Details of a Researcher - Hashimoto, Hisayuki

Papers - Hashimoto, Hisayuki

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Endothelial-fibroblast interactions during Scarb1 accelerate heart failure

Toshiomi Katsuki, Dai Kusumoto, Yohei Akiba, Mai Kimura, Jin Komuro, Takahiro Nakamura, Hisayuki Hashimoto, Thukaa Kuoka, Yutaka Suzuki, Yoshiaki Kubota, Keiichi Fukuda, Shinsuke Yuasa, Masaki Ieda

(Cold Spring Harbor Laboratory) 2023.09

Summary

Endothelial cells (ECs) maintain cardiac homeostasis and EC dysfunction causes heart failure progression. Moreover, pathological changes occur via interactions between multiple cells, including ECs. Here, we conducted single-cell RNA-seq analysis of non-cardiomyocytes in mouse hearts during heart failure progression to elucidate the pathological changes in ECs and fibroblasts (FBs) mediated by cell-cell interactions. We show that capillary and arterial ECs exhibit mesenchymal gene expression changes with heart failure progression, indicating that endothelial-to-mesenchymal transition (EndMT) is a major pathological alteration in ECs. We also found that the interaction between ECs and FBs was enriched during heart failure, particularly when involving Scavenger Receptor Class B Member 1 (Scarb1) in ECs. FBs induce mesenchymal gene alterations in ECs in the EC-FB co-culture system, which is inhibited by blocking SCARB1. RNA-seq analysis showed that administration of a SCARB1 inhibitor blocked mesenchymal gene expression, and inflammatory changes, suggesting that the EC-FB interaction via Scarb1 is important for EndMT induction in ECs. Systemic administration of a SCARB1 inhibitor attenuated heart failure progression and cardiac fibrosis. EC-specificScarb1knockout mouse showed improved cardiac function, suggesting a crucial role of Scarb1 in heart failure progression. Our results suggest that Scarb1 is a promising candidate for novel heart failure treatments that target ECs.

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Machine learning in cardiology: Clinical application and basic research

Jin Komuro, Dai Kusumoto, Hisayuki Hashimoto, Shinsuke Yuasa

Journal of Cardiology (Elsevier BV) 82 ( 2 ) 128 - 133 2023.08

ISSN 0914-5087

Machine learning is a subfield of artificial intelligence. The quality and versatility of machine learning have been rapidly improving and playing a critical role in many aspects of social life. This trend is also observed in the medical field. Generally, there are three main types of machine learning: supervised, unsupervised, and reinforcement learning. Each type of learning is adequately selected for the purpose and type of data. In the field of medicine, various types of information are collected and used, and research using machine learning is becoming increasingly relevant. Many clinical studies are conducted using electronic health and medical records, including in the cardiovascular area. Machine learning has also been applied in basic research. Machine learning has been widely used for several types of data analysis, such as clustering of microarray analysis and RNA sequence analysis. Machine learning is essential for genome and multi-omics analyses. This review summarizes the recent advancements in the use of machine learning in clinical applications and basic cardiovascular research.

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MEF2C/p300-mediated epigenetic remodeling promotes the maturation of induced cardiomyocytes

Hidenori Kojima, Taketaro Sadahiro, Naoto Muraoka, Hiroyuki Yamakawa, Hisayuki Hashimoto, Ryota Ishii, Masahiko Gosho, Yuto Abe, Yu Yamada, Koji Nakano, Seiichiro Honda, Ryo Fujita, Tatsuya Akiyama, Yoichi Sunagawa, Tatsuya Morimoto, Toshifumi Tsukahara, Hiroyuki Hirai, Keiichi Fukuda, Masaki Ieda

Stem Cell Reports (Elsevier BV) 18 ( 6 ) 1274 - 1283 2023.06

ISSN 2213-6711

Cardiac transcription factors (TFs) directly reprogram fibroblasts into induced cardiomyocytes (iCMs), where MEF2C acts as a pioneer factor with GATA4 and TBX5 (GT). However, the generation of functional and mature iCMs is inefficient, and the molecular mechanisms underlying this process remain largely unknown. Here, we found that the overexpression of transcriptionally activated MEF2C via fusion of the powerful MYOD transactivation domain combined with GT increased the generation of beating iCMs by 30-fold. Activated MEF2C with GT generated iCMs that were transcriptionally, structurally, and functionally more mature than those generated by native MEF2C with GT. Mechanistically, activated MEF2C recruited p300 and multiple cardiogenic TFs to cardiac loci to induce chromatin remodeling. In contrast, p300 inhibition suppressed cardiac gene expression, inhibited iCM maturation, and decreased the beating iCM numbers. Splicing isoforms of MEF2C with similar transcriptional activities did not promote functional iCM generation. Thus, MEF2C/p300-mediated epigenetic remodeling promotes iCM maturation.

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Development of non-bias phenotypic drug screening for cardiomyocyte hypertrophy by image segmentation using deep learning

Jin Komuro, Yuta Tokuoka, Tomohisa Seki, Dai Kusumoto, Hisayuki Hashimoto, Toshiomi Katsuki, Takahiro Nakamura, Yohei Akiba, Thukaa Kuoka, Mai Kimura, Takahiro Yamada, Keiichi Fukuda, Akira Funahashi, Shinsuke Yuasa

Biochemical and Biophysical Research Communications (Elsevier BV) 632 181 - 188 2022.12

ISSN 0006-291X

The number of patients with heart failure and related deaths is rapidly increasing worldwide, making it a major problem. Cardiac hypertrophy is a crucial preliminary step in heart failure, but its treatment has not yet been fully successful. In this study, we established a system to evaluate cardiomyocyte hypertrophy using a deep learning-based high-throughput screening system and identified drugs that inhibit it. First, primary cultured cardiomyocytes from neonatal rats were stimulated by both angiotensin II and endothelin-1, and cellular images were captured using a phase-contrast microscope. Subsequently, we used a deep learning model for instance segmentation and established a system to automatically and unbiasedly evaluate the cardiomyocyte size and perimeter. Using this system, we screened 100 FDA-approved drugs library and identified 12 drugs that inhibited cardiomyocyte hypertrophy. We focused on ezetimibe, a cholesterol absorption inhibitor, that inhibited cardiomyocyte hypertrophy in a dose-dependent manner in vitro. Additionally, ezetimibe improved the cardiac dysfunction induced by pressure overload in mice. These results suggest that the deep learning-based system is useful for the evaluation of cardiomyocyte hypertrophy and drug screening, leading to the development of new treatments for heart failure.

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Single cell解析に基づく心不全特異的な線維芽細胞集団を標的とした心不全の新規治療戦略

小室仁, 橋本寿之, 勝木俊臣, 楠本大, 湯浅慎介, 福田恵一

心臓 ((公財)日本心臓財団) 54 ( 12 ) 1405 - 1405 2022.12

ISSN 0586-4488

The complement C3-complement factor D-C3a receptor signalling axis regulates cardiac remodelling in right ventricular failure.

