BACKGROUND: Cardiac fibrosis, a hallmark of heart failure and an unmet clinical need, arises from pathological activation of preexisting cardiac fibroblasts (CFs), but the contribution of CF heterogeneity to this process remains unclear. METHODS: Murine models were used to lineage trace or deplete a specific sub-population of CFs at baseline and after myocardial infarction. Transcriptional and epigenetic differences between fibroblast subsets were assessed using next-generation sequencing. Conservation in humans was evaluated through single-cell RNA-seq data sets and histological examination. RESULTS: In mice, fibroblasts were the sole cardiac cell type expressing the signaling-capable isoform of the LepR (leptin receptor). LepR+ CFs emerged neonatally, occupied a defined niche in the coronary adventitia, exhibited enhanced hedgehog signaling, and responded to leptin. After myocardial infarction, LepR-Cre+ CFs proliferated more than interstitial CFs, became a predominant fibroblast lineage in the scar, and their genetic ablation reduced fibrosis while improving function. LepR+ CFs were also detected in the human heart, where they were embedded in an adipocyte-rich niche. CONCLUSIONS: These findings identify adventitial fibroblasts as key drivers of pathological remodeling and demonstrate that fibroblasts, rather than cardiomyocytes, are the principal responders to leptin in the heart, redefining how this major endocrine pathway influences cardiac remodeling and disease.

AIMS:This study investigated whether TBX18, a transcription factor known for its critical roles in cardiovascular and urogenital development, but never previously studied in the context of the aorta, contributes to the normal development and homeostasis of this major artery. METHODS AND RESULTS:Histological analyses revealed Tbx18 expression in smooth muscle cells (SMCs) of the adult and embryonic aorta. Following this observation, transgenic mouse models were used to promote ablation of Tbx18 in SMCs from early embryogenesis or in adulthood. Phenotypes were assessed by quantitative imaging and histological analyses. Embryonic conditional ablation of Tbx18 resulted in severe aortic malformations and lethality, whereas adult ablation resulted in milder phenotypes. However, when adult Tbx18 ablation was combined with a Marfan-causing mutation, it promoted degradation of aortic ultrastructure, aortic root dilation, and lethality. Multiomics analyses at the transcriptomic and translatomic levels revealed up-regulation of immediate early genes encoding critical transcription factors such as EGR1, FOS, and JUNB in SMCs from Marfan aortae, a response further exacerbated by concomitant ablation of Tbx18. ChIP-seq in primary human aortic SMCs revealed that TBX18 directly binds to several of the genes that were misexpressed in mutant aortae, suggesting direct regulation. Finally, transcriptomic and histological analyses of human patient samples revealed that TBX18 expression was down-regulated in aneurysms, with the extent of down-regulation correlating with lesion severity. CONCLUSION:These results demonstrate that TBX18 in SMCs is essential not only for normal aortic development, but also for preventing gene expression programmes linked to adverse remodelling in adulthood. These findings enhance our understanding of the function of this transcription factor and of molecular mechanisms underlying aneurysm formation, a pathology responsible for a significant number of fatalities in developed countries.

Background Cardiac fibrosis, a hallmark of heart failure and an unmet clinical need, arises from pathological activation of pre-existing cardiac fibroblasts (CFs), but the contribution of CF heterogeneity to this process remains unclear. Methods Murine models were used to lineage trace or deplete a specific sub-population of CFs at baseline and after myocardial infarction (MI). Transcriptional and epigenetic differences between fibroblast subsets were assessed using next-generation sequencing. Conservation in humans was evaluated through single-cell RNA-seq datasets and histological examination. Results In mice, fibroblasts were the sole cardiac cell type expressing the signaling-capable isoform of the leptin receptor (LepR). LepR+ CFs emerged neonatally, occupied a defined niche in the coronary adventitia, exhibited enhanced hedgehog signaling, and responded to leptin. After MI, LepR-Cre+ CFs proliferated more than interstitial CFs, became a predominant fibroblast lineage in the scar, and their genetic ablation reduced fibrosis while improving function. LepR+ CFs were also detected in the human heart, where they were embedded in an adipocyte-rich niche. Conclusions These findings identify adventitial fibroblasts as key drivers of pathological remodeling and demonstrate that fibroblasts, rather than cardiomyocytes, are the principal responders to leptin in the heart, redefining how this major endocrine pathway influences cardiac remodeling and disease. ### Competing Interest Statement The authors have declared no competing interest.

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. , 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. , 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.). , 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.). , 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. , 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. , 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. , 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. , Cecilia CuitinoCecilia Cuitino Department of Radiation Oncology, Arthur G. James Hospital/Ohio State Comprehensive Cancer Center, Columbus (C.C.). , Gustavo W. LeoneGustavo W. Leone Medical College of Wisconsin Cancer Center, Department of Biochemistry, Medical College of Wisconsin, Wauwatosa (G.W.L.). , 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. 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. 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. Authors of the article cited in the comment will be invited to reply, as appropriate.Comments and feedback on AHA/ASA Scientific Statements and Guidelines should be directed to the AHA/ASA Manuscript Oversight Committee via its Correspondence page.Sign In to Submit a Response to This Article Previous Back to top Next FiguresReferencesRelatedDetailsCited ByDing D and Braun T (2023) A Tedious Journey: Cardiomyocyte Proliferation Requires More Than S-Phase Entry and Loss of Polyploidization, Circulation, 147:2, (154-157), Online publication date: 10-Jan-2023.Related articlesA Tedious Journey: Cardiomyocyte Proliferation Requires More Than S-Phase Entry and Loss of PolyploidizationDong Ding, et al. 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

Mechanisms by which specific histone modifications regulate distinct gene networks remain little understood. We investigated how H3K79me2, a modification catalyzed by DOT1L and previously considered a general transcriptional activation mark, regulates gene expression during cardiogenesis. Embryonic cardiomyocyte ablation of Dot1l revealed that H3K79me2 does not act as a general transcriptional activator, but rather regulates highly specific transcriptional networks at two critical cardiogenic junctures: embryonic cardiogenesis, where it was particularly important for left ventricle-specific genes, and postnatal cardiomyocyte cell cycle withdrawal, with Dot1L mutants having more mononuclear cardiomyocytes and prolonged cardiomyocyte cell cycle activity. Mechanistic analyses revealed that H3K79me2 in two distinct domains, gene bodies and regulatory elements, synergized to promote expression of genes activated by DOT1L. Surprisingly, H3K79me2 in specific regulatory elements also contributed to silencing genes usually not expressed in cardiomyocytes. These results reveal mechanisms by which DOT1L successively regulates left ventricle specification and cardiomyocyte cell cycle withdrawal.

