Publications by authors named "Seitaro Nomura"

40 Publications

Activation of Sympathetic Signaling in Macrophages Blocks Systemic Inflammation and Protects against Renal Ischemia-Reperfusion Injury.

J Am Soc Nephrol 2021 Apr 19. Epub 2021 Apr 19.

Division of CKD Pathophysiology, University of Tokyo Graduate School of Medicine, Tokyo, Japan.

Background: The sympathetic nervous system regulates immune cell dynamics. However, the detailed role of sympathetic signaling in inflammatory diseases is still unclear because it varies according to the disease situation and responsible cell types. This study focused on identifying the functions of sympathetic signaling in macrophages in LPS-induced sepsis and renal ischemia-reperfusion injury (IRI).

Methods: We performed RNA sequencing of mouse macrophage cell lines to identify the critical gene that mediates the anti-inflammatory effect of 2-adrenergic receptor (Adrb2) signaling. We also examined the effects of salbutamol (a selective Adrb2 agonist) in LPS-induced systemic inflammation and renal IRI. Macrophage-specific conditional knockout (cKO) mice and the adoptive transfer of salbutamol-treated macrophages were used to assess the involvement of macrophage Adrb2 signaling.

Results: , activation of Adrb2 signaling in macrophages induced the expression of T cell Ig and mucin domain 3 (), which contributes to anti-inflammatory phenotypic alterations. , salbutamol administration blocked LPS-induced systemic inflammation and protected against renal IRI; this protection was mitigated in macrophage-specific cKO mice. The adoptive transfer of salbutamol-treated macrophages also protected against renal IRI. Single-cell RNA sequencing revealed that this protection was associated with the accumulation of -expressing macrophages in the renal tissue.

Conclusions: The activation of Adrb2 signaling in macrophages induces anti-inflammatory phenotypic alterations partially via the induction of expression, which blocks LPS-induced systemic inflammation and protects against renal IRI.
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http://dx.doi.org/10.1681/ASN.2020121723DOI Listing
April 2021

Prediction of single-cell mechanisms for disease progression in hypertrophic remodelling by a trans-omics approach.

Sci Rep 2021 Apr 14;11(1):8112. Epub 2021 Apr 14.

Department of Bioscience and Bioinformatics, Faculty of Computer Science and Systems Engineering, Kyushu Institute of Technology, 680-4 Kawazu, Iizuka, Fukuoka, 820-8502, Japan.

Heart failure is a heterogeneous disease with multiple risk factors and various pathophysiological types, which makes it difficult to understand the molecular mechanisms involved. In this study, we proposed a trans-omics approach for predicting molecular pathological mechanisms of heart failure and identifying marker genes to distinguish heterogeneous phenotypes, by integrating multiple omics data including single-cell RNA-seq, ChIP-seq, and gene interactome data. We detected a significant increase in the expression level of natriuretic peptide A (Nppa), after stress loading with transverse aortic constriction (TAC), and showed that cardiomyocytes with high Nppa expression displayed specific gene expression patterns. Multiple NADH ubiquinone complex family, which are associated with the mitochondrial electron transport system, were negatively correlated with Nppa expression during the early stages of cardiac hypertrophy. Large-scale ChIP-seq data analysis showed that Nkx2-5 and Gtf2b were transcription factors characteristic of high-Nppa-expressing cardiomyocytes. Nppa expression levels may, therefore, represent a useful diagnostic marker for heart failure.
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http://dx.doi.org/10.1038/s41598-021-86821-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8047020PMC
April 2021

CXCR7 ameliorates myocardial infarction as a β-arrestin-biased receptor.

Sci Rep 2021 Feb 9;11(1):3426. Epub 2021 Feb 9.

Department of Cardiovascular Medicine, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-8655, Japan.

Most seven transmembrane receptors (7TMRs) are G protein-coupled receptors; however, some 7TMRs evoke intracellular signals through β-arrestin as a biased receptor. As several β-arrestin-biased agonists have been reported to be cardioprotective, we examined the role of the chemokine receptor CXCR7 as a β-arrestin-biased receptor in the heart. Among 510 7TMR genes examined, Cxcr7 was the most abundantly expressed in the murine heart. Single-cell RNA-sequencing analysis revealed that Cxcr7 was abundantly expressed in cardiomyocytes and fibroblasts. Cardiomyocyte-specific Cxcr7 null mice showed more prominent cardiac dilatation and dysfunction than control mice 4 weeks after myocardial infarction. In contrast, there was no difference in cardiac phenotypes between fibroblast-specific Cxcr7-knockout mice and control mice even after myocardial infarction. TC14012, a specific agonist of CXCR7, significantly recruited β-arrestin to CXCR7 in CXCR7-expressing cells and activated extracellular signal-regulated kinase (ERK) in neonatal rat cardiomyocytes. Cxcr7 expression was significantly increased and ERK was activated in the border zone of the heart in control, but not Cxcr7 null mice. These results indicate that the abundantly expressed CXCR7 in cardiomyocytes may play a protective role in the heart as a β-arrestin-biased receptor and that CXCR7 may be a novel therapeutic target for myocardial infarction.
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http://dx.doi.org/10.1038/s41598-021-83022-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7873251PMC
February 2021

Precision medicine for heart failure based on molecular mechanisms: The 2019 ISHR Research Achievement Award Lecture.

J Mol Cell Cardiol 2021 03 1;152:29-39. Epub 2020 Dec 1.

