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• W2912091946 abstract "•Creation of a mouse conditionally expressing active YAP called YAP5SA•YAP5SA in adult cardiomyocytes (CMs) induces a more primitive transcriptional state•YAP5SA activates developmental enhancers•YAP5SA expression in CMs causes CM hyperplasia and overall heart hypercellularity Specialized adult somatic cells, such as cardiomyocytes (CMs), are highly differentiated with poor renewal capacity, an integral reason underlying organ failure in disease and aging. Among the least renewable cells in the human body, CMs renew approximately 1% annually. Consistent with poor CM turnover, heart failure is the leading cause of death. Here, we show that an active version of the Hippo pathway effector YAP, termed YAP5SA, partially reprograms adult mouse CMs to a more fetal and proliferative state. One week after induction, 19% of CMs that enter S-phase do so twice, CM number increases by 40%, and YAP5SA lineage CMs couple to pre-existing CMs. Genomic studies showed that YAP5SA increases chromatin accessibility and expression of fetal genes, partially reprogramming long-lived somatic cells in vivo to a primitive, fetal-like, and proliferative state. Specialized adult somatic cells, such as cardiomyocytes (CMs), are highly differentiated with poor renewal capacity, an integral reason underlying organ failure in disease and aging. Among the least renewable cells in the human body, CMs renew approximately 1% annually. Consistent with poor CM turnover, heart failure is the leading cause of death. Here, we show that an active version of the Hippo pathway effector YAP, termed YAP5SA, partially reprograms adult mouse CMs to a more fetal and proliferative state. One week after induction, 19% of CMs that enter S-phase do so twice, CM number increases by 40%, and YAP5SA lineage CMs couple to pre-existing CMs. Genomic studies showed that YAP5SA increases chromatin accessibility and expression of fetal genes, partially reprogramming long-lived somatic cells in vivo to a primitive, fetal-like, and proliferative state. Organs such as the heart and brain contain long-lived, poorly renewable parenchymal cells such as cardiomyocytes (CMs) and most neurons (Bergmann et al., 2015Bergmann O. Zdunek S. Felker A. Salehpour M. Alkass K. Bernard S. Sjostrom S.L. Szewczykowska M. Jackowska T. Dos Remedios C. et al.Dynamics of cell generation and turnover in the human heart.Cell. 2015; 161: 1566-1575Abstract Full Text Full Text PDF PubMed Scopus (650) Google Scholar, Frisén, 2016Frisén J. Neurogenesis and gliogenesis in nervous system plasticity and repair.Annu. Rev. Cell Dev. Biol. 2016; 32: 127-141Crossref PubMed Scopus (54) Google Scholar, Sorrells et al., 2018Sorrells S.F. Paredes M.F. Cebrian-Silla A. Sandoval K. Qi D. Kelley K.W. James D. Mayer S. Chang J. Auguste K.I. et al.Human hippocampal neurogenesis drops sharply in children to undetectable levels in adults.Nature. 2018; 555: 377-381Crossref PubMed Scopus (785) Google Scholar). Carbon-14 dating experiments revealed that a complete supply of human CMs is established within the first month of life, and adult human CMs renew at a rate of approximately 1% per year (Bergmann et al., 2015Bergmann O. Zdunek S. Felker A. Salehpour M. Alkass K. Bernard S. Sjostrom S.L. Szewczykowska M. Jackowska T. Dos Remedios C. et al.Dynamics of cell generation and turnover in the human heart.Cell. 2015; 161: 1566-1575Abstract Full Text Full Text PDF PubMed Scopus (650) Google Scholar). In adult mice, CMs have similarly low rates of renewal (Alkass et al., 2015Alkass K. Panula J. Westman M. Wu T.D. Guerquin-Kern J.L. Bergmann O. No evidence for cardiomyocyte number expansion in preadolescent mice.Cell. 2015; 163: 1026-1036Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar, Soonpaa and Field, 1997Soonpaa M.H. Field L.J. Assessment of cardiomyocyte DNA synthesis in normal and injured adult mouse hearts.Am. J. Physiol. Heart Circ. Physiol. 