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Distinguishing Cardiomyocyte Division From Binucleation

2018; Lippincott Williams & Wilkins; Volume: 123; Issue: 9 Linguagem: Inglês

10.1161/circresaha.118.313971

1524-4571

Zachary A. Kadow, James F. Martin,

RNA and protein synthesis mechanisms

HomeCirculation ResearchVol. 123, No. 9Distinguishing Cardiomyocyte Division From Binucleation Free AccessEditorialPDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyReddit Jump toFree AccessEditorialPDF/EPUBDistinguishing Cardiomyocyte Division From BinucleationA Rigorous Examination of the Midbody Provides Clarity Zachary A. Kadow and James F. Martin Zachary A. KadowZachary A. Kadow From the Program in Developmental Biology (Z.A.K., J.F.M.), Baylor College of Medicine, One Baylor Plaza, Houston, TX and James F. MartinJames F. Martin Correspondence to James F. Martin, MD, PhD, Department of Molecular Physiology and Biophysics, Baylor College of Medicine, One Baylor Plaza 512E, Houston, TX 77030, Email E-mail Address: [email protected] From the Program in Developmental Biology (Z.A.K., J.F.M.), Baylor College of Medicine, One Baylor Plaza, Houston, TX Department of Molecular Physiology and Biophysics (J.F.M.), Baylor College of Medicine, One Baylor Plaza, Houston, TX Cardiomyocyte Renewal Laboratory, Texas Heart Institute, Houston (J.F.M.). Originally published11 Oct 2018https://doi.org/10.1161/CIRCRESAHA.118.313971Circulation Research. 2018;123:1012–1014This article is a commentary on the followingMidbody Positioning and Distance Between Daughter Nuclei Enable Unequivocal Identification of Cardiomyocyte Cell Division in MiceThe inability of the adult cardiomyocyte to readily divide in homeostasis and after injury contributes to the high morbidity and mortality of heart disease. It is well documented that adult cardiomyocytes renew at a rate of <1% per year during homeostasis and have little capacity to increase their renewal rate after cardiac injury.1,2 Unsurprisingly, this has generated a large community of investigators interested in studying the cardiomyocyte cell cycle with the translational goal of creating therapies capable of stimulating cardiomyocyte renewal in patients with heart disease. Regardless of the experimental approach, it is essential to rigorously characterize cardiomyocyte division events when investigating cardiomyocyte proliferation. Current methods include immunofluorescence studies of the M-phase markers AURKB (Aurora B kinase) and pHH3 (phospho-histone H3) that are widely used as markers of cardiomyocyte proliferation.3 Another approach, rigorous but labor intensive, is stereology in which all cardiomyocytes in the heart are manually counted.1,2 In this issue of Circulation Research, Hesse et al4 provide data showing that positioning of the AURKB+ midbody and physical distance between daughter nuclei are novel markers of the dividing cardiomyocyte.Article, see p 1039Clearly identifying cardiomyocyte division has been a continued challenge in cardiac regeneration research largely because cell cycle reentry is not synonymous with cardiomyocyte cellular division. In fact, cardiomyocytes undergo many cell cycle variants other than division regularly throughout both development and disease (Figure [A]). These variants include polyploidization, where cardiomyocytes prematurely exit the cell cycle after S phase, and binucleation, in which cardiomyocytes progress through mitosis and karyokinesis but fail to complete cytokinesis. Both cell cycle variants progress through DNA synthesis and, therefore, will show incorporation of nucleotides analogs (EdU, BrdU) and Ki-67 expression. Furthermore, both cellular division and binucleation progress through mitosis and, therefore, demonstrate pHH3 expression. The ambiguity of cell cycle reentry and authentic cardiomyocyte division has led to disagreements in the literature concerning cardiomyocyte proliferation during mouse heart development and after cardiac injury.5,6 Although stereology is a reliable method to count cardiomyocyte and thereby provide evidence of proliferation, more direct methods of measuring cardiomyocyte division are needed.Download figureDownload PowerPointFigure. Midbody position reliably differentiates cardiomyocyte binucleation and cellular division events. A, The postnatal cardiomyocyte is capable of progressing through multiple cell cycle variants. If the cardiomyocyte (CM) exits after DNA synthesis, a process known as polyploidization, it remains mononucleated and contains a tetraploid (4n) nucleus. Furthermore, CMs can complete mitosis but not cytokinesis, leading to the formation of two diploid (2n) nuclei, known as binucleation. True cellular division, the goal of cardiac regenerative therapies, only occurs if the CM is able to fully complete cytokinesis, resulting in 2 daughter CMs. B, Hesse et al4 demonstrate that there are 2 major criteria for differentiating cellular division and binucleation: (1) The midbody positioning, here depicted in green, differs between the 2 events. Specifically, the midbody is found to be located symmetrically between