Published papers

Cardiovascular disease is a leading cause of death worldwide. Mammalian cardiomyocytes (CMs) proliferate during embryonic development, whereas they largely lose their regenerative capacity after birth. Defined factors expressed in cardiac progenitors or embryonic CMs may activate the cell cycle and induce CM proliferation in postnatal and adult hearts. Here, we report that the overexpression of Tbx6, enriched in the cardiac mesoderm (progenitor cells), induces CM proliferation in postnatal and adult mouse hearts. By screening 24 factors enriched in cardiac progenitors or embryonic CMs, we found that only Tbx6 could induce CM proliferation in primary cultured postnatal rat CMs. Intriguingly, it did not induce the proliferation of cardiac fibroblasts. We next generated a recombinant adeno-associated virus serotype 9 vector encoding Tbx6 (AAV9-Tbx6) for transduction into mouse CMs in vivo. The subcutaneous injection of AAV9-Tbx6 into neonatal mice induced CM proliferation in postnatal and adult mouse hearts. Mechanistically, Tbx6 overexpression upregulated multiple cell cycle activators including Aurkb, Mki67, Ccna1, and Ccnb2 and suppressed the tumor suppressor Rb1. Thus, Tbx6 promotes CM proliferation in postnatal and adult mouse hearts by modifying the expression of cell cycle regulators.

＜文献概要＞心不全治療において,現在の薬物療法や心臓移植の限界を克服する新たな手法として,心筋ダイレクトリプログラミングが注目されている.この手法は,iPS細胞などの未分化細胞を介さずに,線維芽細胞から直接心筋細胞を作り出す画期的な方法である.Gata4,Mef2c,Tbx5(GMT)という3因子をリプログラミング因子として同定し,心臓線維芽細胞を心筋細胞へ直接転換することに成功し,その後,Hand2を加えた4因子(MGTH)での効率改善や,遺伝子組換えマウスを用いた薬物投与による慢性心筋梗塞モデルでの有効性も実証した.慢性心筋梗塞モデルでは心臓線維芽細胞の約2%が心筋細胞へと誘導され,心臓の収縮能も回復した.特筆すべき発見として,シングルセル解析により,心筋細胞への転換だけでなく,転換しなかった線維芽細胞においても抗線維化作用が誘導されることを解明し,さらに,近年増加している収縮力の保たれた心不全(HFpEF)モデルにおいても,心機能改善と線維化抑制効果を確認した.HFpEFモデルでは,Gata4単独の発現でも線維化関連遺伝子の発現低下が示された.この手法は細胞移植を必要としない画期的な治療法として期待されており,今後は遺伝性心筋症モデルや大型動物での検証が求められている.

iPS細胞による再生医療に続く新たな手法として,心臓内の線維芽細胞を直接心筋細胞に誘導する「心筋ダイレクトリプログラミング」が注目されている.我々はマウス線維芽細胞に3つの心筋特異的な遺伝子であるGata4,Mef2c,Tbx5(GMT)を導入することで,誘導心筋細胞(induced Cardiomyocyte:iCM細胞)が誘導できることを世界で初めて発見し,マウス急性心筋梗塞モデルにおいて生体内の線維芽細胞からiCM細胞への誘導に成功した.最近では,慢性心筋梗塞モデルにおいてもiCM細胞への誘導に成功し心機能の改善,さらには,心筋梗塞後の瘢痕組織形成に関与する線維芽細胞の性質を変換することで心臓線維化の改善にも成功した.一方で,臨床応用に向けてはいまだ解決すべき問題も存在する.本稿では新たな心臓再生医療である心筋ダイレクトリプログラミングと,最新の研究成果について概説する.(著者抄録)

＜文献概要＞高齢化に伴い心不全患者は増加し,心不全パンデミックが切迫した課題となっている.近年はアンジオテンシン受容体ネプリライシン阻害薬(ARNI)やSGLT2阻害薬の登場,弁膜症や不整脈に対するカテーテル治療の進歩により予後改善が示されているが,依然として難治性心不全患者が存在する.心臓移植は唯一の根治療法であるものの,ドナー不足や厳格な適応基準により限界がある.心筋細胞は再生能力に乏しく,壊死や線維化によりポンプ機能低下をきたすため,再生医療が有望視されてきた.iPS細胞由来心筋細胞を用いた移植治療は治験段階に達したが,コスト,長期生着,侵襲的手技など課題も多い.これを克服する革新的戦略として,筆者らは"心筋ダイレクトリプログラミング"に基づく遺伝子治療を展開しており,本稿では臨床応用の実現化を目指した生体内心筋リプログラミングの成果と,今後の展望について概説する.