As the native regenerative potential of adult cardiac tissue is limited post-injury, stimulating endogenous repair mechanisms in the mammalian myocardium is a potential goal of regenerative medicine therapeutics. Injection of myocardial matrix hydrogels into the heart post-myocardial infarction (MI) has demonstrated increased cardiac muscle and promotion of pathways associated with cardiac development, suggesting potential promotion of cardiomyocyte turnover. In this study, the myocardial matrix hydrogel was shown to have native capability as an effective reactive oxygen species scavenger and protect against oxidative stress induced cell cycle inhibition in vitro. Encapsulation of cardiomyocytes demonstrated an enhanced turnover in in vitro studies, and in vivo assessments of myocardial matrix hydrogel treatment post-MI showed increased thymidine analog uptake in cardiomyocyte nuclei compared to saline controls. Overall, this study provides evidence that properties of the myocardial matrix material provide a microenvironment mitigating oxidative damage and supportive of cardiomyocytes undergoing DNA synthesis, toward possible DNA repair or cell cycle activation. STATEMENT OF SIGNIFICANCE: Loss of adult mammalian cardiomyocyte turnover is influenced by shifts in oxidative damage, which represents a potential mechanism for improving restoration of cardiac muscle after myocardial infarction (MI). Injection of a myocardial matrix hydrogel into the heart post-MI previously demonstrated increased cardiac muscle and promotion of pathways associated with cardiac development, suggesting potential in promoting proliferation of cardiomyocytes. In this study, the myocardial matrix hydrogel was shown to protect cells from oxidative stress and increase proliferation in vitro. In a rat MI model, greater presence of tissue free thiol content spared from oxidative damage, lesser mitochondrial superoxide content, and increased thymidine analog uptake in cardiomyocytes was found in matrix injected animals compared to saline controls. Overall, this study provides evidence that properties of the myocardial matrix material provide a microenvironment supportive of cardiomyocytes undergoing DNA synthesis, toward possible DNA repair or cell cycle activation.

ABSTRACT Mechanisms by which specific histone modifications regulate distinct gene regulatory networks remain little understood. We investigated how H3K79me2, a modification catalyzed by DOT1L and previously considered a general transcriptional activation mark, regulates gene expression in mammalian cardiogenesis. Early embryonic cardiomyocyte ablation of Dot1l revealed that H3K79me2 does not act as a general transcriptional activator, but rather regulates highly specific gene regulatory networks at two critical cardiogenic junctures: left ventricle patterning and postnatal cardiomyocyte cell cycle withdrawal. Mechanistic analyses revealed that H3K79me2 in two distinct domains, gene bodies and regulatory elements, synergized to promote expression of genes activated by DOT1L. Surprisingly, these analyses also revealed that H3K79me2 in specific regulatory elements contributed to silencing genes usually not expressed in cardiomyocytes. As DOT1L mutants had increased numbers of postnatal mononuclear cardiomyocytes and prolonged cardiomyocyte cell cycle activity, controlled inhibition of DOT1L might be a strategy to promote cardiac regeneration post-injury.

Rationale: Extraembryonic tissues, including the yolk sac and placenta, and the heart within the embryo, work to provide crucial nutrients to the embryo. The association of congenital heart defects with extraembryonic tissue defects further supports the potential developmental relationship between the heart and extraembryonic tissues. Although the development of early cardiac lineages has been well-studied, the developmental relationship between cardiac lineages, including epicardium, and extraembryonic mesoderm remains to be defined. Objective: To explore the developmental relationships between cardiac and extraembryonic lineages. Methods and Results: Through high-resolution single-cell and genetic lineage/clonal analyses, we show an unsuspected clonal relationship between extraembryonic mesoderm and cardiac lineages. Single-cell transcriptomics and trajectory analyses uncovered 2 mesodermal progenitor sources contributing to left ventricular cardiomyocytes, 1 embryonic and the other with an extraembryonic gene expression signature. Additional lineage-tracing studies revealed that the extraembryonic-related progenitors reside at the embryonic/extraembryonic interface in gastrulating embryos and produce distinct cell types forming the pericardium, septum transversum, epicardium, dorsolateral regions of the left ventricle and atrioventricular canal myocardium, and extraembryonic mesoderm. Clonal analyses demonstrated that these progenitors are multipotent, giving rise to not only cardiomyocytes and serosal mesothelial cell types but also, remarkably, extraembryonic mesoderm. Conclusions: Overall, our results reveal the location of previously unknown multipotent cardiovascular progenitors at the embryonic/extraembryonic interface and define the earliest embryonic origins of serosal mesothelial lineages, including the epicardium, which contributes fibroblasts and vascular support cells to the heart. The shared lineage relationship between embryonic cardiovascular lineages and extraembryonic mesoderm revealed by our studies underscores an underappreciated blurring of boundaries between embryonic and extraembryonic mesoderm. Our findings suggest unexpected underpinnings of the association between congenital heart disease and placental insufficiency anomalies and the potential utility of extraembryonic cells for generating cardiovascular cell types for heart repair.