Shogo Ito, Hisayuki Hashimoto, Hiroyuki Yamakawa, Dai Kusumoto, Yohei Akiba, Takahiro Nakamura, Mizuki Momoi, Jin Komuro, Toshiomi Katsuki, Mai Kimura, Yoshikazu Kishino, Shin Kashimura, Akira Kunitomi, Mark Lachmann, Masaya Shimojima, Gakuto Yozu, Chikaaki Motoda, Tomohisa Seki, Tsunehisa Yamamoto, Yoshiki Shinya, Takahiro Hiraide, Masaharu Kataoka, Takashi Kawakami, Kunimichi Suzuki, Kei Ito, Hirotaka Yada, Manabu Abe, Mizuko Osaka, Hiromi Tsuru, Masayuki Yoshida, Kenji Sakimura, Yoshihiro Fukumoto, Michisuke Yuzaki, Keiichi Fukuda, Shinsuke Yuasa

Nature communications (Nature Communications) 13 ( 1 ) 5409 - 5409 2022.09

Failure of the right ventricle plays a critical role in any type of heart failure. However, the mechanism remains unclear, and there is no specific therapy. Here, we show that the right ventricle predominantly expresses alternative complement pathway-related genes, including Cfd and C3aR1. Complement 3 (C3)-knockout attenuates right ventricular dysfunction and fibrosis in a mouse model of right ventricular failure. C3a is produced from C3 by the C3 convertase complex, which includes the essential component complement factor D (Cfd). Cfd-knockout mice also show attenuation of right ventricular failure. Moreover, the plasma concentration of CFD correlates with the severity of right ventricular failure in patients with chronic right ventricular failure. A C3a receptor (C3aR) antagonist dramatically improves right ventricular dysfunction in mice. In summary, we demonstrate the crucial role of the C3-Cfd-C3aR axis in right ventricular failure and highlight potential therapeutic targets for right ventricular failure.

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Single cell解析に基づく心不全特異的な線維芽細胞集団を標的とした心不全の新規治療戦略

小室仁, 橋本寿之, 楠本大, 湯浅慎介

大和証券ヘルス財団研究業績集 ((公財)大和証券ヘルス財団) ( 45 ) 36 - 41 2022.03

心臓線維芽細胞に発現する転写因子c-Mycの心臓における役割を、single cell解析に基づいて調べ、心臓線維芽細胞が心不全の治療標的となり得るか検証した。心不全時に特異的に出現する心臓線維芽細胞集団を見つけ、その細胞集団ではc-Mycが特徴的に発現していることを見出した。さらに、その線維芽細胞においてc-Mycは心不全の進行に寄与していることが示唆された。一方、c-Mycを心臓線維芽細胞で過剰発現させ、その遺伝子発現を網羅的に解析したところ、その細胞自体は炎症や線維化に関連した遺伝子の発現が低下しており、心不全の進行を抑制する働きが予想された。これらのことから、in vivoではc-Myc陽性心不全特異的心臓線維芽細胞は別の細胞と相互作用することにより、心不全の進行に寄与しているという仮説が立てられた。

The histone reader PHF7 cooperates with the SWI/SNF complex at cardiac super enhancers to promote direct reprogramming.

Glynnis A Garry, Svetlana Bezprozvannaya, Kenian Chen, Huanyu Zhou, Hisayuki Hashimoto, Maria Gabriela Morales, Ning Liu, Rhonda Bassel-Duby, Eric N Olson

Nature cell biology (Nature Cell Biology) 23 ( 5 ) 467 - 475 2021.05

ISSN 1465-7392

Direct cardiac reprogramming of fibroblasts to cardiomyocytes presents an attractive therapeutic strategy to restore cardiac function following injury. Cardiac reprogramming was initially achieved through overexpression of the transcription factors Gata4, Mef2c and Tbx5; later, Hand2 and Akt1 were found to further enhance this process1-5. Yet, staunch epigenetic barriers severely limit the ability of these cocktails to reprogramme adult fibroblasts6,7. We undertook a screen of mammalian gene regulatory factors to discover novel regulators of cardiac reprogramming in adult fibroblasts and identified the histone reader PHF7 as the most potent activating factor8. Mechanistically, PHF7 localizes to cardiac super enhancers in fibroblasts, and through cooperation with the SWI/SNF complex, it increases chromatin accessibility and transcription factor binding at these sites. Furthermore, PHF7 recruits cardiac transcription factors to activate a positive transcriptional autoregulatory circuit in reprogramming. Importantly, PHF7 achieves efficient reprogramming in the absence of Gata4. Here, we highlight the underexplored necessity of cardiac epigenetic readers, such as PHF7, in harnessing chromatin remodelling and transcriptional complexes to overcome critical barriers to direct cardiac reprogramming.

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Anti-senescent drug screening by deep learning-based morphology senescence scoring.

Dai Kusumoto, Tomohisa Seki, Hiromune Sawada, Akira Kunitomi, Toshiomi Katsuki, Mai Kimura, Shogo Ito, Jin Komuro, Hisayuki Hashimoto, Keiichi Fukuda, Shinsuke Yuasa

Nature communications (Nature Communications) 12 ( 1 ) 257 - 257 2021.01

Advances in deep learning technology have enabled complex task solutions. The accuracy of image classification tasks has improved owing to the establishment of convolutional neural networks (CNN). Cellular senescence is a hallmark of ageing and is important for the pathogenesis of ageing-related diseases. Furthermore, it is a potential therapeutic target. Specific molecular markers are used to identify senescent cells. Moreover senescent cells show unique morphology, which can be identified. We develop a successful morphology-based CNN system to identify senescent cells and a quantitative scoring system to evaluate the state of endothelial cells by senescence probability output from pre-trained CNN optimised for the classification of cellular senescence, Deep Learning-Based Senescence Scoring System by Morphology (Deep-SeSMo). Deep-SeSMo correctly evaluates the effects of well-known anti-senescent reagents. We screen for drugs that control cellular senescence using a kinase inhibitor library by Deep-SeSMo-based drug screening and identify four anti-senescent drugs. RNA sequence analysis reveals that these compounds commonly suppress senescent phenotypes through inhibition of the inflammatory response pathway. Thus, morphology-based CNN system can be a powerful tool for anti-senescent drug screening.

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Dermal fibroblast-like cells reprogrammed directly from adipocytes in mouse.

Mitsunobu Toyosaki, Koichiro Homma, Sayuri Suzuki, Naoto Muraoka, Hisayuki Hashimoto, Naoki Goshima, Masaki Ieda, Junichi Sasaki

Scientific reports (Scientific Reports) 10 ( 1 ) 21467 - 21467 2020.12

In deep burns, early wound closure is important for healing, and skin grafting is mainly used for wound closure. However, it is difficult to achieve early wound closure in extensive total body surface area deep burns due to the lack of donor sites. Dermal fibroblasts, responsible for dermis formation, may be lost in deep burns. However, fat layers composed of adipocytes, lying underneath the dermis, are retained even in such cases. Direct reprogramming is a novel method for directly reprograming some cells into other types by introducing specific master regulators; it has exhibited appreciable success in various fields. In this study, we aimed to assess whether the transfection of master regulators (ELF4, FOXC2, FOXO1, IRF1, PRRX1, and ZEB1) could reprogram mouse adipocytes into dermal fibroblast-like cells. Our results indicated the shrinkage of fat droplets in reprogrammed mouse adipocytes and their transformation into spindle-shaped dermal fibroblasts. Reduced expression of PPAR-2, c/EBP, aP2, and leptin, the known markers of adipocytes, in RT-PCR, and enhanced expression of anti-ER-TR7, the known anti-fibroblast marker, in immunocytochemistry, were confirmed in the reprogrammed mouse adipocytes. The dermal fibroblast-like cells, reported here, may open up a new treatment mode for enabling early closure of deep burn wounds.

