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. Author manuscript; available in PMC: 2026 Jan 7.
Published in final edited form as: Circulation. 2024 Oct 11;151(1):76–93. doi: 10.1161/CIRCULATIONAHA.123.066004

YAP Overcomes Mechanical Barriers to Induce Mitotic Rounding and Adult Cardiomyocyte Division

Yuka Morikawa 1, Jong H Kim 1, Rich Gang Li 1, Lin Liu 1, Shijie Liu 1, Vaibhav Deshmukh 2, Matthew C Hill 4,5, James F Martin 1,2,3,6,7,
PMCID: PMC11671297  NIHMSID: NIHMS2026295  PMID: 39392007

Abstract

Background:

Many specialized cells in adult organs acquire a state of cell cycle arrest and quiescence through unknown mechanisms. Our limited understanding of mammalian cell cycle arrest is derived primarily from cell culture models. Adult mammalian cardiomyocytes (CMs), a classic example of cell cycle arrested cells, exit the cell cycle postnatally and remain in an arrested state for the life of the organism. CMs can be induced to re-enter the cell cycle by YAP5SA, an active form of the Hippo signaling pathway effector YAP.

Methods:

We performed clonal analyses to determine the cell kinetics of YAP5SA CMs. We also performed single-cell RNA sequencing, marker gene analysis, and functional studies to examine how YAP5SA CMs progress through the cell cycle.

Results:

We discovered that YAP5SA-expressing CMs divided efficiently, with more than 20% of YAP5SA CM clones containing two or more CMs. YAP5SA CMs re-entered cell cycle at the G1/S transition and had an S-phase lasting approximately 48 hours. Sarcomere disassembly is required for CM progression from S to G2 phase and the induction of mitotic rounding. While oscillatory Cdk expression was induced in YAP5SA CMs, these cells inefficiently progressed through G2 phase. This is improved by inhibiting P21 function, implicating checkpoint activity as an additional barrier to YAP5SA-induced CM division.

Conclusions:

Our data reveal that YAP5SA overcomes the mechanically constrained myocardial microenvironment to induce mitotic rounding with CM division, thus providing new insights into the in vivo mechanisms that maintain cell cycle quiescence in adult mammals.

Keywords: myocyte proliferation, YAP, Hippo pathway, cell cycle, sarcomere disassembly, mitotic rounding, P21

INTRODUCTION

CMs, specialized and long-lived cells with little renewal capacity, age concurrently with the organism. Carbon-14 isotope studies on postmortem CMs from people who lived during atmospheric nuclear testing revealed that adult CM renewal occurs at approximately 1% per year 1. These low CM renewal rates are similar to the 1% CM renewal per year in mice 2,3. It remains unknown how adult mammalian CMs or other differentiated cell types are maintained in an arrested cell cycle state.

Soon after birth, CMs arrest at the G1/S transition 4. One hypothesis is that CM cell cycle arrest is secondary to fundamental CM structural and functional characteristics, including mechanical barriers 5. Adult CMs have a rigid sarcomeric cytoskeleton, which is required for cardiac pump function and survival 6. In CMs, sarcomere rigidity inhibits spherical shape formation, which is required for mitotic rounding, correct mitotic spindle formation, and cell division 7. During mitotic rounding, enlarging mitotic cells impinge on neighboring cells to acquire a spherical shape. Within constrained myocardium, CMs are surrounded by equally stiff CMs, further inhibiting sphere formation and mitotic rounding. Moreover, the ventricular myocardium is subject to high wall tension, which is required to pump blood against physiologic afterload, adding an additional mechanical barrier to CM sphere formation. The adult myocardial extracellular matrix (ECM) also inhibits CM division 8. Together, the mechanically constrained myocardial microenvironment inhibits CM mitotic rounding, an essential preliminary step to cytokinesis known as “structural block” 9.

CMs preferentially use oxidative metabolism to meet the high energy demands of continuous pumping, exposing CMs to oxygen radicals and DNA damage in an antiproliferative, metabolic cell cycle block 10. Adult mammalian CMs are also polyploid, another barrier to cell division 11. Evidence also suggests that adult mammalian CMs lack essential cell cycle apparatus components, resulting in mitotic arrest 12.

The Hippo Signaling Pathway inhibits nuclear localization and transcriptional activity of the transcriptional co-factor YAP 13. Hippo signaling prevents CM renewal post-myocardial infarction (MI) and during ischemic heart failure (HF) in mice and pigs 14,15. Expression of YAP5SA, a version of YAP resistant to Hippo inhibition, induces cellular division of adult CMs 16. Here, we show in adult mouse hearts that YAP5SA CMs re-enter the cell cycle from the G1/S transition and progress into an extended S-phase lasting 48 hours. Moreover, progression from S-phase into G2/M requires sarcomere disassembly followed by mitotic rounding. YAP5SA CMs progression through mitosis and cytokinesis was slowed by P21 induction, indicating that the metabolic cell cycle block and DNA damage response was only partially overcome by YAP5SA. Together, our data indicate that YAP overcomes the mechanically constrained myocardial microenvironment to induce the reemergence of adult CMs from cell cycle quiescence.

METHODS

Detailed methods are available in Supplemental materials.

Data and Codes Availability

All codes used in the article are deposited in GitHub (github.com/jhkim818/GSE152856_YAP_promotes_cardiomyocyte_cell_cycle_entry). Single cell RNA-seq data has been deposited at the Gene Expression Omnibus under the ID code GSE152856. Other data are available upon reasonable request from the lead contact.

Animals

All animal protocols and procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of Baylor College of Medicine in Houston, Texas. Mice were sacrificed by CO2 gas then bilateral opening of thorax was used to ensure death. Both sexes were used in this study in a blind selection manner. We studied sex differences in our previous study 16, and found no differences.

The MCM; YAP5SA line 16 was used for cardiomyocyte-specific expression of YAP5SA. The transgene was induced by a single intraperitoneal administration of tamoxifen (40 mg/kg animal).

Quantification and Statistical Analysis

Quantification was performed via ImageJ software. Observers were blinded to genotypes. All bar graphs represent mean +/− s.e.m. Mann-Whitney analysis (for 2 sample groups) or One-way Anova (for 3 or more sample groups) were used for statistical analysis.