Department of Cardiovascular Medicine, The University of Tokyo, Japan. Electronic address:

Heart failure is a leading cause of death, and the number of patients with heart failure continues to increase worldwide. To realize precision medicine for heart failure, its underlying molecular mechanisms must be elucidated. In this review summarizing the "The Research Achievement Award Lecture" of the 2019 XXIII ISHR World Congress held in Beijing, China, we would like to introduce our approaches for investigating the molecular mechanisms of cardiac hypertrophy, development, and failure, as well as discuss future perspectives.
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http://dx.doi.org/10.1016/j.yjmcc.2020.11.010DOI Listing
March 2021

Review of Single-Cell RNA Sequencing in the Heart.

Int J Mol Sci 2020 Nov 6;21(21). Epub 2020 Nov 6.

Department of Cardiovascular Medicine, Graduate School of Medicine, The University of Tokyo, Tokyo 113-8654, Japan.

Single-cell RNA sequencing (scRNA-seq) technology is a powerful, rapidly developing tool for characterizing individual cells and elucidating biological mechanisms at the cellular level. Cardiovascular disease is one of the major causes of death worldwide and its precise pathology remains unclear. scRNA-seq has provided many novel insights into both healthy and pathological hearts. In this review, we summarize the various scRNA-seq platforms and describe the molecular mechanisms of cardiovascular development and disease revealed by scRNA-seq analysis. We then describe the latest technological advances in scRNA-seq. Finally, we discuss how to translate basic research into clinical medicine using scRNA-seq technology.
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http://dx.doi.org/10.3390/ijms21218345DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7664385PMC
November 2020

Trends and Limitations in the Assessment of the Contractile Properties of Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes From Patients With Dilated Cardiomyopathy.

Front Cardiovasc Med 2020 3;7:154. Epub 2020 Sep 3.

Department of Cardiovascular Medicine, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan.

The application of human induced pluripotent stem cell-derived cardiomyocytes (hiPSCMs) from patients is expected in disease modeling and drug screening . Dilated cardiomyopathy (DCM) is an intractable disease characterized by the impairment of systolic function and leads to severe heart failure. A number of researchers have focused on disease modeling of DCM and reproduced its pathologic phenotypes in hiPSCMs, but a robust method to evaluate the contractile properties of cardiomyocytes has not been standardized. In addition, it is unknown whether the throughput of measurements and analyses could be increased sufficiently for compound screening. Here, we reviewed the articles in which the contractile abnormalities of DCM hiPSCMs were recapitulated and assessed the trends and problems in sample preparation and evaluation. We found that single-cell level analysis was ineffective in some cases, and a tissue engineering approach has become dominant recently because of its increased efficiency in reproducing impaired contractility. We also examined two commercially available automated measurement devices with moderate throughput for motion analysis using two-dimensional hiPSCM sheets composed of originally established DCM hiPSCMs. As a result, both of the tested devices, an impedance analyzer and a video image-based cell motion analyzer, were not effective in detecting the expected reduction of contractility in the DCM clone. These findings collectively suggest that a tissue engineering approach could expand the potential of disease modeling with hiPSCMs, and so far, appropriate methods for force measurement with sufficient throughput, but without sacrificing physiological fidelity, are awaited.
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http://dx.doi.org/10.3389/fcvm.2020.00154DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7494730PMC
September 2020

Population-specific and trans-ancestry genome-wide analyses identify distinct and shared genetic risk loci for coronary artery disease.

Nat Genet 2020 11 5;52(11):1169-1177. Epub 2020 Oct 5.

Department of Cardiovascular Medicine, Osaka University Graduate School of Medicine, Suita, Japan.

To elucidate the genetics of coronary artery disease (CAD) in the Japanese population, we conducted a large-scale genome-wide association study of 168,228 individuals of Japanese ancestry (25,892 cases and 142,336 controls) with genotype imputation using a newly developed reference panel of Japanese haplotypes including 1,781 CAD cases and 2,636 controls. We detected eight new susceptibility loci and Japanese-specific rare variants contributing to disease severity and increased cardiovascular mortality. We then conducted a trans-ancestry meta-analysis and discovered 35 additional new loci. Using the meta-analysis results, we derived a polygenic risk score (PRS) for CAD, which outperformed those derived from either Japanese or European genome-wide association studies. The PRS prioritized risk factors among various clinical parameters and segregated individuals with increased risk of long-term cardiovascular mortality. Our data improve the clinical characterization of CAD genetics and suggest the utility of trans-ancestry meta-analysis for PRS derivation in non-European populations.
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http://dx.doi.org/10.1038/s41588-020-0705-3DOI Listing
November 2020

Single-cell genomics to understand disease pathogenesis.

Authors:
Seitaro Nomura

J Hum Genet 2021 Jan 19;66(1):75-84. Epub 2020 Sep 19.

Department of Cardiovascular Medicine, Graduate School of Medicine, University of Tokyo, Tokyo, Japan.

Cells are minimal functional units in biological phenomena, and therefore single-cell analysis is needed to understand the molecular behavior leading to cellular function in organisms. In addition, omics analysis technology can be used to identify essential molecular mechanisms in an unbiased manner. Recently, single-cell genomics has unveiled hidden molecular systems leading to disease pathogenesis in patients. In this review, I summarize the recent advances in single-cell genomics for the understanding of disease pathogenesis and discuss future perspectives.
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http://dx.doi.org/10.1038/s10038-020-00844-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7728598PMC
January 2021

Cardiac dopamine D1 receptor triggers ventricular arrhythmia in chronic heart failure.

