Increasing Mononuclear Diploid Cardiomyocytes by Loss of E2F Transcription Factor 7/8 Fails to Improve Cardiac Regeneration After Infarct.

HomeCirculationVol. 147, No. 2Increasing Mononuclear Diploid Cardiomyocytes by Loss of E2F Transcription Factor 7/8 Fails to Improve Cardiac Regeneration After Infarct Free AccessLetterPDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyReddit Jump toFree AccessLetterPDF/EPUBIncreasing Mononuclear Diploid Cardiomyocytes by Loss of E2F Transcription Factor 7/8 Fails to Improve Cardiac Regeneration After Infarct Zhe Yu, Lunfeng Zhang, Paola Cattaneo, Nuno Guimarães-Camboa, Xi Fang, Yusu Gu, Kirk L. Peterson, Julius Bogomolovas, Cecilia Cuitino, Gustavo W. Leone, Ju Chen and Sylvia M. Evans Zhe YuZhe Yu Skaggs School of Pharmacy and Pharmaceutical Sciences (Z.Y., L.Z., S.M.E.), University of California at San Diego, La Jolla. *Z. Yu and L. Zhang contributed equally. Search for more papers by this author , Lunfeng ZhangLunfeng Zhang Skaggs School of Pharmacy and Pharmaceutical Sciences (Z.Y., L.Z., S.M.E.), University of California at San Diego, La Jolla. *Z. Yu and L. Zhang contributed equally. Search for more papers by this author , Paola CattaneoPaola Cattaneo Institute of Genetic and Biomedical Research (IRGB), UOS of Milan, National Research Council of Italy (P.C.). Humanitas Clinical and Research Center-IRCCS, Rozzano, Italy (P.C.). Institute of Cardiovascular Regeneration, Goethe University, Frankfurt, Germany (P.C., N.G.-C.). German Center for Cardiovascular Research, Berlin (partner site Frankfurt Rhine-Main) (P.C., N.G.-C.). Search for more papers by this author , Nuno Guimarães-CamboaNuno Guimarães-Camboa Institute of Cardiovascular Regeneration, Goethe University, Frankfurt, Germany (P.C., N.G.-C.). German Center for Cardiovascular Research, Berlin (partner site Frankfurt Rhine-Main) (P.C., N.G.-C.). Search for more papers by this author , Xi FangXi Fang https://orcid.org/0000-0001-7816-8821 Department of Medicine (X.F., Y.G., K.L.P., J.B., J.C., S.M.E.), University of California at San Diego, La Jolla. Search for more papers by this author , Yusu GuYusu Gu Department of Medicine (X.F., Y.G., K.L.P., J.B., J.C., S.M.E.), University of California at San Diego, La Jolla. Search for more papers by this author , Kirk L. PetersonKirk L. Peterson Department of Medicine (X.F., Y.G., K.L.P., J.B., J.C., S.M.E.), University of California at San Diego, La Jolla. Search for more papers by this author , Julius BogomolovasJulius Bogomolovas Department of Medicine (X.F., Y.G., K.L.P., J.B., J.C., S.M.E.), University of California at San Diego, La Jolla. Search for more papers by this author , Cecilia CuitinoCecilia Cuitino Department of Radiation Oncology, Arthur G. James Hospital/Ohio State Comprehensive Cancer Center, Columbus (C.C.). Search for more papers by this author , Gustavo W. LeoneGustavo W. Leone Medical College of Wisconsin Cancer Center, Department of Biochemistry, Medical College of Wisconsin, Wauwatosa (G.W.L.). Search for more papers by this author , Ju ChenJu Chen https://orcid.org/0000-0001-7674-4776 Department of Medicine (X.F., Y.G., K.L.P., J.B., J.C., S.M.E.), University of California at San Diego, La Jolla. Search for more papers by this author and Sylvia M. EvansSylvia M. Evans Correspondence to: Sylvia M. Evans, PhD, Medicine, 9500 Gilman Dr, BRF II, Room 2A16, University of California San Diego, La Jolla, CA 92093. Email E-mail Address: [email protected] https://orcid.org/0000-0001-5035-3697 Skaggs School of Pharmacy and Pharmaceutical Sciences (Z.Y., L.Z., S.M.E.), University of California at San Diego, La Jolla. Department of Medicine (X.F., Y.G., K.L.P., J.B., J.C., S.M.E.), University of California at San Diego, La Jolla. Department of Pharmacology (S.M.E.), University of California at San Diego, La Jolla. Search for more papers by this author Originally published9 Jan 2023https://doi.org/10.1161/CIRCULATIONAHA.122.061018Circulation. 