Complex organs are composed of a multitude of specialized cell types which assemble to form functional biological structures. How these cell types are created and organized remains to be elucidated for many organs including the heart, the first organ to form during embryogenesis. Here, we show the ontogeny of mammalian mesoderm at high-resolution single cell and genetic lineage/clonal analyses, which revealed an unexpected complexity of the contribution and multi-potentiality of mesodermal progenitors to cardiac lineages creating distinct cell types forming specific regions of the heart. Single-cell transcriptomics of Mesp1 lineage-traced cells during embryogenesis and corresponding trajectory analyses uncovered unanticipated developmental relationships between these progenitors and lineages including two mesodermal progenitor sources contributing to the first heart field (FHF), an intraembryonic and a previously uncharacterized extraembryonic-related source, that produce distinct cardiac lineages creating the left ventricle. Lineage-tracing studies revealed that these extraembryonic-related FHF progenitors reside at the extraembryonic-intraembryonic interface in gastrulating embryos and generate cardiac cell types that form the epicardium and the dorsolateral regions of the left ventricle and atrioventricular canal myocardium. Clonal analyses further showed that these progenitors are multi-potent, creating not only cardiomyocytes and epicardial cell types but also extraembryonic mesoderm. Overall, these results reveal unsuspected multiregional origins of the heart fields, and provide new insights into the relationship between intraembryonic cardiac lineages and extraembryonic tissues and the associations between congenital heart disease and placental insufficiency anomalies.

Enhancer RNAs (eRNAs) are a subset of long noncoding RNA generated from genomic enhancers: they are thought to act as potent promoters of the expression of nearby genes through interaction with the transcriptional and epigenomic machineries. In the present work, we describe two eRNAs transcribed from the enhancer of Nkx2-5-a gene specifying a master cardiomyogenic lineage transcription factor (TF)-which we call Intergenic Regulatory Element Nkx2-5 Enhancers (IRENEs). The IRENEs are encoded, respectively, on the same strand (SS) and in the divergent direction (div) respect to the nearby gene. Of note, these two eRNAs have opposing roles in the regulation of Nkx2-5: IRENE-SS acts as a canonical promoter of transcription, whereas IRENE-div represses the activity of the enhancer through recruitment of the histone deacetylase sirtuin 1. Thus, we have identified an autoregulatory loop controlling expression of the master cardiac TF NKX2-5, in which one eRNA represses transcription.

Novel therapeutics have sought to stimulate the endogenous repair mechanisms in the mammalian myocardium as the native regenerative potential of the adult cardiac tissue is limited. In particular, a myocardial matrix derived injectable hydrogel has shown efficacy and safety in various animal myocardial infarction (MI) including evidence of increased myocardium. In this study, investigation on the properties of this myocardial matrix material demonstrated its native capability as an effective reactive oxygen species (ROS) scavenger that can protect against oxidative stress and maintain cardiomyocyte proliferation in vitro. In vivo assessment of of myocardial matrix hydrogel treatment post-MI demonstrated increased thymidine analog uptake in cardiomyocytes compared to saline controls along with co-staining with cell cycle progression marker, phospho-histone H3. Overall, this study provides further evidence that properties of the myocardial matrix hydrogel promote an environment supportive of cardiomyocytes undergoing cell cycle progression.

Despite decades of studies suggesting that the in vivo adipocyte progenitor resides within the vascular niche, the exact nature of this progenitor remains controversial because distinct studies have attributed adipogenic properties to multiple vascular cell types. Using Cre recombinases labeling distinct vascular lineages, we conduct parallel lineage tracing experiments to assess their degree of contribution to de novo adipogenesis. Although we detect occasional adipocytes that were lineage traced by endothelial or mural recombinases, these are rare events. On the other hand, platelet-derived growth factor receptor alpha (PDGFRα)-expressing adventitial or capsular fibroblasts make a significant contribution to adipocytes in all depots and experimental settings tested. Our data also suggest that fibroblasts transition to an intermediate beige adipocyte phenotype prior to differentiating to a mature white adipocyte. These observations, together with histological analyses revealing that adipose tissue fibroblasts express the mural cell marker PDGFRβ, harmonize a highly controversial field with implications for multiple human diseases, including the pandemic of obesity.

Background: Membrane contact sites are fundamental for transmission and translation of signals in multicellular organisms. The junctional membrane complexes in the cardiac dyads, where transverse (T) tubules are juxtaposed to the sarcoplasmic reticulum, are a prime example. T-tubule uncoupling and remodeling are well-known features of cardiac disease and heart failure. Even subtle alterations in the association between T-tubules and the junctional sarcoplasmic reticulum can cause serious cardiac disorders. NEXN (nexilin) has been identified as an actin-binding protein, and multiple mutations in the NEXN gene are associated with cardiac diseases, but the precise role of NEXN in heart function and disease is still unknown. Methods: Nexn global and cardiomyocyte-specific knockout mice were generated. Comprehensive phenotypic and RNA sequencing and mass spectrometry analyses were performed. Heart tissue samples and isolated single cardiomyocytes were analyzed by electron and confocal microscopy. Results: Global and cardiomyocyte-specific loss of Nexn in mice resulted in a rapidly progressive dilated cardiomyopathy. In vivo and in vitro analyses revealed that NEXN interacted with junctional sarcoplasmic reticulum proteins, was essential for optimal calcium transients, and was required for initiation of T-tubule invagination and formation. Conclusions: These results demonstrated that NEXN is a pivotal component of the junctional membrane complex and is required for initiation and formation of T-tubules, thus providing insight into mechanisms underlying cardiomyopathy in patients with mutations in NEXN.