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Stem Cell Aging in Skeletal Muscle Regeneration and Disease.

Yamakawa H, Kusumoto D, Hashimoto H, Yuasa S

International journal of molecular sciences 21 ( 5 ) 2020.03

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補体副経路を標的とした右心不全および心室性不整脈に対する新たな治療法の開発

伊藤章吾, 湯浅慎介, 小室仁, 勝木俊臣, 木村舞, 岸野喜一, 楠本大, 橋本寿之, 鈴木邦道, 柚崎通介, 福田恵一

補体 ((一社)日本補体学会) 56 ( 1 ) 55 - 56 2019.07

ISSN 2188-8205

Cardiac Reprogramming Factors Synergistically Activate Genome-wide Cardiogenic Stage-Specific Enhancers.

Hashimoto H, Wang Z, Garry GA, Malladi VS, Botten GA, Ye W, Zhou H, Osterwalder M, Dickel DE, Visel A, Liu N, Bassel-Duby R, Olson EN

Cell stem cell (Elsevier BV) 25 ( 1 ) 69 - 86.e5 2019.05

Research paper (scientific journal), Joint Work, Accepted, ISSN 1934-5909

The cardiogenic transcription factors (TFs) Mef2c, Gata4, and Tbx5 can directly reprogram fibroblasts to induced cardiac-like myocytes (iCLMs), presenting a potential source of cells for cardiac repair. While activity of these TFs is enhanced by Hand2 and Akt1, their genomic targets and interactions during reprogramming are not well studied. We performed genome-wide analyses of cardiogenic TF binding and enhancer profiling during cardiac reprogramming. We found that these TFs synergistically activate enhancers highlighted by Mef2c binding sites and that Hand2 and Akt1 coordinately recruit other TFs to enhancer elements. Intriguingly, these enhancer landscapes collectively resemble patterns of enhancer activation during embryonic cardiogenesis. We further constructed a cardiac reprogramming gene regulatory network and found repression of EGFR signaling pathway genes. Consistently, chemical inhibition of EGFR signaling augmented reprogramming. Thus, by defining epigenetic landscapes these findings reveal synergistic transcriptional activation across a broad landscape of cardiac enhancers and key signaling pathways that govern iCLM reprogramming.

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Therapeutic approaches for cardiac regeneration and repair.

Hashimoto H, Olson EN, Bassel-Duby R

Nature reviews. Cardiology (Springer Science and Business Media LLC) 15 ( 10 ) 585 - 600 2018.10

Research paper (scientific journal), Joint Work, Accepted, ISSN 17595002

Ischaemic heart disease is a leading cause of death worldwide. Injury to the heart is followed by loss of the damaged cardiomyocytes, which are replaced with fibrotic scar tissue. Depletion of cardiomyocytes results in decreased cardiac contraction, which leads to pathological cardiac dilatation, additional cardiomyocyte loss, and mechanical dysfunction, culminating in heart failure. This sequential reaction is defined as cardiac remodelling. Many therapies have focused on preventing the progressive process of cardiac remodelling to heart failure. However, after patients have developed end-stage heart failure, intervention is limited to heart transplantation. One of the main reasons for the dramatic injurious effect of cardiomyocyte loss is that the adult human heart has minimal regenerative capacity. In the past 2 decades, several strategies to repair the injured heart and improve heart function have been pursued, including cellular and noncellular therapies. In this Review, we discuss current therapeutic approaches for cardiac repair and regeneration, describing outcomes, limitations, and future prospects of preclinical and clinical trials of heart regeneration. Substantial progress has been made towards understanding the cellular and molecular mechanisms regulating heart regeneration, offering the potential to control cardiac remodelling and redirect the adult heart to a regenerative state.

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Epigenetic barrier against the propagation of fluctuating gene expression in embryonic stem cells.

Saito Y, Kunitomi A, Seki T, Tohyama S, Kusumoto D, Takei M, Kashimura S, Hashimoto H, Yozu G, Motoda C, Shimojima M, Egashira T, Oda M, Fukuda K, Yuasa S

FEBS letters (WILEY) 591 ( 18 ) 2879 - 2889 2017.09

Research paper (scientific journal), Joint Work, Accepted, ISSN 0014-5793

The expression of pluripotency genes fluctuates in a population of embryonic stem (ES) cells and the fluctuations in the expression of some pluripotency genes correlate. However, no correlation in the fluctuation of Pou5f1, Zfp42, and Nanog expression was observed in ES cells. Correlation between Pou5f1 and Zfp42 fluctuations was demonstrated in ES cells containing a knockout in the NuRD component Mbd3. ES cells containing a triple knockout in the DNA methyltransferases Dnmt1, Dnmt3a, and Dnmt3b showed correlation between the fluctuation of Pou5f1, Zfp42, and Nanog gene expression. We suggest that an epigenetic barrier is key to preventing the propagation of fluctuating pluripotency gene expression in ES cells.

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ZNF281 enhances cardiac reprogramming by modulating cardiac and inflammatory gene expression.

Zhou H, Morales MG, Hashimoto H, Dickson ME, Song K, Ye W, Kim MS, Niederstrasser H, Wang Z, Chen B, Posner BA, Bassel-Duby R, Olson EN

Genes & development (COLD SPRING HARBOR LAB PRESS, PUBLICATIONS DEPT) 31 ( 17 ) 1770 - 1783 2017.09

Research paper (scientific journal), Joint Work, Accepted, ISSN 0890-9369

Direct reprogramming of fibroblasts to cardiomyocytes represents a potential means of restoring cardiac function following myocardial injury. AKT1 in the presence of four cardiogenic transcription factors, GATA4, HAND2, MEF2C, and TBX5 (AGHMT), efficiently induces the cardiac gene program in mouse embryonic fibroblasts but not adult fibroblasts. To identify additional regulators of adult cardiac reprogramming, we performed an unbiased screen of transcription factors and cytokines for those that might enhance or suppress the cardiogenic activity of AGHMT in adult mouse fibroblasts. Among a collection of inducers and repressors of cardiac reprogramming, we discovered that the zinc finger transcription factor 281 (ZNF281) potently stimulates cardiac reprogramming by genome-wide association with GATA4 on cardiac enhancers. Concomitantly, ZNF281 suppresses expression of genes associated with inflammatory signaling, suggesting the antagonistic convergence of cardiac and inflammatory transcriptional programs. Consistent with an inhibitory influence of inflammatory pathways on cardiac reprogramming, blockade of these pathways with anti-inflammatory drugs or components of the nucleosome remodeling deacetylase (NuRD) complex, which associate with ZNF281, stimulates cardiac gene expression. We conclude that ZNF281 acts at a nexus of cardiac and inflammatory gene programs, which exert opposing influences on fibroblast to cardiac reprogramming.

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Emerin plays a crucial role in nuclear invagination and in the nuclear calcium transient.