Nat Commun 2020 08 31;11(1):4364. Epub 2020 Aug 31.

Department of Cardiovascular Medicine, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan.

Pathophysiological roles of cardiac dopamine system remain unknown. Here, we show the role of dopamine D1 receptor (D1R)-expressing cardiomyocytes (CMs) in triggering heart failure-associated ventricular arrhythmia. Comprehensive single-cell resolution analysis identifies the presence of D1R-expressing CMs in both heart failure model mice and in heart failure patients with sustained ventricular tachycardia. Overexpression of D1R in CMs disturbs normal calcium handling while CM-specific deletion of D1R ameliorates heart failure-associated ventricular arrhythmia. Thus, cardiac D1R has the potential to become a therapeutic target for preventing heart failure-associated ventricular arrhythmia.
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http://dx.doi.org/10.1038/s41467-020-18128-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7459304PMC
August 2020

Transethnic Meta-Analysis of Genome-Wide Association Studies Identifies Three New Loci and Characterizes Population-Specific Differences for Coronary Artery Disease.

Circ Genom Precis Med 2020 06 29;13(3):e002670. Epub 2020 May 29.

Department of Cardiovascular Medicine, Osaka University Graduate School of Medicine, Suita (S. Suna, Yasushi Sakata).

Background: Genome-wide association studies provided many biological insights into coronary artery disease (CAD), but these studies were mainly performed in Europeans. Genome-wide association studies in diverse populations have the potential to advance our understanding of CAD.

Methods: We conducted 2 genome-wide association studies for CAD in the Japanese population, which included 12 494 cases and 28 879 controls and 2808 cases and 7261 controls, respectively. Then, we performed transethnic meta-analysis using the results of the coronary artery disease genome-wide replication and meta-analysis plus the coronary artery disease 1000 Genomes meta-analysis with UK Biobank. We then explored the pathophysiological significance of these novel loci and examined the differences in CAD-susceptibility loci between Japanese and Europeans.

Results: We identified 3 new loci on chromosome 1q21 (), 10q26 (), and 11q22 (-). Quantitative trait locus analyses suggested the association of and - with atherosclerotic immune cells. Tissue/cell type enrichment analysis showed the involvement of arteries, adrenal glands, and fat tissues in the development of CAD. We next compared the odds ratios of lead variants for myocardial infarction at 76 genome-wide significant loci in the transethnic meta-analysis and a moderate correlation between Japanese and Europeans, where 8 loci showed a difference. Finally, we performed tissue/cell type enrichment analysis using East Asian-frequent and European-frequent variants according to the risk allele frequencies and identified significant enrichment of adrenal glands in the East Asian-frequent group while the enrichment of arteries and fat tissues was found in the European-frequent group. These findings indicate biological differences in CAD susceptibility between Japanese and Europeans.

Conclusions: We identified 3 new loci for CAD and highlighted the genetic differences between the Japanese and European populations. Moreover, our transethnic analyses showed both shared and unique genetic architectures between the Japanese and Europeans. While most of the underlying genetic bases for CAD are shared, further analyses in diverse populations will be needed to elucidate variations fully.
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http://dx.doi.org/10.1161/CIRCGEN.119.002670DOI Listing
June 2020

Quantification of DNA Damage in Heart Tissue as a Novel Prediction Tool for Therapeutic Prognosis of Patients With Dilated Cardiomyopathy.

JACC Basic Transl Sci 2019 Oct 25;4(6):670-680. Epub 2019 Sep 25.

Department of Cardiovascular Medicine, Graduate School of Medicine, University of Tokyo, Tokyo, Japan.

This study evaluated myocardial nuclear staining for the DNA damage markers poly(ADP-ribose) (PAR) and γ-H2A.X in 58 patients with dilated cardiomyopathy. Patients with left ventricular reverse remodeling (LVRR) showed a significantly smaller proportion of PAR-positive nuclei and γ-H2A.X-positive nuclei in biopsy specimens compared with those without LVRR. Propensity analysis showed that the proportion of both PAR-positive and γ-H2A.X-positive nuclei were independent prognostic factors for LVRR. In conclusion, we showed the utility of DNA damage-marker staining to predict the probability of LVRR, thus revealing a novel prognostic predictor of medical therapy for dilated cardiomyopathy.
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http://dx.doi.org/10.1016/j.jacbts.2019.05.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6834953PMC
October 2019

Cooperation of PU.1 With IRF8 and NFATc1 Defines Chromatin Landscapes During RANKL-Induced Osteoclastogenesis.

J Bone Miner Res 2019 06 28;34(6):1143-1154. Epub 2019 Feb 28.

Department of Orthopaedic Surgery, Faculty of Medicine, The University of Tokyo, Tokyo, Japan.