RESULTS

YAP5SA adult cardiomyocytes enter all cell cycle phases and divide

Using the R26R-Confetti mouse line for clonal analysis, we determined how frequently YAP5SA CMs complete the cell cycle and divide 17 (Fig. 1A–C). MCM; R26R-Confetti mice were injected with low-dose tamoxifen to induce sparse labeling of individual CMs with fluorescent reporters and then infected with an AAV9 virus expressing YAP5SA (AAV9: YAP5SA) (Fig. 1A). By necessity, this method uses low dose tamoxifen to sparsely label CMs and therefore underreports the true frequency of YAP5SA CM cell division. Hearts were imaged for GFP, RFP, and YFP encoded by R26R-Confetti (Fig. 1B, Fig. S1A–I, Suppl Movie). We quantified each CM clone, which consisted of either a single labeled CM or a cluster of two or more CMs (Fig. 1C). In control hearts, all labeled CMs occurred as single CMs, while more than 20% of AAV9: YAP5SA labeled CMs contained clusters containing two or more CMs, supporting the conclusion that YAP5SA CMs divide and generate new daughter CMs (Fig. 1C).

Fig 1. Analysis of YAP5SA-induced cell cycle.

Fig 1.

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A-C: Analysis of cytokinesis using R26F-Confetti line. A: (Left) Timing of the injections and sample harvesting. Low dose tamoxifen (Tm) was injected into MCM; R26R-Confetti mice, then AAV9-YAP5SA were injected 2 days after. Saline was injected as control. Hearts were harvested in 2 weeks after virus injection. (Right) Schematic presentation of clonal analysis using R26R-Confetti mouse line. Low dose tamoxifen results in single cell labeling of fluorescent proteins. Cell proliferation results in clusters of 2 or more cells while non-proliferative cell remain as a clone of single cell. B: Representative images of control and YAP5SA hearts for YFP, WGA, and merged. Sections were stained with WGA to delineate cell boundary. Arrow show YFP-expressing CMs. C: Quantification of clonal formation in the control and YAP5SA hearts. Groups were compared using Chi-square test for distribution. Double and 3+ clones within treatments were compared using Fisher’s exact test (n=5 for control, n=6 for YAP5SA). **p<0.01, ****P<0.0001. D-I: Expression of cell cycle markers in adult heart were determined by immunohistochemistry. D: Timing of tamoxifen (Tm) injection and sample harvesting for panels E-I. YAP5SA and control mouse lines were used. E: Representative images of PCNA and cTNT expression in control and YAP5SA expressing hearts in transverse sections. Yellow arrowheads are PCNA positive nuclei in CMs. F: Quantification of PCNA positive nuclei. Groups were compared using the Mann-Whitney U test (n=5 each). **p<0.01. G: Timing of cyclin D1 (CCND1), CDK2, CYCLIN A2 (CCNA2), CDK1, and PHH3 expression during cell cycle. H: Quantification of CCND1, CDK2, CCNA2, CDK1, and PHH3 positive CM nuclei (n=5). I: Representative images of CCND1, CDK2, CCNA2, and CDK1 in YAP5SA expressing hearts. Representative images of PHH3 staining are in Fig. S2H, I.

Cell cycle progression in YAP5SA CMs was further investigated six days after YAP5SA induction. Using a single injection protocol, we observed that YAP5SA expression is restricted to CMs (Fig. S2A). Roughly 50% of CMs express YAP5SA by Day 2 (Fig. S2B). Cell cycling CMs were quantified via proliferating nuclear antigen (PCNA) that marks CMs in S and early G2 phase 18 (Fig. 1D–F). 18.3+/−1.0% of YAP5SA CMs express PCNA, indicating cell cycle induction in adult YAP5SA CMs. Non-CMs also expressed PCNA in YAP5SA hearts, as reported previously (Fig. 1F) 16. Consistent with recent studies indicating that subendocardial CMs are more likely to enter the cell cycle than other CMs 19, subendocardial YAP5SA CMs had more PCNA positive (+) CMs compared to subepicardial YAP5SA CMs. Nonetheless, over 10% of subepicardial YAP5SA CMs were PCNA+, indicating that YAP5SA CMs throughout the heart re-enter the cell cycle (Fig. S2C–G). For consistency, we focused on subendocardial CMs.

We used immunofluorescence (IF) to determine YAP5SA CMs cell cycle dynamics using Cyclin D1(CCND1, G1), cyclin-dependent kinase 2 (CDK2, S to early G2), Cyclin A2 (CCNA2, late S to early G2), cyclin-dependent kinase 1 (CDK1, G2 to M), and phosphorylated histone H3 (PHH3, M phase) (Fig. 1G–I, Fig. S2H, I). Our findings indicate that YAP5SA CMs are competent to enter all cell cycle phases (Fig. 1G). Of YAP5SA CMs in the cell cycle, approximately 20–30% were in S to early G2 (overlapping CDK2/CCNA2) at any one time (Fig. 1H–I).

YAP5SA cardiomyocytes re-enter the cell cycle at the G1-S transition

We investigated YAP5SA CM cell cycle reentry by examining G1 and S marker expression shortly after YAP5SA induction. Control CMs were consistently CCND1 and PCNA negative (data not shown). Interestingly, G1 cyclin CCND1 was first detected at day five post-YAP5SA induction, while PCNA (S phase) was detected at day two post-YAP5SA induction, indicating that YAP5SA CMs bypass G1 upon cell cycle re-entry (Fig. 2A–E). Both CCND1 and PCNA were expressed from day two post-induction in non-myocytes of YAP5SA hearts (Fig. 2C–E).

Fig 2. Cell cycle re-entry in adult cardiomyocytes.

Fig 2.

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A: Overview of the proteins expressed in G1 and G1/S phase analyzed in this figure. # are Yap targets. B: Timing of tamoxifen (Tm) injection and sample harvesting for panels C-G. YAP5SA mouse line was used and hearts were harvested 1–5 days post tamoxifen. C-E: Expression of cyclin D1 (CCND1) and PCNA was analyzed 1–5 days after YAP5SA induction. C: Representative image of YAP5SA heart at day3 show non-myocyte CCND1 expression (white arrows). PCNA expressed in both CMs (yellow arrowheads), and non-myocyte (white arrowheads). D, E: Quantification of CCND1 (D) and PCNA (E) expressing nuclei in CMs. Groups were compared using one-way ANOVA with the Tukey post-hoc test for pairwise comparisons (n=5 each time point). *p<0.05, ***p<0.001, ****p<0.0001. F, G: Expression of CDK2, CCNE1 (cyclin E1) and P-RB were analyzed at 2 and 3 days after tamoxifen injection. F: Representative images of CDK2, CCNE1, and P-RB in YAP5SA expressing hearts at day 3. Arrows show positive CM nuclei. G: Quantification of CDK2, CCNE, and P-RB expressing nuclei in CMs. Groups were compared using Mann-Whitney analysis. **p<0.01. H-J: Flow analysis of DNA synthesis. H: Timing of injections and harvesting (Left), and steps of flow analysis (Right). I: Representative FACS image of PCM1 positive nuclei (top) and EdU-stained nuclei in PCM1 population. J: Quantification of EdU incorporation in PCM1-positive CM nuclei of control (n=5 experiments) and YAP5SA expressing hearts (n=6 experiments). Groups were compared using Mann-Whitney test. **p<0.01.

