantly increased during follow-up. Among these phenotypes, AA was significantly associated with MVA. CCD was significantly associated with LVD. LVD, meanwhile, was significantly associated with CCD and MVA. Male sex was significantly associated with MVA. Furthermore, during follow-up, 17 patients died: 12 end-stage heart failure, 4 SCD and 1 stroke. LVD was the only independent predictor for all-cause death (OR: 41.7, 95% CI: 4.1-422.3; P=0.0016).Several cardiac phenotypes were age-dependently increased in LMNA mutation carriers, suggesting that ICD or CRT-D could suppress SCD after middle age; however, LVD leading to end-stage heart failure was the only independent predictor for total mortality.

  View details for DOI 10.1253/circj.CJ-18-0339

  View details for Web of Science ID 000450971300006

  View details for PubMedID 30078822

• Complex aberrant splicing in the induced pluripotent stem cell-derived cardiomyocytes from a patient with long QT syndrome carrying KCNQ1-A344Aspl mutation HEART RHYTHM Wuriyanghai, Y., Makiyama, T., Sasaki, K., Kamakura, T., Yamamoto, Y., Hayano, M., Harita, T., Nishiuchi, S., Chen, J., Kohjitani, H., Hirose, S., Yokoi, F., Gao, J., Chonabayashi, K., Watanabe, K., Ohno, S., Yoshida, Y., Kimura, T., Horie, M. 2018; 15 (10): 1566-1574

  Abstract

  Long QT syndrome type 1 (LQT1) is caused by mutations in KCNQ1, which encodes the α subunit of the slow delayed rectifier potassium current channel. We previously reported that a synonymous mutation, c.1032G>A, p.A344Aspl, in KCNQ1 is most commonly identified in genotyped patients with LQT1 in Japan and the aberrant splicing was analyzed in the lymphocytes isolated from patients' blood samples. However, the mechanisms underlying the observed processes in human cardiomyocytes remain unclear.The purpose of this study was to establish and analyze patient-specific human-induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM) model carrying KCNQ1-A344Aspl.We generated hiPSCs from the peripheral blood mononuclear cells obtained from a patient with LQT1 carrying KCNQ1-A344Aspl. Using the differentiated cardiomyocytes, we analyzed splicing variants and performed electrophysiology studies.We identified 7 aberrant RNA variants in A344Aspl hiPSC-CMs, which were more complex compared with those in peripheral lymphocytes. Multielectrode array analysis revealed that 1 μM isoproterenol significantly prolonged the duration of the corrected field potential in A344Aspl hiPSC-CMs as compared with that in control hiPSC-CMs. In addition, 100 nM E-4031, which inhibits the rapid component of the delayed rectifier potassium current, was shown to induce early afterdepolarization-like waveforms in A344Aspl hiPSC-CMs. Action potential durations (APDs) did not significantly differ between the hiPSC-CM groups. After administering 500 nM isoproterenol, APDs of A344Aspl hiPSC-CMs were significantly longer than those of the controls. (R)-N-(4-(4-Methoxyphenyl)thiazol-2-yl)-1-tosylpiperidine-2-carboxamide and phenylboronic acid, slow delayed rectifier potassium current activators, ameliorated the APDs of hiPSC-CMs.We identified complex aberrant messenger RNA variants in the A344Aspl hiPSC-CM model and successfully recapitulated the clinical phenotypes of the patient with concealed LQT1. This model allows the investigation of the underlying mechanisms and development of novel therapies.

