Progesterone, via yes‐associated protein, promotes cardiomyocyte proliferation and cardiac repair

Abstract Objectives The mechanisms responsible for the postnatal loss of mammalian cardiac regenerative capacity are not fully elucidated. The aim of the present study is to investigate the role of progesterone in cardiac regeneration and explore underlying mechanism. Materials and Methods Effect of progesterone on cardiomyocyte proliferation was analysed by immunofluorescent staining. RNA sequencing was performed to screen key target genes of progesterone, and yes‐associated protein (YAP) was knocked down to demonstrate its role in pro‐proliferative effect of progesterone. Effect of progesterone on activity of YAP promoter was measured by luciferase assay and interaction between progesterone receptor and YAP promoter by electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP). Adult mice were subjected to myocardial infarction, and then, effects of progesterone on adult cardiac regeneration were analysed. Results Progesterone supplementation enhanced cardiomyocyte proliferation in a progesterone receptor‐dependent manner. Progesterone up‐regulated YAP expression and knockdown of YAP by small interfering RNA reduced progesterone‐mediated cardiomyocyte proliferative effect. Progesterone receptor interacted with the YAP promoter, determined by ChIP and EMSA; progesterone increased luciferase activity of YAP promoter and up‐regulated YAP target genes. Progesterone administration also promoted adult cardiomyocyte proliferation and improved cardiac function in myocardial infarction. Conclusion Our data uncover a role of circulating progesterone withdrawal as a novel mechanism for the postnatal loss of mammalian cardiac regenerative potential. Progesterone promotes both neonatal and adult cardiomyocyte proliferation by up‐regulating YAP expression.


| INTRODUC TI ON
Heart failure, caused by ischemic heart disease, as in myocardial infarction (MI), is one of the major causes of mortality worldwide. 1 Despite remarkable advances in the development of pharmacological and technological treatments for MI, irreversible loss of cardiomyocytes (CMs) makes the incidence of heart failure remain high. 2 Thus, intensive research has focused on the development of regenerative therapies to replace lost myocardium. 3 One paradigm-breaking concept has recently emerged that the adult mammalian CM is still endowed with proliferative potential, although the capacity is insufficient to replenish lost CMs. 4 In particular, a 14 C dating study showed an annual CM renewal rate of 0.45%-1% in adult humans. 5 Therefore, regenerating CMs in situ, via stimulation of endogenous CM proliferation, is an appealing approach to treat myocardial diseases associated with cell death, for example, MI. Large amount of evidences have demonstrated that foetal and neonatal CMs possess robust proliferative capacity and injured CMs during this period can be fully replaced by newly generated CMs. 6,7 However, this regenerative capacity quickly diminishes by postnatal day 7 (P7) in mice.
Thus, discovery of regulators that account for the withdrawal of CM's regenerative property after birth could help uncover new approaches to induce cell cycle re-entry in adult CMs.
Circulatory hormones, especially progesterone, change drastically after birth. 8 During foetal development, when CMs are rapidly proliferating, the foetus is supplied with progesterone produced by the placenta and/or the ovarian corpus luteum. 9 Increased circulating level of progesterone plays a crucial role in the maintenance of pregnancy through inhibition of oxytocin-induced myometrial activity. 10 Notably, progesterone is also important in the regulation of foetal development and growth. 11 However, the relationship between the decrease in foetal progesterone level and the postnatal withdrawal of the CM's regenerative property is unknown. In the current study, we first show that serum progesterone level drastically declines from foetal to neonatal mice, which is in parallel with the withdrawal of cardiac regenerative capacity. Furthermore, progesterone supplementation, by up-regulating Yes-associated protein (YAP) expression,  In the neonatal mice studies, progesterone was intraperitoneally injected daily from postnatal day 1 (P1) to postnatal day 6 (P6); the hearts were harvested at P7 to analyse CM proliferation. Eight-to 10-week-old male C57Bl/6J mice were anaesthetized by inhalation of isoflurane (2%) and subjected to MI by ligation of the left anterior descending (LAD) coronary artery, as previously published. 12 After the surgery, the mice were allowed to recover with free access to food and water for 48 hours and progesterone was intraperitoneally injected daily from day 2 until the heart was collected at day 7 and day 35 for analysis. The dose of progesterone for both neonatal and adult mice was 8 mg/kg/d, which has been used to investigate its neuroprotective effect in young adult and aged mice. 13 Animals were euthanized using overdose of isoflurane (5%) and cervical dislocation before heart extraction.