To define YAP5SA CM cell cycle re-entry more accurately, we examined the G1/S factors cyclin E1 (CCNE1), CDK2, and phospho-RB. All three markers were expressed at days two and three post-YAP5SA induction (Fig. 2F–G). To validate that YAP5SA CMs were in S phase, we used 5-ethyl-2’-deoxyuridine (EdU) to label newly synthesized DNA three days post YAP5SA CM induction (Fig. 2H–J). Mice were injected with a single EdU dose two hours before harvest and EdU incorporation was quantified by flow cytometry. Since EdU lasts approximately 30 minutes, the short EdU pulse only labeled a small subset of S-phase YAP5SA CMs 20. Consistent with IF data, 5% of YAP5SA CMs had increased EdU incorporation. Together, these results show that adult YAP5SA CMs re-enter the cell cycle at the G1/S phase transition and, combined with clonal analysis, support the conclusion that CCND1-positive CMs at day five had progressed through mitosis and entered G1 in a second cell cycle.

Adult YAP5SA cardiomyocytes have an S phase lasting 48 hours

To study cell cycle progression in YAP5SA CMs after S phase entry, we injected a single EdU dose three days post YAP5SA induction and collected hearts at 24, 48, and 72 hours post-EdU injection (Fig. 3A). We analyzed cell cycle markers in EdU-positive YAP5SA CMs (YAP5SA; EdU+), including CCNA2 (late S to early G2), CDK1 (late G2 to M), and CCND1 (G1) (Fig. 3B). Approximately 50% of YAP5SA; EdU+ CMs were CCNA2 positive at 24 hours post-EdU labeling followed by decreased CCNA2 expression at 48 hours, suggesting that most YAP5SA; EdU+ CMs exit S phase 48 hours post-EdU labeling (Fig. 3C). YAP5SA; EdU+ CMs in late G2/M (CDK1+) appeared 48 hours after EdU labeling, supporting the conclusion that YAP5SA; EdU+ CMs enter G2 phase 48 hours after S phase entry (Fig. 3D). Of the 50% of YAP5SA CMs that entered S-phase, approximately 2–3% were CDK1 positive at 48 hours post-EdU injection. The low percentage of CDK1-positive YAP5SA CMs suggests that a subset of YAP5SA; EdU+ CMs transition from S to G2 within 48 hours while other YAP5SA CMs arrest at G2/M (Fig. 3D).

Fig 3. Cell cycle analysis of adult cardiomyocytes by EdU pulse and cell cycle markers.

Fig 3.

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A: Timing of tamoxifen (Tm) and EdU injection, and sample harvesting. EdU was injected 3 days after tamoxifen injection and hearts were harvested 24, 48, and 72 hours after EdU injection. B: Markers used to determine cell cycle length in pulse-labelled cells. CCNA2 (late S to early G2 phase), CDK1 (late G2 to M phase), and CCND (G1 phase) were co-labelled with EdU. C, D: Representative images and quantification of double positive nuclei for EdU and cell cycle markers CCNA2 (C) and CDK1(D) in YAP5SA-expressing CMs. Groups were compared by using one-way ANOVA with the Tukey post-hoc test for pairwise comparisons (n=5 each time point). ****p<0.0001.

E-H: Nucleation and ploidy analysis. E: Timing of tamoxifen (Tm) and EdU administration, and sample harvesting. EdU was given by Alzet pump at 3 days for 24 hr and hearts were harvested at day 4 and 6 of tamoxifen injection. F: Representative images of isolated CMs from day 4 and 6. CMs were stained for DAPI, EdU and cTNT. G: Nucleation of EdU+ CMs. Proportion of mono-nucleated (MonoNuc) and binucleated (BiNuc) CMs were compared between CMs from day 4 and day 6. Chi-square test was used to compare two groups (n=3 each group). **** p<0.0001. H: Ploidy of EdU+ CM nuclei. Day 4 MonoNuc (47 nuclei), Day 4 BiNuc (288 nuclei), Day 6 MonoNuc (136 nuclei), and Day 6 BiNuc (181 nuclei) were analyzed. Percentage of ploidy in individual samples were shown in Fig. S3I. Chi-square test was used to compare groups. *p<0.05, *** p<0.001, **** p<0.0001.

I: Representative images and quantification of double positive nuclei for EdU and CCND1 in YAP5SA-expressing CMs. Groups were compared by using one-way ANOVA with the Tukey post-hoc test for pairwise comparisons (n=5 each time point). ****p<0.0001. J, K: Analysis of EdU twin spots. J: Representative images of the EdU and CCND1 double positive twin spot (arrows) at 72 hours post EdU injection. K: Number of EdU twin spots, and CCND1 positive nuclei among EdU twin spots (n=5). Chi-square test was used. *p<0.05. Percentage of twin spots in each time point, and percentage of CCND1 positive EdU twin spot per sample are shown in Fig. S3J and K.

To estimate cytokinesis of YAP5SA; EdU+ CMs, we conducted nucleation and ploidy analysis on isolated CMs (Fig. 3E–H). Using Alzet pump delivery, YAP5SA mice were exposed to EdU for 24 hours starting on Day three after tamoxifen injection, and then CMs were isolated on Days four and six after tamoxifen (Fig. 3E, Fig. S3A–H). One day after EdU labeling (Day four after tamoxifen), approximately 20% of EdU+ CMs were mononucleated (monoNuc), while 80% of EdU+ CMs were binucleated (BiNuc) (Fig. 3F–G). In contrast, six days after tamoxifen injection, the proportion of mononucleated EdU+ CMs increased to 60%, consistent with our previous observation that YAP5SA hearts have more mononucleated CMs (Fig. 3F–G) 16.

Using DAPI intensity to measure nuclear ploidy, we found that EdU+ CM nuclei four days after tamoxifen injection were primarily greater than 2N, consistent with S phase, G2 phase, or polyploid nuclei (Fig. 3H, Fig. S3I). Conversely, six days after tamoxifen, greater than 40% of mononucleated EdU+ CMs had 2N nuclear ploidy, supporting the conclusion that EdU+ CMs divide into two daughters. Moreover, binucleated CM nuclei also had increased 2N ploidy, suggesting mononucleated 4N CMs at day six undergo karyokinesis to form binucleated CMs with 2N nuclei. We also observed that nuclei within binucleated CMs were occasionally unsynchronized and were in different cell cycle phases (Fig. S3C, G, H).

