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Homotypic Fusion Generates Multinucleated Cardiomyocytes in the Murine Heart

2020; Lippincott Williams & Wilkins; Volume: 141; Issue: 23 Linguagem: Inglês

10.1161/circulationaha.119.043530

1524-4539

Shah R. Ali, Ivan Menendez-Montes, Jane N. Warshaw, Feng Xiao, Hesham A. Sadek,

RNA Research and Splicing

HomeCirculationVol. 141, No. 23Homotypic Fusion Generates Multinucleated Cardiomyocytes in the Murine Heart Free AccessLetterPDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyReddit Jump toFree AccessLetterPDF/EPUBHomotypic Fusion Generates Multinucleated Cardiomyocytes in the Murine Heart Shah R. Ali, MD, Ivan Menendez-Montes, PhD, Jane Warshaw, BA, Feng Xiao, PhD and Hesham A. Sadek, MD, PhD Shah R. AliShah R. Ali Departments of Internal Medicine/Cardiology (S.R.A., I.M.-M., J.W., F.X., H.A.S.), University of Texas Southwestern Medical Center, Dallas. , Ivan Menendez-MontesIvan Menendez-Montes Departments of Internal Medicine/Cardiology (S.R.A., I.M.-M., J.W., F.X., H.A.S.), University of Texas Southwestern Medical Center, Dallas. , Jane WarshawJane Warshaw Departments of Internal Medicine/Cardiology (S.R.A., I.M.-M., J.W., F.X., H.A.S.), University of Texas Southwestern Medical Center, Dallas. , Feng XiaoFeng Xiao Departments of Internal Medicine/Cardiology (S.R.A., I.M.-M., J.W., F.X., H.A.S.), University of Texas Southwestern Medical Center, Dallas. and Hesham A. SadekHesham A. Sadek Hesham A. Sadek, MD, PhD, Department of Internal Medicine, Division of Cardiology, University of Texas Southwestern Medical Center, 6000 Harry Hines Blvd, Dallas, TX 75390. Email E-mail Address: [email protected] Departments of Internal Medicine/Cardiology (S.R.A., I.M.-M., J.W., F.X., H.A.S.), University of Texas Southwestern Medical Center, Dallas. Radiation Oncology, Molecular Biology (H.A.S.), University of Texas Southwestern Medical Center, Dallas. Biophysics (H.A.S.), University of Texas Southwestern Medical Center, Dallas. Center for Regenerative Science and Medicine (H.A.S.), University of Texas Southwestern Medical Center, Dallas. Originally published8 Jun 2020https://doi.org/10.1161/CIRCULATIONAHA.119.043530Circulation. 2020;141:1940–1942Cardiomyocytes in mammalian hearts generally harbor >1 nucleus, which varies by species and strain.1 Approximately 25% of human cardiomyocytes are binucleated after the first decade of life, whereas ≈90% of cardiomyocytes in mice are multinucleated. The source of binucleated and multinucleated cardiomyocytes has long been attributed to failed cytokinesis after mitosis in the early postnatal period. However, pulse-chase experiments reveal that not all binucleated cardiomyocytes incorporate markers of DNA synthesis in the first week of life, a discrepancy that has evaded scrutiny.1,2 Homotypic cell fusion is the predominant mechanism for generating many multinucleated cell types, for example, myoblasts, and previous reports have revealed that cardiomyocytes can fuse with nonmyocytes.3 However, it remains unknown whether multinucleated cardiomyocytes can arise from fusion between cardiomyocytes. Here, we provide the first clonal evidence that homotypic cell fusion generates a fraction of multinucleated cardiomyocytes.We generated Myh6-MerCreMer;Confetti+/− (MCM;Confetti) mice in which a single dose of tamoxifen leads to stochastic, indelible labeling of cardiomyocytes with 1 of 4 fluorescent proteins (Figure [A]).4 By pulsing tamoxifen at postnatal day (P) 0 to P1, before significant binucleation or polyploidization occurs,1,2 we ensured that labeled cardiomyocytes would have only 1 fluorescent label (rather than 2 labels that could theoretically arise in a binucleated or polyploid cardiomyocyte.) Because each cardiomyocyte could adopt only 1 label, we hypothesized that MCM;Confetti could be used to screen for homotypic fusion of cardiomyocytes; fused cardiomyocytes could acquire 2 (or more) unique labels (Figure [A through C]).Download figureDownload