| Serum progesterone determination
Animals were euthanized using overdose of isoflurane (5%) before blood collection. Blood samples from embryonic day 16 (E16), P1, P7 and P14 mice were collected and placed at room temperature for 2 hours and then centrifuged at 300 g for 30 minutes to obtain the serum. The serum progesterone concentration was quantified using commercial progesterone ELISA kits (ADI-900-011, Enzo Life Sciences), following the manufacturer's instructions. Briefly, 100 μL progesterone stands (5, 2.5, 1.25, 0.62, 0.31, 0.15 and 0.075 ng/mL) and diluted serum samples (1:10) were pipetted into 96-well plates (anti-mouse IgG-coated) in duplicate. Then, 50 μL progesterone alkaline phosphatase conjugate and 50 μL solution of a monoclonal antibody to progesterone were added, gently mixed and incubated at room temperature on a plate shaker for 2 hours.
After that, contents of the wells were removed and washed twice and 200 μL of the pNpp substrate solution was added to every well and incubated for 45 minutes at room temperature. Lastly, 50 μL of stop solution was added to each well, optical density was read immediately at 405 nm and progesterone concentration was calculated.

| Echocardiographic evaluation
Similar to our previous study, 12 28 days after MI, mice were anaesthetized with isoflurane and cardiac function was determined by echocardiography, using GE Vivid 9-Dimension Ultrasound. Two-dimensionally guided M-mode images of the short axis between the two papillary muscles were recorded to measure left ventricular (LV) wall structure and evaluate cardiac function by calculating LV fraction shortening (FS %) and LV ejection fraction (EF %). All measurements were carried out by a technician who was unaware of the experimental groups. The average of at least three measurements was used in all calculations.

| Isolation and culture of neonatal rat cardiomyocytes
Neonatal CMs were isolated by enzymatic disassociation of hearts from 7-day-old SD rats according to our published procedure. 14 Briefly, rats were euthanized using overdose of isoflurane (5%) and hearts were extracted. Then, hearts were washed with ice-cold saline and minced into pieces smaller than 1 mm 3 before enzymatic digestion using 1.25 mg/ mL trypsin for 3 minutes. The heart pieces were collected further digested using 0.8 mg/mL collagenase II for 30 minutes at 37°C. The digestion supernatant was collected and added with an equal volume of Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FBS before being filtered through a cellular strainer. Then, cell suspension was incubated at 37°C for 90 minutes for differential attachment.
After that, cells in the suspension were plated and cultured in DMEM with 10% foetal bovine serum at 37°C in atmosphere with 5% CO 2 for 24 hours. Then, progesterone at various concentrations (10 −9 -10 −4 M) with or without the progesterone blocker RU486 (10 −6 M) was added to the medium to incubate for 24 hours, and then, the cells were fixed with 4% polyformaldehyde for 20 minutes followed by immunostain-

| Isolation of rod-shaped cardiomyocytes
Rod-shaped CMs were isolated from P7 and adult mice according to the protocol described previously 15

| Immunostaining
The hearts were harvested and fixed with 4% polyformaldehyde, followed by dehydration and embedding into paraffin to cut 4-μm-thick sections. Fixed cells or tissue sections were permeabilized with Triton (0.1%) for 10 minutes and blocked in PBS with 5% BSA for 1 hours. Then, the samples were incubated with primary antibodies overnight at 4°C. After three washes with PBS, the samples were incubated with fluorescent secondary antibodies for 1 hour at room temperature, followed by 10 minutes of DAPI staining for nuclei visualization. After staining, the slides were mounted with Immu-Mount and viewed under an Olympus confocal microscope (FluoView 1000). Cardiac troponin T (cTnT) and pan-cadherin were immunostained to show intact CMs, and Ki67 + , PH3 + and Aurora B + CMs were counted to evaluate CM proliferation, as specified in the