We next analyzed CCND1; EdU double positive YAP5SA CMs (CCND1+; YAP5SA EdU+), which had completed the cell cycle, divided, and entered G1. CCND1+; YAP5SA EdU+ CMs, comprising 12% of all YAP5SA; EdU+ CMs, appeared 72 hours post-EdU injection, suggesting that 12% of YAP5SA; EdU+ CMs divide 72 hours after EdU injection or approximately 5–6 days post YAP5SA induction, which is also consistent with our R26R; Confetti data (Fig. 3I and Fig. 1C). To investigate this further, we analyzed CCND1+ twin spots, which are two daughter CMs derived from the same mother CM (Fig. 3J). 24–48 hours after EdU injection, YAP5SA; EdU+ CM were found side by side and were CCND1 negative, suggesting that these rare CMs were neighboring YAP5SA; EdU+ CMs that simultaneously entered S phase rather than clonal twin spot CMs. 72 hours after EdU injection (completion of the predicted YAP5SA cell cycle), the number of side-by-side YAP5SA EdU+ CMs increased, and 50% were CCND1 positive in both nuclei indicating a clonal twin spot (Fig. 3K, Fig. S3J, K)., Taken together, our twin spot analysis, R26R;Confetti data, and the ploidy/nucleation analysis, support the conclusion that YAP5SA; EdU+ CMs go through cytokinesis and divide within 72 hours of S phase entry.

Single-cell RNA sequencing reveals YAP5SA-enriched CM cell states

We used single cell RNA-sequencing (sc RNA-seq) data 21 to investigate YAP5SA CM cell state and cell cycle dynamics in greater depth (Fig. S4). We profiled 21,706 single cells from control and YAP5SA hearts and identified 6 distinct cell clusters: epicardium (EpiC), endothelial cells (EC), mural cells (mural), CMs, cardiac fibroblasts (CF), and macrophages (MAC) (Fig. S5A–C, Table S1). After re-clustering, we identified 5 CM clusters: CM A-D and CM G2/M (Fig. S5D–F; Table S2). Since YAP5SA CMs were enriched for CM B and CM G2/M (Fig. S4E), we re-clustered CM B and CM G2/M from both control and YAP5SA hearts and uncovered 5 CM cell states; CM-1 – CM-5 (Fig. 4A–E). Control CMs were composed of CM-1 and CM-3, while YAP5SA CMs were enriched for CM-2, CM-4, and CM-5 cell states (Fig. 4B, Fig. S6A, B).

Fig 4. Single cell analysis in YAP5SA heart.

Fig 4.

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A-E: Analysis of YAP5SA and control CM drop-seq 6 days after TAM injection. A: UMAP of re-clustered CMs. B: Cell composition graph of 5 clusters. C: Top 10 Differentially expressed genes, ranked by descending fold change from each cluster. D: Representative GO for 5 clusters. GO analysis from each cluster is shown in the Fig. S6C–G. E: Cell trajectory analysis of clusters 2, 4, and 5 colored by pseudotime (top). Cellular density of each cluster along Comp1 axis (middle). Patterning of trajectory determining genes along pseudotime (bottom). F-H: Comparison between adult and P2 CM datasets. F: Volcano plots of differentially expressed genes between adult control and P2 sham CMs from the CM cluster shown in Fig. S6. G: UMAP of CM clusters from the drop-seq analysis. Same image as in Fig. S5D. H: P2 CM score. P2 data from the Fig. 4F was mapped on the Fig. 4G.

Gene ontology (GO) analysis revealed that control-enriched CM-1 and CM-3 associate with terms such as TCA cycle and Oxidative Phosphorylation, which are characteristic of the adult CM metabolic state (Fig. 4C, D, Fig. S6C–G, Table S3). GO analysis of YAP5SA-enriched CMs revealed that CM-2 associates with terms such as actin cytoskeleton, CM-4 included terms such as SCF(Skp2)-mediated degradation of P27/21, and CM-5 had terms for G2/M phase (Fig. 4D, Fig. S6C–G, Table S3). The CM-5 cell state is consistent with previous data indicating that G2/M genes, regulated by the B-MYB-MuvB (MMB) complex 22, are also activated by YAP in combination with the MMB complex 23. Yap and MMB target genes that function in mitosis, such as anln, ccna2, cdc20, cenpf, hmmr, nusap1, and top2a are differentially expressed in adult G2/M CMs (cluster 5).

Pseudo-time trajectory analysis for CM-2, CM-4, and CM-5 revealed that the CM-2 cell state, characterized by sarcomere disassembly, occurs before CM-4 and CM-5. Together, the single cell data support the conclusion that sarcomere disassembly is the first cell cycle arrest point overcome by YAP5SA (Fig. 4E, Table S3).

To extend our analysis further, we compared adult YAP5SA and control neonatal CM cell states using available neonatal CM single nuclei data 24. After data integration and CM re-clustering, we extracted differentially expressed genes between postnatal (P) 2 and adult control CMs to obtain P2 and adult CM gene expression modules (Fig. 4F, Fig. S7A, B, Table S4). We scored adult control CMs and YAP5SA CMs for the P2 CM gene expression module and found CMs scoring highly for the P2 CM genes were found in CM B, CM D, and CM G2/M, which are YAP5SA CMs (Fig. 4G, H). Moreover, YAP5SA CMs were more enriched for P2 CM genes compared to adult CM genes, suggesting that YAP5SA CMs acquire an immature cell state (Fig. S7C, D).

Sarcomere disassembly precedes cardiomyocyte G2/M phase entry

To determine when sarcomere disassembly occurred in the YAP5SA CM cell cycle, we evaluated sarcomere disassembly after YAP5SA induction in adult CMs (Fig. 5A). Distribution of the sarcomere marker cardiac troponin (cTNT) was distinct in control and YAP5SA CMs (Fig. 5B–C). In contrast to tightly packed and oriented sarcomeres in control CMs, YAP5SA CM sarcomeres localize to cell periphery, leaving parts of the CM cytoplasm partially devoid of sarcomeres (Fig. 5C). Importantly, sarcomere disassembly initiated at four days after YAP5SA induction, one day before completion of the YAP5SA CM cell cycle (Fig. 5D). We concurrently assessed CDK1 expression and sarcomere disassembly in YAP5SA CMs to investigate sarcomere disassembly in G2/M (Fig. 5E). We observed CDK1 expression in YAP5SA CMs five days after YAP5SA induction (one day after initiation of sarcomere disassembly in YAP5SA CMs, Fig. 5D, E) indicating that sarcomere disassembly precedes G2/M in YAP5SA CMs. Moreover, CDK1-positive CMs had a higher sarcomere disassembly score (Fig. 5F–H), consistent with our scRNA-seq pseudotime analysis and suggesting that 1) sarcomere disassembly is permissive for YAP5SA CM G2/M cell cycle progression and 2) following sarcomere disassembly initiation, cell cycle progression and CM division occur within 48 hours.