  View details for DOI 10.1016/j.hrthm.2018.05.028

  View details for Web of Science ID 000445914700028

  View details for PubMedID 29857160

• Gene-Based Risk Stratification for Cardiac Disorders in LMNA Mutation Carriers CIRCULATION-CARDIOVASCULAR GENETICS Nishiuchi, S., Makiyama, T., Aiba, T., Nakajima, K., Hirose, S., Kohjitani, H., Yamamoto, Y., Harita, T., Hayano, M., Wuriyanghai, Y., Chen, J., Sasaki, K., Yagihara, N., Ishikawa, T., Onoue, K., Murakoshi, N., Watanabe, I., Ohkubo, K., Watanabe, H., Ohno, S., Doi, T., Shizuta, S., Minamino, T., Saito, Y., Oginosawa, Y., Nogami, A., Aonuma, K., Kusano, K., Makita, N., Shimizu, W., Horie, M., Kimura, T. 2017; 10 (6)

  Abstract

  Mutations in LMNA (lamin A/C), which encodes lamin A and C, typically cause age-dependent cardiac phenotypes, including dilated cardiomyopathy, cardiac conduction disturbance, atrial fibrillation, and malignant ventricular arrhythmias. Although the type of LMNA mutations have been reported to be associated with susceptibility to malignant ventricular arrhythmias, the gene-based risk stratification for cardiac complications remains unexplored.The multicenter cohort included 77 LMNA mutation carriers from 45 families; cardiac disorders were retrospectively analyzed. The mean age of patients when they underwent genetic testing was 45±17, and they were followed for a median 49 months. Of the 77 carriers, 71 (92%) were phenotypically affected and showed cardiac conduction disturbance (81%), low left ventricular ejection fraction (<50%; 45%), atrial arrhythmias (58%), and malignant ventricular arrhythmias (26%). During the follow-up period, 9 (12%) died, either from end-stage heart failure (n=7) or suddenly (n=2). Genetic analysis showed truncation mutations in 58 patients from 31 families and missense mutations in 19 patients from 14 families. The onset of cardiac disorders indicated that subjects with truncation mutations had an earlier occurrence of cardiac conduction disturbance and low left ventricular ejection fraction, than those with missense mutations. In addition, the truncation mutation was found to be a risk factor for the early onset of cardiac conduction disturbance and the occurrence of atrial arrhythmias and low left ventricular ejection fraction, as estimated using multivariable analyses.The truncation mutations were associated with manifestation of cardiac phenotypes in LMNA-related cardiomyopathy, suggesting that genetic analysis might be useful for diagnosis and risk stratification.

  View details for DOI 10.1161/CIRCGENETICS.116.001603

  View details for Web of Science ID 000418461400005

  View details for PubMedID 29237675

• Development of a Patient-Derived Induced Pluripotent Stem Cell Model for the Investigation of SCN5A-D1275N-Related Cardiac Sodium Channelopathy CIRCULATION JOURNAL Hayano, M., Makiyama, T., Kamakura, T., Watanabe, H., Sasaki, K., Funakoshi, S., Wuriyanghai, Y., Nishiuchi, S., Harita, T., Yamamoto, Y., Kohjitani, H., Hirose, S., Yokoi, F., Chen, J., Baba, O., Horie, T., Chonabayashi, K., Ohno, S., Toyoda, F., Yoshida, Y., Ono, K., Horie, M., Kimura, T. 2017; 81 (12): 1783-+

  Abstract

  TheSCN5Agene encodes the α subunit of the cardiac voltage-gated sodium channel, NaV1.5. The missense mutation, D1275N, has been associated with a range of unusual phenotypes associated with reduced NaV1.5 function, including cardiac conduction disease and dilated cardiomyopathy. Curiously, the reported biophysical properties ofSCN5A-D1275N channels vary with experimental system.Methods and Results:First, using a human embryonic kidney (HEK) 293 cell-based heterologous expression system, theSCN5A-D1275N channels showed similar maximum sodium conductance but a significantly depolarizing shift of activation gate (+10 mV) compared to wild type. Second, we generated human-induced pluripotent stem cells (hiPSCs) from a 24-year-old female who carried heterozygousSCN5A-D1275N and analyzed the differentiated cardiomyocytes (CMs). AlthoughSCN5Atranscript levels were equivalent between D1275N and control hiPSC-CMs, both the total amount of NaV1.5 and the membrane fractions were reduced approximately half in the D1275N cells, which were rescued by the proteasome inhibitor MG132 treatment. Electrophysiological assays revealed that maximum sodium conductance was reduced to approximately half of that in control hiPSC-CMs in the D1275N cells, and maximum upstroke velocity of action potential was lower in D1275N, which was consistent with the reduced protein level of NaV1.5.This study successfully demonstrated diminished sodium currents resulting from lower NaV1.5 protein levels, which is dependent on proteasomal degradation, using a hiPSC-based model forSCN5A-D1275N-related sodium channelopathy.