PowerPointFigure. Homotypic fusion between cardiomyocytes occurs in the postnatal mouse heart.A, Myh6-MerCreMer;R26-Confetti+/− schematic showing the outcome of each recombination event. B, List of all possible fusion outcomes. C, Cartoon schematic of detectable fusion outcomes. D, Experimental strategy. E, A zoomed-out view of a heart section from a labeled heart showing single-color recombinations and a red fluorescent protein (RFP)/nuclear (n) green fluorescent protein (GFP) fused cardiomyocyte (white box). Scale bar, 50 μm. E´, Zoom view of the fused RFP/nGFP cardiomyocyte from E. Scale bar, 10 μm. E´´, Imaris 3-dimensional (3D) reconstruction of the fused cell from E´. F, Example of YFP-nGFP fused cardiomyocyte. Scale bar, 10 μm. F´, Imaris 3D reconstruction of F based on confocal imaging shows that both nGFP nuclei are harbored within the yellow fluorescent protein (YFP) cytoplasm. G, Example of YFP-RFP fused cardiomyocyte. Scale bar, 10 μm. G´, Imaris 3D reconstruction of G. H, Isolated cardiomyocyte nucleation count at different time points with representative images of each type of cardiomyocyte. I, Overall percentage of fused cardiomyocytes (as a fraction of labeled cardiomyocytes). J, Overall frequency of each Confetti recombination outcome at different time points. K, Overall schematic highlighting the main finding of the study. Error bars show mean±SD. One-way ANOVA tests were used to calculate significance unless otherwise noted. e Indicates embryonic day; and P, postnatal day.We analyzed the mice at 1 week after injection and at 1 month after injection (Figure [D]). At P7 and P28, ≈1% to 5% of all cardiomyocytes had undergone Cre recombination and expressed a fluorescent label (Figure [E]). There was an equal propensity to achieve a nuclear green fluorescent protein, cytoplasmic red fluorescent protein, or cytoplasmic yellow fluorescent protein label, consistent with previous reports; cyan fluorescent protein could not be detected with a standard epifluorescent microscope and was not counted (Figure [J]). Intriguingly, we observed a small fraction of cardiomyocytes that appeared to be double-labeled, which can occur upon fusion between cardiomyocytes with 2 different labels (Figure [E through G]). Analysis of these cells with confocal microscopy and 3D image reconstruction confirmed that double-labeled cells contain both labels (Figure [E´ through G´]). Assessment of the nucleation count in double-labeled cells was limited by the thin sections, which often did not contain the entire body of each cardiomyocyte. We observed double-labeled cells with 1, 2, or more nuclei. Moreover, double-labeled cells were present in all 4 chambers of the heart at similar frequencies.We next analyzed 1-month-old MCM;Confetti mice (labeled at P0–P1), which had a frequency of double-labeling similar to that of P7 mice (0.43% [n=5] versus 0.72% [n=3]; P=0.45 by t test; Figure [I]). This suggests that homotypic fusion of cardiomyocytes in postnatal mice occurs by the first week of life and remains stable at 1 month. However, our model cannot detect abscission of fused cells; therefore, this model cannot address whether rates of fusion (or abscission, if it occurs) change over time.Although the number of polyploid and binucleated cells at the time of Cre induction (P0–P1) is exceedingly low (Figure [H]), we injected tamoxifen at embryonic day 16.5 to induce recombination when the binucleation rate is zero (n=3).1 At P14, these embryonically-labeled mice exhibited a labeling efficiency of 3% to 8%. The percentage of double-labeled cells (0.65%) was similar to that of the P0 injected hearts, further validating the P0 recombination findings (Figure [I]).There are 10 possible double-labeled fusion outcomes, of which only 3 are detectable (Figure [B and C]). Therefore, the average double-labeling frequency of 0.6% was divided by 0.3 to account for the uncountable double-labeled outcomes (which assumes that all outcomes are equally likely); ≈2% of