Fig 5. Verification of single cell analysis by immunohistochemistry.

Fig 5.

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A: Timing of tamoxifen (Tm) injection and sample harvesting for panels B-E. B-C: Sarcomere disassembly was determined by cTNT staining. Representative images of the control, and YAP5SA day 2, day 4, and day 5 after tamoxifen are shown. Asterisks show CMs with sarcomere disassembled. C: Representative images of sarcomere structure imaged by SIM microscopy. D: Quantification of sarcomere disassembly. Disassembly score is a percentage of the area devoid of cTNT staining. Score of each CMs were measured. Groups were compared by using nested one-way ANOVA with the Tukey post-hoc test for multiple comparisons (n=5 each time point, 20 CMs each time point). **p<0.01, ***p<0.001. E: Quantification of CDK1 positive CMs from day 2 to 5. Groups were compared by using one-way ANOVA with the Tukey post-hoc test for pairwise comparisons (n=5 each time point). ***p<0.001. F: Timing of tamoxifen (Tm) injection and sample harvesting for panels G-J. G: Representative image of cTNT staining in CDK1 positive CM at Day 6. H: Quantification of disassembly score in Flag-positive CMs and CDK1-positive CMs. ****p<0.0001. Groups were compared by using nested t-test (n=5 each group). I, J: Sarcomere disassembly and SPTAN1 expression. I: Representative images of control and YAP5SA hearts stained with SPTAN1, cTNT, and DAPI. J: Quantification of SPTAN1 intensity and sarcomere disassembly score. Each group was analyzed with simple linear regression (n=5 each). Only YAP5SA group is significant and R2 is shown. ****P<0.0001.

K-M: Effect of SPTAN1 haploinsufficiency in YAP5SA-induced CMs. K: Timing of tamoxifen (Tm) injection and sample harvesting for panels L, M. L: Representative images of YAP5SA and YAP5SA; Sptan1 het for cTNT, WGA, and DAPI. M: Sarcomere disassembly and CDK1 expression of Sptan1 het (control), YAP5SA, and YAP5SA; Sptan1 het CMs were quantified. Groups were compared by using one-way ANOVA with the Tukey post-hoc test for pairwise comparisons (n=3 each genotype). **p<0.01, ****p<0.0001.

Spectrin alpha, non-erythrocytic 1 (SPTAN1), a direct YAP target gene, was differentially expressed and upregulated in YAP5SA CM cluster 2 (Table S2) 16. IF revealed increased SPTAN1 levels in Yap5SA CMs, localizing to inner plasmalemma (Fig. 5I). Quantification of SPTAN1 intensity revealed increased sarcomere disassembly in YAP5SA CMs (Fig. 5J). To functionally investigate SPTAN1 in sarcomere disassembly and G2/M, we used a Sptan1 conditional null allele and MCM to delete one copy of Sptan1 in YAP5SA CMs (Fig. 5K–M). Both sarcomere disassembly and G2/M entry (Fig. 5M) were less frequent in YAP5SA CMs with genetically reduced Sptan1 dosage, revealing an essential role of SPTAN1 in YAP5SA CM sarcomere disassembly.

YAP5SA cardiomyocytes assume a spherical shape for mitotic rounding

To progress through the cell cycle, cells must take on the spherical shape of mitotic rounding for correct spindle formation and mitosis fidelity 7. To investigate mitotic rounding in YAP5SA CMs, we developed a mitotic rounding gene expression module using available, unbiased data identifying mitotic rounding genes and scored YAP5SA CMs using our scRNA-seq data 25. Among mitotic rounding genes, 45 were differentially expressed among CM-1 to CM-5 clusters (Fig. 6A, E). The strongest mitotic rounding score was observed in CM-2, enriched in YAP5SA hearts, and associated with sarcomere disassembly genes (Fig. 6B, C). Feature plots of mitotic rounding genes, myh9, actn4, and F2r, revealed enrichment in CM-2 (Fig. 6D). To determine whether mitotic rounding genes are directly regulated by YAP, we analyzed YAP chromatin accessibility for mitotic rounding genes using our previously generated Assay for Transpose Accessible Chromatin (ATAC) data 16. Multiple mitotic rounding genes including Clic5, Pik3cb, and Camk2a had newly accessible chromatin with TEAD elements in YAP5SA CMs (Fig. S8A–K).

Fig 6. Mitotic Rounding in YAP5SA CMs.

Fig 6.

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A-E: Expression of mitotic rounding genes in control and YAP5SA CMs. A-Expression of 45 mitotic rounding genes were examined in CM re-cluster 1–5. B: UMAP of re-clustered CMs. Same image as in Fig. 4A. C: Mitotic rounding score. D: Feature plots of representative genes, Myh9, Actn4, and F2r. E: Quantification of rounding score. Groups were compared by one-way ANOVA. *p<0.05, ***p<0.001, ****p<0.0001. F, G: Solidity of the control and YAP5SA CMs. Random CMs from control and FLAG-positive YAP5SA CMs were analyzed for solidity. Groups were compared by using nested t-test (n=5 each group, 20 CMs each heart). ****P<0.0001. H: Solidity of the CMs from YAP5SA hearts. CMs of Flag(−), Sptan1(+), and CDK1 (+), were analyzed for solidity. Groups were compared by using one-way Anova (n=5). *P<0.05, **P<0.01, ***P<0.001. ****P<0.0001. I, J: Solidity of YAP5SA; Sptan1 het CMs. Same samples as Fig. 5K–M were used. I: Representative images of YAP5SA and YAP5SA; sptan1 het CMs. J: Solidity of control, YAP5SA, Yap5SA; Sptan1 het CMs. Groups were compared by using one-way Anova (n=3 each genotype). ***P<0.001, ****P<0.0001.