  View details for DOI 10.1253/circj.CJ-17-0064

  View details for Web of Science ID 000416524000009

  View details for PubMedID 28637969

• Patient-Specific Human Induced Pluripotent Stem Cell Model Assessed with Electrical Pacing Validates S107 as a Potential Therapeutic Agent for Catecholaminergic Polymorphic Ventricular Tachycardia PLOS ONE Sasaki, K., Makiyama, T., Yoshida, Y., Wuriyanghai, Y., Kamakura, T., Nishiuchi, S., Hayano, M., Harita, T., Yamamoto, Y., Kohjitani, H., Hirose, S., Chen, J., Kawamura, M., Ohno, S., Itoh, H., Takeuchi, A., Matsuoka, S., Miura, M., Sumitomo, N., Horie, M., Yamanaka, S., Kimura, T. 2016; 11 (10): e0164795

  Abstract

  Human induced pluripotent stem cells (hiPSCs) offer a unique opportunity for disease modeling. However, it is not invariably successful to recapitulate the disease phenotype because of the immaturity of hiPSC-derived cardiomyocytes (hiPSC-CMs). The purpose of this study was to establish and analyze iPSC-based model of catecholaminergic polymorphic ventricular tachycardia (CPVT), which is characterized by adrenergically mediated lethal arrhythmias, more precisely using electrical pacing that could promote the development of new pharmacotherapies.We generated hiPSCs from a 37-year-old CPVT patient and differentiated them into cardiomyocytes. Under spontaneous beating conditions, no significant difference was found in the timing irregularity of spontaneous Ca2+ transients between control- and CPVT-hiPSC-CMs. Using Ca2+ imaging at 1 Hz electrical field stimulation, isoproterenol induced an abnormal diastolic Ca2+ increase more frequently in CPVT- than in control-hiPSC-CMs (control 12% vs. CPVT 43%, p<0.05). Action potential recordings of spontaneous beating hiPSC-CMs revealed no significant difference in the frequency of delayed afterdepolarizations (DADs) between control and CPVT cells. After isoproterenol application with pacing at 1 Hz, 87.5% of CPVT-hiPSC-CMs developed DADs, compared to 30% of control-hiPSC-CMs (p<0.05). Pre-incubation with 10 μM S107, which stabilizes the closed state of the ryanodine receptor 2, significantly decreased the percentage of CPVT-hiPSC-CMs presenting DADs to 25% (p<0.05).We recapitulated the electrophysiological features of CPVT-derived hiPSC-CMs using electrical pacing. The development of DADs in the presence of isoproterenol was significantly suppressed by S107. Our model provides a promising platform to study disease mechanisms and screen drugs.

  View details for DOI 10.1371/journal.pone.0164795

  View details for Web of Science ID 000386204500066

  View details for PubMedID 27764147

  View details for PubMedCentralID PMC5072719

• Cardiac sodium channel mutation associated with epinephrine-induced QT prolongation and sinus node dysfunction HEART RHYTHM Chen, J., Makiyama, T., Wuriyanghai, Y., Ohno, S., Sasaki, K., Hayano, M., Harita, T., Nishiuchi, S., Yamamoto, Y., Ueyama, T., Shimizu, A., Horie, M., Kimura, T. 2016; 13 (1): 289-298

