Overexpression of Gata4, Mef2c, and Tbx5 Generates Induced Cardiomyocytes Via Direct Reprogramming and Rare Fusion in the Heart
2021; Lippincott Williams & Wilkins; Volume: 143; Issue: 21 Linguagem: Inglês
10.1161/circulationaha.120.052799
ISSN1524-4539
AutoresMari Isomi, Taketaro Sadahiro, Hiroyuki Yamakawa, Ryo Fujita, Yu Yamada, Yuto Abe, Yoshiko Murakata, Tatsuya Akiyama, Tsugumine Shu, Hiroaki Mizukami, Keiichi Fukuda, Masaki Ieda,
Tópico(s)Signaling Pathways in Disease
ResumoHomeCirculationVol. 143, No. 21Overexpression of Gata4, Mef2c, and Tbx5 Generates Induced Cardiomyocytes Via Direct Reprogramming and Rare Fusion in the Heart Free AccessLetterPDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyRedditDiggEmail Jump toFree AccessLetterPDF/EPUBOverexpression of Gata4, Mef2c, and Tbx5 Generates Induced Cardiomyocytes Via Direct Reprogramming and Rare Fusion in the Heart Mari Isomi, BVSc Taketaro Sadahiro, MD, PhD Hiroyuki Yamakawa, MD, PhD Ryo Fujita, PhD Yu Yamada, MD Yuto Abe, MD Yoshiko Murakata, BS Tatsuya Akiyama, MD Tsugumine Shu, PhD Hiroaki Mizukami, MD, PhD Keiichi Fukuda, MD, PhD Masaki IedaMD, PhD Mari IsomiMari Isomi Departments of Cardiology (M. Isomi, T. Sadahiro, R.F., Y.Y., Y.A., Y.M., T.A., M. Ieda), University of Tsukuba, Ibaraki, Japan. Search for more papers by this author , Taketaro SadahiroTaketaro Sadahiro Departments of Cardiology (M. Isomi, T. Sadahiro, R.F., Y.Y., Y.A., Y.M., T.A., M. Ieda), University of Tsukuba, Ibaraki, Japan. Search for more papers by this author , Hiroyuki YamakawaHiroyuki Yamakawa https://orcid.org/0000-0003-3100-5077 Department of Cardiology, Keio University School of Medicine, Tokyo, Japan (H.Y., K.F.). Search for more papers by this author , Ryo FujitaRyo Fujita https://orcid.org/0000-0002-8392-0122 Departments of Cardiology (M. Isomi, T. Sadahiro, R.F., Y.Y., Y.A., Y.M., T.A., M. Ieda), University of Tsukuba, Ibaraki, Japan. Faculty of Medicine, and Division of Regenerative Medicine, Transborder Medical Research Center (R.F.), University of Tsukuba, Ibaraki, Japan. Search for more papers by this author , Yu YamadaYu Yamada Departments of Cardiology (M. Isomi, T. Sadahiro, R.F., Y.Y., Y.A., Y.M., T.A., M. Ieda), University of Tsukuba, Ibaraki, Japan. Search for more papers by this author , Yuto AbeYuto Abe Departments of Cardiology (M. Isomi, T. Sadahiro, R.F., Y.Y., Y.A., Y.M., T.A., M. Ieda), University of Tsukuba, Ibaraki, Japan. Search for more papers by this author , Yoshiko MurakataYoshiko Murakata Departments of Cardiology (M. Isomi, T. Sadahiro, R.F., Y.Y., Y.A., Y.M., T.A., M. Ieda), University of Tsukuba, Ibaraki, Japan. Search for more papers by this author , Tatsuya AkiyamaTatsuya Akiyama Departments of Cardiology (M. Isomi, T. Sadahiro, R.F., Y.Y., Y.A., Y.M., T.A., M. Ieda), University of Tsukuba, Ibaraki, Japan. Respiratory Medicine (T.A.), University of Tsukuba, Ibaraki, Japan. Search for more papers by this author , Tsugumine ShuTsugumine Shu ID Pharma Co, Ltd, R&D Center, Techno Park Oho, Tsukuba, Ibaraki, Japan (T. Shu). Search for more papers by this author , Hiroaki MizukamiHiroaki Mizukami Division of Genetic Therapeutics, Center for Molecular Medicine, Jichi Medical University, Tochigi, Japan (H.M.). Search for more papers by this author , Keiichi FukudaKeiichi Fukuda Department of Cardiology, Keio University School of Medicine, Tokyo, Japan (H.Y., K.F.). Search for more papers by this author , Masaki IedaMasaki Ieda Masaki Ieda, MD, PhD, Department of Cardiology, Faculty of Medicine, University of Tsukuba, 1-1-1 Tennoudai, Tsukuba City, Ibaraki 305-8575, Japan. Email E-mail Address: [email protected] https://orcid.org/0000-0003-4724-3299 Departments of Cardiology (M. Isomi, T. Sadahiro, R.F., Y.Y., Y.A., Y.M., T.A., M. Ieda), University of Tsukuba, Ibaraki, Japan. Search for more papers by this author Originally published24 May 2021https://doi.org/10.1161/CIRCULATIONAHA.120.052799Circulation. 