We also measured CM solidity, a measure of cell membrane regularity, which is increased as cells assume a spherical shape (Fig. 6F–J). Cells that have a more regular shape are scored as 1.0 26. Cell solidity was higher in YAP5SA CMs than in control CMs. In addition, YAP5SA CMs positive for SPTAN1 and CDK1 had higher solidity scores compared to FLAG (−) CMs, suggesting that YAP5SA CMs attain the spherical shape required for progression through cell division (Fig. 6K) 27. Moreover, YAP5SA CMs with disassembled sarcomeres displayed expanded expression of Vinculin, which modulates focal adhesion turnover, connects sarcomeres to the plasma membrane, and increases adhesion of rounding cells to adjacent cells during mitotic rounding (Fig. S9A–D) 28, 29.

Spectrins, cytoskeletal network components, support the plasmalemmal structure in mechanically stressed cells, such as red blood cells, suggesting a similar role in rounding YAP5SA CMs 30. To obtain functional support for this notion, we measured solidity in YAP5SA; Sptan1-heterozygous CMs and compared to YAP5SA and control CMs (Fig. 6L-N). In YAP5SA; Sptan1-heterozygous CMs, solidity was reduced to control levels. Under mechanical stress conditions (including mitotic rounding), maintaining the plasmalemmal structure requires surface tension with limited surface irregularities, as measured by solidity. These data support the conclusion that SPTAN1 maintains plasmalemmal stability during sarcomere disassembly and mitotic rounding in YAP5SA CMs, consistent with known functions of the spectrin cytoskeletal network 31.

Stabilized sarcomeres inhibit cell cycle progression

To disrupt sarcomere stability, we leveraged our previous finding that Hippo signaling phosphorylation of YAP on S127 (P-YAP S127) promotes YAP interaction with the Dystrophin Glycoprotein Complex (DGC) and stabilization of sarcomere structure 32. We injected an AAV9 virus encoding a YAP phospho-mimetic (YAPS127D) into YAP5SA CMs (Fig. 7A). YAPS127D models the inhibitory YAP S127 phosphorylation induced by Hippo signaling. YAP5SA: AAV9-YAPS127D CMs were compared to YAP5SA CMs injected with control AAV9-GFP virus (YAP5SA: AAV9-GFP). We also injected AAV9-YAPS127D into wild-type control CMs (“control”, Fig. 7C–E). Sarcomere structure analysis revealed that compared to AAV9-GFP, AAV9-YAPS127D inhibits sarcomere disassembly in YAP5SA CMs (Fig. 7B–C) and induces lower solidity (Fig. 7D), consistent with the conclusion that P-YAP inhibits sarcomere disassembly.

Fig 7. Phospho-mimetic YAP blocks cell cycle progression in YAP5SA-expressing cardiomyocytes.

Fig 7.

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A: Timing of AAV9-YAP127D, tamoxifen (Tm), and EdU injection, and sample harvesting for the panels B-F. B, C: Sarcomere disassembly was determined by cTNT staining in Flag-positive CMs. B: Representative images of the YAP5SA + AAV9-GFP, and YAP5SA + AAV9-YAPS127D are shown. Asterisks show flag-positive CMs. C: Quantification of sarcomere disassembly in the control (MCM + AAV9-S127S) and YAP5SA plus GFP or YAPS127D. Groups were compared by nested one-way ANOVA with the Tukey post-hoc test for pairwise comparisons (n=5 each). *p<0.05, ***p<0.001, ****p<0.0001. D: Solidity of control, YAP5SA, and YAP5SA; YAPs127D CMs. Groups were compared by nested one-way ANOVA with the Tukey post-hoc test for pairwise comparisons (n=5 each). ****p<0.0001. E: Cell cycle stages were determined in control, YAP5SA, and YAP5SA; YAPs127D CMs. CDK2, CCNA2, CDK1, PHH3, and CCND1 expressing CMs were quantified. Groups were compared by using one-way ANOVA with the Tukey post-hoc test for pairwise comparisons (n=5 each). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. F: EdU pulse labelled CMs at 72 hours after EdU injection. Double positives for EdU and CDK2 or CCND1 were examined. Groups were compared using the Mann-Whitney U test (n=5 each). **p<0.01. G-I: Confetti analysis with YAP5SA and YAP5SA + YAPS127D hearts. G: Timing of AAV9-YAP127D and Tm injection, and sample harvesting for the panels H, I. H: Representative images of YAP5SA; confetti and YAP5SA; confetti with AAV-YAP S127D. I: Quantification of clonal formation in the YAP5SA and YAP5SA+YAPS127D hearts. Groups were compared using Chi-square test for distribution (n=4). ****p<0.0001.

J-O: Inhibition of sarcomere disassembly by YAP-S127D during endogenous regeneration. J: Timing of injections, surgery, echo analysis and sample harvest for panels K-O. K: Representative images of trichrome stained hearts for sham and MI hearts at 28 dpmi. L: Quantification of scar size at 28 dpmi. Groups were compared by using one-way ANOVA with the Tukey post-hoc test for pairwise comparisons (n=12 for both sham combined, n=6 for saline MI, n=6 for S127D MI). **p<0.01, ****p<0.0001. M: Ejection fraction and fractional shortening at 8 and 28 dpmi. Groups were compared by using two-way ANOVA (n=6 each treatment). ***p<0.001, ****p<0.0001. N-O: Sarcomere disassembly and PHH3 expression at 4 dpmi. N: Representative images of MI heart with saline and S127D injection. O: (Left) Quantification of sarcomere disassembly. Groups were compared by nested t-test (n=6 each). (Right)Quantification of PHH3 expressing CMs. Groups were compared using the Mann-Whitney U test (n=6 each). **p<0.01.

In addition to increased sarcomere stability, YAPS127D slowed progression through S-phase. YAP5SA: AAV9-YAPS127D CMs more frequently expressed the early S phase marker CDK2 compared with YAP5SA:AAV9-GFP CMs (Fig. 7E). In contrast, expression of CCNA2, CDK1, PHH3 and CCND1 (Fig. 7E) were reduced in YAP5SA: AAV9-YAPS127D CMs compared to YAP5SA:AAV9-GFP CMs, indicating that YAPS127D stabilizes sarcomeres and prolongs S phase in YAP5SA CMs.

We next labeled S phase CMs with EdU 72 hours before harvest (Fig. 7A). Compared to EdU+ YAP5SA:AAV9-GFP CMs, which had progressed beyond S phase, most EdU+ YAP5SA: AAV9-YAPS127D CMs remained in S phase at 72 hours after EdU injection (Fig. 7F). Similarly, compared to EdU+ YAP5SA:AAV9-GFP CMs, fewer EdU+YAP5SA:AAV9-YAPS127D CMs entered the second cell cycle as determined by CCND1 expression (Fig. 7F). Confetti analysis revealed that YAP-S127D inhibits CM cell division (Fig. 7G–I). Together, these findings support our model that sarcomere disassembly is required for YAP5SA CMs to progress through G2/M and that P-YAP, which stabilizes sarcomeres, slows S-phase progression and delays G2/M entry.