Receptor activator of nuclear factor κB ligand (RANKL) induces osteoclast (OC) differentiation from bone marrow-derived macrophages (BMMs). The transcription factors nuclear factor of activated T cells 1 (NFATc1) and interferon regulatory factor (IRF) 8 play positive and negative roles, respectively, in this process. However, genomewide mapping of the active cis-regulatory elements regulating OC differentiation has not been performed, and little is known about the global landscape of OC-specific gene regulation. We used chromatin immunoprecipitation and formaldehyde-assisted isolation of regulatory elements followed by sequencing to show that PU.1 transcription factor binding motifs were overrepresented at active cis-regulatory regions in both murine BMMs and OCs, while IRF and NFAT binding motifs were selectively enriched at these regions in BMMs and OCs, respectively. We also found that RANKL induced the downregulation of Irf8 and upregulation of Nfatc1 expression, which was associated with dramatic alterations in histone modification. BMM-specific PU.1 binding sites were observed to overlap with IRF8 binding sites in BMMs, and this also occurred for OC-specific PU.1 binding sites and NFATc1 binding sites in OCs. The expression of genes with IRF8 peaks within BMM-specific PU.1 binding sites was significantly higher in BMMs than in OCs, while that of genes with NFATc1 peaks within OC-specific PU.1 binding sites was significantly higher in OCs than in BMMs. Our results suggest that PU.1 switches its transcription partner from IRF8 to NFATc1 and alters the binding regions during RANKL-induced osteoclastogenesis, which is associated with changes in epigenetic profiles and the control of cell type-specific gene expression. © 2019 American Society for Bone and Mineral Research.
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http://dx.doi.org/10.1002/jbmr.3689DOI Listing
June 2019

Characterization of a small molecule that promotes cell cycle activation of human induced pluripotent stem cell-derived cardiomyocytes.

J Mol Cell Cardiol 2019 03 23;128:90-95. Epub 2019 Jan 23.

Department of Cardiovascular Medicine, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, Japan. Electronic address:

Background: Since regenerative capacity of adult mammalian myocardium is limited, activation of the endogenous proliferative capacity of existing cardiomyocytes is a potential therapeutic strategy for treating heart diseases accompanied by cardiomyocyte loss. Recently, we performed a compound screening and developed a new drug named TT-10 (CHFNOS) which promotes the proliferation of murine cardiomyocytes via enhancement of YES-associated protein (YAP)-transcriptional enhancer factor domain (TEAD) activity and improves cardiac function after myocardial infarction in adult mice.

Methods And Results: To test whether TT-10 can also promote the proliferative capacity of human cardiomyocytes, we investigated the efficacy of TT-10 on human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes (hiPSCMs). The hiPSCs were established from monocytes obtained from healthy donors and cardiac differentiation was performed using a chemically defined protocol. As was observed in murine cardiomyocytes, TT-10 markedly promoted cell cycle activation and increased cell division of hiPSCMs. We then evaluated other effects of TT-10 on the functional properties of hiPSCMs by gene expression and cell motion analyses. We observed that TT-10 had no unfavorable effects on the expression of functional and structural genes or the contractile properties of hiPSCMs.

Conclusions: Our results suggest that the novel drug TT-10 effectively activated the cell cycle of hiPSCMs without apparent functional impairment of myocardium, suggesting the potential of clinical usefulness of this drug.
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http://dx.doi.org/10.1016/j.yjmcc.2019.01.020DOI Listing
March 2019

High-throughput single-molecule RNA imaging analysis reveals heterogeneous responses of cardiomyocytes to hemodynamic overload.

J Mol Cell Cardiol 2019 03 3;128:77-89. Epub 2019 Jan 3.

Department of Cardiovascular Medicine, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan. Electronic address:

Background: The heart responds to hemodynamic overload through cardiac hypertrophy and activation of the fetal gene program. However, these changes have not been thoroughly examined in individual cardiomyocytes, and the relation between cardiomyocyte size and fetal gene expression remains elusive. We established a method of high-throughput single-molecule RNA imaging analysis of in vivo cardiomyocytes and determined spatial and temporal changes during the development of heart failure.

Methods And Results: We applied three novel single-cell analysis methods, namely, single-cell quantitative PCR (sc-qPCR), single-cell RNA sequencing (scRNA-seq), and single-molecule fluorescence in situ hybridization (smFISH). Isolated cardiomyocytes and cross sections from pressure overloaded murine hearts after transverse aortic constriction (TAC) were analyzed at an early hypertrophy stage (2 weeks, TAC2W) and at a late heart failure stage (8 weeks, TAC8W). Expression of myosin heavy chain β (Myh7), a representative fetal gene, was induced in some cardiomyocytes in TAC2W hearts and in more cardiomyocytes in TAC8W hearts. Expression levels of Myh7 varied considerably among cardiomyocytes. Myh7-expressing cardiomyocytes were significantly more abundant in the middle layer, compared with the inner or outer layers of TAC2W hearts, while such spatial differences were not observed in TAC8W hearts. Expression levels of Myh7 were inversely correlated with cardiomyocyte size and expression levels of mitochondria-related genes.

Conclusions: We developed a new image-analysis pipeline to allow automated and unbiased quantification of gene expression at the single-cell level and determined the spatial and temporal regulation of heterogenous Myh7 expression in cardiomyocytes after pressure overload.
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http://dx.doi.org/10.1016/j.yjmcc.2018.12.018DOI Listing
March 2019

Genetic and non-genetic determinants of clinical phenotypes in cardiomyopathy.

Authors:
Seitaro Nomura

J Cardiol 2019 03 5;73(3):187-190. Epub 2018 Dec 5.

Department of Cardiovascular Medicine, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan. Electronic address:

Cardiomyopathy, a leading cause of death worldwide, is etiologically and phenotypically heterogeneous and is caused by a combination of genetic and non-genetic factors. Major genomic determinants of dilated cardiomyopathy (DCM) are titin truncating mutations and lamin A/C mutations. Patients with these two genotypes show critically different phenotypes, including penetrance, coexistence with a conduction system abnormality, cardiac prognosis, and treatment response. The transcriptomic and epigenomic characteristics of DCM include activation of the DNA damage response, metabolic reprogramming, and dedifferentiation. The proteomic and metabolomic signatures of the DCM heart include a rigorous dependency for free fatty acids, activation of the stress response, and metabolic reprogramming. Proteomic and metabolomic analyses of blood show a distinct immune response and an unexpected link with pathology-specific microbiota in DCM. The direct integration of multi-omics data will not only elucidate inter-omics associations but also enable omics-based patient stratification, which will lead to a deeper understanding of cardiomyopathy and the development of precision medicine in cardiology.
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http://dx.doi.org/10.1016/j.jjcc.2018.11.001DOI Listing
March 2019

Discovery of a Small Molecule to Increase Cardiomyocytes and Protect the Heart After Ischemic Injury.