＜文献概要＞心不全に対して近年,薬物療法のみならずカテーテル・デバイス治療も大幅に開発が進んでいるが,一定数に治療抵抗性を示す予後不良な心不全が存在する。そのような難治性心不全には心移植が検討されるが,ドナー不足による心移植待機期間の長期化,その間のケアギバーの不足,移植後の免疫学的拒絶反応,生涯にわたる免疫抑制薬服薬など数多くの問題を抱えており,新たな治療法として再生治療が注目を集めている。

心血管疾患は世界的に主要な死因のひとつであり,日本では悪性腫瘍に次ぐ第2位となっている.成熟した心筋細胞は自己複製能がないため,虚血性心疾患や心不全などの心疾患で壊死した心筋細胞は,線維芽細胞に置換され瘢痕化する.心臓の線維化・瘢痕化は心機能の低下および致死的不整脈の発症につながる.薬物療法などによる治療で改善が見込めない場合は心臓移植が最終手段となるが,わが国ではドナー不足の問題で広く普及していない現状があり,新たな治療戦略として心筋再生に注目が集まっている.人工多能性幹細胞(iPS細胞)を用いた心筋再生が期待されているが,幹細胞による腫瘍形成の可能性や生着率の低さが問題点として存在する.遺伝子治療によって線維芽細胞から心筋細胞へ直接誘導することができれば,これらの問題点を解決することができる.筆者らは,iPS細胞とは異なるアプローチの心筋再生法として,心臓線維芽細胞を心筋細胞に直接分化転換させる"心筋ダイレクトリプログラミング"の研究を行っている(図1).(著者抄録)

＜文献概要＞心臓における幹細胞研究は,基礎研究手法の発展により大きなパラダイムシフトを迎えた。系統追跡技術の進歩により,発生過程において多様な細胞集団が時間・空間的に複雑な分化を繰り返し,心臓が形成されることが明らかとなり,シングルセル解析によって,その細胞集団の全貌が解明されはじめている。一方で,これらの技術の進歩は,組織幹細胞としての心臓幹細胞の存在を覆し,基礎研究で得られた知見が,なぜ有効な臨床応用に結びつかなかったかを明らかにした。本稿では,その変遷を幅広い視点から述べていくこととする。

＜ここがポイント!＞▼治療を始める前に前負荷,後負荷,心機能,心拍数を評価する.▼モニタリングは非侵襲的評価方法があるが,限界を理解し,評価が難しい場合には侵襲的評価を行う.▼不全心における高血圧は,心拍出量を減らすことから,放置せずに介入する.▼過剰な頻脈は是正することを検討し,頻脈性心房細動の場合には除細動も考慮する.▼エコーは検査へのアクセスが容易であり,経時的変化をみながら管理を行う.(著者抄録)

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.

Heart disease remains a leading cause of death worldwide. Owing to the limited regenerative capacity of heart tissue, cardiac regenerative therapy has emerged as an attractive approach. Direct reprogramming of human cardiac fibroblasts (HCFs) into cardiomyocytes may hold great potential for this purpose. We reported previously that induced cardiomyocyte-like cells (iCMs) can be directly generated from mouse cardiac fibroblasts in vitro and vivo by transduction of three transcription factors: Gata4, Mef2c, and Tbx5, collectively termed GMT. In the present study, we sought to determine whether human fibroblasts also could be converted to iCMs by defined factors. Our initial finding that GMT was not sufficient for cardiac induction in HCFs prompted us to screen for additional factors to promote cardiac reprogramming by analyzing multiple cardiac-specific gene induction with quantitative RT-PCR. The addition of Mesp1 and Myocd to GMT up-regulated a broader spectrum of cardiac genes in HCFs more efficiently compared with GMT alone. The HCFs and human dermal fibroblasts transduced with GMT, Mesp1, and Myocd (GMTMM) changed the cell morphology from a spindle shape to a rod-like or polygonal shape, expressed multiple cardiac-specific proteins, increased a broad range of cardiac genes and concomitantly suppressed fibroblast genes, and exhibited spontaneous Ca2+ oscillations. Moreover, the cells matured to exhibit action potentials and contract synchronously in coculture with murine cardiomyocytes. A 5-ethynyl-2′-deoxyuridine assay revealed that the iCMs thus generated do not pass through a mitotic cell state. These findings demonstrate that human fibroblasts can be directly converted to iCMs by defined factors, which may facilitate future applications in regenerative medicine.

