rest regulates the cell cycle for cardiac development and

ARTICLE

REST regulates the cell cycle for cardiacdevelopment and regenerationDonghong Zhang1, Yidong Wang1, Pengfei Lu1, Ping Wang1, Xinchun Yuan2, Jianyun Yan3, Chenleng Cai3,

Ching-Pin Chang4, Deyou Zheng 5, Bingruo Wu1 & Bin Zhou 6,7

Despite the importance of cardiomyocyte proliferation in cardiac development and regen-

eration, the mechanisms that promote cardiomyocyte cell cycle remain incompletely

understood. RE1 silencing transcription factor (REST) is a transcriptional repressor of neu-

ronal genes. Here we show that REST also regulates the cardiomyocyte cell cycle. REST binds

and represses the cell cycle inhibitor gene p21 and is required for mouse cardiac development

and regeneration. Rest deletion de-represses p21 and inhibits the cardiomyocyte cell cycle and

proliferation in embryonic or regenerating hearts. By contrast, REST overexpression in cul-

tured cardiomyocytes represses p21 and increases proliferation. We further show that p21

knockout rescues cardiomyocyte cell cycle and proliferation defects resulting from Rest

deletion. Our study reveals a REST-p21 regulatory axis as a mechanism for cell cycle pro-

gression in cardiomyocytes, which might be exploited therapeutically to enhance cardiac

regeneration.

Corrected: Author correction

DOI: 10.1038/s41467-017-02210-y OPEN

1 Department of Genetics, Albert Einstein College of Medicine, Bronx, NY 10461, USA. 2Department of Medical Ultrasound, The First Affiliated Hospital ofNanchang University, Nanchang 330006, China. 3 Department of Developmental and Regenerative Biology, The Black Family Stem Cell Institute, and TheMindich Child Health and Development Institute, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA. 4Department of Medicine, IndianUniversity School of Medicine, Indianapolis, IN 46202, USA. 5Departments of Genetics, Neurology and Neuroscience, Albert Einstein College of Medicine,Bronx, NY 10461, USA. 6 Departments of Genetics, Pediatrics, and Medicine (Cardiology), The Wilf Cardiovascular Research Institute, The Institute for AgingResearch, Albert Einstein College of Medicine, Bronx, NY 10461, USA. 7Department of Cardiology of First Affiliated Hospital and State Key Laboratory ofReproductive Medicine, Nanjing Medical University, Nanjing, Jiangsu 210029, China. Correspondence and requests for materials should be addressed toB.Z. (email: [email protected])

NATURE COMMUNICATIONS |8: 1979 |DOI: 10.1038/s41467-017-02210-y |www.nature.com/naturecommunications 1

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https://doi.org/10.1038/s41467-017-02617-7

Cardiomyocyte proliferation is required for generatingmyocardial mass and building a functional four-chamberheart during embryonic development1–3. After birth, car-

diomyocytes continue to proliferate in a short neonatal period,which is crucial for the final cardiac growth surge as well as forregeneration of injured mouse neonatal hearts4–6. The vastmajority of cardiomyocytes then exits the cell cycle and stopsproliferating after preadolescence7–10. The inability of

cardiomyocytes to proliferate prevents the replenishment of lostor dysfunctional cells in a diseased heart11. Because heart diseasesare the number one cause of death worldwide11, it is important toidentify the regulatory factors of the cardiomyocyte cell cycle,which may be used as therapeutic targets for these devastatingconditions.

Several transcription factors, such as GATA412, TBX2013,BRG114, YAP15,16, ERBB217, PITX218, and MEIS119 have been

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Fig. 1 Rest is required for myocardial development and cardiomyocyte proliferation. a, b Immunostaining and western blot analyses showing the efficientdeletion of Rest in cardiomyocytes in TnTCre;RestGT/GT (RestmKO) hearts. Controls were RestGT/GT mice. c A summarizing graph of the distribution andnumber of embryos at different stages indicates death of Restmko embryos between E12.5 and E16.5. d, e H&E-stained sections of E10.5 and E12.5 heartsindicate thin ventricular wall resulting from Rest deletion. f Quantitative immunostaining of cell cycle markers showing reduced proliferation of RestmKO

cardiomyocytes. n= 4/group, mean± s.d., *p< 0.05 by unpaired two-tailed Student’s t test. Scale bars= 40 µm

ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/s41467-017-02210-y

2 NATURE COMMUNICATIONS |8: 1979 |DOI: 10.1038/s41467-017-02210-y |www.nature.com/naturecommunications


shown to be essential for cardiomyocyte proliferation duringdevelopment and for regeneration following neonatal heartinjury. Transcriptional repressor element-1 silencing transcrip-tion factor (REST), also known as neuron-restrictive silencerfactor (NRSF), is widely expressed in the embryonic tissues20,21. It

binds a cis-element of 21 nucleotide base pairs, named RE1motif22, to silence the expression of neuronal genes in the non-neuronal cells required for neurogenesis23,24. We have recentlyreported REST acting as a transcription repressor in mouseembryonic hearts20. In this study, we reveal that suppression of

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Fig. 2 Rest is required for neonatal cardiomyocyte proliferation and cardiac function. a A graph of experimental design for generating myocardial Restknockout (RestimKO) at different postnatal (P) stages using the inducible TnTMerCreMer deleter mice. b Western blots showing effective deletion of Rest. cSurvival curve of control and RestimKO mice, p< 0.001, log-rank (Mantel–Cox) testing between mice with Rest deletion at different stages and control (Tam-treated Rest+/+;TnTMerCreMer/+ mice). d Representative H&E staining and heart/body weight ratio revealing underdeveloped RestimKO hearts at P3 after Restdeletion at P1. n= 5/group. Scale bar= 100 µm. e–g Echocardiography showing structure defects and decreased left ventricle function of P3 RestimKO. n= 6for RestimKO; n= 8 for Control. h–j FACS shows reduced percentage of cardiomyocytes isolated from P3 RestimKO hearts (h) and arrested cell cycle of theRestimKO cardiomyocytes at G0/G1 phase (i, j). k Immunostaining for cell cycle markers indicating reduced proliferation of Rest null cardiomyocytes. n= 4/group for FACS and immunostaining. Scale bar= 40 µm. Mean± s.d., *p< 0.05, unpaired two-tailed Student’s t test

NATURE COMMUNICATIONS | DOI: 10.1038/s41467-017-02210-y ARTICLE




the cell cycle inhibitor gene p21 by REST is critically requiredduring cardiac development and regeneration to maintain car-diomyocyte proliferation. We show that REST binds andrepresses the cell cycle inhibitor gene p21, which is required formouse cardiac development and regeneration. Rest deletion de-represses p21 and inhibits the cardiomyocyte cell cycle and pro-liferation in embryonic or regenerating mouse hearts. We alsoshow that p21 knockout rescues the cardiomyocyte cell cycle andproliferation defects resulting from Rest deletion. By elucidatingthe REST-p21 genetic mechanism underlying the cell cycle reg-ulation of proliferating cardiomyocytes during cardiac develop-ment and regeneration, our study provides an opportunity fordeveloping cell-based therapeutics for heart disease.

ResultsRest is required for embryonic cardiomyocyte proliferation.Our recent studies showed that REST represses transcription inmouse embryonic hearts20. Based upon this, we did a time courseof REST expression levels at various developmental stages. Bywestern blot, we found that REST was downregulated in theventricles of neonatal hearts (Supplementary Fig. 1a, b). Further,immunostaining showed that REST was expressed in the majorityof cardiomyocytes between embryonic day (E) 11.5 and postnatalday (P) 3, whereas the number of REST-expressing cardiomyo-cytes was drastically reduced from P3 to P28 (SupplementaryFig. 1c, d). The downregulation of REST protein level was notaccompanied by a change in mRNA level (SupplementaryFig. 1e), and was possibly due to the REST proteindegradation21,25.

Given that downregulation of REST coincided with the cellcycle exit of cardiomyocytes5, we examined the relationship of

REST expression with cardiomyocyte proliferation by immunos-taining. The results revealed that the majority of EdU+proliferating cells expressed REST (Supplementary Fig. 2a–c).We next inactivated Rest in the myocardium (RestmKO) usingTnTCre and RestGT/GT mice23 to determine its role in cardio-myocytes and confirmed that Rest was effectively deleted in themyocardium by immunostaining and western blot (Fig. 1a, b).RestmKO embryos were runted and >80% of them were dead byE16.5 (Fig. 1c). RestmKO hearts at E10.5–12.5 had thin ventricularwalls and defective trabeculae (Fig. 1d, e). Notably, there wassignificantly reduced percentage of RestmKO cardiomyocytes thatwere expressing the cell cycle markers (Ki67 for cell cycle activity,EdU for DNA synthesis, pH3 for mitosis, and Aurora B forcytokinesis) (Fig. 1f). Such proliferation defect was not associatedwith changes in myocardial differentiation and apoptosis(Supplementary Fig. 3a–g). These observations demonstrate thatRest is essential for embryonic cardiomyocyte proliferation andchamber development.