JACC Basic Transl Sci 2018 Oct 12;3(5):639-653. Epub 2018 Nov 12.

Department of Cardiovascular Medicine, The University of Tokyo Hospital, Tokyo, Japan.

Accumulating data suggest that new cardiomyocytes in adults are generated from existing cardiomyocytes throughout life. To enhance the endogenous cardiac regeneration, we performed chemical screenings to identify compounds that activate pro-proliferative YES-associated protein and transcriptional enhancer factor domain activities in cardiomyocytes. We synthesized a novel fluorine-containing TT-10 (CHFNOS) from the biologically hit compound. TT-10 promoted cardiomyocyte proliferation and simultaneously exerted antioxidant and antiapoptotic effects in vitro. TT-10 treatment in mice ameliorated myocardial infarction-induced cardiac dysfunction at least in part via enhancing clonal expansion of existing cardiomyocytes with nuclear YES-associated protein expression. Stimulating cardiomyocyte proliferation and/or protection with TT-10 might complement current therapies for myocardial infarction.
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http://dx.doi.org/10.1016/j.jacbts.2018.07.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6234526PMC
October 2018

Cardiomyocyte gene programs encoding morphological and functional signatures in cardiac hypertrophy and failure.

Nat Commun 2018 10 30;9(1):4435. Epub 2018 Oct 30.

Department of Cardiovascular Medicine, Graduate School of Medicine, The University of Tokyo, Tokyo, 113-8655, Japan.

Pressure overload induces a transition from cardiac hypertrophy to heart failure, but its underlying mechanisms remain elusive. Here we reconstruct a trajectory of cardiomyocyte remodeling and clarify distinct cardiomyocyte gene programs encoding morphological and functional signatures in cardiac hypertrophy and failure, by integrating single-cardiomyocyte transcriptome with cell morphology, epigenomic state and heart function. During early hypertrophy, cardiomyocytes activate mitochondrial translation/metabolism genes, whose expression is correlated with cell size and linked to ERK1/2 and NRF1/2 transcriptional networks. Persistent overload leads to a bifurcation into adaptive and failing cardiomyocytes, and p53 signaling is specifically activated in late hypertrophy. Cardiomyocyte-specific p53 deletion shows that cardiomyocyte remodeling is initiated by p53-independent mitochondrial activation and morphological hypertrophy, followed by p53-dependent mitochondrial inhibition, morphological elongation, and heart failure gene program activation. Human single-cardiomyocyte analysis validates the conservation of the pathogenic transcriptional signatures. Collectively, cardiomyocyte identity is encoded in transcriptional programs that orchestrate morphological and functional phenotypes.
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http://dx.doi.org/10.1038/s41467-018-06639-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6207673PMC
October 2018

Author Correction: Activation of endothelial β-catenin signaling induces heart failure.

Sci Rep 2018 Oct 29;8(1):15858. Epub 2018 Oct 29.

Department of Cardiovascular Medicine, Graduate School of Medicine, The University of Tokyo, Bunkyo-ku, Tokyo, 113-8655, Japan.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has not been fixed in the paper.
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http://dx.doi.org/10.1038/s41598-018-34062-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6206138PMC
October 2018

Phenotypic Screening Using Patient-Derived Induced Pluripotent Stem Cells Identified Pyr3 as a Candidate Compound for the Treatment of Infantile Hypertrophic Cardiomyopathy.

Int Heart J 2018 Sep 11;59(5):1096-1105. Epub 2018 Aug 11.

Department of Cardiovascular Medicine, The University of Tokyo Graduate School of Medicine.

Hypertrophic cardiomyopathy (HCM) is a genetic disorder that is characterized by hypertrophy of the myocardium. Some of the patients are diagnosed for HCM during infancy, and the prognosis of infantile HCM is worse than general HCM. Nevertheless, pathophysiology of infantile HCM is less investigated and remains largely unknown. In the present study, we generated induced pluripotent stem cells (iPSCs) from two patients with infantile HCM: one with Noonan syndrome and the other with idiopathic HCM. We found that iPSC-derived cardiomyocytes (iPSC-CMs) from idiopathic HCM patient were significantly larger and showed higher diastolic intracellular calcium concentration compared with the iPSC-CMs from healthy subject. Unlike iPSC-CMs from the adult/adolescent HCM patient, arrhythmia was not observed as a disease-related phenotype in iPSC-CMs from idiopathic infantile HCM patient. Phenotypic screening revealed that Pyr3, a transient receptor potential channel 3 channel inhibitor, decreased both the cell size and diastolic intracellular calcium concentration in iPSC-CMs from both Noonan syndrome and idiopathic infantile HCM patients, suggesting that the target of Pyr3 may play a role in the pathogenesis of infantile HCM, regardless of the etiology. Further research may unveil the possibility of Pyr3 or its derivatives in the treatment of infantile HCM.
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http://dx.doi.org/10.1536/ihj.17-730DOI Listing
September 2018

Cardiomyopathy with LMNA Mutation.