labeled cardiomyocytes are the result of homotypic fusion. Because Cre recombination in MCM;Confetti is a stochastic event, we infer that this percentage reflects the degree of fusion in cardiomyocytes globally. Because of the low labeling efficiency in our transgenic system, we speculate that our calculated global fusion rate of 2% reflects the lower limit of this phenomenon.In summary, we provide evidence for homotypic cardiomyocyte fusion in the mouse heart using MCM;Confetti. Our transgenic system reveals that, at minimum, 2% of cardiomyocytes with >1 nucleus arise from cell-cell fusion rather than from failed cytokinesis, which invites a re-examination of dogma in the field of postnatal cardiac growth (Figure [K]). Although embryonic zebrafish cardiomyocytes were recently shown to undergo transient homotypic fusion,5 this does not appear to contribute to binucleation. Future studies and alternative transgenic systems are needed to determine whether this phenomenon is evolutionarily conserved in higher mammals and whether pathological states or aging can modulate the rate of fusion. Our findings raise the provocative question of whether the fused cardiomyocyte fraction may serve as a pool for increase cardiomyocyte number through a nonmitotic mechanism (abscission) after injury, which would represent a novel regenerative mechanism in the adult heart.All mouse experiments were conducted in accordance with protocols approved by the Institutional Animal Care and Use Committee of the University of Texas Southwestern Medical Center and were in compliance with the relevant ethics regulations for animal research.Sources of FundingDr Sadek is supported by grants from the National Institutes of Health (1R01HL11527, R01H2131778, R01HL147276 R01HL149137), American Heart Association (16EIA27740034), Cancer Prevention and Research Institute of Texas (RP190435), Hamon Center for Regenerative Science and Medicine, and Fondation Leducq.DisclosuresNone.Footnoteshttps://www.ahajournals.org/journal/circThe data, analytical methods, and study materials will be made available to other researchers for purposes of reproducing the results by contacting the corresponding author.Guest Editor for this article was William Sessa, PhD.Hesham A. Sadek, MD, PhD, Department of Internal Medicine, Division of Cardiology, University of Texas Southwestern Medical Center, 6000 Harry Hines Blvd, Dallas, TX 75390. Email hesham.[email protected]eduReferences1. Soonpaa MH, Kim KK, Pajak L, Franklin M, Field LJ. Cardiomyocyte DNA synthesis and binucleation during murine development.Am J Physiol. 1996; 271(pt 2):H2183–H2189. doi: 10.1152/ajpheart.1996.271.5.H2183MedlineGoogle Scholar2. Alkass K, Panula J, Westman M, Wu TD, Guerquin-Kern JL, Bergmann O. No evidence for cardiomyocyte number expansion in preadolescent mice.Cell. 2015; 163:1026–1036. doi: 10.1016/j.cell.2015.10.035CrossrefMedlineGoogle Scholar3. Alvarez-Dolado M, Pardal R, Garcia-Verdugo JM, Fike JR, Lee HO, Pfeffer K, Lois C, Morrison SJ, Alvarez-Buylla A. Fusion of bone-marrow-derived cells with Purkinje neurons, cardiomyocytes and hepatocytes.Nature. 2003; 425:968–973. doi: 10.1038/nature02069CrossrefMedlineGoogle Scholar4. 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Kirillova A, Han L, Liu H and Kühn B (2021) Polyploid cardiomyocytes: implications for heart regeneration, Development, 10.1242/dev.199401, 148:14, Online publication date: 15-Jul-2021. Ganassi M and Zammit P (2022) Involvement of muscle satellite cell dysfunction in neuromuscular disorders: Expanding the portfolio of satellite cell-opathies, European Journal of Translational Myology, 10.4081/ejtm.2022.10064, 32:1 June 9, 2020Vol 141, Issue 23 Advertisement Article InformationMetrics © 2020 American Heart Association, Inc.https://doi.org/10.1161/CIRCULATIONAHA.119.043530PMID: 32510998 Originally publishedJune 8, 2020 KeywordsmyocytesCre recombinasepostnatal developmentcell fusionmitosiscardiacmicecell nucleusPDF download Advertisement