1997; 272: H220-H226Crossref PubMed Google Scholar). Many long-lived cells are highly specialized, such as CMs and neurons, and express cell-type-specific proteins essential for function. CMs have a structured contractile apparatus, called the sarcomere, that is essential for contractility. It is thought that the sarcomere poses a physical barrier that prevents CM cytokinesis (Tzahor and Poss, 2017Tzahor E. Poss K.D. Cardiac regeneration strategies: staying young at heart.Science. 2017; 356: 1035-1039Crossref PubMed Scopus (202) Google Scholar). The metabolic state of CMs, which utilize oxidative phosphorylation, also contributes to poor renewal (Puente et al., 2014Puente B.N. Kimura W. Muralidhar S.A. Moon J. Amatruda J.F. Phelps K.L. Grinsfelder D. Rothermel B.A. Chen R. Garcia J.A. et al.The oxygen-rich postnatal environment induces cardiomyocyte cell-cycle arrest through DNA damage response.Cell. 2014; 157: 565-579Abstract Full Text Full Text PDF PubMed Scopus (494) Google Scholar). Another barrier to CM renewal likely occurs at the epigenetic level. During development, the fate of differentiated cells is determined by the gradual restriction of the chromatin landscape from the embryonic state to that of lineage-restricted and differentiated cell (Nord et al., 2013Nord A.S. Blow M.J. Attanasio C. Akiyama J.A. Holt A. Hosseini R. Phouanenavong S. Plajzer-Frick I. Shoukry M. Afzal V. et al.Rapid and pervasive changes in genome-wide enhancer usage during mammalian development.Cell. 2013; 155: 1521-1531Abstract Full Text Full Text PDF PubMed Scopus (246) Google Scholar, Stergachis et al., 2013Stergachis A.B. Neph S. Reynolds A. Humbert R. Miller B. Paige S.L. Vernot B. Cheng J.B. Thurman R.E. Sandstrom R. et al.Developmental fate and cellular maturity encoded in human regulatory DNA landscapes.Cell. 2013; 154: 888-903Abstract Full Text Full Text PDF PubMed Scopus (239) Google Scholar). The chromatin state of a differentiated CM is considered to be stable and irreversible. Reversion to a more developmental cell state, as defined by chromatin accessibility, has been described in cancer but not in tissue renewal (Denny et al., 2016Denny S.K. Yang D. Chuang C.H. Brady J.J. Lim J.S. Grüner B.M. Chiou S.H. Schep A.N. Baral J. Hamard C. et al.Nfib promotes metastasis through a widespread increase in chromatin accessibility.Cell. 2016; 166: 328-342Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar, Stergachis et al., 2013Stergachis A.B. Neph S. Reynolds A. Humbert R. Miller B. Paige S.L. Vernot B. Cheng J.B. Thurman R.E. Sandstrom R. et al.Developmental fate and cellular maturity encoded in human regulatory DNA landscapes.Cell. 2013; 154: 888-903Abstract Full Text Full Text PDF PubMed Scopus (239) Google Scholar, Zhu et al., 2013Zhu J. Adli M. Zou J.Y. Verstappen G. Coyne M. Zhang X. Durham T. Miri M. Deshpande V. De Jager P.L. et al.Genome-wide chromatin state transitions associated with developmental and environmental cues.Cell. 2013; 152: 642-654Abstract Full Text Full Text PDF PubMed Scopus (385) Google Scholar). The long-lived and specialized nature of CMs led to the notion that improving endogenous CM renewal was an ineffective strategy to treat heart disease. Recent work revealed that CMs can be induced to renew by expressing exogenous factors, such as cell-cycle components or microRNAs (Mohamed et al., 2018Mohamed T.M.A. Ang Y.S. Radzinsky E. Zhou P. Huang Y. Elfenbein A. Foley A. Magnitsky S. Srivastava D. Regulation of cell cycle to stimulate adult cardiomyocyte proliferation and cardiac regeneration.Cell. 2018; 173: 104-116Abstract Full Text Full Text PDF PubMed Scopus (298) Google Scholar, Wang and Martin, 2014Wang J. Martin J.F. Macro advances in microRNAs and myocardial regeneration.Curr. Opin. Cardiol. 