To investigate YAP-S127D in cardiac regeneration, we expressed AAV9-YAPS127D in neonatal mice and performed MI (Fig. 7J–O). YAP-S127D expressing hearts had larger scars (Fig. 6K, L) and reduced EF and FS after 28 days compared to controls (Fig. 7M). Both sarcomere disassembly and M phase entry were reduced in YAP-S127D CMs (Fig. 6N, O), supporting the conclusion that stabilizing sarcomere inhibits endogenous CM division.

P21 inhibition promotes G2/M transition in YAP5SA cardiomyocytes

The cell cycle checkpoint protein P21 is activated in response to cellular replicative and oxidative stress 33,34. Our scRNA-seq data indicate that genes encoding proteins that degrade p21 and cdkn1a, which encodes P21, are highly expressed in YAP5SA CM-4. Moreover, the CM-4 cell state occurs after sarcomere disassembly, suggesting that P21 activity inhibits the progression of YAP5SA CMs into G2 after sarcomere disassembly (Fig. 4C–E). Using IF, we detected nuclear P21 expression in YAP5SA CMs at days three through five after YAP5SA induction, suggesting that P21 inhibits YAP5SA CM progression through G2/M (Fig. 8A–C).

Fig 8. Function of P21 in YAP5SA-expressing CMs.

Fig 8.

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A-C: Timing of P21 nuclear accumulation. A: Timing of tamoxifen (Tm) injection and sample harvesting for panels B, C. B, B: Representative images of P21 immunostaining in YAP5SA hearts. Arrows are P21 positive CM nuclei. C: Quantification of P21-expressing CM nuclei of the control and YAP5SA from day2 to 5. Groups were compared by using one-way ANOVA with the Tukey post-hoc test for pairwise comparisons (n=5 each time point).: **p<0.01, ****p<0.0001. E-N: Inhibition of P21 in YAP5SA-expressing animals. D: P21 inhibitor UC2288 inhibits gene expression of cdkn1a. E: Injection protocol of tamoxifen (Tm) and P21 inhibitor, and sample harvesting timing for panels F-H. F: Survival of vehicle (n=10) and P21 inhibitor (n=12)-treated YAP5SA animals at day 5. Groups were compared with qui square test. *p<0.05. G: Representative images of P21 and CDK1 immunostaining in YAP5SA+vehicle and YAP5SA+P21 inhibitor are shown. Arrows are P21 positive or CDK1 positive CM nuclei. H: Quantification of P21, CDK1, and PHH3 positive CM nuclei. Groups were compared by using one-way ANOVA with the Tukey post-hoc test for pairwise comparisons (n=5 each treatment). **p<0.01, ****p<0.0001. I-K: Confetti analysis of YAP5SA vehicle and P21 inhibitor (P21 inh) treated hearts. I: Timing of Tamoxifen and viral injection, and sample collection for panels J, K. J: Representative images of vehicle and P21 inh treated YAP5SA hearts. K: Quantification of clonal formation in the vehicle and P21 inh treated YAP5SA hearts. Groups were compared using Chi-square test for distribution (n=5). ****p<0.0001.

L: Model for YAP5SA-induced adult CM cell cycle.

To investigate P21 in YAP5SA CMs, we modulated P21 activity using pharmacologic agents. To separate the inhibitory P21 function from sarcomere disassembly, we administered the P21 inhibitor (UC2288) at day four post-YAP5SA induction, after CMs entered S phase and sarcomere disassembly initiation (Fig. 8D, E). We previously reported that YAP5SA expression in adult CMs induces lethality in mice seven days post-YAP5SA induction 16. P21 inhibition induced earlier lethality compared to vehicle-treated YAP5SA mice (Fig. 8F). IF revealed that a P21 inhibitor reduces P21 expression while increasing CDK1 and PHH3 expression (Fig. 8G–H) and confetti analysis revealed that P21 inhibition increases YAP5SA CM division (Fig. 8I–K).

In the converse experiment, we focused on SKP2, an E3 ubiquitin ligase that degrades P21 35. We administered the SKP2 inhibitor SZL P1–41 to stabilize P21 in YAP5SA CMs at day four post-YAP5SA induction (after sarcomere disassembly initiation, Fig. S10A, B). SKP2 inhibitor treatment resulted in increased survival of YAP5SA mice, marked by frequent detection of P21 in CMs compared to vehicle-treated YAP5SA controls (Fig. S10C, D). CDK1 and PHH3 positive YAP5SA CMs were decreased in the SKP2 inhibitor treatment group, indicating reduced progression of YAP5SA CMs into G2/M (Fig. S10E, F). Since we treated YAP5SA mice after CM sarcomere disassembly and S phase entry, the number of YAP5SA CMs expressing CDK2 was unchanged by SKP2 inhibitor treatment, as expected (Fig. S10G).

DISCUSSION

We discovered that YAP overcomes cell cycle barriers in adult CMs to induce reemergence from quiescence. YAP activates a transcriptional program for cell cycle re-entry at the G1/S transition, also called the restriction point. Induction of S phase entry by YAP5SA was efficient with more than 10% of YAP5SA CMs expressing Cdk2 72 hours after YAP5SA induction. Moreover, YAP5SA CMs exhibited other hallmarks of cycling cells, including oscillatory Cdk expression and mitotic rounding. Sarcomere disassembly, a process specific to highly specialized CMs, is required to progress from S into the G2 phase. Transit through G2/M and cytokinesis in YAP5SA CMs is enhanced by inhibiting P21 checkpoint activity. Our data collectively suggest that Hippo pathway repression of YAP sustains adult CM cell cycle quiescence (Fig. 8L).

YAP5SA activates sarcomere disassembly and mitotic rounding

To progress through the cell cycle, all cells must undergo dramatic cytoskeletal reorganization to acquire the spherical shape of mitotic rounding. Mitotic cells push against surrounding cells, concurrently reducing adhesion to ECM and increasing adhesion to adjacent cells 7. Rounding. which is characterized by increased actomyosin cortical tension and intracellular hydrostatic pressure, typically places mechanical strain on surrounding cells in the microenvironment. The highly confined myocardial microenvironment, which includes side-by-side sarcomere-filled CMs and a stiff ECM, presents a strong barrier to spherical shape acquisition. In quiescent CMs, vinculins are found in adherens junctions of the Intercalated Disc, which connect CMs to adjacent CMs 36. During mitotic rounding, connections to surrounding cells are strengthened, while cell-ECM connections are reduced to allow mitotic rounding 29. Increased adhesion to adjacent cells is strengthened via integrins and vinculins 29. Consistently, we found that rounding YAP5SA CMs have expanded expression of Vinculin, providing evidence that YAP5SA CMs undergo mitotic rounding.