HomeCirculationVol. 139, No. 12Kindlin-2 Is Essential for Preserving Integrity of the Developing Heart and Preventing Ventricular Rupture Free AccessLetterPDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyReddit Jump toFree AccessLetterPDF/EPUBKindlin-2 Is Essential for Preserving Integrity of the Developing Heart and Preventing Ventricular Rupture Zhiyuan Zhang, MD, PhD, Yongxin Mu, PhD, Jianlin Zhang, PhD, Yangzhao Zhou, MD, Paola Cattaneo, PhD, Jennifer Veevers, PhD, Angela K. Peter, PhD, Ana Maria Manso, PhD, Kirk U. Knowlton, MD, Xinmin Zhou, MD, PhD, Sylvia M. Evans, PhD, Robert S. Ross, MD and Ju Chen, PhD Zhiyuan ZhangZhiyuan Zhang Department of Medicine-Cardiology, University of California San Diego, La Jolla (Z.Z., Y.M., J.Z., Y.Z., J.V., A.K.P., A.M.M., K.U.K., S.M.E., R.S.R., J.C.). Department of Cardiovascular Surgery, The Second Xiangya Hospital, Central South University, Changsha, Hunan, China (Z.Z., Y.Z., X.Z.). *Drs Z. Zhang and Y. Mu contributed equally. Search for more papers by this author , Yongxin MuYongxin Mu Department of Medicine-Cardiology, University of California San Diego, La Jolla (Z.Z., Y.M., J.Z., Y.Z., J.V., A.K.P., A.M.M., K.U.K., S.M.E., R.S.R., J.C.). Department of Cardiovascular Surgery, The Second Xiangya Hospital, Central South University, Changsha, Hunan, China (Z.Z., Y.Z., X.Z.). *Drs Z. Zhang and Y. Mu contributed equally. Search for more papers by this author , Jianlin ZhangJianlin Zhang Department of Medicine-Cardiology, University of California San Diego, La Jolla (Z.Z., Y.M., J.Z., Y.Z., J.V., A.K.P., A.M.M., K.U.K., S.M.E., R.S.R., J.C.). Search for more papers by this author , Yangzhao ZhouYangzhao Zhou Department of Medicine-Cardiology, University of California San Diego, La Jolla (Z.Z., Y.M., J.Z., Y.Z., J.V., A.K.P., A.M.M., K.U.K., S.M.E., R.S.R., J.C.). Search for more papers by this author , Paola CattaneoPaola Cattaneo National Research Council, Institute of Genetics and Biomedical Research, Milan Unit, Italy (P.C.). Humanitas Clinical and Research Center, Rozzano (MI), Italy (P.C.). Search for more papers by this author , Jennifer VeeversJennifer Veevers Department of Medicine-Cardiology, University of California San Diego, La Jolla (Z.Z., Y.M., J.Z., Y.Z., J.V., A.K.P., A.M.M., K.U.K., S.M.E., R.S.R., J.C.). Search for more papers by this author , Angela K. PeterAngela K. Peter Department of Medicine-Cardiology, University of California San Diego, La Jolla (Z.Z., Y.M., J.Z., Y.Z., J.V., A.K.P., A.M.M., K.U.K., S.M.E., R.S.R., J.C.). Department of Molecular, Cellular, and Developmental Biology and BioFrontiers Institute, University of Colorado, Boulder (A.K.P.). Search for more papers by this author , Ana Maria MansoAna Maria Manso Department of Medicine-Cardiology, University of California San Diego, La Jolla (Z.Z., Y.M., J.Z., Y.Z., J.V., A.K.P., A.M.M., K.U.K., S.M.E., R.S.R., J.C.). Veterans Administration Healthcare, Medicine/Cardiology, San Diego, CA (A.M.M., R.S.R.). Search for more papers by this author , Kirk U. KnowltonKirk U. Knowlton Department of Medicine-Cardiology, University of California San Diego, La Jolla (Z.Z., Y.M., J.Z., Y.Z., J.V., A.K.P., A.M.M., K.U.K., S.M.E., R.S.R., J.C.). Intermountain Heart Institute Intermountain Medical Center, Salt Lake City, UT (K.U.K.). Search for more papers by this author , Xinmin ZhouXinmin Zhou Department of Cardiovascular Surgery, The Second Xiangya Hospital, Central South University, Changsha, Hunan, China (Z.Z., Y.Z., X.Z.). Search for more papers by this author , Sylvia M. EvansSylvia M. Evans Department of Medicine-Cardiology, University of California San Diego, La Jolla (Z.Z., Y.M., J.Z., Y.Z., J.V., A.K.P., A.M.M., K.U.K., S.M.E., R.S.R., J.C.). Search for more papers by this author , Robert S. RossRobert S. Ross Ju Chen, PhD, Department of Medicine-Cardiology, University of California San Diego, 9500 Gilman Dr, Mail Code 0613-C, La Jolla, CA 92093-0613. Email E-mail Address: [email protected] Department of Medicine-Cardiology, University of California San Diego, La Jolla (Z.Z., Y.M., J.Z., Y.Z., J.V., A.K.P., A.M.M., K.U.K., S.M.E., R.S.R., J.C.). Veterans Administration Healthcare, Medicine/Cardiology, San Diego, CA (A.M.M., R.S.R.). Search for more papers by this author and Ju ChenJu Chen Ju Chen, PhD, Department of Medicine-Cardiology, University of California San Diego, 9500 Gilman Dr, Mail Code 0613-C, La Jolla, CA 92093-0613. Email E-mail Address: [email protected] Department of Medicine-Cardiology, University of California San Diego, La Jolla (Z.Z., Y.M., J.Z., Y.Z., J.V., A.K.P., A.M.M., K.U.K., S.M.E., R.S.R., J.C.). Search for more papers by this author Originally published18 Mar 2019https://doi.org/10.1161/CIRCULATIONAHA.118.038383Circulation. 2019;139:1554–1556Myocardial wall integrity is essential for normal heart development and for preservation of normal postnatal cardiac function. Congenital ventricular aneurysms can lead to rupture, heart failure, or heart rhythm disturbance.1 Weakened myocardial wall can also result in post–myocardial infarction rupture, frequently leading to death.Kindlin-2 is the only Kindlin family member expressed in the mammalian heart. Kindlin-2 regulates integrin activation2 and integrin-independent pathways.3 A potential role for Kindlin-2 in developing cardiomyocytes, and whether any requirement for Kindlin-2 reflects an integrin-dependent or integrin-independent role for Kindlin-2, remains to be addressed. Accordingly, we generated Kindlin-2 cardiomyocyte-specific knockout (KN2-cKO) mice by crossing floxed Kindlin-2 mice (Kindlin-2f/f)3 with TnT-Cre mice.4 KN2-cKOs began to die at embryonic day 11.5 (E11.5), and no viable KN2-cKOs were observed at E12.5. No morphological differences were evident between KN2-cKO and control hearts at E10.5. However, morphological abnormalities were evident in E11.5 KN2-cKO hearts (Figure, A and B). Histological analysis revealed hemorrhage within the E11.5 KN2-cKO myocardial wall of all 9 embryos analyzed (Figure B). Results from immunostaining for α-actinin (cardiomyocytes) and CD31 (endothelium) revealed that, at E10.5, KN2-cKO hearts were indistinguishable from controls. In contrast, at E11.5, KN2-cKO hearts displayed discontinuity of α-actinin–positive myocardium that was replaced with CD31-positive thrombi (Figure C).Download figureDownload PowerPointFigure. Kindlin-2/β1 integrin pathway in cardiomyocytes is essential for preserving the integrity of the developing heart and preventing ventricular rupture.A, Whole-mount microscopic assessment of control (ctrl) and cardiomyocyte-specific knockout (KN2-cKO) embryos (left lateral view) at embryonic day (E) 10.5, E11.5, and E12.5. Arrow indicates pericardial effusion. B, Top, Microscopic assessment of ctrl and KN2-cKO hearts at E10.5 and E11.5. Middle, Histological evaluation with hematoxylin and eosin staining