RATIONALE: After myocardial infarction (MI), massive cell death in the myocardium initiates fibrosis and scar formation, leading to heart failure. We recently found that a combination of 3 cardiac transcription factors, Gata4, Mef2c, and Tbx5 (GMT), reprograms fibroblasts directly into functional cardiomyocytes in vitro. OBJECTIVE: To investigate whether viral gene transfer of GMT into infarcted hearts induces cardiomyocyte generation. METHODS AND RESULTS: Coronary artery ligation was used to generate MI in the mouse. In vitro transduction of GMT retrovirus converted cardiac fibroblasts from the infarct region into cardiomyocyte-like cells with cardiac-specific gene expression and sarcomeric structures. Injection of the green fluorescent protein (GFP) retrovirus into mouse hearts, immediately after MI, infected only proliferating noncardiomyocytes, mainly fibroblasts, in the infarct region. The GFP expression diminished after 2 weeks in immunocompetent mice but remained stable for 3 months in immunosuppressed mice, in which cardiac induction did not occur. In contrast, injection of GMT retrovirus into α-myosin heavy chain (αMHC)-GFP transgenic mouse hearts induced the expression of αMHC-GFP, a marker of cardiomyocytes, in 3% of virus-infected cells after 1 week. A pooled GMT injection into the immunosuppressed mouse hearts induced cardiac marker expression in retrovirus-infected cells within 2 weeks, although few cells showed striated muscle structures. To transduce GMT efficiently in vivo, we generated a polycistronic retrovirus expressing GMT separated by 2A "self-cleaving" peptides (3F2A). The 3F2A-induced cardiomyocyte-like cells in fibrotic tissue expressed sarcomeric α-actinin and cardiac troponin T and had clear cross striations. Quantitative RT-PCR also demonstrated that FACS-sorted 3F2A-transduced cells expressed cardiac-specific genes. CONCLUSIONS: GMT gene transfer induced cardiomyocyte-like cells in infarcted hearts.

ssDirect reprogramming is a promising approach in regenerative medicine. Overexpression of the cardiac transcription factors Gata4, Mef2c, and Tbx5 (GMT) or GMT plus Hand2 (GHMT) directly reprogram fibroblasts into cardiomyocyte-like cells (iCMs). However, the critical timing of transgene expression and the molecular mechanisms for cardiac reprogramming remain unclear. The conventional doxycycline (Dox)-inducible temporal transgene expression systems require simultaneous transduction of two vectors (pLVX-rtTA/pLVX-cDNA) harboring the reverse tetracycline transactivator (rtTA) and the tetracycline response element (TRE)-controlled transgene, respectively, leading to inefficient cardiac reprogramming. Herein, we developed a single-construct-based polycistronic Dox-inducible vector (pDox-cDNA) expressing both the rtTA and TRE-controlled transgenes. Fluorescence activated cell sorting (FACS) analyses, quantitative RT-PCR, and immunostaining revealed that pDox-GMT increased cardiac reprogramming three-fold compared to the conventional pLVX-rtTA/pLVX-GMT. After four weeks, pDox-GMT-induced iCMs expressed multiple cardiac genes, produced sarcomeric structures, and beat spontaneously. Co-transduction of pDox-Hand2 with retroviral pMX-GMT increased cardiac reprogramming three-fold compared to pMX-GMT alone. Temporal Dox administration revealed that Hand2 transgene expression is critical during the first two weeks of cardiac reprogramming. Microarray analyses demonstrated that Hand2 represses cell cycle-promoting genes and enhances cardiac reprogramming. Thus, we have developed an efficient temporal transgene expression system, which could be invaluable in the study of cardiac reprogramming.