Shimojima M, Yuasa S, Motoda C, Yozu G, Nagai T, Ito S, Lachmann M, Kashimura S, Takei M, Kusumoto D, Kunitomi A, Hayashiji N, Seki T, Tohyama S, Hashimoto H, Kodaira M, Egashira T, Hayashi K, Nakanishi C, Sakata K, Yamagishi M, Fukuda K

Scientific reports (NATURE PUBLISHING GROUP) 7 44312 - 44312 2017.03

Research paper (scientific journal), Joint Work, Accepted, ISSN 2045-2322

Alteration of the nuclear Ca2+ transient is an early event in cardiac remodeling. Regulation of the nuclear Ca2+ transient is partly independent of the cytosolic Ca2+ transient in cardiomyocytes. One nuclear membrane protein, emerin, is encoded by EMD, and an EMD mutation causes Emery-Dreifuss muscular dystrophy (EDMD). It remains unclear whether emerin is involved in nuclear Ca2+ homeostasis. The aim of this study is to elucidate the role of emerin in rat cardiomyocytes by means of hypertrophic stimuli and in EDMD induced pluripotent stem (iPS) cell-derived cardiomyocytes in terms of nuclear structure and the Ca2+ transient. The cardiac hypertrophic stimuli increased the nuclear area, decreased nuclear invagination, and increased the half-decay time of the nuclear Ca2+ transient in cardiomyocytes. Emd knockdown cardiomyocytes showed similar properties after hypertrophic stimuli. The EDMD-iPS cell-derived cardiomyocytes showed increased nuclear area, decreased nuclear invagination, and increased half-decay time of the nuclear Ca2+ transient. An autopsied heart from a patient with EDMD also showed increased nuclear area and decreased nuclear invagination. These data suggest that Emerin plays a crucial role in nuclear structure and in the nuclear Ca2+ transient. Thus, emerin and the nuclear Ca2+ transient are possible therapeutic targets in heart failure and EDMD.

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Notch Inhibition Enhances Cardiac Reprogramming by Increasing MEF2C Transcriptional Activity.

Abad M, Hashimoto H, Zhou H, Morales MG, Chen B, Bassel-Duby R, Olson EN

Stem cell reports (CELL PRESS) 8 ( 3 ) 548 - 560 2017.03

Research paper (scientific journal), Joint Work, Accepted, ISSN 22136711

Conversion of fibroblasts into functional cardiomyocytes represents a potential means of restoring cardiac function after myocardial infarction, but so far this process remains inefficient and little is known about its molecular mechanisms. Here we show that DAPT, a classical Notch inhibitor, enhances the conversion of mouse fibroblasts into induced cardiac-like myocytes by the transcription factors GATA4, HAND2, MEF2C, and TBX5. DAPT cooperates with AKT kinase to further augment this process, resulting in up to 70% conversion efficiency. Moreover, DAPT promotes the acquisition of specific cardiomyocyte features, substantially increasing calcium flux, sarcomere structure, and the number of spontaneously beating cells. Transcriptome analysis shows that DAPT induces genetic programs related to muscle development, differentiation, and excitation-contraction coupling. Mechanistically, DAPT increases binding of the transcription factor MEF2C to the promoter regions of cardiac structural genes. These findings provide mechanistic insights into the reprogramming process and may have important implications for cardiac regeneration therapies.

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Flecainide ameliorates arrhythmogenicity through NCX flux in Andersen-Tawil syndrome-iPS cell-derived cardiomyocytes.

Kuroda Y, Yuasa S, Watanabe Y, Ito S, Egashira T, Seki T, Hattori T, Ohno S, Kodaira M, Suzuki T, Hashimoto H, Okata S, Tanaka A, Aizawa Y, Murata M, Aiba T, Makita N, Furukawa T, Shimizu W, Kodama I, Ogawa S, Kokubun N, Horigome H, Horie M, Kamiya K, Fukuda K

Biochemistry and biophysics reports (Biochemistry and Biophysics Reports) 9 245 - 256 2017.03

Research paper (scientific journal), Joint Work, Accepted, ISSN 2405-5808

Andersen-Tawil syndrome (ATS) is a rare inherited channelopathy. The cardiac phenotype in ATS is typified by a prominent U wave and ventricular arrhythmia. An effective treatment for this disease remains to be established. We reprogrammed somatic cells from three ATS patients to generate induced pluripotent stem cells (iPSCs). Multi-electrode arrays (MEAs) were used to record extracellular electrograms of iPSC-derived cardiomyocytes, revealing strong arrhythmic events in the ATS-iPSC-derived cardiomyocytes. Ca2+ imaging of cells loaded with the Ca2+ indicator Fluo-4 enabled us to examine intracellular Ca2+ handling properties, and we found a significantly higher incidence of irregular Ca2+ release in the ATS-iPSC-derived cardiomyocytes than in control-iPSC-derived cardiomyocytes. Drug testing using ATS-iPSC-derived cardiomyocytes further revealed that antiarrhythmic agent, flecainide, but not the sodium channel blocker, pilsicainide, significantly suppressed these irregular Ca2+ release and arrhythmic events, suggesting that flecainide's effect in these cardiac cells was not via sodium channels blocking. A reverse-mode Na+/Ca2+exchanger (NCX) inhibitor, KB-R7943, was also found to suppress the irregular Ca2+ release, and whole-cell voltage clamping of isolated guinea-pig cardiac ventricular myocytes confirmed that flecainide could directly affect the NCX current (INCX). ATS-iPSC-derived cardiomyocytes recapitulate abnormal electrophysiological phenotypes and flecainide suppresses the arrhythmic events through the modulation of INCX.

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Embryonic type Na⁺ channel β-subunit, SCN3B masks the disease phenotype of Brugada syndrome.

Okata S, Yuasa S, Suzuki T, Ito S, Makita N, Yoshida T, Li M, Kurokawa J, Seki T, Egashira T, Aizawa Y, Kodaira M, Motoda C, Yozu G, Shimojima M, Hayashiji N, Hashimoto H, Kuroda Y, Tanaka A, Murata M, Aiba T, Shimizu W, Horie M, Kamiya K, Furukawa T, Fukuda K

Scientific reports (NATURE PUBLISHING GROUP) 6 34198 - 34198 2016.09

Research paper (scientific journal), Joint Work, Accepted, ISSN 2045-2322

SCN5A is abundant in heart and has a major role in I-Na. Loss-of-function mutation in SCN5A results in Brugada syndrome (BrS), which causes sudden death in adults. It remains unclear why disease phenotype does not manifest in the young even though mutated SCN5A is expressed in the young. The aim of the present study is to elucidate the timing of the disease manifestation in BrS. A gain-of-function mutation in SCN5A also results in Long QT syndrome type 3 (LQTS3), leading to sudden death in the young. Induced pluripotent stem cells (iPSCs) were generated from a patient with a mixed phenotype of LQTS3 and BrS with the E1784K SCN5A mutation. Here we show that electrophysiological analysis revealed that LQTS3/BrS iPSC-derived cardiomyocytes recapitulate the phenotype of LQTS3 but not BrS. Each beta-subunit of the sodium channel is differentially expressed in embryonic and adult hearts. SCN3B is highly expressed in embryonic hearts and iPSC-derived cardiomyocytes. A heterologous expression system revealed that I-Na of mutated SCN5A is decreased and SCN3B augmented INa of mutated SCN5A. Knockdown of SCN3B in LQTS3/BrS iPSC-derived cardiomyocytes successfully unmasked the phenotype of BrS. Isogenic control of LQTS3/BrS (corrected-LQTS3/BrS) iPSC-derived cardiomyocytes gained the normal electrophysiological properties.