  Abstract

  Long-QT syndrome (LQTS) is an inherited arrhythmia characterized by prolonged ventricular repolarization and malignant tachyarrhythmias. LQT1, LQT2, and LQT3 are caused by mutations in KCNQ1 (LQT1), KCNH2 (LQT2), and SCN5A (LQT3), which account for approximately 90% of genotyped LQTS patients. Most cardiac events in LQT1 patients occur during exercise, whereas patients with LQT3 tend to have arrhythmic events during rest or asleep.The study aimed to identify a genetic mutation in a Japanese man who presented with sinus node dysfunction and prolonged QT interval on exercise and epinephrine stress tests, as well as to clarify the electrophysiological properties of mutant channels.LQTS-related genes were screened in this patient. Electrophysiological functional assays were conducted with a heterologous expression system.We identified a heterozygous missense SCN5A mutation, V2016M, which changes the last amino acid of the cardiac sodium channel. Electrophysiological analyses revealed that the mutant channels exhibited a loss-of-function feature, decreased peak sodium current densities (wild type 175.2 ± 17.6 pA/pF; V2016M 97.2 ± 16.0 pA/pF; P < .01). In addition, the mutant channels showed gain-of-function features: increased late sodium currents by protein kinase A activation (wild type 0.07 ± 0.01%; V2016M 0.17 ± 0.03%; P < .05) and impaired inactivation of sodium channels by protein kinase A or C activation.We identified an SCN5A mutation in a patient with sinus node dysfunction and epinephrine-induced QT prolongation, which was an atypical phenotype for LQT3. The electrophysiological properties of the mutant channels might be associated with the overlapping clinical features of the patient.

  View details for DOI 10.1016/j.hrthm.2015.08.021

  View details for Web of Science ID 000367394200044

  View details for PubMedID 26282245

• Comparative effects of amlodipine and benazepril on Left Atrial Pressure in Dogs with experimentally-induced Mitral Valve Regurgitation BMC VETERINARY RESEARCH Suzuki, S., Fukushima, R., Ishikawa, T., Yamamoto, Y., Hamabe, L., Kim, S., Yoshiyuki, R., Machida, N., Tanaka, R. 2012; 8: 166

  Abstract

  One of the purposes of treatment for dogs with mitral regurgitation (MR) is lowering left atrial pressure (LAP). There has been few study of the amlodipine in dogs with MR and amlodipine's effect on LAP has not been fully evaluated in a quantitative manner because of difficulties in directly measuring LAP. The objective of our study was to compare the short-term effects of amlodipine (0.2 mg/kg PO q12h) vs benazepril (0.5 mg/kg PO q12h), on LAP and echocardiographic parameters in five beagle dogs with experimentally-induced MR. LAP of eight dogs that has own control were measured using radiotelemetry system at baseline and again on days 1, 2, 3, 4, 5, 6, 7 of the drug administration.Mean LAP decreased significantly after amlodipine (11.20 ± 4.19 mmHg vs 14.61 ± 3.81 mmHg at baseline, p < .01) but not after benazepril treatment (13.19 ± 3.47 mmHg, p > .05). LAP was lower after 7 days of amlodipine treatment than after 7 days of benazepril treatment. Significant reduction was seen for the first time 4 days after the administration amlodipine. The rate of the maximal area of the regurgitant jet signals to the left atrium area (ARJ/LAA) of the amlodipine treatment was significantly lower (p < .05) after 7 days compared to baseline. Other echocardiographic parameters did not change significantly.LAP was significantly decreased after amlodipine treatment in dogs with surgically-induced MR but not after benazepril treatment. Although this study did not focus on adverse effects, amlodipine may be an effective drug for helping the patients with acute onset of severe MR, such as rupture of chordae tendinae or end stage patients were the LAP is likely to be elevated. Additional studies in clinical patients with degenerative mitral valve disease and acute chordal rupture are warranted because the blood-pressure lowering effects of amlodipine can decrease renal perfusion and this can further activate the RAAS.

  View details for DOI 10.1186/1746-6148-8-166

  View details for Web of Science ID 000311231000001

  View details for PubMedID 22989022

  View details for PubMedCentralID PMC3489586