2023;147:183–186This article is commented on by the following:A Tedious Journey: Cardiomyocyte Proliferation Requires More Than S-Phase Entry and Loss of PolyploidizationThe heart is among the least regenerative of organs. Transition of cardiomyocytes (CMs) from hyperplasic to hypertrophic growth occurs after birth, accompanied by binucleation and polyploidization, suggested as a barrier to cardiac regeneration. Cardiac regeneration and functional recovery after myocardial infarction (MI) have been correlated with an increased baseline frequency of mononuclear diploid (MND) CMs.1 However, the capacity of MND adult CMs to undergo proliferation is still controversial. RNA-sequencing analyses of fluorescence-activated cell sorting–separated CMs suggested transcriptional differences between mononuclear and binuclear CMs,2 yet other studies using scRNA-sequencing3 or bulk RNA-sequencing4 analyses found similar transcriptomes between mononuclear and multinuclear CMs in both normal and postinjury conditions. Our aim was to further investigate the regenerative potential of MND CMs.Editorial, see p 154Liver-specific ablation of E2f7 and E2f8 diminishes nucleation and ploidy in hepatocytes,5 and the E2F pathway is implicated in CM binucleation.2 Therefore, we ablated E2f7 and E2f8 in CMs (XMLC2-78dKO mice) using double-floxed controls (E2f7f/f; E2f8f/f). Mouse protocols were approved by the Institutional Animal Care and Use Committee. XMLC2-78dKO mice were viable and fertile. Isolation of CMs (Figure [A]) demonstrated a 2.7-fold increase in mononuclear CMs from the left ventricle of XMLC2-78dKO mice compared with controls (Figure [B]). Within the left ventricle, mutants exhibited a 10-fold increase in MND CMs (Figure [C]). Increased MND CMs were also observed in the interventricular septum and right ventricle of mutants relative to controls (Figure [B and C]).Download figureDownload PowerPointFigure. Increased frequency of mononuclear diploid CMs fails to promote cardiac regeneration after MI. A, Representative images of major types of cardiomyocytes (CMs) with different nucleation and ploidy levels. CM nuclei and sarcomeric F-actin were labeled by DAPI and phalloidin, respectively. Scale bar, 10 μm. B, XMLC2-78dKO mice exhibited a significantly increased percentage of mononuclear CMs in the left ventricle (LV), right ventricle (RV), and interventricular septum (IVS; 5.1% to 14% in LV; 17% in mice used by Patterson et al1). No differences in sarcomere structure or size of CMs were observed (data not shown). C, Ploidy levels of mononuclear CMs as determined by integrated fluorescence intensity of DAPI. Significantly increased mononuclear diploid (MND) CMs were observed in the LV, RV, and IVS of mutants (0.5% to 5.1% in the LV). CMs were isolated from 8-week-old mice (n=5 for each; 200 CMs per animal). D, Echocardiography data showed that 8-week-old XMLC2-78dKO mice had normal cardiac function (n=40 for each). E, Regional radial strain analyses showed no significant differences in cardiac function between XMLC2-78dKO mice and controls on day 2 (n=4 for each sham; n=15 for control myocardial infarction [MI]; n=16 for 78dKO MI) and day 7 or 28 (n=8 for control sham; n=20 for control MI; n=7 for 78dKO sham; n=24 for 78dKO MI) after MI. F, Representative images of sham and MI hearts in both XMLC2-78dKO mice and controls. Hearts were sectioned every 500 μm from suture. Sirius red staining was performed to label the infarct zone (IZ). Scale bar, 1 mm. G, XMLC2-78dKO mice and controls had similar infarct sizes on day 28 (n=6 for each) and day 42 (n=7 for control; n=10 for 78dKO) after MI. Infarct size was calculated as total infarct circumference divided by total LV circumference from all sections. H, Schematic of experimental protocol. Mice were injected once a day with 200 μg EdU from days 