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H1foo Has a Pivotal Role in Qualifying Induced Pluripotent Stem Cells.

Kunitomi A, Yuasa S, Sugiyama F, Saito Y, Seki T, Kusumoto D, Kashimura S, Takei M, Tohyama S, Hashimoto H, Egashira T, Tanimoto Y, Mizuno S, Tanaka S, Okuno H, Yamazawa K, Watanabe H, Oda M, Kaneda R, Matsuzaki Y, Nagai T, Okano H, Yagami KI, Tanaka M, Fukuda K

Stem cell reports (CELL PRESS) 6 ( 6 ) 825 - 833 2016.06

Research paper (scientific journal), Joint Work, Accepted, ISSN 22136711

Embryonic stem cells (ESCs) are a hallmark of ideal pluripotent stem cells. Epigenetic reprogramming of induced pluripotent stem cells (iPSCs) has not been fully accomplished. iPSC generation is similar to somatic cell nuclear transfer (SCNT) in oocytes, and this procedure can be used to generate ESCs (SCNT-ESCs), which suggests the contribution of oocyte-specific constituents. Here, we show that the mammalian oocyte-specific linker histone H1foo has beneficial effects on iPSC generation. Induction of H1foo with Oct4, Sox2, and Klf4 significantly enhanced the efficiency of iPSC generation. H1foo promoted in vitro differentiation characteristics with low heterogeneity in iPSCs. H1foo enhanced the generation of germline-competent chimeric mice from iPSCs in a manner similar to that for ESCs. These findings indicate that H1foo contributes to the generation of higher-quality iPSCs.

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Analysis of cardiomyocyte movement in the developing murine heart.

Hashimoto H, Yuasa S, Tabata H, Tohyama S, Seki T, Egashira T, Hayashiji N, Hattori F, Kusumoto D, Kunitomi A, Takei M, Kashimura S, Yozu G, Shimojima M, Motoda C, Muraoka N, Nakajima K, Sakaue-Sawano A, Miyawaki A, Fukuda K

Biochemical and biophysical research communications (ACADEMIC PRESS INC ELSEVIER SCIENCE) 464 ( 4 ) 1000 - 1007 2015.09

Research paper (scientific journal), Joint Work, Accepted, ISSN 0006-291X

The precise assemblage of several types of cardiac precursors controls heart organogenesis. The cardiac precursors show dynamic movement during early development and then form the complicated heart structure. However, cardiomyocyte movements inside the newly organized mammalian heart remain unclear. We previously established the method of ex vivo time-lapse imaging of the murine heart to study cardiomyocyte behavior by using the Fucci (fluorescent ubiquitination-based cell cycle indicator) system, which can effectively label individual G1, S/G2/M, and G1/S-transition phase nuclei in living cardiomyocytes as red, green, and yellow, respectively. Global analysis of gene expression in Fucci green positive ventricular cardiomyocytes confirmed that cell cycle regulatory genes expressed in G1/S, S, G2/M, and M phase transitions were upregulated. Interestingly, pathway analysis revealed that many genes related to the cell cycle were significantly upregulated in the Fucci green positive ventricular cardiomyocytes, while only a small number of genes related to cell motility were upregulated. Time-lapse imaging showed that murine proliferating cardiomyocytes did not exhibit dynamic movement inside the heart, but stayed on site after entering the cell cycle.

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Erratum: G-CSF supports long-term muscle regeneration in mouse models of muscular dystrophy.

Hayashiji N, Yuasa S, Miyagoe-Suzuki Y, Hara M, Ito N, Hashimoto H, Kusumoto D, Seki T, Tohyama S, Kodaira M, Kunitomi A, Kashimura S, Takei M, Saito Y, Okata S, Egashira T, Endo J, Sasaoka T, Takeda S, Fukuda K

Nature communications (NATURE PUBLISHING GROUP) 6 7295 - 7295 2015.06

Joint Work, Accepted, ISSN 2041-1723

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Patients with reduced heart rate response to adenosine infusion have low myocardial flow reserve in N-13-ammonia PET studies

Takeshi Tomiyama, Shin-ichiro Kumita, Keiichi Ishihara, Masaya Suda, Minoru Sakurai, Kenta Hakozaki, Hidenobu Hashimoto, Naoto Takahashi, Hitoshi Takano, Yasuhiro Kobayashi, Tomonari Kiriyama, Yoshimitsu Fukushima, Wataru Shimizu

INTERNATIONAL JOURNAL OF CARDIOVASCULAR IMAGING (SPRINGER) 31 ( 5 ) 1089 - 1095 2015.06

Research paper (scientific journal), Joint Work, Accepted, ISSN 1569-5794

To assess the effect of adenosine infusion by evaluating the relationship between heart rate (HR) response to adenosine and myocardial flow reserve (MFR) of remote regions supplied by normal coronary arteries in N-13-ammonia PET. Thirty-one consecutive subjects (20 known coronary artery disease patients, 4 chronic heart failure patients, and 7 normal volunteers) except cases having 3-vessel disease underwent rest and adenosine stress N-13-ammonia myocardial perfusion PET. Semi-quantitative, quantitative, and gated analyses were performed. Subjects were divided into two groups with regard to HR response to adenosine. Twenty-two subjects had normal HR response (peak/rest HR > 1.20), while reduced HR response (a parts per thousand currency sign1.20) was observed in nine subjects. There were no differences in rest myocardial blood flow (MBF) of remote regions between the groups. Subjects with reduced HR response had significantly lower stress MBF and MFR of remote regions than those with normal HR response (stress MBF: 1.559 +/- A 0.517 vs. 2.279 +/- A 0.530, p = 0.004, MFR: 1.59 +/- A 0.36 vs. 2.35 +/- A 0.53, p = 0.001). There were no significant differences between the groups by means of semi-quantitative scoring. Rest and stress ejection fraction (EF) in the reduced HR response group was lower than that in the normal HR response group. In a multiple stepwise regression analysis, HR ratio, dyslipidemia, and Brinkman index were identified as predictors of the change in MFR of remote regions. Subjects with reduced HR response to adenosine had lower stress MBF and MFR of remote regions and lower EF. Moreover, HR response was one of the predictors of the change in MFR of remote regions.

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G-CSF supports long-term muscle regeneration in mouse models of muscular dystrophy.

Nature communications (NATURE PUBLISHING GROUP) 6 6745 - 6745 2015.04

Research paper (scientific journal), Joint Work, Accepted, ISSN 2041-1723

Duchenne muscular dystrophy (DMD) is a chronic and life-threatening disease that is initially supported by muscle regeneration but eventually shows satellite cell exhaustion and muscular dysfunction. The life-long maintenance of skeletal muscle homoeostasis requires the satellite stem cell pool to be preserved. Asymmetric cell division plays a pivotal role in the maintenance of the satellite cell pool. Here we show that granulocyte colony-stimulating factor receptor (G-CSFR) is asymmetrically expressed in activated satellite cells. G-CSF positively affects the satellite cell population during multiple stages of differentiation in ex vivo cultured fibres. G-CSF could be important in developing an effective therapy for DMD based on its potential to modulate the supply of multiple stages of regenerated myocytes. This study shows that the G-CSF-G-CSFR axis is fundamentally important for long-term muscle regeneration, functional maintenance and lifespan extension in mouse models of DMD with varying severities.