Int Heart J 2018;59(3):462-464

Department of Cardiovascular Medicine, Graduate School of Medicine, The University of Tokyo.

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http://dx.doi.org/10.1536/ihj.18-214DOI Listing
August 2018

Distinct variants affecting differential splicing of TGFBR1 exon 5 cause either Loeys-Dietz syndrome or multiple self-healing squamous epithelioma.

Eur J Hum Genet 2018 08 30;26(8):1151-1158. Epub 2018 Apr 30.

Department of Cardiovascular Medicine, The University of Tokyo Hospital, 7-3-1 Hongo, Tokyo, 113-8655, Japan.

Variants in TGFBR1 have been reported to induce two completely distinct diseases, namely Loeys-Dietz syndrome (LDS) and multiple self-healing squamous epithelioma (MSSE). However, detailed mechanisms underlying this effect remain unknown. We report a Japanese familial case of LDS with a novel splice donor site variant in TGFBR1 gene (c.973 + 1 G > A; NG_007461.1). The intronic variant was predicted to mediate in-frame exon 5 skipping within the serine/threonine kinase (STK) domain, which may also be mediated by a similar TGFBR1 variant of a splice acceptor site in intron 4 (c.806-2 A > C), identified in a British familial case of MSSE. Therefore, ex vivo splicing and functional assays were performed in mammalian cells to evaluate the effect of these sequence variants. The MSSE variant activated a cryptic acceptor site at 76 bp downstream of the 3' natural splice acceptor site, which produced an out-of-frame transcript (r.807_882del, p.Asn270Thrfs*8). In contrast, the LDS variant generated two types of in-frame transcription products, r.[806_973del, 965_973 del], and produced two functionally inactivated proteins, p.[Asp269_Gln324del, Thr323_Gly325del], as a result of exon 5 skipping and the activation of a cryptic donor splice site at 9 bp upstream of the 5' natural splice donor site, respectively. Our results support the previously proposed but not yet approved mechanism that dominant-negative and truncating variants in STK domain induce LDS and MSSE, respectively.
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http://dx.doi.org/10.1038/s41431-018-0127-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6057981PMC
August 2018

Genetic basis of cardiomyopathy and the genotypes involved in prognosis and left ventricular reverse remodeling.

Sci Rep 2018 01 31;8(1):1998. Epub 2018 Jan 31.

Department of Cardiovascular Medicine, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan.

Dilated cardiomyopathy (DCM) and hypertrophic cardiomyopathy (HCM) are genetically and phenotypically heterogeneous. Cardiac function is improved after treatment in some cardiomyopathy patients, but little is known about genetic predictors of long-term outcomes and myocardial recovery following medical treatment. To elucidate the genetic basis of cardiomyopathy in Japan and the genotypes involved in prognosis and left ventricular reverse remodeling (LVRR), we performed targeted sequencing on 120 DCM (70 sporadic and 50 familial) and 52 HCM (15 sporadic and 37 familial) patients and integrated their genotypes with clinical phenotypes. Among the 120 DCM patients, 20 (16.7%) had TTN truncating variants and 13 (10.8%) had LMNA variants. TTN truncating variants were the major cause of sporadic DCM (21.4% of sporadic cases) as with Caucasians, whereas LMNA variants, which include a novel recurrent LMNA E115M variant, were the most frequent in familial DCM (24.0% of familial cases) unlike Caucasians. Of the 52 HCM patients, MYH7 and MYBPC3 variants were the most common (12 (23.1%) had MYH7 variants and 11 (21.2%) had MYBPC3 variants) as with Caucasians. DCM patients harboring TTN truncating variants had better prognosis than those with LMNA variants. Most patients with TTN truncating variants achieved LVRR, unlike most patients with LMNA variants.
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http://dx.doi.org/10.1038/s41598-018-20114-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5792481PMC
January 2018

Identification of MYLK3 mutations in familial dilated cardiomyopathy.

Sci Rep 2017 12 13;7(1):17495. Epub 2017 Dec 13.

Department of Cardiovascular Medicine, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan.

Dilated cardiomyopathy (DCM) is a primary cause of heart failure, life-threatening arrhythmias, and cardiac death. Pathogenic mutations have been identified at the loci of more than 50 genes in approximately 50% of DCM cases, while the etiologies of the remainder have yet to be determined. In this study, we applied whole exome sequencing in combination with segregation analysis to one pedigree with familial DCM, and identified a read-through mutation (c.2459 A > C; p.*820Sext*19) in the myosin light chain kinase 3 gene (MYLK3). We then conducted MYLK3 gene screening of 15 DCM patients (7 familial and 8 sporadic) who were negative for mutation screening of the previously-reported cardiomyopathy-causing genes, and identified another case with a MYLK3 frameshift mutation (c.1879_1885del; p.L627fs*41). In vitro experiments and immunohistochemistry suggested that the MYLK3 mutations identified in this study result in markedly reduced levels of protein expression and myosin light chain 2 phosphorylation. This is the first report that MYLK3 mutations can cause DCM in humans. The clinical phenotypes of DCM patients were consistent with MYLK3 loss-of-function mouse and zebrafish models in which cardiac enlargement and heart failure are observed. Our findings highlight an essential role for cardiac myosin light chain kinase in the human heart.
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http://dx.doi.org/10.1038/s41598-017-17769-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5727479PMC
December 2017

Echinomycin inhibits adipogenesis in 3T3-L1 cells in a HIF-independent manner.