10 to 18 after MI. Hearts were harvested on day 28 after MI and either sectioned or border zone (BZ)/IZ CM isolation was performed. I, Representative images of EdU+ CM nuclei in sections of BZ/IZ. CM nuclei were labeled by Tnnt2 RNA scope intronic probes. Scale bar, 100 μm. J, Significantly increased numbers of EdU+ CM nuclei were observed in sections of BZ/IZ of XMLC2-78dKO mice (n=4 for each sham; n=6 for each MI; total count of CM nuclei in control/78dKO MI hearts: 14 099/14 390 in BZ, 2462/2582 in IZ, 4536/6497 in LV, 8822/10 092 in RV, and 8595/10 666 in IVS; total count of CM nuclei in control/78dKO sham hearts: 7595/7769 in LV, 9018/8878 in RV, and 9639/10 014 in IVS). Coimmunostaining with Ki67 and PCM1 confirmed increased cell-cycle reentry in sections of BZ/IZ of XMLC2-78dKO mice (data not shown). K, Representative images of major types of EdU+ CMs with different nucleation and ploidy levels isolated from BZ/IZ tissue. Scale bar, 10 μm. L, Mononuclear CMs were overrepresented in EdU+ CMs relative to their baseline frequencies (see B) for both control and XMLC2-78dKO mice (n=7 for each; total count of mononuclear/binuclear EdU+ CMs: 95/102 in controls; 592/56 in 78dKO mice). M, EdU+ mononuclear CMs were predominantly polyploid in XMLC2-78dKO mice and controls. Increased frequency of 4n+ mononuclear CMs was observed in controls compared with mutants. N, Distribution of ploidy levels in EdU+ binuclear CMs was similar in XMLC2-78dKO mice and controls. O, For pulse labeling, each mouse was given a single injection of 200 μg EdU on day 13 after MI (maximal EdU incorporation; data not shown). Hearts were harvested 1 hour later, and BZ/IZ CMs were isolated. P, Comparison of frequencies of ploidy categories of mononuclear CMs at baseline (a) and after EdU pulse labeling (b; n=6 for each; total EdU+ mononuclear CMs: 14 in controls; 40 in 78dKO mice) suggested that MND CMs preferentially incorporated EdU. Comparison of frequencies of ploidy categories of mononuclear CMs after pulse labeling (b) and on day 28 (M) suggested that preferentially labeled MND CMs became polyploid. Proliferation of initially labeled MND CMs would be expected to result in increased frequency of EdU-labeled MND CMs on day 28, which was not observed. Instead, decreased frequency of EdU-labeled MND CMs and increased frequency of EdU-labeled mononuclear polyploid CMs were observed on day 28, thus supporting polyploidization of EdU+ MND CMs. P (a), Distinct representation of C to facilitate comparison with M/P(b). Q, Similar comparison as in P but for binuclear CMs. EdU pulse labeling (b; total EdU+ binuclear CMs: 25 in controls; 9 in 78dKO mice) suggested that EdU incorporation by binuclear CMs was likely to result in polyploidization. R, Schematic summary. XMLC2-78dKO mice exhibited a 10-fold baseline increase in MND CMs. After MI, EdU was preferentially incorporated by MND CMs in BZ/IZ. Proliferation of EdU+ MND CMs would result in increased frequency of EdU+ MND CMs, which was not observed. Polyploidization of EdU+ MND CMs would result in increased frequency of EdU+ mononuclear polyploid CMs, which was observed, thus supporting polyploidization rather than proliferation. Consistent with these results, we found that 99% of mononuclear CMs in both controls and mutants had perinuclear PCM1 (inability to proliferate; data not shown). No improvement in cardiac function or reduction in infarct scar was observed between controls and mutants after MI. Data are presented as mean±SD. To determine statistical significance, negative binomial regression was performed for B, C, J, and L; Conway-Maxwell-Poisson regression was performed for M, N, P, and Q; Student t test was used for D; and mixed ANOVA and 2-way ANOVA were used for E and G, respectively. Conway-Maxwell-Poisson regression was performed with the mpcmp package in R. Other statistics were performed by SPSS 28. Graphs