2014; 29: 207-213Crossref PubMed Scopus (28) Google Scholar, Xin et al., 2013Xin M. Olson E.N. Bassel-Duby R. Mending broken hearts: cardiac development as a basis for adult heart regeneration and repair.Nat. Rev. Mol. Cell Biol. 2013; 14: 529-541Crossref PubMed Scopus (333) Google Scholar), and through physiologic means such as exercise (Vujic et al., 2018Vujic A. Lerchenmüller C. Wu T.D. Guillermier C. Rabolli C.P. Gonzalez E. Senyo S.E. Liu X. Guerquin-Kern J.L. Steinhauser M.L. et al.Exercise induces new cardiomyocyte generation in the adult mammalian heart.Nat. Commun. 2018; 9: 1659Crossref PubMed Scopus (94) Google Scholar). To develop effective therapies, it is important to uncover intrinsic molecular mechanisms that inhibit CM renewal (Tzahor and Poss, 2017Tzahor E. Poss K.D. Cardiac regeneration strategies: staying young at heart.Science. 2017; 356: 1035-1039Crossref PubMed Scopus (202) Google Scholar). The Hippo pathway, a kinase cascade, suppresses transcriptional activity of YAP by phosphorylating serine (S) residues at five nuclear Dbf2-related (NDR) kinase family motifs (HXRXXS) (Halder and Johnson, 2011Halder G. Johnson R.L. Hippo signaling: growth control and beyond.Development. 2011; 138: 9-22Crossref PubMed Scopus (797) Google Scholar) via large tumor suppressor (Lats) 1 and Lats2. Deletion of upstream Hippo pathway genes or expressing an active YAP with a single serine (S) to alanine (A) mutation results in increased CM renewal (Heallen et al., 2013Heallen T. Morikawa Y. Leach J. Tao G. Willerson J.T. Johnson R.L. Martin J.F. Hippo signaling impedes adult heart regeneration.Development. 2013; 140: 4683-4690Crossref PubMed Scopus (298) Google Scholar, Lin et al., 2014Lin Z. von Gise A. Zhou P. Gu F. Ma Q. Jiang J. Yau A.L. Buck J.N. Gouin K.A. van Gorp P.R. et al.Cardiac-specific YAP activation improves cardiac function and survival in an experimental murine MI model.Circ. Res. 2014; 115: 354-363Crossref PubMed Scopus (251) Google Scholar, Morikawa et al., 2015Morikawa Y. Zhang M. Heallen T. Leach J. Tao G. Xiao Y. Bai Y. Li W. Willerson J.T. Martin J.F. Actin cytoskeletal remodeling with protrusion formation is essential for heart regeneration in Hippo-deficient mice.Sci. Signal. 2015; 8: ra41Crossref PubMed Scopus (135) Google Scholar, Xin et al., 2013Xin M. Olson E.N. Bassel-Duby R. Mending broken hearts: cardiac development as a basis for adult heart regeneration and repair.Nat. Rev. Mol. Cell Biol. 2013; 14: 529-541Crossref PubMed Scopus (333) Google Scholar). Recent work revealed that the Hippo pathway was upregulated in human heart failure (HF) and Hippo pathway deficiency in mice reversed systolic HF, supporting the notion that the Hippo pathway is an endogenous genetic pathway that can be manipulated to improve CM renewal (Leach et al., 2017Leach J.P. Heallen T. Zhang M. Rahmani M. Morikawa Y. Hill M.C. Segura A. Willerson J.T. Martin J.F. Hippo pathway deficiency reverses systolic heart failure after infarction.Nature. 2017; 550: 260-264Crossref PubMed Scopus (231) Google Scholar). Previous experiments inactivating Hippo pathway components left considerable residual CM Hippo activity. We hypothesized that more completely bypassing Hippo pathway negative regulation would more efficiently increase adult CM renewal and afford us the opportunity to study Yap activity in greater depth. To circumvent the physiologic inputs that activate Hippo and inhibit Yap, we generated mice that conditionally overexpress YAP5SA, a version of YAP that has all LATS1/2 phosphorylation sites mutated from S to A, in adult CMs (Zhao et al., 2010Zhao B. Li L. Tumaneng K. Wang C.