2021;143:2123–2125After myocardial infarction (MI), the lost myocardium is replaced by fibrotic tissues, leading to cardiac dysfunction. The overexpression of cardiac transcription factors Gata4, Mef2c, and Tbx5 (GMT) has been shown to directly reprogram cardiac fibroblasts (CFs) into induced cardiomyocytes (iCMs) in vitro, generate new iCMs in vivo, and improve cardiac function after MI in mice.1–3 Thus, direct cardiac reprogramming holds great potential for cardiac regeneration, if iCMs truly arise from cardiac reprogramming in the heart. In this regard, c-kit+ progenitor cells were initially reported to produce new cardiomyocytes in the heart; however, recent lineage-tracing studies have revealed that these cells fuse with the surrounding cardiomyocytes and minimally contribute to cardiac regeneration.4,5 Thus, it is critical to determine whether in vivo iCMs originate from bona fide cardiac reprogramming or cell fusion with cardiomyocytes. We provide the first evidence that Sendai virus (SeV) vectors expressing GMT generate iCMs through largely bona fide cardiac reprogramming in adult mouse hearts after MI.We generated Tcf21iCre/R26mTmG mice constitutively expressing a membrane-targeted fluorescent tdTomato from the Rosa26 locus. On fibroblast-lineage Cre-mediated recombination, tdTomato was lost, and a membrane-targeted eGFP (enhanced green fluorescent protein) was expressed to assess cardiac reprogramming and fusion events.3,4 In these mice, cell fusion between CFs and cardiomyocytes is indicated by the presence of both tdTomato and eGFP signals (fusion), whereas direct reprogramming of cardiomyocytes from CFs is indicated by the presence of eGFP signal alone (bona fide cardiac reprogramming; Figure, A). All animal procedures were performed in accordance with institutional guidelines. The Tcf21iCre/R26mTmG mice were treated with tamoxifen for 5 days to label the TCF21-expressing CFs and coronary artery ligation was performed 7 days after the last tamoxifen treatment. Immunohistochemistry results detected numerous eGFP+ fibroblasts within the infarct areas at 4 weeks after MI (Figure, B). Next, we directly injected mock or SeV-GMT into the Tcf21iCre/R26mTmG mouse hearts immediately after MI, and immunohistochemistry analysis was performed after 4 weeks. SeV vectors are nonintegrating vectors that do not introduce insertional mutagenesis and primarily infect CFs in infarcted mouse hearts.3 In the mock group, only ≈0.3% of eGFP+ cells expressed both α-actinin and tdTomato at the border infarct area, suggesting the occurrence of rare fusion events between CFs and cardiomyocytes, which was not observed before MI (Figure, C, F, and G). eGFP+/tdTomato− cardiomyocytes were not detected in the mock group, indicating that CFs were not converted into cardiomyocytes. In contrast, 1.0 to 1.5% eGFP+ cells expressed α-actinin but not tdTomato (cardiac reprogramming) and cell fusion rates were unchanged with SeV-GMT treatment, suggesting that the majority (≈80%) of new iCMs were generated through bona fide cardiac reprogramming (Figure, D and G). We confirmed that bona fide, matured iCMs exhibited clear cross-striations and expressed eGFP and α-actinin by 3-dimensional analyses (Figure, D and E). Thus, rare fusion events between cardiomyocytes and CFs were observed in mouse hearts after MI, and SeV-GMT treatment mostly generated new iCMs through bona fide cardiac reprogramming. The data that support the findings of this study and research materials, as well as experimental procedures and protocols, are available from the corresponding author on reasonable request.Download figureDownload PowerPointFigure. SeV–GMT generated induced cardiomyocytes through bona fide cardiac reprogramming and cell fusion.A, Schematic diagram shows the genetic fate mapping method to trace the lineage of resident cardiac fibroblasts (CFs) and fusion analyses using Tcf21iCre/mTmG mice. GFP+ (green fluorescent protein) cardiomyocytes indicate bona fide cardiac reprogramming, whereas GFP+ tdTomato+ cardiomyocytes indicate cell fusion between CFs and cardiomyocytes. B, Section of the Tcf21iCre/mTmG mouse heart 4 weeks after myocardial infarction (MI). Immunohistochemistry for tdTomato, GFP, and DAPI (4′,6-diamidino-2-phenylindole) is shown. C, Immunohistochemistry for GFP, tdTomato, and α-actinin 4 weeks after MI (mock group). The GFP+ cells did not colocalize with tdTomato and α-actinin (upper panel). An example of a GFP+ cardiomyocyte that contains tdTomato, indicating fusion, is presented in the lower panel. D and E, Immunohistochemistry for GFP, tdTomato, and α-actinin 4 weeks after MI in Tcf21iCre/mTmG mouse hearts (SeV-GMT group). SeV-GMT was directly injected into the infarct myocardium. Examples of cardiac reprogramming, in which GFP and α-actinin are expressed and tdTomato is lost, are presented (D, upper and middle panels). An example of a GFP+ cardiomyocyte that contains tdTomato, indicating fusion, is presented (D, lower panel). High-magnification views in insets show the sarcomeric organization of induced cardiomyocytes. Z-stack images for bona fide induced cardiomyocytes are shown in E. F, Immunohistochemistry for GFP and tdTomato in Tcf21iCre/mTmG mouse hearts 7 days after finishing the tamoxifen treatment and before MI (day 0) is shown. G, Quantitative analyses are shown (5 independent triplicate experiments). Representative images are shown. All data are presented as the mean±SD. Data were analyzed using 1-way analysis of variance and a Tukey multiple comparisons test. **P<0.01 versus the relevant control. Scale bars = 50 μm. GMT indicates Gata4, Mef2c, and Tbx5; ND, not detected; NS, not significant; and SeV, Sendai virus.Previous studies have used αMHC (α-myosin heavy chain)–MerCreMer/reporter transgenic mice and excluded fusion between cardiomyocytes and CFs during in vivo cardiac reprogramming.1,2 However, αMHC-MerCreMer/reporter lines can label only 60% to 80% of endogenous cardiomyocytes, which may underestimate the fusion events between cardiomyocytes and CFs. Here, we used Tcf21iCre/R26mTmG mice that have been extensively used for fibroblast lineage tracing and fusion analyses.3,4 The R26mTmG detection system could precisely differentiate between de novo cardiomyocyte generation and cell fusion in the hearts. Using c-kit–MerCreMer/R26mTmG mice, Van Berlo et al.4 revealed that the majority (80% to 88%) of c-kit+ lineage cardiomyocytes were derived from fusion with the surrounding cardiomyocytes. With this system, we detected rare fusion events between CFs and cardiomyocytes