were generated with GraphPad Prism 9. EdU+ 4n+ indicates EdU+ nuclei with ploidy level higher than 4n; EDV, end-diastolic volume; EF, ejection fraction; ESV, end-systolic volume; FS, fractional shortening; LVIDd, left ventricular internal diameter at end diastole; LVIDs, left ventricular internal diameter at end systole; LVPWd, left ventricular posterior wall end diastole; and RZ, remote zone. All mice were males and maintained on FVB/NJ background. *P<0.05. **P<0.01. ***P<0.001.To determine whether increased MND CMs led to improved cardiac function before or after MI, we performed echocardiography on 8-week-old XMLC2-78dKO mice and controls before and after left anterior descending artery ligation. There were no preinjury differences (Figure [D]). Cardiac functional parameters, including ejection fraction, end-diastolic volume, and end-systolic volume, were measured on days 2, 7, and 28 after MI. Both groups displayed similar cardiac functional impairment (Figure [E, top]). A regional radial strain analysis also showed no differences between mutants and controls (Figure [E, bottom]). Scar sizes were similar on days 28 and 42 after MI for both groups (Figure [F and G]).To explore potential CM proliferation in border/infarct zones, 8-week-old XMLC2-78dKO mice and controls were subjected to left anterior descending artery ligation. Mice were injected with 200 μg EdU once a day between days 10 and 18 after MI, and hearts were harvested on day 28 (Figure [H]). Hearts were sectioned, and Tnnt2 intronic RNA scope probes were used to label CM nuclei (Figure [I]). We observed a 1.7-fold or 1.5-fold increase in EdU+ CM nuclei within border or infarct zones, respectively, in XMLC2-78dKO mice relative to controls (Figure [J]).To determine nucleation and ploidy levels of EdU+ CMs, hearts were Langendorff perfused, and single CM suspensions from dissected border/infarct zones were stained with EdU, DAPI, and phalloidin (Figure [K]). Although at baseline, mononuclear CMs represented only 5% to 14% of total CMs (Figure [B]), mononuclear CMs represented 50% to 92% of EdU-labeled CMs in controls and XMLC2-78dKOs (Figure [L]), suggesting preferential uptake of EdU by mononuclear CMs. Nuclei of EdU+ mononuclear or binuclear CMs were predominantly tetraploid (Figure [M and N]). XMLC2-78dKO mice exhibited an increased frequency of EdU+ 4n CMs and a reduced frequency of EdU+ 4n+ CMs relative to controls (Figure [M]). Ploidy levels of EdU+ binuclear CMs were similar in both groups (Figure [N]). The significantly increased frequency of EdU+ mononuclear tetraploid CMs in XMLC2-78dKO ventricles after infarct might reflect preferential uptake of EdU by MND CMs to result in polyploidization.To investigate this, we performed pulse labeling to capture initial ploidy levels of EdU+ CMs (Figure [O]). On day 13 after MI, mice were injected with 200 μg EdU. One hour later, CMs from border/infarct zones were isolated, and ploidy levels were analyzed. On average, 29% or 57% of EdU+ mononuclear CMs were 2n in controls or XMLC2-78dKO mice, respectively (Figure [P, b]). Because ploidy of an initial MND CM in the late S phase would be greater than 2n, the actual frequency of MND CMs labeled by EdU should be higher. A comparison of ploidy distributions in mononuclear CMs at baseline and after pulse labeling (Figure [P]) suggested that MND CMs were the most prone to undergo DNA synthesis after cardiac injury. Comparison of ploidy distributions after pulse labeling (Figure [P, b]) and 28 days after infarct (Figure [M]) suggested that EdU incorporation by MND CMs resulted in polyploidization, not proliferation (no increase in proportion of MND CMs by day 28). For binuclear CMs, most pulse-labeled EdU+ nuclei were diploid (Figure [Q]). On day 28, the majority of EdU-labeled nuclei were tetraploid, suggesting that the originally labeled diploid nuclei had undergone polyploidization (Figure [N]).In summary, XMLC2-78dKO mice exhibited significantly increased MND CMs at baseline (Figure [R]). After infarction, MND CMs within border/infarct zones have an increased capacity to undergo DNA replication compared with mononuclear polyploid or binuclear CMs. However, DNA replication results in polyploidization, not proliferation. Increased MND CM frequency had no impact on cardiac function or infarct size after MI.The data, analytical methods, and study materials that support the findings of this study will be available to other researchers from the corresponding authors on reasonable request.Article InformationAcknowledgmentsThe authors thank Zhiheng Mai, Yushen Feng, and Chumo Chen (University of California, San Diego, La Jolla) for their assistance in mouse husbandry and CM isolation. The authors also thank Joshua A. Fong (University of California, San Diego, La Jolla) for analyses of echo data.Sources of FundingDr Evans is funded by the National Institutes of Health, National Heart, Lung, and Blood Institute. All confocal images were captured in UCSD microscopy core, supported by grant NINDS P30NS047101.Disclosures None.Footnotes*Z. Yu and L. Zhang contributed equally.For Sources of Funding and Disclosures, see page 186.Circulation is available at www.ahajournals.org/journal/circCorrespondence to: Sylvia M. Evans, PhD, Medicine, 9500 Gilman Dr, BRF II, Room 2A16, University of California San Diego, La Jolla, CA 92093. Email syevans@ucsd.eduReferences1. Patterson M, Barske L, Van Handel B, Rau CD, Gan P, Sharma A, Parikh S, Denholtz M, Huang Y, Yamaguchi Y, et al. Frequency of mononuclear diploid cardiomyocytes underlies natural variation in heart regeneration. Nat Genet. 2017; 49:1346–1353. doi: 10.1038/ng.3929CrossrefMedlineGoogle Scholar2. Windmueller R, Leach JP, Babu A, Zhou S, Morley MP, Wakabayashi A, Petrenko NB, Viatour P, Morrisey EE. Direct comparison of mononucleated and binucleated cardiomyocytes reveals molecular mechanisms underlying distinct proliferative competencies. Cell Rep. 2020; 30:3105–3116.e4. doi: 10.1016/j.celrep.2020.02.034CrossrefMedlineGoogle Scholar3. Yekelchyk M, Guenther S, Preussner J, Braun T. Mono- and multi-nucleated ventricular cardiomyocytes constitute a transcriptionally homogenous cell population. Basic Res Cardiol. 2019; 114:36. doi: 10.1007/s00395-019-0744-zCrossrefMedlineGoogle Scholar4. Hesse M, Bednarz R, Carls E, Becker C, Bondareva O, Lother A, Geisen C, Dressen M, Krane M, Roell W, et al. Proximity to injury, but neither number of nuclei nor ploidy define pathological adaptation and plasticity in cardiomyocytes. J Mol Cell Cardiol. 2021; 152:95–104. doi: 10.1016/j.yjmcc.2020.11.012CrossrefMedlineGoogle Scholar5. Chen HZ, Ouseph MM, Li J, Pecot T, Chokshi V, Kent L, Bae S, Byrne M, Duran C, Comstock G, et al. Canonical and atypical E2Fs regulate the mammalian endocycle. Nat Cell Biol. 2012; 14:1192–1202. doi: 10.1038/ncb2595CrossrefMedlineGoogle Scholar eLetters(0)eLetters should relate to an article recently published in the journal and are not a forum for providing unpublished data. Comments are reviewed for appropriate use of tone and language. Comments are not peer-reviewed. Acceptable comments are posted to the journal website only. Comments are not published in an issue and are not indexed in PubMed. Comments should be no longer than 500 words and will only be posted online. References are limited to 10. 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Circulation. 2023;147:154-157 January 10, 2023Vol 147, Issue 2 Advertisement Article InformationMetrics © 2023 American Heart Association, Inc.https://doi.org/10.1161/CIRCULATIONAHA.122.061018PMID: 36622904 Originally publishedJanuary 9, 2023 KeywordsE2f7E2f8mononuclear diploid cardiomyocytesheart regenerationproliferationpolyploidizationPDF download Advertisement SubjectsBasic Science ResearchMyocardial Regeneration