-Y. Guan K.-L. A coordinated phosphorylation by Lats and CK1 regulates YAP stability through SCF(beta-TRCP).Genes Dev. 2010; 24: 72-85Crossref PubMed Scopus (933) Google Scholar). YAP5SA globally reprograms adult CM chromatin accessibility at select genomic regions to promote a more renewable state. We made a Cre-inducible YAP5SA transgene containing a LoxP-flanked eGFP-STOP cassette, a FLAG-tagged YAP5SA, and IRES LacZ reporter for lineage tracing that directed eGFP expression in embryonic and postnatal hearts before Cre recombination (Figures 1A–1E ). We crossed YAP5SA mice to tamoxifen (tam)-inducible, CM-specific Cre driver, αMyHC-Cre-ERT2 (MCM) to generate mice with YAP5SA overexpressing (OE) CMs (Sohal et al., 2001Sohal D.S. Nghiem M. Crackower M.A. Witt S.A. Kimball T.R. Tymitz K.M. Penninger J.M. Molkentin J.D. Temporally regulated and tissue-specific gene manipulations in the adult and embryonic heart using a tamoxifen-inducible Cre protein.Circ. Res. 2001; 89: 20-25Crossref PubMed Scopus (471) Google Scholar). To activate YAP5SA in adult CMs, we performed daily tam injections (40 μg/g) for 4 consecutive days in a “high dose” injection regimen. Western blots showed a 5-fold increase in YAP levels in YAP5SA OE hearts compared to controls (Figures 1F and 1G) 1 day after the fourth tam injection. We noted an increase in endogenous P-YAP levels that we discuss below (Figures 1F and 1H). Yap immunofluorescence (IF) revealed increased nuclear and cytoplasmic YAP with varying intensities, suggesting that not all YAP5SA-expressing CMs had constitutive nuclear Yap activity (Figures 1I and 1J). Since CMs expressing low levels of nuclear Yap may be non-transgenic CMs, we performed FLAG IF to look at YAP5SA CM expression. In YAP5SA CMs, nuclear FLAG expression also varied over a wide range and was increased on average approximately 3.2-fold over cytoplasmic FLAG (Figure 1K). We conclude that the YAP5SA OE mice had an increase in nuclear and cytoplasmic Yap in adult CMs but that YAP5SA subcellular localization was still regulated through Hippo-independent mechanisms such as interaction with the intercalated disc (ICD), as we found previously (Morikawa et al., 2017Morikawa Y. Heallen T. Leach J. Xiao Y. Martin J.F. Dystrophin-glycoprotein complex sequesters Yap to inhibit cardiomyocyte proliferation.Nature. 2017; 547: 227-231Crossref PubMed Scopus (166) Google Scholar). One day after the fourth tam injection, echocardiography (ECHO) showed thickened ventricular walls, decreased chamber size, and increased ejection fraction in YAP5SA OE hearts compared to the same mice pre-tam and tam-injected MCM controls (Figures 1L and S1A; Videos S1 and S2). Compared to before tam, YAP5SA OE hearts increased ejection fraction (59.1 +/− 3.5% to 72.5 +/− 4.7%) and fractional shortening (30.9 +/− 1.4% to 41.4 +/−3.5%) while the left ventricular (LV) chamber diameters decreased from 2.66 +/− 0.15 to 1.87 +/− 0.28 mm in systole (ESD) and 3.84 +/− 0.17 to 3.14 +/− 0.25 mm in diastole (EDD), resulting in decreased cardiac output. YAP5SA OE hearts had increased posterior free wall width in systole (FWWS) from 1.05 +/− 0.03 to 1.38 +/− 0.06 mm and in diastole (FWWD) from 0.78 +/− 0.03 to 1.01 +/− 0.04 mm (Figure S1A). Two days after the final tam injection, the LV chambers were reduced (Figure 1M). Histology of YAP5SA OE hearts 3 days after the fourth tam injection also revealed an increased LV wall thickness and smaller chamber (Figure 