| WGA and Masson's trichrome staining
Tissue sections of P7 hearts were subjected to anti-wheat germ agglutinin (WGA) immunofluorescence staining following the manufacturer's protocol, and cross-sectional area of the CMs was analysed as previously described. 14 Briefly, after deparaffinization and rehydration, WGA was added to tissue sections and incubated for 30 minutes at room temperature. Then, images were taken with Olympus confocal microscope (FluoView 1000). Similar to previous studies, 16 80-100 cells were randomly selected per section and four sections were measured per heart. Tissue sections from apex to base of post-MI hearts were subjected to Masson's trichrome staining following the manufacturer's protocol to analyse the size of the fibrotic scars.
Briefly, tissue sections were subjected to deparaffinization and rehydration, and incubation with Bouin's solution at room temperature overnight. Then, the slides were washed with distilled water and sequentially stained with working haematoxylin for 15 minutes, Biebrich scarlet for 5 minutes, phosphotungstic/phosphomolybdic acid for 10 minutes, aniline for blue for 5 minutes and 1% acetic acid solution for 3 minutes. Lastly, images were acquired to quantify scar size using ImageJ software (Wayne Rasband, NIH), which was calculated as relative to total left ventricle wall size.

| Real-time RT-PCR
Total RNAs of cells or tissues were extracted using RNAiso Plus (9109, TAKARA), following standard protocols. The extracted RNA was reverse-transcribed (RT-PCR) with the TOYOBO RT-PCR Kit.
Quantitative real-time PCRs of GAPDH, cell cycle genes CCNA1, CDK1, CDK4, CCNB1, CCND1, YAP and YAP target genes ANKRD1, CYR61 and CTGF, and regulators of CM proliferation NRG1, Postn, GATA4, TBX20 and FSTL1 were carried out. Bio-Rad software was utilized to analyse melting curves, and a comparative cycle threshold method was used to calculate relative mRNA expression. All experiments were repeated at least three times, and the expression of mRNA was normalized by GAPDH. The primers used are listed in Table 1.

| RNA sequencing
We isolated and cultured P7 CMs using 7-day-old SD rats and treated cells with/without progesterone (10 −7 M) for 24 hours to prepare sample (three biological replicates for each group) for RNA sequencing (RNA-Seq). Libraries were constructed with the NEBNext Ultra™ RNA Library Prep Kit for the Illumina system according to the manufacturer's instructions, and sequencing was performed using the Illumina HiSeq platform by the Novogene Bioinformatics Institute (Beijing, China). GO and KEGG pathway analysis were performed using the OmicShare tools, a free online platform for data analysis (http://www.omics hare.com/tools). The hierarchical clustering heatmap was generated with the ggplot library.

| Luciferase assay
Primary neonatal CMs were cultured in 24-well plates and transfected with luciferase reporter plasmids, including pGL3-basic (firefly luciferase reporter), pRL-TK (Renilla luciferase reporter) and YAP-p  input. 17 The primers used are listed in Table 2.

| Electrophoretic mobility shift assay
Electrophoretic mobility shift assay (EMSA) was performed as previously published. 18 Nuclear extracts were prepared using NE-PER™ Nuclear and Cytoplasmic Extraction Kit (78835, Thermo Fish Scientific), following standard procedures. Briefly, cells were harvested using trypsin-EDTA and centrifuged at 500 g for 5 minutes.
Ice-cold CER I was added to cell pellet and vortexed vigorously to fully suspend prior to addition of CER II. Then, the tube was vor-

| Statistical analysis
All data are expressed as means ± SEM, and statistical analysis was performed using SPSS 18.0 statistical package (International Business Machines Corporation, New York, USA). Significant differences between and among groups were determined by one-way ANOVA and Student-Newman-Keuls post hoc test for groups >2 or Student's t test for groups = 2. P < .05 was considered significant.