the 2 daughter nuclei if the cell will complete cytokinesis and divide but asymmetrically if instead the CM will binucleated. The authors demonstrate the midbody can be visualized by either anillin or AURKB (Aurora B kinase) immunofluorescence. (2) The distance between the daughter nuclei varies, with the distance between the nuclei being greater preceding cellular division than binucleation.Previous work in neonatal rat cardiomyocyte culture systems demonstrated that two critical components for cytokinesis, AURKB, and anillin, were differentially localized within the contractile ring and midbody of cardiomyocytes undergoing division and binucleation.7 Hesse et al4 build upon this previous observation through the use of live-imaging technologies to study mouse cardiomyocyte division and binucleation in both in vitro and in vivo systems.Hesse et al4 use a neonatal cardiomyocyte culture in vitro system, culturing cardiomyocytes from a double transgenic Myh6-H2B-mCherry/CAG-eGFP-anillin transgenic mouse created previously by the group.8,9 This transgenic mouse expresses both the mCherry-H2B fusion protein to label cardiomyocyte nuclei and an ubiquitously expressed eGFP (enhanced green fluorescent protein)-tagged anillin protein that is differentially located throughout the cell cycle. Both fluorophores are bright enough to be captured through live imaging, allowing for real-time observation of cell cycle activity in cultured cardiomyocytes.Using this approach, they were able to stimulate cell cycle reentry through treatment with miR-199 (microRNA-199) and p21 knockdown with siRNA (small interfering RNA), two previously published methods of inducing cardiomyocyte proliferation.10,11 Although treatments increased the number of both cardiomyocyte binucleation and division events as expected, more interesting is the observation that the localization of the midbody (marked by eGFP-anillin fusion protein) always predicts whether the cardiomyocyte will undergo binucleation or a true cellular division. Specifically, symmetrical midbody positioning, where anillin localization is detected centrally between the two nuclei, indicated true cardiomyocyte division was to follow. Alternatively, an asymmetrical midbody, where anillin localization is seen at the periphery of the cleavage furrow, indicated that only binucleation would occur.The transgenic cardiomyocytes were then fixed, and the cellular location of AURKB was investigated through immunofluorescence. Each midbody identified by eGFP-anillin also stained positive for AURKB. The colocalization of both proteins at the midbody indicated that AURKB localization was also capable of differentiating between cardiomyocyte division and binucleation.Hesse et al4 next investigate whether the midbody positioning is also predictive of cardiomyocyte cell cycle fate in vivo. To do this, the group generated another transgenic mouse, Myh6-eGFP-anillin, to restrict the eGFP-tagged anillin expression only to cardiomyocytes. Live imaging of both neonatal ventricular and atrial cardiomyocytes was performed using acute heart slices and two-photon microscopy. Both binucleation and division events were captured in real time, and anillin localization was again found to be symmetrical in cardiomyocytes undergoing division and asymmetrical when undergoing binucleation. Additionally, the distance between the daughter nuclei, when the midbody is present, is consistently greater in cellular division than binucleation. Immunofluorescent labeling of AURKB in heart sections demonstrates similar results, both in terms of midbody positioning and distance between cardiomyocyte daughter nuclei (Figure [B]).Through the use of the endogenous midbody reporter, eGFP-anillin, and live imaging, Hesse et al demonstrate that AURKB expression does not only indicate cardiomyocyte division but also cardiomyocyte binucleation. Importantly, they were also able to establish AURKB + midbody symmetry and distance between daughter nuclei as criteria for differentiating these two cell cycle events. Specifically, their data leads the group to recommend colabeling of the midbody with AURKB, cardiomyocyte nuclear lamina with PCM-1 (pericentriolar material 1), and the cellular membrane with wheat germ agglutinin to quantify unequivocal cardiomyocyte division events in heart tissue from any regenerative model.Importantly, it should be noted that the amount of binucleation events in both the in vitro and in vivo systems studied here were significant and often outnumbered the cardiomyocyte undergoing authentic cellular division. Furthermore, the ratio between the two events depended upon the specific treatment and developmental stage of the cardiomyocytes. Both of these findings highlight the importance of quantifying authentic cardiomyocyte division events separately from binucleation when investigating cardiomyocyte proliferation.Moving forward, the work of Hesse et al4 challenges the widely used quantification of AURKB + cardiomyocytes as an assay for proliferation. Instead, this work suggests that more careful analysis must be done when performing AURKB