1N). eyJraWQiOiI4ZjUxYWNhY2IzYjhiNjNlNzFlYmIzYWFmYTU5NmZmYyIsImFsZyI6IlJTMjU2In0.eyJzdWIiOiI1MWYyOTBlNzQ0MDNlYmIxNjhhMjRlOTljNjllOTNlNCIsImtpZCI6IjhmNTFhY2FjYjNiOGI2M2U3MWViYjNhYWZhNTk2ZmZjIiwiZXhwIjoxNjc4NTkxNzIxfQ.FisUIUil4C8QqiXOvWYELTIpAnKTE8N2xPmauduneAIrmnLd22pcluAmdVGrN76XRz6AgYF-XZEiLYGP9gSRh1LAxIsUI-2eGUsGspazYZkhBUd10mUWrSL03PyhADWot0mKlS6JhqeWDkFaXYrMAGQ4tHaZt0TtjJQZTqRcicdnx89khfaTJ7vYREwzMEYOIYazLcVqgWpi7uqSuMljiDDPNQII79roS4nVzD8vX8P7SxQzihkFKKhD7C3rmzqD690pAQ2FhnU9zX9_tZOeYbuPEaiaCoEoO4yY_m1hHHzOK9h9NY2MxPgvexNlWsOJM2ItPrHgKAqqoAEhOi87xw Download .mp4 (3.57 MB) Help with .mp4 files Video S1. Short-Axis B-Mode Echocardiography, Related to Figure 1(A) YAP5SA heart before tamoxifen administration (control).(B) YAP5SA overexpressing heart 2 days after the final tamoxifen dose. eyJraWQiOiI4ZjUxYWNhY2IzYjhiNjNlNzFlYmIzYWFmYTU5NmZmYyIsImFsZyI6IlJTMjU2In0.eyJzdWIiOiI1NTk5ZWYxZThlNWE4ZGI3NmNkYmY2YzIxMGM4MDQ1NiIsImtpZCI6IjhmNTFhY2FjYjNiOGI2M2U3MWViYjNhYWZhNTk2ZmZjIiwiZXhwIjoxNjc4NTkxNzIxfQ.oHSx8lWNZUOP7D2UzkvJ4tXTZHPYJIZ6o4MfbZ8_QzD_0P5Cej_nuaopFjgqj7wK4tWrvCjllBRsnXYByrdj4EGL8U4WY6Pls0LAz2nWYaiPKteG_3g-3TCoQyh-SlG-TgAild5XNCYicvYVgJcv5WfR_3RLWDCMHIIFSqd2pghCTtzqm-K2gHP-J3fNmVX_q0Bnk-xQKLDiUcnNKWK3_upQIBbd6x-2nE6gxETq-_XqtvTV8zScwXZhbShnhDEnMbf8UF1x9Wksn_md2yyn26kJRT5ZqRj4GB-RG7d9bQZqmZATP9JIFBfnSs5Y3GNx6xJfnbB-PNgog1r8ol_FZQ Download .mp4 (1.31 MB) Help with .mp4 files Video S2. Long-Axis B-Mode Echocardiography, Related to Figure 1(A) YAP5SA heart before tamoxifen administration (control).(B) YAP5SA overexpressing heart 2 days after the final tamoxifen dose. YAP5SA OE mice died within 4 days after the last tam dose as a result of HF. Necropsy showed that YAP5SA OE mice had signs of HF including pleural effusion, ascites, and interstitial pulmonary edema (Figure S1B). Controls, which included MCM; YAP5SA treated with oil and MCM treated with tam, did not exhibit HF (Figure 1O). There was no difference in CM size, number, or heart size between MCM; YAP5SA and MCM control mice before tam administration (Figures S1C–S1E). To quantify S-phase entry in adult YAP5SA OE CMs, we provided nucleotide analog, EdU (5-ethynyl-2′-deoxyuridine), ad libitum in drinking water for 2 days following the high-dose tam regimen. EdU incorporation was undetectable in control MCM and MCM; YAP5SA CMs without tam, consistent with previous data (Figures 2A and 2B ) (Alkass et al., 2015Alkass K. Panula J. Westman M. Wu T.D. Guerquin-Kern J.L. Bergmann O. No evidence for cardiomyocyte number expansion in preadolescent mice.Cell. 2015; 163: 1026-1036Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar, Soonpaa and Field, 1997Soonpaa M.H. Field L.J. Assessment of cardiomyocyte DNA synthesis in normal and injured adult mouse hearts.Am. J. Physiol. Heart Circ. Physiol. 1997; 272: H220-H226Crossref PubMed Google Scholar). Approximately 16% of YAP5SA OE CMs were EdU positive (Figures 2A and 2B). We also administered EdU and performed FLAG IF experiments to visualize YAP5SA. Two days after the last tam injection, for a total of six EdU injection days, 40% of CMs expressed nuclear YAP5SA as determined by FLAG IF. Of the nuclear FLAG-positive CMs, 10% were also EdU-positive. Interestingly, 20% of CMs that were negative for nuclear FLAG were EdU-positive (Figures S1F and S1G), indicating a population of YAP5SA OE CMs that had entered S-phase and extinguished nuclear YAP5SA. Alternatively, there may be a paracrine effect of YAP5SA OE CMs on neighboring CMs that do not express YAP5SA. We investigated mitotic marker expression 2 days after the fourth tam dose. Phosphorylation of Histone H3-Ser10 (pHH3) by Aurora Kinase B (AURKB) occurs during chromosome condensation (prophase) and persists throughout metaphase before a decline at anaphase and later stages of mitosis. In contrast, the chromosomal passenger protein AURKB is detected more broadly throughout M-phase and cytokinesis (Sawicka and Seiser, 2012Sawicka A. Seiser C. Histone H3 phosphorylation - a versatile chromatin modification for different occasions.Biochimie. 