Sarcomere disassembly has been observed in two contexts of endogenous CM regeneration: zebrafish and neonatal mouse CMs 37–39. In both contexts, the myocardial microenvironment is less constrained than the adult mouse heart either due to sarcomere immaturity in neonatal mice or the low-pressure cardiovascular system in zebrafish. Our data reveal that sarcomere disassembly, required for transition through S phase, is a regulated process in YAP5SA CMs and is the first step towards mitotic rounding and CM division.

Several YAP target genes are activated in YAP5SA CMs and encode proteins implicated in sarcomere disassembly. Among these genes are Sptan1, which encodes the Spectrin SPTAN1, and Capn2 encoding Calpain 2 16. Notably, Spectrins have been identified as sarcomere disassembly factors, and Calpains are important in sarcomere turnover 40,41. Based on functional and genetic loss of function studies, we also posit that SPTAN1 stabilizes the CM plasmalemma during mitotic rounding in YAP5SA CMs.

Our scRNA-seq data revealed that genes expressed in CM cluster 2, enriched in YAP5SA hearts before G2 entry, are associated with actin cytoskeleton remodeling. Multiple CM cluster 2 genes are direct YAP targets, including ezr, ect2, and rock2. The ERM family protein Ezrin, encoded by ezr, links the actomyosin skeleton to the plasma membrane to drive mitotic rounding 42,43. ect2 encodes Ect2, also a direct YAP target gene in CMs, which has been implicated in CM proliferation in human congenital heart disease and zebrafish regeneration16,44. Ect2 is also important for mitotic rounding in the Drosophila imaginal disk and mammalian cultured cells 25,45. Likewise, the YAP target gene rock2 was directly implicated in mitotic rounding 46. Our data indicate a direct role for YAP target genes in CM sarcomere disassembly and mitotic rounding.

YAP5SA CMs reenter the cell cycle at G1/S transition

Adult YAP5SA CMs re-enter the cell cycle at restriction (R) point when cells are irreversibly committed to DNA synthesis and transition from G1 into S phase. Our data reveal that adult CMs are arrested at the G1/S boundary and progress through the S phase by YAP5SA is consistent with other work, which reported that postnatal CMs arrest at the G1/S boundary and are maintained in a quiescent state through adulthood 4.

In neonatal hearts, YAP is required for CM proliferation and heart regeneration 47, and YAP activity declines concurrently with CM cell cycle exit at G1/S. Moreover, YAP directly regulates genes important for progression through S phase, including CCNE and E2Fs 16. Cell cycle quiescence in adult CMs is associated with downregulated expression of cell cycle regulators. In postnatal CMs, CDKs and Cyclins are downregulated by postnatal day 23, consistent with acquiring a quiescent cell state. Moreover, binucleated CMs, the majority of postnatal mouse CMs, do not express S phase genes, providing further evidence that adult CMs are quiescent and arrested at G1/S 48.

As cells progress through G1/S transition, the CDK2 and Cyclin E complex phosphorylates and inactivates Rb and derepresses transcription of E2F transcription factors which induce expression of S phase gene expression. Since S phase regulators and genes encoding E2F transcription factors are direct YAP targets 16, our findings are consistent with the model that YAP induces S-phase entry by activating transcription of S phase genes.

YAP induced P21 degradation mediates transition to G2/M phase

In YAP5SA CMs, the P21 degradation pathway is activated after sarcomere disassembly and before G2/M. One component of the P21 degradation pathway, SKP2, which is differentially expressed in YAP5SA CMs, is a direct YAP target gene 49. P21 inhibits multiple CDKs to regulate transitions between the cell cycle stages, notably G1/S and G2/M. At the G2/M transition, increased P21 results in G2 arrest in response to DNA damage, which is induced postnatally in CMs by oxidative stress 10.

Previous work implicated P21 in CM cell cycle inhibition in neonatal CMs. Meis1 gain of function in neonatal CMs promoted CM cell cycle arrest through direct activation of CDK inhibitors, including P21 50. Furthermore, telomere dysfunction-induced cell cycle arrest in postnatal CMs is mediated through P21 activation 33. These findings are consistent with the inhibitory role of P21 in YAP5SA-activated CMs in the adult heart and provide insight into potential therapies to improve CM renewal in the adult heart.

Supplementary Material

Supplemental Publication Material
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Clinical Perspective.

What is new?

YAP activation induces adult cardiomyocyte division by promoting cytoskeleton disassembly and mitotic rounding. Adult cardiomyocytes enter the cell cycle at the G1-S transition and progress through DNA damage checkpoints to enter M phase.

What are the clinical implications?

Sarcomere disassembly to enable mitotic rounding and inhibiting P21 activity promotes adult cardiomyocyte division and heart regeneration.

Acknowledgments

We thank the Developmental Disabilities Research Center Neuroconnectivity Core at Baylor College of Medicine for AAV9 viral vectors and the Texas Heart Institute Flow Cytometry Core Facility for Flow analysis.

Sources of Funding

This study was supported by grants from the National Institutes of Health (HL 127717, HL 130804, and HL 118761 to J.F.M.), the Vivian L. Smith Foundation (J.F.M), and HL 5T32HL007208–42 (M.C.H). American Heart Association the AHA Career Development Award (849706 to S.L.)

Nonstandard Abbreviations and Acronyms

AAV

adeno-associated virus

ATAC

assay for transpose accessible chromatin

CCNA2

cyclin A2

CCND1

cyclin D1

CCNE1

cyclin E1

CDK1

cyclin-dependent kinase 1

CDK2

cyclin-dependent kinase 2

cTNT

cardiac troponin T

EdU

5-ethyl-2’-deoxyuridine

GO

gene ontology

IF

immunofluorescence

MI

myocardial infarction

P

postnatal

PCNA

proliferating nuclear antigen

PHH3

phosphorylated histone H3

scRNA-seq

single cell RNA-sequencing

SPTAN1

spectrin alpha, non-erythrocytic

Footnotes

Conflict of Interest Disclosures

James Martin is a co-founder of and owns shares in Yap Therapeutics, a company with the goal of treating heart failure using gene therapy.

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