of transverse sections from hearts. Bottom, High-magnification views of boxed areas. Arrow indicates rupture site. C, Immunomicroscopic analysis of transverse sections of hearts of ctrl and KN2-cKO embryos at E10.5 and E11.5, staining for sarcomeric α-actinin (red), CD31 (green), and DAPI (blue). Arrow indicates rupture site. D, Immunomicroscopic analysis of organization and distribution of fibronectin, laminin, and perlecan in hearts of ctrls and KN2-cKOs at E10.5, staining for fibronectin/laminin/ perlecan (green), sarcomeric α-actinin (red), and DAPI (blue). Arrows point to the well-organized layer of fibronectin, laminin, and perlecan in ctrl hearts, whereas arrowheads indicate the loss of well-delineated fibronectin, laminin, and perlecan on the epicardial surface of the compact myocardial layer. Transmission electron microscopy (TEM) analysis confirmed the loss of basement membrane in KN2-cKO hearts. Arrows point to well-formed basement membrane in ctrl hearts, whereas arrow heads point to severely diminished membrane in KN2-cKO hearts at E11.0. E, Western blot analysis of β1 integrin in ctrl and KN2-cKO hearts at E10.5. GAPDH served as a loading control (Top left). The graphs show corresponding quantitative densitometric analysis where protein pixel density is normalized to the level of GAPDH (bottom left). n=4. Stainings for total β1 integrin (green), sarcomeric α-actinin (red), and DAPI (blue) are shown (middle). Stainings for ligand-bound confirmation/activated form of β1 integrin using the activation-specific antibody 9EG7 (green), sarcomeric α-actinin (red), and DAPI (blue) are shown (right). F, Immunomicroscopic analysis of ctrl and INT-cKO mouse hearts at E11.5 to E13.5, staining for β1 integrin (green), sarcomeric α-actinin (red), and DAPI (blue). G, Immunomicroscopic analysis of the organization and distribution of fibronectin, laminin, and perlecan in the hearts of ctrl and INT-cKO at E10.5, staining for fibronectin/laminin/ perlecan (green), sarcomeric α-actinin (red), and DAPI (blue). Arrows point to the well-organized layer of fibronectin, laminin, and perlecan in ctrl hearts, whereas arrowheads indicate the loss of well-delineated fibronectin, laminin, and perlecan on the epicardial surface of the compact myocardial layer. TEM analysis confirmed the loss of basement membrane in INT-cKO hearts. Arrows point to well-formed basement membrane in ctrl hearts, whereas arrowheads point to severely diminished basement membrane in INT-cKO hearts at E11.0. H, Real-time PCR analysis of relative mRNA levels of target genes of interest in ctrl and KN2-cKO hearts at E10.5. Data are normalized to corresponding 18S rRNA levels, and cKO values are expressed as fold change vs ctrls. n=3. All genes tested were not statistically different between control and knockout samples, when evaluated using Student t test (P>0.05). I, RNA-sequencing data for (i) yap-related genes, (ii) TGFβ-related genes, and (iii) sox9 in ctrl and KN2-cKO hearts at E10.5. n=3. No genes were expressed differentially between ctrl and KN2-cKO samples when evaluated using Student t test (P>0.05). DAPI indicates 4′,6-diamidino-2-phenylindole; PCR, polymerase chain reaction; and TGFβ, transforming growth factor β.Because ablation of the basement membrane protein, perlecan, also leads to embryonic heart rupture,5 we investigated extracellular matrix organization. Indeed, extracellular matrix components, fibronectin, laminin, and perlecan were severely reduced in compact myocardium of KN2-cKO hearts (Figure D). Transmission electron microscopy analysis confirmed the loss of basement membrane in KN2-cKO hearts (Figure D). We next investigated how the loss of Kindlin-2 from cardiomyocytes might affect integrin abundance and localization. RNA-sequencing data from isolated embryonic cardiomyocytes revealed that β1 integrin was the dominant β isoform (reads per kilobase per million mapped reads ≈1000), with β3 and β5 also being expressed, but at much lower levels (reads per kilobase per million mapped reads ≈30). Thus, we focused our attention on β1 integrin. Western blot analysis revealed that the level of β1 integrin was slightly decreased in E10.5 KN2-cKO hearts in comparison with controls, although immunomicroscopic analysis showed no evident differences between KN2-cKO and control samples (Figure E). We then evaluated integrin activation using an antibody (9EG7) that detects ligand-bound β1 integrin, a state judged to represent the activated form. β1 integrin activation was dramatically reduced in KN2-cKO hearts versus controls (Figure E), indicating that Kindlin-2 was required for β1 integrin activation in cardiomyocytes.To investigate whether disruption of β1 integrin activation might account for the loss of myocardial integrity and hemorrhage observed in KN2-cKO embryos, we generated β1 integrinf/–;TnT-Cre mice (INT-cKO). INT-cKO embryos began to die at E13.5 with hemorrhage evident in the chest region, and no viable INT-cKO embryos were observed at E14.5. Like KN2-cKO hearts, discontinuity of the compact layer of the myocardium was clearly evident, with hemorrhage through the myocardial wall into the pericardium of INT-cKO embryos (Figure F). Another striking similarity between KN2-cKO and INT-cKO embryos included severely reduced expression of extracellular matrix proteins fibronectin, laminin, and perlecan in the developing heart (Figure G). Potential compensation by β3 or β5 integrin for the loss of β1 integrin may explain why myocardial rupture and subsequent embryonic lethality occurred ≈2 days later in INT-cKO embryos in comparison with KN2-cKO embryos.In addition to its integrin-dependent role, Kindlin-2 can also play a role in transforming growth factor β pathway function, and can regulate expression of sox9 and yap/taz.3 Thus, we evaluated these pathways in our KN2-cKO mice. Real-time polymerase chain reaction and RNA-sequencing analyses were performed and showed that neither expression of yap1/taz, sox9, or target genes of the transforming growth factor β pathway was affected by the loss of Kindlin-2 (Figure H and I).In summary, our data demonstrate a previously unknown essential role for a Kindlin-2/integrin pathway in extracellular matrix organization and myocardial integrity of the midgestation heart. These results critically provide a new understanding of basic processes of cardiogenesis, but also have potential implications for postnatal heart disease, because the integrity of the myocardium is essential for both normal heart development and for the preservation of normal postnatal cardiac function, prevention of cardiomyopathy, and, most dramatically, protection from myocardial wall rupture from congenital or postnatal causes.All procedures were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and approved by the Institutional Animal Care and Use Committee of the