2012; 94: 2193-2201Crossref PubMed Scopus (149) Google Scholar). Approximately 1% of YAP5SA OE CMs were pHH3-positive (Figures 2C and 2D) and 11% nuclear AURKB-positive (Figures 2E and 2F), providing support that YAP5SA OE CMs were proliferative. While most AURKB expression was nuclear, we found AURKB in dividing CMs in the cytokinesis remnant, albeit more rarely (Figures 2E–2G). To test whether YAP5SA CMs progressed through the cell cycle multiple times, we performed a double-labeling experiment using a single low-dose tam injection to induce YAP5SA in a low number of CMs (STAR Methods). After the single tam injection, mice were pulsed with EdU for 3 days, with a 2-day washout, followed by another 3-day BrdU (5-Bromo-2′-deoxyuridine) pulse (Figure 2H). To identify CM nuclei, we used wheat germ agglutinin (WGA) (Figure 2I), Pericentriolar material 1 (PCM-1) (Figure S1H), or cardiac troponin T (cTnT) (Figure S1I). We detected 4.25% EdU-labeled and 5.04% BrdU-labeled CM nuclei, indicating comparable labeling for both analogs (Figures 2H–2J, S1H, and S1I). Of EdU-labeled CM nuclei, approximately 19% were also BrdU-positive (0.83% total double positive) (Figure 2J). These findings support the conclusion that proliferative YAP5SA OE CMs are capable of cycling more than once. To estimate CM number in LV, we used stereology on hearts harvested 2 days after the fourth tam injection of the high-dose regimen. Hearts were sectioned from apex to base in 7-μm increments and LV area measured at different tissue depths. LV volume was calculated by plotting LV area as a function of location and integrating the area under the curve. Compared with controls, YAP5SA OE hearts had increased LV muscle volume and decreased chamber volume (Figures 2K–2M and 1M). YAP5SA OE hearts had an increased ratio of LV weight to body weight, but CMs were smaller (Figures 2N and 2O). Controls were MCM mice (with and without) tam and MCM; YAP5SA mice without tam. To estimate LV CM number, we calculated the density of PCM-1-positive CM nuclei in control and YAP5SA OE hearts (73,000 +/− 3,000 CM nuclei/mm3 versus 70,600 +/− 1,300 CM nuclei/mm3, respectively) (Alkass et al., 2015Alkass K. Panula J. Westman M. Wu T.D. Guerquin-Kern J.L. Bergmann O. No evidence for cardiomyocyte number expansion in preadolescent mice.Cell. 2015; 163: 1026-1036Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar, Bergmann et al., 2015Bergmann O. Zdunek S. Felker A. Salehpour M. Alkass K. Bernard S. Sjostrom S.L. Szewczykowska M. Jackowska T. Dos Remedios C. et al.Dynamics of cell generation and turnover in the human heart.Cell. 2015; 161: 1566-1575Abstract Full Text Full Text PDF PubMed Scopus (650) Google Scholar). Our CM nuclei density data were consistent with mouse stereology data previously reported (Alkass et al., 2015Alkass K. Panula J. Westman M. Wu T.D. Guerquin-Kern J.L. Bergmann O. No evidence for cardiomyocyte number expansion in preadolescent mice.Cell. 2015; 163: 1026-1036Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar). We multiplied CM density by total heart volume and corrected for CM nucleation (see below) (Alkass et al., 2015Alkass K. Panula J. Westman M. Wu T.D. Guerquin-Kern J.L. Bergmann O. No evidence for cardiomyocyte number expansion in preadolescent mice.Cell. 2015; 163: 1026-1036Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar, Bergmann et al., 2015Bergmann O. Zdunek S. Felker A. Salehpour M. Alkass