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Impaired respiratory function in MELAS-induced pluripotent stem cells with high heteroplasmy levels.

Kodaira M, Hatakeyama H, Yuasa S, Seki T, Egashira T, Tohyama S, Kuroda Y, Tanaka A, Okata S, Hashimoto H, Kusumoto D, Kunitomi A, Takei M, Kashimura S, Suzuki T, Yozu G, Shimojima M, Motoda C, Hayashiji N, Saito Y, Goto Y, Fukuda K

FEBS open bio (ELSEVIER SCIENCE LONDON) 5 219 - 225 2015

Research paper (scientific journal), Joint Work, Accepted, ISSN 22115463

Mitochondrial diseases are heterogeneous disorders, caused by mitochondrial dysfunction. Mitochondria are not regulated solely by nuclear genomic DNA but by mitochondrial DNA. It is difficult to develop effective therapies for mitochondrial disease because of the lack of mitochondrial disease models. Mitochondrial myopathy, encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS) is one of the major mitochondrial diseases. The aim of this study was to generate MELAS-specific induced pluripotent stem cells (iPSCs) and to demonstrate that MELAS-iPSCs can be models for mitochondrial disease. We successfully established iPSCs from the primary MELAS-fibroblasts carrying 77.7% of m.3243A>G heteroplasmy. MELAS-iPSC lines ranged from 3.6% to 99.4% of m.3243A>G heteroplasmy levels. The enzymatic activities of mitochondrial respiratory complexes indicated that MELAS-iPSC-derived fibroblasts with high heteroplasmy levels showed a deficiency of complex I activity but MELAS-iPSC-derived fibroblasts with low heteroplasmy levels showed normal complex I activity. Our data indicate that MELAS-iPSCs can be models for MELAS but we should carefully select MELAS-iPSCs with appropriate heteroplasmy levels and respiratory functions for mitochondrial disease modeling.

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MiR-133 promotes cardiac reprogramming by directly repressing Snai1 and silencing fibroblast signatures.

Muraoka N, Yamakawa H, Miyamoto K, Sadahiro T, Umei T, Isomi M, Nakashima H, Akiyama M, Wada R, Inagawa K, Nishiyama T, Kaneda R, Fukuda T, Takeda S, Tohyama S, Hashimoto H, Kawamura Y, Goshima N, Aeba R, Yamagishi H, Fukuda K, Ieda M

The EMBO journal (WILEY-BLACKWELL) 33 ( 14 ) 1565 - 1581 2014.07

Research paper (scientific journal), Joint Work, Accepted, ISSN 0261-4189

Fibroblasts can be directly reprogrammed into cardiomyocyte-like cells (iCMs) by overexpression of cardiac transcription factors or microRNAs. However, induction of functional cardiomyocytes is inefficient, and molecular mechanisms of direct reprogramming remain undefined. Here, we demonstrate that addition of miR-133a (miR-133) to Gata4, Mef2c, and Tbx5 (GMT) or GMT plus Mesp1 and Myocd improved cardiac reprogramming from mouse or human fibroblasts by directly repressing Snai1, a master regulator of epithelial-to-mesenchymal transition. MiR-133 overexpression with GMT generated sevenfold more beating iCMs from mouse embryonic fibroblasts and shortened the duration to induce beating cells from 30 to 10 days, compared to GMT alone. Snai1 knockdown suppressed fibroblast genes, upregulated cardiac gene expression, and induced more contracting iCMs with GMT transduction, recapitulating the effects of miR-133 overexpression. In contrast, overexpression of Snai1 in GMT/miR-133-transduced cells maintained fibroblast signatures and inhibited generation of beating iCMs. MiR-133-mediated Snai1 repression was also critical for cardiac reprogramming in adult mouse and human cardiac fibroblasts. Thus, silencing fibroblast signatures, mediated by miR-133/Snai1, is a key molecular roadblock during cardiac reprogramming.

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Time-lapse imaging of cell cycle dynamics during development in living cardiomyocyte.

Hisayuki Hashimoto, Shinsuke Yuasa, Hidenori Tabata, Shugo Tohyama, Nozomi Hayashiji, Fumiyuki Hattori, Naoto Muraoka, Toru Egashira, Shinichiro Okata, Kojiro Yae, Tomohisa Seki, Takahiko Nishiyama, Kazunori Nakajima, Asako Sakaue-Sawano, Atsushi Miyawaki, Keiichi Fukuda

Journal of molecular and cellular cardiology (ACADEMIC PRESS LTD- ELSEVIER SCIENCE LTD) 72 241 - 9 2014.07

Research paper (scientific journal), Joint Work, Accepted, ISSN 0022-2828

Mammalian cardiomyocytes withdraw from the cell cycle shortly after birth, although it remains unclear how cardiomyocyte cell cycles behave during development. Compared to conventional immunohistochemistry in static observation, time-lapse imaging can reveal comprehensive data in hard-to-understand biological phenomenon. However, there are no reports of an established protocol of successful time-lapse imaging in mammalian heart. Thus, it is valuable to establish a time-lapse imaging system to enable the observation of cell cycle dynamics in living murine cardiomyocytes. This study sought to establish time-lapse imaging of murine heart to study cardiomyocyte cell cycle behavior. The Fucci (fluorescent ubiquitination-based cell cycle indicator) system can effectively label individual G1, S/G2/M, and G1/S-transition phase nuclei red, green and yellow, respectively, in living mammalian cells, and could therefore be useful to visualize the real-time cell cycle transitions in living murine heart. To establish a similar system for time-lapse imaging of murine heart, we first developed an ex vivo culture system, with the culture conditions determined in terms of sample state, serum concentration, and oxygen concentration. The optimal condition (slice culture, oxygen concentration 20%, serum concentration 10%) successfully mimicked physiological cardiomyocyte proliferation in vivo. Time-lapse imaging of cardiac slices from E11.5, E14.5, E18.5, and P1 Fucci-expressing transgenic mice revealed an elongated S/G2/M phase in cardiomyocytes during development. Our time-lapse imaging of murine heart revealed a gradual elongation of the S/G2/M phase during development in living cardiomyocytes.

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Generation and characterization of functional cardiomyocytes derived from human T cell-derived induced pluripotent stem cells.

