Published papers

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.

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.

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.

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.

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。

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.