University of California, San Diego.Sources of FundingDrs Chen, Evans, and Ross are supported by grants from the National Institutes of Health. Dr Chen is the American Heart Association (AHA) Endowed Chair in Cardiovascular Research. Dr Zhang is supported by Hunan Provincial Natural Science Foundation of China (2018JJ3722).DisclosuresNone.Footnotes*Drs Z. Zhang and Y. Mu contributed equally.https://www.ahajournals.org/journal/circData sharing: RNAseq raw data can be accessed at GEO using GSE123280. Other data, analytic methods, and study materials will be/have been made available to other researchers for purposes of reproducing results or replicating procedures.Ju Chen, PhD, Department of Medicine-Cardiology, University of California San Diego, 9500 Gilman Dr, Mail Code 0613-C, La Jolla, CA 92093-0613. Email [email protected]eduRobert S. Ross, MD, Department of Medicine-Cardiology, University of California San Diego, 9500 Gilman Dr, Mail Code 0613-C, La Jolla, CA 92093-0613. Email [email protected]eduReferences1. Ohlow MA, von Korn H, Lauer B. Characteristics and outcome of congenital left ventricular aneurysm and diverticulum: analysis of 809 cases published since 1816.Int J Cardiol. 2015; 185:34–45. doi: 10.1016/j.ijcard.2015.03.050CrossrefMedlineGoogle Scholar2. Montanez E, Ussar S, Schifferer M, Bösl M, Zent R, Moser M, Fässler R. Kindlin-2 controls bidirectional signaling of integrins.Genes Dev. 2008; 22:1325–1330. doi: 10.1101/gad.469408CrossrefMedlineGoogle Scholar3. Wu C, Jiao H, Lai Y, Zheng W, Chen K, Qu H, Deng W, Song P, Zhu K, Cao H, Galson DL, Fan J, Im HJ, Liu Y, Chen J, Chen D, Xiao G. Kindlin-2 controls TGF-β signalling and Sox9 expression to regulate chondrogenesis.Nat Commun. 2015; 6:7531. doi: 10.1038/ncomms8531CrossrefMedlineGoogle Scholar4. Jiao K, Kulessa H, Tompkins K, Zhou Y, Batts L, Baldwin HS, Hogan BL. An essential role of Bmp4 in the atrioventricular septation of the mouse heart.Genes Dev. 2003; 17:2362–2367. doi: 10.1101/gad.1124803CrossrefMedlineGoogle Scholar5. Costell M, Gustafsson E, Aszódi A, Mörgelin M, Bloch W, Hunziker E, Addicks K, Timpl R, Fässler R. Perlecan maintains the integrity of cartilage and some basement membranes.J Cell Biol. 1999; 147:1109–1122.CrossrefMedlineGoogle Scholar Previous Back to top Next FiguresReferencesRelatedDetailsCited By Peng D, Fu M, Wang M, Wei Y and Wei X (2022) Targeting TGF-β signal transduction for fibrosis and cancer therapy, Molecular Cancer, 10.1186/s12943-022-01569-x, 21:1, Online publication date: 1-Dec-2022. Gao H, Zhou L, Zhong Y, Ding Z, Lin S, Hou X, Zhou X, Shao J, Yang F, Zou X, Cao H and Xiao G (2022) Kindlin-2 haploinsufficiency protects against fatty liver by targeting Foxo1 in mice, Nature Communications, 10.1038/s41467-022-28692-z, 13:1, Online publication date: 1-Dec-2022. Chen S, Wu X, Lai Y, Chen D, Bai X, Liu S, Wu Y, Chen M, Lai Y, Cao H, Shao Z and Xiao G (2022) Kindlin-2 inhibits Nlrp3 inflammasome activation in nucleus pulposus to maintain homeostasis of the intervertebral disc, Bone Research, 10.1038/s41413-021-00179-5, 10:1, Online publication date: 1-Dec-2022. Wu X, Lai Y, Chen S, Zhou C, Tao C, Fu X, Li J, Tong W, Tian H, Shao Z, Liu C, Chen D, Bai X, Cao H and Xiao G (2022) Kindlin-2 preserves integrity of the articular cartilage to protect against osteoarthritis, Nature Aging, 10.1038/s43587-021-00165-w, 2:4, (332-347), Online publication date: 1-Apr-2022. Bang M, Bogomolovas J and Chen J (2022) Understanding the molecular basis of cardiomyopathy, American Journal of Physiology-Heart and Circulatory Physiology, 10.1152/ajpheart.00562.2021, 322:2, (H181-H233), Online publication date: 1-Feb-2022. Wang W, Rana P, Alkrekshi A, Bialkowska K, Markovic V, Schiemann W, Plow E, Pluskota E and Sossey-Alaoui K (2022) Targeted Deletion of Kindlin-2 in Mouse Mammary Glands Inhibits Tumor Growth, Invasion, and Metastasis Downstream of a TGF-β/EGF Oncogenic Signaling Pathway, Cancers, 10.3390/cancers14030639, 14:3, (639) Wu X, Qu M, Gong W, Zhou C, Lai Y and Xiao G (2022) Kindlin-2 deletion in osteoprogenitors causes severe chondrodysplasia and low-turnover osteopenia in mice, Journal of Orthopaedic Translation, 10.1016/j.jot.2021.08.005, 32, (41-48), Online publication date: 1-Jan-2022. Qin L, Fu X, Ma J, Lin M, Zhang P, Wang Y, Yan Q, Tao C, Liu W, Tang B, Chen D, Bai X, Cao H and Xiao G (2021) Kindlin-2 mediates mechanotransduction in bone by regulating expression of Sclerostin in osteocytes, Communications Biology, 10.1038/s42003-021-01950-4, 4:1, Online publication date: 1-Dec-2021. Gao H, Zhong Y, Ding Z, Lin S, Hou X, Tang W, Zhou X, Zou X, Shao J, Yang F, Bai X, Liu C, Cao H and Xiao G (2021) Pinch Loss Ameliorates Obesity, Glucose Intolerance, and Fatty Liver by Modulating Adipocyte Apoptosis in Mice, Diabetes, 10.2337/db21-0392, 70:11, (2492-2505), Online publication date: 1-Nov-2021. Chen K, Guo L and Wu C (2021) How signaling pathways link extracellular mechano‐environment to proline biosynthesis: A hypothesis, BioEssays, 10.1002/bies.202100116, 43:9, (2100116), Online publication date: 1-Sep-2021. Chen D, Zhang C, Chen J, Yang M, Afzal T, An W, Maguire E, He S, Luo J, Wang X, Zhao Y, Wu Q and Xiao Q (2020) miRNA ‐200c‐ 3p promotes endothelial to mesenchymal transition and neointimal hyperplasia in artery bypass grafts , The Journal of Pathology, 10.1002/path.5574, 253:2, (209-224), Online publication date: 1-Feb-2021. Fu X, Zhou B, Yan Q, Tao C, Qin L, Wu X, Lin S, Chen S, Lai Y, Zou X, Shao Z, Wang M, Chen D, Jin W, Song Y, Cao H, Zhang G and Xiao G (2020) Kindlin-2 regulates skeletal homeostasis by modulating PTH1R in mice, Signal Transduction and Targeted Therapy, 10.1038/s41392-020-00328-y, 5:1, Online publication date: 1-Dec-2020. Yang F, Huang L, Tso A, Wang H, Cui L, Lin L, Wang X, Ren M, Fang X, Liu J, Han Z, Chen J, Ouyang K and Firulli A (2020) Inositol 1,4,5-trisphosphate receptors are essential for fetal-maternal connection and embryo viability, PLOS Genetics, 10.1371/journal.pgen.1008739, 16:4, (e1008739) Mu Y, Yu H, Wu T, Zhang J, Evans S, Chen J and Firulli A (2020) O-linked β-N-acetylglucosamine transferase plays an essential role in heart development through regulating angiopoietin-1, PLOS Genetics, 10.1371/journal.pgen.1008730, 16:4, (e1008730) Zhou Y, Chen Z, Zhang L, Zhu M, Tan C, Zhou X, Evans S, Fang X, Feng W and Chen J (2020) Loss of Filamin C Is Catastrophic for Heart Function, Circulation, 141:10, (869-871), Online publication date: 10-Mar-2020. March 19, 2019Vol 139, Issue 12 Advertisement Article InformationMetrics © 2019 American Heart Association, Inc.https://doi.org/10.1161/CIRCULATIONAHA.118.038383PMID: 30883226 Originally publishedMarch 18, 2019 Keywordsheartextracellular matrixintegrinmyocardiumaneurysmbasement membranePDF download Advertisement SubjectsBasic Science ResearchCell Signaling/Signal TransductionMyocardial Biology