Seki T, Yuasa S, Kusumoto D, Kunitomi A, Saito Y, Tohyama S, Yae K, Kishino Y, Okada M, Hashimoto H, Takei M, Egashira T, Kodaira M, Kuroda Y, Tanaka A, Okata S, Suzuki T, Murata M, Fujita J, Fukuda K

PloS one (PUBLIC LIBRARY SCIENCE) 9 ( 1 ) e85645 2014

Research paper (scientific journal), Joint Work, Accepted, ISSN 1932-6203

Induced pluripotent stem cells (iPSCs) have been proposed as novel cell sources for genetic disease models and revolutionary clinical therapies. Accordingly, human iPSC-derived cardiomyocytes are potential cell sources for cardiomyocyte transplantation therapy. We previously developed a novel generation method for human peripheral T cell-derived iPSCs (TiPSCs) that uses a minimally invasive approach to obtain patient cells. However, it remained unknown whether TiPSCs with genomic rearrangements in the T cell receptor (TCR) gene could differentiate into functional cardiomyocyte in vitro. To address this issue, we investigated the morphology, gene expression pattern, and electrophysiological properties of TiPSC-derived cardiomyocytes differentiated by floating culture. RT-PCR analysis and immunohistochemistry showed that the TiPSC-derived cardiomyocytes properly express cardiomyocyte markers and ion channels, and show the typical cardiomyocyte morphology. Multiple electrode arrays with application of ion channel inhibitors also revealed normal electrophysiological responses in the TiPSC-derived cardiomyocytes in terms of beating rate and the field potential waveform. In this report, we showed that TiPSCs successfully differentiated into cardiomyocytes with morphology, gene expression patterns, and electrophysiological features typical of native cardiomyocytes. TiPSCs-derived cardiomyocytes obtained from patients by a minimally invasive technique could therefore become disease models for understanding the mechanisms of cardiac disease and cell sources for revolutionary cardiomyocyte therapies.

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Testosterone induces cardiomyocyte differentiation from embryonic stem cells.

Hashimoto H, Yuasa S

Journal of molecular and cellular cardiology (ACADEMIC PRESS LTD- ELSEVIER SCIENCE LTD) 62 69 - 71 2013.09

Joint Work, Accepted, ISSN 0022-2828

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Distinct metabolic flow enables large-scale purification of mouse and human pluripotent stem cell-derived cardiomyocytes.

Tohyama S, Hattori F, Sano M, Hishiki T, Nagahata Y, Matsuura T, Hashimoto H, Suzuki T, Yamashita H, Satoh Y, Egashira T, Seki T, Muraoka N, Yamakawa H, Ohgino Y, Tanaka T, Yoichi M, Yuasa S, Murata M, Suematsu M, Fukuda K

Cell stem cell (Cell Stem Cell) 12 ( 1 ) 127 - 137 2013.01

Research paper (scientific journal), Joint Work, Accepted, ISSN 1934-5909

Heart disease remains a major cause of death despite advances in medical technology. Heart-regenerative therapy that uses pluripotent stem cells (PSCs) is a potentially promising strategy for patients with heart disease, but the inability to generate highly purified cardiomyocytes in sufficient quantities has been a barrier to realizing this potential. Here, we report a nongenetic method for mass-producing cardiomyocytes from mouse and human PSC derivatives that is based on the marked biochemical differences in glucose and lactate metabolism between cardiomyocytes and noncardiomyocytes, including undifferentiated cells. We cultured PSC derivatives with glucose-depleted culture medium containing abundant lactate and found that only cardiomyocytes survived. Using this approach, we obtained cardiomyocytes of up to 99% purity that did not form tumors after transplantation. We believe that our technological method broadens the range of potential applications for purified PSC-derived cardiomyocytes and could facilitate progress toward PSC-based cardiac regenerative therapy.

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Distinct iPS Cells Show Different Cardiac Differentiation Efficiency.

Ohno Y, Yuasa S, Egashira T, Seki T, Hashimoto H, Tohyama S, Saito Y, Kunitomi A, Shimoji K, Onizuka T, Kageyama T, Yae K, Tanaka T, Kaneda R, Hattori F, Murata M, Kimura K, Fukuda K

Stem cells international (HINDAWI PUBLISHING CORPORATION) 2013 659739 - 659739 2013

Research paper (scientific journal), Joint Work, Accepted, ISSN 1687-966X

Patient-specific induced pluripotent stem (iPS) cells can be generated by introducing transcription factors that are highly expressed in embryonic stem (ES) cells into somatic cells. This opens up new possibilities for cell transplantation-based regenerative medicine by overcoming the ethical issues and immunological problems associated with ES cells. Despite the development of various methods for the generation of iPS cells that have resulted in increased efficiency, safety, and general versatility, it remains unknown which types of iPS cells are suitable for clinical use. Therefore, the aims of the present study were to assess (1) the differentiation potential, time course, and efficiency of different types of iPS cell lines to differentiate into cardiomyocytes in vitro and (2) the properties of the iPS cell-derived cardiomyocytes. We found that high-quality iPS cells exhibited better cardiomyocyte differentiation in terms of the time course and efficiency of differentiation than low-quality iPS cells, which hardly ever differentiated into cardiomyocytes. Because of the different properties of the various iPS cell lines such as cardiac differentiation efficiency and potential safety hazards, newly established iPS cell lines must be characterized prior to their use in cardiac regenerative medicine.

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Disease characterization using LQTS-specific induced pluripotent stem cells.

Egashira T, Yuasa S, Suzuki T, Aizawa Y, Yamakawa H, Matsuhashi T, Ohno Y, Tohyama S, Okata S, Seki T, Kuroda Y, Yae K, Hashimoto H, Tanaka T, Hattori F, Sato T, Miyoshi S, Takatsuki S, Murata M, Kurokawa J, Furukawa T, Makita N, Aiba T, Shimizu W, Horie M, Kamiya K, Kodama I, Ogawa S, Fukuda K

Cardiovascular research (OXFORD UNIV PRESS) 95 ( 4 ) 419 - 429 2012.09

Research paper (scientific journal), Joint Work, Accepted, ISSN 0008-6363

AIMS: Long QT syndrome (LQTS) is an inheritable and life-threatening disease; however, it is often difficult to determine disease characteristics in sporadic cases with novel mutations, and more precise analysis is necessary for the successful development of evidence-based clinical therapies. This study thus sought to better characterize ion channel cardiac disorders using induced pluripotent stem cells (iPSCs). METHODS AND RESULTS: We reprogrammed somatic cells from a patient with sporadic LQTS and from controls, and differentiated them into cardiomyocytes through embryoid body (EB) formation. Electrophysiological analysis of the LQTS-iPSC-derived EBs using a multi-electrode array (MEA) system revealed a markedly prolonged field potential duration (FPD). The IKr blocker E4031 significantly prolonged FPD in control- and LQTS-iPSC-derived EBs and induced frequent severe arrhythmia only in LQTS-iPSC-derived EBs. The IKs blocker chromanol 293B did not prolong FPD in the LQTS-iPSC-derived EBs, but significantly prolonged FPD in the control EBs, suggesting the involvement of IKs disturbance in the patient. Patch-clamp analysis and immunostaining confirmed a dominant-negative role for 1893delC in IKs channels due to a trafficking deficiency in iPSC-derived cardiomyocytes and human embryonic kidney (HEK) cells. CONCLUSIONS: This study demonstrated that iPSCs could be useful to characterize LQTS disease as well as drug responses in the LQTS patient with a novel mutation. Such analyses may in turn lead to future progress in personalized medicine.

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miR-142-3p is essential for hematopoiesis and affects cardiac cell fate in zebrafish.