Cardiovascular diseases are a common cause of death worldwide. Adult cardiomyocytes have limited regenerative capacity after injury, and there is growing interest in cardiac regeneration as a new therapeutic strategy. There are several limitations of induced pluripotent stem cell-based transplantation therapy with respect to efficiency and risks of tumorigenesis. Direct reprogramming enables the conversion of terminally differentiated cells into target cell types using defined factors. In most cardiac diseases, activated fibroblasts proliferate in the damaged heart and contribute to the progression of heart failure. In vivo cardiac reprogramming, in which resident cardiac fibroblasts are converted into cardiomyocytes in situ, is expected to become a new cardiac regenerative therapy. Indeed, we and other groups have demonstrated that in vivo reprogramming improves cardiac function and reduces fibrosis after myocardial infarction. In this review, we summarize recent discoveries and developments related to in vivo reprogramming. In addition, issues that need to be resolved for clinical application are described.

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.

OBJECTIVE: Myocardial fibrosis related to non-specific inflammation can be detected using late gadolinium-enhancement cardiovascular MR (LGE-CMR), which is an important prognostic indicator for dilated cardiomyopathy (DCM). The aims of this study were to define the prognostic factors for DCM with LGE-CMR, and to evaluate the impact of the prognostic factors on adverse effects. METHODS: We performed a retrospective analysis of a prospectively maintained single centre registry. We analysed the data from 76 patients with DCM who had been admitted for acute heart failure. The primary combined end point was defined as all-cause mortality and rehospitalisation. RESULTS: LGE-CMR was present in 39 patients (51%), and the mean follow-up period was 813±54 days. The primary end point occurred in 20 patients (5 (13.5%) patients without LGE-CMR and 15 (38.5%) patients with LGE-CMR, p=0.006). Sixteen of 39 patients with LGE-CMR exhibited elevated high-sensitivity C reactive protein (hs-CRP >0.3 mg/dL). Patients with elevated hs-CRP and LGE-CMR had a significantly higher incidence of the primary end point compared with patients with normal hs-CRP and LGE-CMR (62.5%; 10 patients, 22.7%; 5 patients, respectively, p=0.001). Elevated hs-CRP was significantly associated with the primary end point (HR: 4.04; 95% CI 1.67 to 9.76; p=0.002). After elevated hs-CRP was adjusted for known predictors of DCM, it was still associated with the primary end point (HR: 2.91; 95% CI 1.19 to 7.15; p=0.02). CONCLUSIONS: Among patients with DCM, LGE-CMR and elevated hs-CRP are associated with a higher incidence of the long-term combined end point of all-cause mortality and hospitalisation. TRIAL REGISTRATION NUMBER: UMIN000001171.

BACKGROUND: The CRISPR/Cas9 technique has undergone many modifications to decrease the effort and shorten the time needed for efficient production of mutant mice. The use of fresh embryos consumes time and effort during oocytes preparation and fertilization before every experiment, and freeze-thawed embryos overcome this limitation. However, cryopreservation of 1-cell embryos is challenging. NEW METHOD: We introduce a protocol that combines a modified method for cryopreserving 1-cell C57BL/6J embryos with optimized electroporation conditions that were used to deliver CRISPR reagents into embryos, 1 h after thawing. RESULTS: Freeze-thawed 1-cell embryos showed similar survival rates and surprisingly high developmental rates compared to fresh embryos. Using our protocol, we generated several lines of mutant mice: knockout mice via non-homologous end joining (NHEJ) and knock-in mice via homology-directed repair (HDR) with high-efficient mutation rates (100%, 75% respectively) and a low mosaic rate within 4 weeks. COMPARISON WITH EXISTING METHOD (S): Our protocol associates the use of freeze-thawed embryos from an inbred strain and electroporation, and can be performed by laboratory personnel with basic training in embryo manipulation to generate mutant mice within short time periods. CONCLUSION: We developed a simple, economic, and robust protocol facilitating the generation of genetically modified mice, bypassing the need of backcrossing, with a high efficiency and a low mosaic rate. It makes the preparation of mouse models of human diseases a simple task with unprecedented ease, pace, and efficiency.