Sci Rep 2017 07 26;7(1):6516. Epub 2017 Jul 26.

Division of Nephrology and Endocrinology, The University of Tokyo Graduate School of Medicine, Tokyo, Japan.

Obesity is a risk factor for many diseases including diabetes, cancer, cardiovascular disease, and chronic kidney disease. Obesity is characterized by the expansion of white adipose tissue (WAT). Hypertrophy and hyperplasia of adipocytes cause tissue hypoxia followed by inflammation and fibrosis. Its trigger, preadipocyte differentiation into mature adipocytes, is finely regulated by transcription factors, signal molecules, and cofactors. We found that echinomycin, a potent HIF-1 inhibitor, completely inhibited adipogenesis in 3T3-L1 WAT preadipocytes by affecting the early phase of mitotic clonal expansion. The dose required to exert the effect was surprisingly low and the time was short. Interestingly, its inhibitory effect was independent of HIF-1 pathways. Time-course DNA microarray analysis of drug-treated and untreated preadipocytes extracted a major transcription factor, CCAAT/enhancer-protein β, as a key target of echinomycin. Echinomycin also inhibited adipogenesis and body weight gain in high fat diet mice. These findings highlight a novel role of echinomycin in suppressing adipocyte differentiation and offer a new therapeutic strategy against obesity and diabetes.
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http://dx.doi.org/10.1038/s41598-017-06761-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5529514PMC
July 2017

DNA single-strand break-induced DNA damage response causes heart failure.

Nat Commun 2017 04 24;8:15104. Epub 2017 Apr 24.

CREST, Sanbanmachi-building, 5 Sanbanmachi, Tokyo 102-0075, Japan.

The DNA damage response (DDR) plays a pivotal role in maintaining genome integrity. DNA damage and DDR activation are observed in the failing heart, however, the type of DNA damage and its role in the pathogenesis of heart failure remain elusive. Here we show the critical role of DNA single-strand break (SSB) in the pathogenesis of pressure overload-induced heart failure. Accumulation of unrepaired SSB is observed in cardiomyocytes of the failing heart. Unrepaired SSB activates DDR and increases the expression of inflammatory cytokines through NF-κB signalling. Pressure overload-induced heart failure is more severe in the mice lacking XRCC1, an essential protein for SSB repair, which is rescued by blocking DDR activation through genetic deletion of ATM, suggesting the causative role of SSB accumulation and DDR activation in the pathogenesis of heart failure. Prevention of SSB accumulation or persistent DDR activation may become a new therapeutic strategy against heart failure.
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http://dx.doi.org/10.1038/ncomms15104DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5413978PMC
April 2017

Negative feedback loop of bone resorption by NFATc1-dependent induction of Cadm1.

PLoS One 2017 17;12(4):e0175632. Epub 2017 Apr 17.

Department of Orthopaedic Surgery, Faculty of Medicine, The University of Tokyo, Bunkyo-ku, Tokyo, Japan.

Trimethylation of histone H3 lysine 4 and lysine 27 (H3K4me3 and H3K27me3) at gene promoter regions critically regulates gene expression. Key developmental genes tend to exhibit changes in histone modification patterns from the H3K4me3/H3K27me3 bivalent pattern to the H3K4me3 monovalent pattern. Using comprehensive chromatin immunoprecipitation followed by sequencing in bone marrow-derived macrophages (BMMs) and mature osteoclasts, we found that cell surface adhesion molecule 1 (Cadm1) is a direct target of nuclear factor of activated T cells 1 (NFATc1) and exhibits a bivalent histone pattern in BMMs and a monovalent pattern in osteoclasts. Cadm1 expression was upregulated in BMMs by receptor activator of nuclear factor kappa B ligand (RANKL), and blocked by a calcineurin/NFATc1 inhibitor, FK506. Cadm1-deficient mice exhibited significantly reduced bone mass compared with wild-type mice, which was due to the increased osteoclast differentiation, survival and bone-resorbing activity in Cadm1-deficient osteoclasts. These results suggest that Cadm1 is a direct target of NFATc1, which is induced by RANKL through epigenetic modification, and regulates osteoclastic bone resorption in a negative feedback manner.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0175632PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5393607PMC
May 2017

Dysbiosis and compositional alterations with aging in the gut microbiota of patients with heart failure.

PLoS One 2017 22;12(3):e0174099. Epub 2017 Mar 22.

Department of Cardiovascular Medicine, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan.

Emerging evidence has suggested a potential impact of gut microbiota on the pathophysiology of heart failure (HF). However, it is still unknown whether HF is associated with dysbiosis in gut microbiota. We investigated the composition of gut microbiota in patients with HF to elucidate whether gut microbial dysbiosis is associated with HF. We performed 16S ribosomal RNA gene sequencing of fecal samples obtained from 12 HF patients and 12 age-matched healthy control (HC) subjects, and analyzed the differences in gut microbiota. We further compared the composition of gut microbiota of 12 HF patients younger than 60 years of age with that of 10 HF patients 60 years of age or older. The composition of gut microbial communities of HF patients was distinct from that of HC subjects in both unweighted and weighted UniFrac analyses. Eubacterium rectale and Dorea longicatena were less abundant in the gut microbiota of HF patients than in that of HC subjects. Compared to younger HF patients, older HF patients had diminished proportions of Bacteroidetes and larger quantities of Proteobacteria. The genus Faecalibacterium was depleted, while Lactobacillus was enriched in the gut microbiota of older HF patients. These results suggest that patients with HF harbor significantly altered gut microbiota, which varies further according to age. New concept of heart-gut axis has a great potential for breakthroughs in the development of novel diagnostic and therapeutic approach for HF.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0174099PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5362204PMC
August 2017

Hypoxia-Inducible Factor-1α Activates the Transforming Growth Factor-β/SMAD3 Pathway in Kidney Tubular Epithelial Cells.