Rationale: Myocardial infarction is a major cause of adult mortality worldwide. The origin(s) of cardiac fibroblasts that constitute the postinfarct scar remain controversial, in particular the potential contribution of bone marrow lineages to activated fibroblasts within the scar. Objective: The aim of this study was to establish the origin(s) of infarct fibroblasts using lineage tracing and bone marrow transplants and a robust marker for cardiac fibroblasts, the Collagen1a1-green fluorescent protein reporter. Methods and Results: Using genetic lineage tracing or bone marrow transplant, we found no evidence for collagen-producing fibroblasts derived from hematopoietic or bone marrow lineages in hearts subjected to permanent left anterior descending coronary artery ligation. In fact, fibroblasts within the infarcted area were largely of epicardial origin. Intriguingly, collagen-producing fibrocytes from hematopoietic lineages were observed attached to the epicardial surface of infarcted and sham-operated hearts in which a suture was placed around the left anterior descending coronary artery. Conclusions: In this controversial field, our study demonstrated that the vast majority of infarct fibroblasts were of epicardial origin and not derived from bone marrow lineages, endothelial-to-mesenchymal transition, or blood. We also noted the presence of collagen-producing fibrocytes on the epicardial surface that resulted at least in part from the surgical procedure.

RATIONALE:Myocardial infarction is a major cause of adult mortality worldwide. The origin(s) of cardiac fibroblasts that constitute the postinfarct scar remain controversial, in particular the potential contribution of bone marrow lineages to activated fibroblasts within the scar.OBJECTIVE:The aim of this study was to establish the origin(s) of infarct fibroblasts using lineage tracing and bone marrow transplants and a robust marker for cardiac fibroblasts, the Collagen1a1-green fluorescent protein reporter.METHODS AND RESULTS:Using genetic lineage tracing or bone marrow transplant, we found no evidence for collagen-producing fibroblasts derived from hematopoietic or bone marrow lineages in hearts subjected to permanent left anterior descending coronary artery ligation. In fact, fibroblasts within the infarcted area were largely of epicardial origin. Intriguingly, collagen-producing fibrocytes from hematopoietic lineages were observed attached to the epicardial surface of infarcted and sham-operated hearts in which a suture was placed around the left anterior descending coronary artery.CONCLUSIONS:In this controversial field, our study demonstrated that the vast majority of infarct fibroblasts were of epicardial origin and not derived from bone marrow lineages, endothelial-to-mesenchymal transition, or blood. We also noted the presence of collagen-producing fibrocytes on the epicardial surface that resulted at least in part from the surgical procedure.