in the border infarct area after MI; it is notable that the fusion rate did not change with SeV-GMT treatment. However, it is likely that not all CFs were labeled using the Tcf21iCre/R26mTmG mouse and a pulse of tamoxifen for 5 days. Thus, the frequency of fusion is likely context dependent and may not apply to all viral vectors or other cell types. An alternative transgenic system using dual recombinases (Cre and Dre) may achieve more selective genetic tracing and reveal fusion and direct reprogramming events in detail.5 Other groups also reported a higher reprogramming efficiency than that observed in this study, which may be attributed to the use of different mouse lines, viral vectors, or quantification methods.1,2 Our findings also raise the intriguing question of whether increasing cell fusion between CFs and cardiomyocytes in situ may represent a novel approach for cardiac repair. In summary, direct reprogramming may be a promising strategy for cardiac regeneration, and further studies are needed to accelerate its potential clinical translation for therapy.Sources of FundingThis work was supported by research grants from the Practical Research Project for Rare/Intractable Diseases (grant JP20ek0109305), the Research Center Network for Realization of Regenerative Medicine (grant JP20bm0704030), the Japan Agency for Medical Research and Development, the Japan Society for the Promotion of Science (grants 19K22613 and 19K17550), and the Takeda Science Foundation.Disclosures None.Footnoteshttps://www.ahajournals.org/journal/circMasaki Ieda, MD, PhD, Department of Cardiology, Faculty of Medicine, University of Tsukuba, 1-1-1 Tennoudai, Tsukuba City, Ibaraki 305-8575, Japan. Email [email protected]tsukuba.ac.jpReferences1. Qian L, Huang Y, Spencer CI, Foley A, Vedantham V, Liu L, Conway SJ, Fu JD, Srivastava D. In vivo reprogramming of murine cardiac fibroblasts into induced cardiomyocytes.Nature. 2012; 485:593–598. doi: 10.1038/nature11044CrossrefMedlineGoogle Scholar2. Song K, Nam YJ, Luo X, Qi X, Tan W, Huang GN, Acharya A, Smith CL, Tallquist MD, Neilson EG, et al.. Heart repair by reprogramming non-myocytes with cardiac transcription factors.Nature. 2012; 485:599–604. doi: 10.1038/nature11139CrossrefMedlineGoogle Scholar3. Miyamoto K, Akiyama M, Tamura F, Isomi M, Yamakawa H, Sadahiro T, Muraoka N, Kojima H, Haginiwa S, Kurotsu S, et al.. Direct in vivo reprogramming with Sendai virus vectors improves cardiac function after myocardial infarction.Cell Stem Cell. 2018; 22:91–103.e5. doi: 10.1016/j.stem.2017.11.010CrossrefMedlineGoogle Scholar4. van Berlo JH, Kanisicak O, Maillet M, Vagnozzi RJ, Karch J, Lin SC, Middleton RC, Marbán E, Molkentin JD. C-kit+ cells minimally contribute cardiomyocytes to the heart.Nature. 2014; 509:337–341. doi: 10.1038/nature13309CrossrefMedlineGoogle Scholar5. Li Y, He L, Huang X, Bhaloo SI, Zhao H, Zhang S, Pu W, Tian X, Li Y, Liu Q, et al.. Genetic lineage tracing of nonmyocyte population by dual recombinases.Circulation. 2018; 138:793–805. doi: 10.1161/CIRCULATIONAHA.118.034250LinkGoogle Scholar Previous Back to top Next FiguresReferencesRelatedDetails May 25, 2021Vol 143, Issue 21Article InformationMetrics Download: 1,495 © 2021 American Heart Association, Inc.https://doi.org/10.1161/CIRCULATIONAHA.120.052799PMID: 34029137 Originally publishedMay 24, 2021 Keywordsmyocytes, cardiacmiceregenerationfibroblastsPDF download SubjectsBasic Science ResearchStem Cells
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