Am J Nephrol 2016 9;44(4):276-285. Epub 2016 Sep 9.

Division of Genome Science, Research Center for Advanced Science and Technology, University of Tokyo, Tokyo, Japan.

Background: Kidney injury, including chronic kidney disease and acute kidney injury, is a worldwide health problem. Hypoxia and transforming growth factor-β (TGF-β) are well-known factors that promote kidney injury. Hypoxia-inducible factor (HIF) and SMAD3 are their main downstream transcriptional factors. Hypoxia-HIF pathway and TGF-β/SMAD3 pathway play a crucial role in the progression of kidney injury. However, reports on their interactions are limited, and the global transcriptional regulation under their control is almost unknown.

Methods: Kidney tubular epithelial cells were cultured and stimulated by hypoxia and TGF-β. We detected global binding sites of HIF-1α and SMAD3 in cells using chromatin immunoprecipitation-sequencing (ChIP-Seq), and measured the gene expression using RNA-sequencing (RNA-Seq). ChIP-quantitative PCR (qPCR) was used to quantitatively evaluate bindings of SMAD3.

Results: ChIP-Seq revealed that 2,065 and 5,003 sites were bound by HIF-1α and SMAD3, respectively, with 614 sites co-occupied by both factors. RNA-Seq showed that hypoxia and TGF-β stimulation causes synergistic upregulation of 249 genes, including collagen type I alpha 1 (COL1A1) and serpin peptidase inhibitor, clade E, member 1, which are well-known to be involved in fibrosis. Ontology of the 249 genes implied that the interaction of HIF-1α and SMAD3 is related to biological processes such as fibrosis. ChIP-qPCR of SMAD3 at HIF-1α binding sites near COL1A1 and SERPINE1 indicated that HIF-1α promotes the bindings of SMAD3, which is induced by TGF-β.

Conclusions: These findings suggest that HIF-1α induced by hypoxia activates the TGF-β/SMAD3 pathway. This mechanism may promote kidney injury, especially by upregulating genes related to fibrosis.
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http://dx.doi.org/10.1159/000449323DOI Listing
January 2018

CellTree: an R/bioconductor package to infer the hierarchical structure of cell populations from single-cell RNA-seq data.

BMC Bioinformatics 2016 Sep 13;17(1):363. Epub 2016 Sep 13.

Graduate School of Frontier Sciences at the University of Tokyo, 5-1-5 Kashiwa-no-ha, Kashiwa, Japan.

Background: Single-cell RNA sequencing is fast becoming one the standard method for gene expression measurement, providing unique insights into cellular processes. A number of methods, based on general dimensionality reduction techniques, have been suggested to help infer and visualise the underlying structure of cell populations from single-cell expression levels, yet their models generally lack proper biological grounding and struggle at identifying complex differentiation paths.

Results: Here we introduce cellTree: an R/Bioconductor package that uses a novel statistical approach, based on document analysis techniques, to produce tree structures outlining the hierarchical relationship between single-cell samples, while identifying latent groups of genes that can provide biological insights.

Conclusions: With cellTree, we provide experimentalists with an easy-to-use tool, based on statistically and biologically-sound algorithms, to efficiently explore and visualise single-cell RNA data. The cellTree package is publicly available in the online Bionconductor repository at: http://bioconductor.org/packages/cellTree/ .
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http://dx.doi.org/10.1186/s12859-016-1175-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5020541PMC
September 2016

Activation of endothelial β-catenin signaling induces heart failure.

Sci Rep 2016 05 5;6:25009. Epub 2016 May 5.

Department of Cardiovascular Medicine, Graduate School of Medicine, The University of Tokyo, Bunkyo-ku, Tokyo 113-8655, Japan.

Activation of β-catenin-dependent canonical Wnt signaling in endothelial cells plays a key role in angiogenesis during development and ischemic diseases, however, other roles of Wnt/β-catenin signaling in endothelial cells remain poorly understood. Here, we report that sustained activation of β-catenin signaling in endothelial cells causes cardiac dysfunction through suppressing neuregulin-ErbB pathway in the heart. Conditional gain-of-function mutation of β-catenin, which activates Wnt/β-catenin signaling in Bmx-positive arterial endothelial cells (Bmx/CA mice) led to progressive cardiac dysfunction and 100% mortality at 40 weeks after tamoxifen treatment. Electron microscopic analysis revealed dilatation of T-tubules and degeneration of mitochondria in cardiomyocytes of Bmx/CA mice, which are similar to the changes observed in mice with decreased neuregulin-ErbB signaling. Endothelial expression of Nrg1 and cardiac ErbB signaling were suppressed in Bmx/CA mice. The cardiac dysfunction of Bmx/CA mice was ameliorated by administration of recombinant neuregulin protein. These results collectively suggest that sustained activation of Wnt/β-catenin signaling in endothelial cells might be a cause of heart failure through suppressing neuregulin-ErbB signaling, and that the Wnt/β-catenin/NRG axis in cardiac endothelial cells might become a therapeutic target for heart failure.
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http://dx.doi.org/10.1038/srep25009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4857119PMC
May 2016