K. Bernard S. Sjostrom S.L. Szewczykowska M. Jackowska T. Dos Remedios C. et al.Dynamics of cell generation and turnover in the human heart.Cell. 2015; 161: 1566-1575Abstract Full Text Full Text PDF PubMed Scopus (650) Google Scholar). Compared with controls, YAP5SA OE hearts had a large increase in the number of LV CMs (1,840,000 +/− 39,000 versus YAP5SA: 2,680,000 +/− 54,000, respectively; Figures 2P and 2Q). Our control CM number data were consistent with previously published mouse data (Bersell et al., 2009Bersell K. Arab S. Haring B. Kühn B. Neuregulin1/ErbB4 signaling induces cardiomyocyte proliferation and repair of heart injury.Cell. 2009; 138: 257-270Abstract Full Text Full Text PDF PubMed Scopus (726) Google Scholar). Controls were MCM mice with and without tam and MCM; YAP5SA mice without tam. Our data suggest that YAP5SA induces the genesis of new CMs. We next examined nucleation and ploidy in YAP5SA CMs. It was reported that an increase in mononuclear CMs improved renewal capacity, while an increase in ploidy was deleterious for CM renewal in mice and zebrafish (González-Rosa et al., 2018González-Rosa J.M. Sharpe M. Field D. Soonpaa M.H. Field L.J. Burns C.E. Burns C.G. Myocardial polyploidization creates a barrier to heart regeneration in zebrafish.Dev. Cell. 2018; 44: 433-446Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar, Kadow and Martin, 2018Kadow Z.A. Martin J.F. A role for ploidy in heart regeneration.Dev. Cell. 2018; 44: 403-404Abstract Full Text Full Text PDF PubMed Scopus (3) Google Scholar, Patterson et al., 2017Patterson M. Barske L. Van Handel B. Rau C.D. 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: 1346-1353Crossref PubMed Scopus (177) Google Scholar). In isolated CMs 2 days after the fourth tam injection, we found an increased proportion of mononuclear CMs in YAP5SA OE hearts but no change in bi-nucleated CMs compared to tam-injected MCM controls. YAP5SA OE hearts also had a reduced proportion of CMs with 4 nuclei (Figure S1J). To determine DNA content of EdU-containing nuclei in YAP5SA CMs, we used the high-dose tam regimen and injected EdU along with the third and fourth tam injections. Quantification of nuclear DNA content by flow cytometry 24 h after the last tam injection revealed that in EdU-positive CM nuclei from YAP5SA OE hearts, approximately 28% of CM nuclei were diploid and 35% tetraploid, revealing that at least 28% of EdU-labeled nuclei divided. A further 35% were in S-phase, most likely intermediate between the diploid and tetraploid states (Figures S1K–S1M). In total CM nuclei, we saw an increase in 2–4N and 4N nuclei but no increase in greater than 4N nuclei compared to tam-injected MCM controls (Figure S1N). These data, along with our double-labeling experiments, reveal productive YAP5SA OE CM proliferation with more mononuclear CMs without evidence of increased CM ploidy. Because YAP5SA OE mice died after high dose tam injection regimen (Figure 1O), we investigated whether YAP5SA mice had arrhythmias. Optical mapping 48 h after the high-dose tam regimen indicated a continuous spread of action potential across the surface myocardium in YAP5SA OE hearts with a mildly reduced conduction velocity in YAP5SA OE LVs, resulting from the thickened ventricular wall, but no evidence of ventricular arrhythmias (Figures S2A and S2B; Video S3). Telemetry electrocardiogram recordings similarly indicated that YAP5SA OE mice did not have severe arrhythmias, even when challenged with tam that causes T-wave inversion, as a consequence of transient Cre or tam toxicity in CMs 24 h after tam (Bersell et al., 2013Bersell K. Choudhury S. Mollova M. Polizzotti B.D. Ganapathy B. Walsh S. Wadugu B. Arab S. Kuhn B. Moderate and high amounts of tamoxifen in αMHC-MerCreMer mice induce a DNA damage response, leading to heart failure and death.Dis. Models Mech. 2013; 6: 1459-1469Crossref PubMed Scopus (94) Google Scholar, Pugach et al., 2015Pugach E.K. Richmond P.A. Azofeifa J.G. Dowell R.D. Leinwand L.A. Prolonged Cre expression driven by the alpha-myosin heavy chain promoter can be cardiotoxic.J. Mol. Cell. Cardiol. 