immunofluorescence, taking into account localization of the midbody and daughter nuclei to separately categorize binucleation and division events. Complementary to the approach described in this work, and stereology to count cardiomyocytes, is the use of the Mosaic Analysis with Double Markers (MADM) mouse model, which is a method of clonal analysis capable of unequivocally labeling cardiomyocyte division events. In the MADM system, the N- and C- terminus of RFP (red fluorescent protein) and GFP are located on reciprocal alleles, such that activation of Cre recombinase will allow for formation of recombinant alleles and fluorophore expression. If cell division is occurring during Cre-mediated recombination, there is a potential for the recombinant alleles to segregate into separate daughter cells, creating separate GFP and RFP labeled progeny, whereas a binucleation event would only result in coexpression of the fluorophores.12The MADM system has successfully been used in the heart, utilizing the inducible cardiomyocyte-specific Myh6CreERT2 Cre recombinase allele to investigate postnatal cardiomyocyte division.13 A clear drawback of this system is the requirement of 3 alleles (1 Cre allele and the 2 MADM alleles) into a single mouse to perform the experiment, even before the genetic alleles of interest are incorporated, which will require extensive mouse breeding schemes. However, if the regenerative therapy can be introduced through viral delivery, such as adenovirus or adeno-associated virus, then the MADM system can be used more conveniently to investigate cardiomyocyte division.14The criteria outlined in this issue of Circulation Research by Hesse et al4 will allow investigators studying cardiomyocyte proliferation to immediately identify unequivocal cardiomyocyte division in their model of interest. As novel regulators of the cardiomyocyte cell cycle are discovered, it is important that these new methodologies become the standard to rigorously differentiate authentic division events from cardiomyocytes exhibiting premature cell cycle exit.AcknowledgmentsWe thank Baylor College of Medicine Medical Scientist Training Program and Baylor Research Advocates for Student Scientists.Sources of FundingJ.F. Martin was supported by LeDucq Foundation Transatlantic Networks of Excellence in Cardiovascular Research Award 14CVD01, National Institutes of Health Grants DE023177, HL127717, HL13084, and HL118761, and the Vivian L. Smith Foundation.DisclosuresNone.FootnotesThe opinions expressed in this article are not necessarily those of the editors or of the American Heart Association.Correspondence to James F. Martin, MD, PhD, Department of Molecular Physiology and Biophysics, Baylor College of Medicine, One Baylor Plaza 512E, Houston, TX 77030, Email [email protected]eduReferences1. Bergmann O, Bhardwaj RD, Bernard S, Zdunek S, Barnabé-Heider F, Walsh S, Zupicich J, Alkass K, Buchholz BA, Druid H, Jovinge S, Frisén J. Evidence for cardiomyocyte renewal in humans.Science. 2009; 324:98–102. doi: 10.1126/science.1164680CrossrefMedlineGoogle Scholar2. Senyo SE, Steinhauser ML, Pizzimenti CL, Yang VK, Cai L, Wang M, Wu TD, Guerquin-Kern JL, Lechene CP, Lee RT. 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Regulation of cell cycle to stimulate adult cardiomyocyte proliferation and cardiac regeneration.Cell. 2018; 173:104.e–116.e12. doi: 10.1016/j.cell.2018.02.014CrossrefGoogle Scholar Previous Back to top Next FiguresReferencesRelatedDetailsCited By Kolluri K, Nazarian T and Ardehali R (2022) Clonal Tracing of Heart Regeneration, Journal of Cardiovascular Development and Disease, 10.3390/jcdd9050141, 9:5, (141) Garbern J and Lee R (2022) Heart regeneration: 20 years of progress and renewed optimism, Developmental Cell, 10.1016/j.devcel.2022.01.012, 57:4, (424-439), Online publication date: 1-Feb-2022. Dookun E, Passos J, Arthur H and Richardson G (2020) Therapeutic Potential of Senolytics in Cardiovascular Disease, Cardiovascular Drugs and Therapy, 10.1007/s10557-020-07075-w, 36:1, (187-196), Online publication date: 1-Feb-2022. Milliron H, Weiland M, Kort E and Jovinge S (2019) Isolation of Cardiomyocytes Undergoing Mitosis With Complete Cytokinesis, Circulation Research, 125:12, (1070-1086), Online publication date: 6-Dec-2019. Broughton K and Sussman M (2019) Adult Cardiomyocyte Cell Cycle Detour: Off-ramp to Quiescent Destinations, Trends in Endocrinology & Metabolism, 10.1016/j.tem.2019.05.006, 30:8, (557-567), Online publication date: 1-Aug-2019. Related articlesMidbody Positioning and Distance Between Daughter Nuclei Enable Unequivocal Identification of Cardiomyocyte Cell Division in MiceMichael Hesse, et al. Circulation Research. 2018;123:1039-1052 October 12, 2018Vol 123, Issue 9 Advertisement Article InformationMetrics © 2018 American Heart Association, Inc.https://doi.org/10.1161/CIRCRESAHA.118.313971PMID: 30355168 Originally publishedOctober 11, 2018 Keywordshistonecell cyclehomeostasisheart diseaseEditorialsPDF download Advertisement SubjectsBasic Science ResearchCell Biology/Structural BiologyDevelopmental BiologyImagingMyocardial Regeneration