Pericytes are widely believed to function as mesenchymal stem cells (MSCs), multipotent tissue-resident progenitors with great potential for regenerative medicine. Cultured pericytes isolated from distinct tissues can differentiate into multiple cell types in vitro or following transplantation in vivo. However, the cell fate plasticity of endogenous pericytes in vivo remains unclear. Here, we show that the transcription factor Tbx18 selectively marks pericytes and vascular smooth muscle cells in multiple organs of adult mouse. Fluorescence-activated cell sorting (FACS)-purified Tbx18-expressing cells behaved as MSCs in vitro. However, lineage-tracing experiments using an inducible Tbx18-CreERT2 line revealed that pericytes and vascular smooth muscle cells maintained their identity in aging and diverse pathological settings and did not significantly contribute to other cell lineages. These results challenge the current view of endogenous pericytes as multipotent tissue-resident progenitors and suggest that the plasticity observed in vitro or following transplantation in vivo arises from artificial cell manipulations ex vivo.

Cardiac fibroblasts produce the extracellular matrix (ECM) scaffold within which the various cellular components of the heart are organized. As well as providing structural support, it is becoming evident that the quality and quantity of ECM is a key factor for determining cardiac cell behavior during development and in pathological contexts such as heart failure involving fibrosis. Cardiac fibroblasts have long remained a poorly characterized cardiac lineage. Well characterized markers are now paving the way for a better understanding of the roles of these cells in various developmental and disease contexts. Notably, the relevance of processes including endothelial-tomesenchymal transition and the recruitment of circulating fibroblast progenitors in heart failure has been challenged. This review describes the latest findings on cardiac fibroblast markers and developmental origins, and discusses their importance in myocardial remodeling. Effective modulation of cardiac fibroblast activity would likely contribute to successful treatment of various cardiac disorders.

No Evidence for Cardiomyocyte Number Expansion in Preadolescent Mice Alkass et alCell . 2015;163:1026–1036.Understanding cardiomyocyte cell cycle regulation after birth is key to optimizing regenerative strategies for the heart post injury, yet poses multiple technical challenges, as evidenced by recent studies that have arrived at divergent conclusions. In a recent publication in Cell , Alkass et al undertook multiple approaches to examine cardiomyocyte cell cycle regulation in the first 3 weeks after birth. Here, we summarize results of Alkass et al and 3 other groups in examining preadolescent cardiomyocyte cell cycle regulation, highlighting the distinct approaches and incumbent caveats .Understanding cardiomyocyte cell cycle activity during the perinatal and preadolescent period is extremely challenging and is a subject of intense debate. From a classical viewpoint, throughout embryonic development, cardiomyocytes progressively lose their ability to divide and proliferate. After the period between postnatal day 5 (P5) and 10 (P10), the second and final wave of nonreplicative DNA synthesis ends with binucleation of existing cardiomyocytes.1 Recently, Naqvi et al proposed that cardiomyocytes undergo an additional burst of synchronized proliferation on postnatal day 15 (P15) that results in a 40% increase in the number of cardiomyocytes.2 However, in a recent study, Alkass et al observed that an increase in cardiomyocyte number after birth is largely restricted to the first postnatal week, with no significant increase in number after postnatal day 11.3 Consistent with Alkass et al, 2 other groups were not able to substantiate a proliferative burst of cardiomyocytes during preadolescence between the second and third postnatal weeks.4,5 Interestingly, Alkass et al observed a peak of polyploidization of binucleated cardiomyocytes between the second and third postnatal weeks, introducing further complexity to the model of cardiomyocyte cell …