| Progesterone promotes postnatal CM proliferation in vivo
To investigate the relationship between serum progesterone and CM proliferation, serums and hearts were harvested in E16, P1, P7 and P14 mice. As shown in Figure 1A

| Progesterone promotes CM proliferation in vitro via a progesterone receptordependent manner
To uncover the mechanism underlying the progesterone-induced CM proliferation, we studied the effect of progesterone in in vitro experiments. expression. 20 Thus, we tested whether progesterone could promote CM proliferation via a receptor-dependent manner. As is shown in Figure 3A-C and S5, progesterone significantly increased the percentage of Ki67 + , PH3 + and Aurora B + CMs, and also total protein levels of these proliferation markers; this effect was blocked by co-treatment with the progesterone receptor inhibitor RU486. Furthermore, RU486 co-treatment also prevented the progesterone-induced up-regulation of the expression of cell cycle genes ( Figure 3D). Taken together, these results support the notion that progesterone promotes CM proliferation in a progesterone receptor-dependent manner. In addition, we also performed RNA-Seq on cultured P7 CMs with or without progesterone treatment to further confirm proliferative effect of progesterone and uncover underlying mechanism, and the results further confirmed up-regulation of cell cycles genes, including CDK1, CDK4 and CCNB1 ( Figure 4A). KEGG pathway analysis of up-regulated genes indicated increased cell cycle activities in progesterone group ( Figure 4B).

| Progesterone promotes CM proliferation through up-regulation of YAP expression
The RNA-Seq showed that there were 8596 transcripts differentially expressed between control and progesterone-treated CMs, with 3448 F I G U R E 1 Serum progesterone concentration declines after birth and is associated with decrease in CM proliferation. A,B, Evaluation of CM proliferation by Ki67 and PH3 immunostaining at E16, P1, P7 and P14 hearts. Representative images with z-stacking are shown in A1 and B1, and quantification of percentages of Ki67 + and PH3 + CMs is shown in A2 and B2, respectively (n = 5). Scale bars are 20 µm. cTnT indicates cardiac troponin T. C, Serum concentrations of progesterone in mice at E16, P1, P7 and P14 detected using ELISA kit (n = 5-8). isolated from P7 heart. Eighty to one hundred cells were randomly selected per heart (n = 6). Scale bars are 20 µm. F, Heart weight and body weight (HW/BW) ratio (n = 10-11). G, The mRNA expression of cell cycle activators in mouse hearts was analysed by real-time PCR (n = 5). CM, cardiomyocyte progesterone treatment ( Figure 5A); after blockade of progesterone receptor by its inhibitor RU486, the stimulatory effect of progesterone on YAP expression was reduced ( Figure 5A,B). The progesterone-induced up-regulation of YAP protein was associated with its functional activation, because progesterone also up-regulated YAP target genes, including ANKRD1, CYR61 and CTGF ( Figure 5C). These results indicated that up-regulation of YAP expression may be the underlying mechanism through which progesterone promotes CM proliferation.
YAP silencing study further confirmed the importance of YAP in the progesterone-mediated CM proliferation, because down-regulation of YAP expression by YAP-specific siRNA ( Figure S6) abolished the pro-proliferative effect of progesterone, evidenced by the decrease in percentage of Ki67 + , PH3 + and Aurora B + CMs and also total protein levels of these proliferation markers ( Figure 5D-F and Figure S7).
These findings suggest that progesterone promotes CM proliferation through up-regulation of YAP expression.
As progesterone receptor is a transcription factor to regulate target gene expression, 22 we, therefore, investigated the effect of progesterone on YAP trans-activation by performing luciferase reporter activity assay. The results showed that progesterone stimulation significantly increased luciferase activity of YAP promoter ( Figure 6A). The progesterone receptor deposition on the YAP promoter was further determined by ChIP assays. We found that progesterone receptor was present at all three regions of the YAP Collectively, these results suggest that progesterone increased YAP expression through the progesterone receptor by binding directly with the YAP promoter and consequently promoting YAP transcription in CMs.