Nishiyama T, Kaneda R, Ono T, Tohyama S, Hashimoto H, Endo J, Tsuruta H, Yuasa S, Ieda M, Makino S, Fukuda K

Biochemical and biophysical research communications (ACADEMIC PRESS INC ELSEVIER SCIENCE) 425 ( 4 ) 755 - 761 2012.09

Research paper (scientific journal), Joint Work, Accepted, ISSN 0006-291X

MicroRNAs (miRNAs) play a pivotal role during embryonic development and are required for proper organogenesis, including hematopoiesis. Recent studies suggest that, in the early mesoderm, there is an interaction between the hematopoietic and cardiac lineages. However, whether miRNAs can affect other lineages remains unknown. Therefore, we investigated whether hematopoietic miR-142-3p modulated the mesoderm formation. We report that knockdown (KD) of miR-142-3p, a hematopoietic-specific miRNA, in zebrafish resulted in loss of hematopoiesis during embryonic development. Intriguingly, we observed abnormal cardiac phenotypes and insufficiency of somitegenesis in KD-morphants. In the early developmental stage, a tiny heart, contractile dysfunction in the ventricle, cardiac arrhythmia (e.g. a 2:1 ratio of atrial:ventricular beating), and bradycardia were consistently observed. Histological examination revealed severe hypoplasia of the ventricle and disrupted muscle alignment. To determine the mechanism, we performed DNA microarray analysis. The results revealed that the expression of several mesodermal genes essential for the formation of cardiac and somatic mesoderm, such as no tail, T-box gene 16, mesoderm posterior a, one eye pinhead, and rho-associated, coiled-coil containing protein kinase (Rock2a), were increased in miR-142-3p KD-morphants. The luciferase reporter assay revealed that miR-142-3p repressed luciferase activity on the Rock2a 3'-UTR. The findings of the present study indicate that miR-142-3p plays a critical role in hematopoiesis, cardiogenesis, and somitegenesis in the early stage of mesoderm formation via regulation of Rock2a.

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RESIDUAL TRANSGENE EXPRESSION OF YAMANAKA FACTORS DETERMINE QUALITY AND DIFFERENTIATION EFFICIENCY OF CARDIOMYOCYTES DERIVED FROM MOUSE INDUCED PLURIPOTENT STEM CELL LINES

Yohei Ohno, Shinsuke Yuasa, Toru Egashira, Tomohisa Seki, Hisayuki Hashimoto, Shugo Tohyama, Masaki Kodaira, Dai Kusumoto, Fumiyuki Hattori, Kojiro Yae, Mitsushige Murata, Keiichi Fukuda

JOURNAL OF THE AMERICAN COLLEGE OF CARDIOLOGY (ELSEVIER SCIENCE INC) 59 ( 13 ) E1001 - E1001 2012.03

Joint Work, Accepted, ISSN 0735-1097

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Generation of induced pluripotent stem cells from human terminally differentiated circulating T cells.

Seki T, Yuasa S, Oda M, Egashira T, Yae K, Kusumoto D, Nakata H, Tohyama S, Hashimoto H, Kodaira M, Okada Y, Seimiya H, Fusaki N, Hasegawa M, Fukuda K

Cell stem cell (CELL PRESS) 7 ( 1 ) 11 - 14 2010.07

Research paper (scientific journal), Joint Work, Accepted, ISSN 1934-5909

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Zac1 is an essential transcription factor for cardiac morphogenesis.

Yuasa S, Onizuka T, Shimoji K, Ohno Y, Kageyama T, Yoon SH, Egashira T, Seki T, Hashimoto H, Nishiyama T, Kaneda R, Murata M, Hattori F, Makino S, Sano M, Ogawa S, Prall OW, Harvey RP, Fukuda K

Circulation research (LIPPINCOTT WILLIAMS & WILKINS) 106 ( 6 ) 1083 - 1091 2010.04

Research paper (scientific journal), Joint Work, Accepted, ISSN 0009-7330

RATIONALE: The transcriptional networks guiding heart development remain poorly understood, despite the identification of several essential cardiac transcription factors. OBJECTIVE: To isolate novel cardiac transcription factors, we performed gene chip analysis and found that Zac1, a zinc finger-type transcription factor, was strongly expressed in the developing heart. This study was designed to investigate the molecular and functional role of Zac1 as a cardiac transcription factor. METHODS AND RESULTS: Zac1 was strongly expressed in the heart from cardiac crescent stages and in the looping heart showed a chamber-restricted pattern. Zac1 stimulated luciferase reporter constructs driven by ANF, BNP, or alphaMHC promoters. Strong functional synergy was seen between Zac1 and Nkx2-5 on the ANF promoter, which carries adjacent Zac1 and Nkx2-5 DNA-binding sites. Zac1 directly associated with the ANF promoter in vitro and in vivo, and Zac1 and Nkx2-5 physically associated through zinc fingers 5 and 6 in Zac1, and the homeodomain in Nkx2-5. Zac1 is a maternally imprinted gene and is the first such gene found to be involved in heart development. Homozygous and paternally derived heterozygous mice carrying an interruption in the Zac1 locus showed decreased levels of chamber and myofilament genes, increased apoptotic cells, partially penetrant lethality and morphological defects including atrial and ventricular septal defects, and thin ventricular walls. CONCLUSIONS: Zac1 plays an essential role in the cardiac gene regulatory network. Our data provide a potential mechanistic link between Zac1 in cardiogenesis and congenital heart disease manifestations associated with genetic or epigenetic defects in an imprinted gene network.

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A novel GDP-dependent pyruvate kinase isozyme from Toxoplasma gondii localizes to both the apicoplast and the mitochondrion.

Saito T, Nishi M, Lim MI, Wu B, Maeda T, Hashimoto H, Takeuchi T, Roos DS, Asai T

The Journal of biological chemistry (AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC) 283 ( 20 ) 14041 - 14052 2008.05

Research paper (scientific journal), Joint Work, Accepted, ISSN 0021-9258

We previously reported a cytosolic pyruvate kinase (EC 2.7.1.40) from Toxoplasma gondii (TgPyKI) that differs from most eukaryotic pyruvate kinases in being regulated by glucose 6-phosphate rather than fructose 1,6-diphosphate. Another putative pyruvate kinase (TgPyKII) was identified from parasite genome, which exhibits 32% amino acid sequence identity to TgPyKI and retains pyruvate kinase signature motifs and amino acids essential for substrate binding and catalysis. Whereas TgPyKI is most closely related to plant/algal enzymes, phylogenetic analysis suggests a proteobacterial origin for TgPyKII. Enzymatic characterization of recombinant TgPyKII shows a high pH optimum at 8.5, and a preference for GDP as a phosphate recipient. Catalytic activity is independent of K+, and no allosteric or regulatory effects were observed in the presence of fructose 1,6-diphosphate, fructose 2,6-diphosphate, glucose 6-phosphate, ribose 5-phosphate, AMP, or ATP. Unlike TgPyKI, native TgPyKII activity was exclusively associated with the membranous fraction of a T. gondii tachyzoite lysate. TgPyKII possesses a long N-terminal extension containing five putative start codons before the conserved region and localizes to both apicoplast and mitochondrion by immunofluorescence assay using native antibody and fluorescent protein fusion to the N-terminal extension. Further deletional and site-directed mutagenesis suggests that a translation product from 1st Met is responsible for the localization to the apicoplast, whereas one from 3rd Met is for the mitochondrion. This is the first study of a potential mitochondrial pyruvate kinase in any system.

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