2015; 86: 54-61Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar) (Figures S2C and S2D). We observed clusters of lineage-traced β-gal-positive YAP5SA OE CMs and 94% of these β-gal-positive CMs were coupled to surrounding CMs at ICDs, as indicated by N-cadherin IF (Figure S2E). Of β-gal-positive CMs that coupled to other CMs, 37% coupled only to other YAP5SA OE lineage β-gal-positive CMs, 22% coupled to β-gal-negative CMs only, and 41% coupled to both β-gal-positive and β-gal-negative CMs (Figure S2F). eyJraWQiOiI4ZjUxYWNhY2IzYjhiNjNlNzFlYmIzYWFmYTU5NmZmYyIsImFsZyI6IlJTMjU2In0.eyJzdWIiOiJjNzJmMzg1Y2U2NjE3NTNjNjhlMDcxZmZiNDRiZWVmMSIsImtpZCI6IjhmNTFhY2FjYjNiOGI2M2U3MWViYjNhYWZhNTk2ZmZjIiwiZXhwIjoxNjc4NTkxNzIxfQ.WnnSxC05OmJc1Lkla8acwJ-zD-Eae3x4eH9S1D_edwhMpYoDqR_SylKMVMzGBS56gXs56zdJs8ZMvMhfZgSfP-AP7xLPqpX_p49smbXQQahCO7cl9vCWBOyt86ZuF0-JS_E_3Z9VundO9ilhRi0oUHPPZdcHeFNItLPgYMygRLjAG-V2x7BxP92RVgHBoNOoH7mrt1BVtlMepP_81tF2F4qVFKWfxZUw7ftEfofxqxMz2zqdOjkuROZOncEC-yOwDYMiv1JRILsYwPWUVLtUf6hjTx8mwGf2A-OjHUwmq0NQOTgxFZnvHcEUAeEy9-RNe0Enqxmysgz_uFVcghsBTw Download .mp4 (5.32 MB) Help with .mp4 files Video S3. Action Potential Propagation across the Surface, Related to Figure S2(A) Control mouse heart, MCM mouse injected with tamoxifen (B) YAP5SA OE heart. These are from 48 h after tamoxifen and with 10 Hz pacing. Plots indicate fluorescence intensity over time at the indicated locations (top: right atrium; middle: left atrium; bottom: left ventricle). To measure YAP5SA OE CM contractility, we isolated CMs 24 h after the final tam injection of the high-dose regimen and measured individual CM contractility. Compared to tam-injected MCM controls, YAP5SA OE CMs had similar resting sarcomere length and contractility in response to field stimulation (Figures S2G–S2I). YAP5SA induction did not induce interstitial fibrosis, although we noted that there was a 20% increase in number and space occupied by non-CMs compared to tam-injected MCM controls (Figures S2J–S2M). The total volume of non-CMs increased as the hearts grew (0.33 × 1010 μm3 to 0.55 × 1010 μm3), but the increase of non-CM volume only accounted for approximately 12% of the total myocardium volume difference between MCM control to YAP5SA OE hearts (Con: 5.38 × 1010 μm3; YAP5SA: 7.22 × 1010 μm3) (Figures 2M and S2L). We performed CM-specific nuclear RNA sequencing (RNA-seq), assay for transpose accessible chromatin (ATAC)-sequencing, and chromatin conformation capture assays (3C and 4C) 2 days after the fourth tam injection of the high-dose regimen (Buenrostro et al., 2013Buenrostro J.D. Giresi P.G. Zaba L.C. Chang H.Y. Greenleaf W.J. Transposition of native chromatin for fast and sensitive epigenomic profiling of open chromatin, DNA-binding proteins and nucleosome position.Nat. Methods. 2013; 10: 1213-1218Crossref PubMed Scopus (3189) Google Scholar, Gilsbach et al., 2014Gilsbach R. Preissl S. Grüning B.A. Schnick T. Burger L. Benes V. Würch A. Böni" @default.
• W2912091946 created "2019-02-21" @default.
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• W2912091946 date "2019-03-01" @default.
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• W2912091946 title "YAP Partially Reprograms Chromatin Accessibility to Directly Induce Adult Cardiogenesis In Vivo" @default.
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