| Progesterone promotes adult CM proliferation and improves cardiac function in MI
Finally, we assessed the effect of progesterone on adult CM proliferation and explored the clinical translational potential of the above studies. MI in adult mice was induced by ligation of the LAD coronary artery, followed by the daily intraperitoneal injection of progesterone ( Figure 7A). At day 7 after MI, the percentages of Ki67 + , PH3 + and Aurora B + CMs in the peri-infarcted area were significantly higher in the progesterone-than the vehicle-treated group, indicating that progesterone could promote adult CM proliferation after MI ( Figure 7B-D). Immunostaining of isolated CMs further showed that percentages of Ki67 + and PH3 + CMs were significantly higher in the progesterone-than the vehicle-treated group ( Figure S9A,B).
Consistent with in vitro results, progesterone also significantly increased YAP expression in CMs after MI ( Figure S10). At day 28 after MI, cardiac function was measured by echocardiography. As shown in Figure 7E,F, progesterone was also able to ameliorate the loss of cardiac function induced by MI, evidenced by the increase in LVEF and LVFS. We also examined cardiac fibrosis at day 35 after MI by Masson's trichrome staining and found that progesterone treatment significantly decreased the fibrotic scars ( Figure 7G). These findings suggest that progesterone can promote adult CM proliferation and cardiac repair after MI.

| D ISCUSS I ON
In recent years, the concept that adult mammalian CM is completely incapable of regeneration has been challenged by many studies. 21,23 Indeed, increasing pieces of evidence have demonstrated that adult mammalian CM undergoes a very low level of proliferation, but this is insufficient to compensate for CM loss and restore cardiac function after injury. 24,25 Thus, understanding the mechanisms by Progesterone is a cholesterol-derived hormone, which plays critical role in mammalian reproduction, including oocyte maturation, implantation of the embryo and quiescence of uterine muscle during foetal development. 29,30 Progesterone also has been demonstrated to possess many other functions, such as neuroprotective effect in ischemic/reperfusion and traumatic brain injury by reducing inflammation and attenuating neuronal death. [31][32][33] Cardiovascular effects of progesterone have also been reported; its anti-atherosclerotic effect occurs by inhibiting proliferation and migration of aortic smooth muscle cells. Furthermore, progesterone-mediated cardioprotective effects against bisphenol A-induced arrhythmogenesis and doxorubicin-induced apoptotic cell death were also observed. 34  plays important roles in many biological processes, including cell proliferation, migration and fate determination. 42 Notably, it is well established that activation of YAP by its overexpression or inhibition of Hippo signalling can provoke CM proliferation, thus enhancing heart regeneration and repair after injury, including MI. 41,[43][44][45] Our data show that increased expression of YAP in progesterone-treated CMs occurred in parallel with the activation of YAP target genes, including ANKRD1, CYR61 and CTGF. Conversely, the pro-proliferative effect of progesterone could be diminished by YAP silencing. Thus, we can conclude that up-regulation of YAP is the main mechanism for the progesterone-mediated promotion of CM proliferation ( Figure 8).
Furthermore, in accordance with progesterone's well-established ability in regulating target gene expression, we showed binding of progesterone receptor in YAP promoter, suggesting progesterone promotes YAP expression via progesterone receptor-mediated transcription activation of the YAP promoter. Herein, we report for the first time that YAP is a direct target gene of the progesterone receptor in CM. As previously reported, 41 YAP plays an important role in cardiomyocyte proliferation, suggesting it as a therapeutic target gene in cardiac regeneration. However, it should be noticed that gene therapy technology is still far from clinical application due to low organ specificity and other drawbacks. 46 Furthermore, sustained activation of YAP may lead to adaptive cardiac hypertrophy upon hypertrophic stress and lead to tumorigenesis in other organs. 47,48 If using progesterone as a therapy method, the duration and dosage can be well controlled, thus avoiding the side effects resulted from sustained and non-specific YAP activation.
In summary, the current report unveiled progesterone withdrawal as a new mechanism for postnatal CM cell cycle arrest. In addition, progesterone supplementation promotes both neonatal and adult CM proliferation through up-regulation of YAP signalling to improve cardiac repair after injury. Our findings may help to advance therapeutics in the field of cardiac regenerative medicine.

ACK N OWLED G EM ENTS
These studies were supported in part by grants from the National

CO N FLI C T O F I NTE R E S T
The authors declare that they have no competing interest. Xu revised the manuscript.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author on reasonable request.