Rest is required for neonatal cardiomyocyte proliferation. Todetermine whether Rest is also requirement for neonatal cardio-myocyte proliferation, we deleted Rest at P1, P3, or P7 using aninducible TnTMerCreMer driver (RestimKO, thereafter) (Fig. 2a, b).Rest inactivation at P1, but not at P3 or P7, resulted in the deathof ~80% of neonates within 1 week after tamoxifen injection(Fig. 2c). Based upon this, we focused on inactivating Rest at P1and investigating the resultant changes in cardiac morphologyand function at P3. H&E staining showed that the RestimKO heartswere underdeveloped with a reduced ratio of the heart/bodyweight (Fig. 2d). Echocardiography revealed a noticeable reduc-tion in the left ventricular mass, left ventricular wall thickness,

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Fig. 3 REST binds p21 to repress its expression. a, b Immunostaining (n= 4/group) showing upregulated p21 protein level in the Rest deleted E12.5 and P3hearts. Scale bar= 40 µm. c Top, Sequence alignment of p21 between mouse and human indicating a conserved RE-1 motif (red vertical line) located at the3′ UTR. Bottom, The p21-RE1 motif and its mutated (Mut) or deleted (Del) counterpart. d Luciferase reporter assays with a 690-bp mouse DNA fragmentcontaining the wild type (WT), mutated, or deleted RE1 motif reveals that the intact p21-RE1 motif is required for repressing the reporter gene transcriptionin primary cultured P1 mouse cardiomyocytes. e qChIP using REST antibodies indicates enrichment of p21-RE1 motif containing DNA fragments in theventricles of E12.5 and P1, but not P21. f qChIP with P2 ventricles using the REST and histone mark antibodies showing the REST-dependent histonemodification of the p21-RE1 motif. n= 3/group for d–f, mean± s.d., *p< 0.05 by one-way ANOVA followed by Tukey’s test in d; unpaired Student’s t test inb, e, f. NS no significance




and contractility (Fig. 2e–g). Flow cytometry assays furtherrevealed a reduced percentage of cardiomyocytes in RestimKO

hearts (Fig. 2h). The null cardiomyocytes were accumulated at theG0/G1 phase of cell cycle, while their presence at the S and G2/Mphases was reduced (Fig. 2i, j). Immunostaining for Ki67 andEdU confirmed the cell cycle defect (Fig. 2k). In contrast, Restdeletion at P3 or P7, when cardiomyocytes were exiting the cellcycle, had no effect on cardiac morphology and function (Sup-plementary Fig. 4a–d). Therefore, Rest is also required for

neonatal cardiomyocyte proliferation through maintaining cellcycle progression, and neonatal cardiac growth and function.

REST binds and represses p21 to regulate cardiomyocyte pro-liferation. To identify the downstream factor(s) of REST involvedin the cell cycle regulation, we re-analyzed our previously pub-lished RNA-seq data from E12.5 control versus RestmKO hearts20

and found that p21 (Cdkn1a) was the only cell cycle inhibitor

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Fig. 4 p21 deletion rescues embryonic heart defects resulting from myocardial Rest deletion. a Survival curve, no difference between control and p21 null(p21KO) or p21/Rest double knockout (DKO) group by log-rank (Mantel–Cox) test. p< 0.001 between RestmKO and each of other groups. b, c H&E-stainedE12.5 heart sections showing that thin ventricular walls of Rest deleted hearts are restored by p21 deletion (n= 3/group). Scale bar= 100 μm. dRepresentative western blot revealing that upregulation of p21 and downregulation of Cyclin B, Cyclin E, and CDK1/2 in E12.5 Rest null hearts are restoredto the normal levels of control hearts by p21 deletion. e Immunostaining for cell cycle markers indicating that p21 inactivation rescues the cell cycle defectresulting from Rest deletion (n= 4/group). Scale bar= 40 μm. f–i H&E staining (f scale bar= 100 μm), ratio of heart/body weight (g), andechocardiography (h, i) showing that P28 Rest/p21 DKO mice have cardiac structure and function comparable to control littermates (n= 6–8/group).Mean± s.d. *p< 0.05 by one-way ANOVA followed by Tukey’s test. NS no significance





gene significantly upregulated in Rest null hearts, whereas theexpression of several cell cycle activators was downregulated(Supplementary Fig. 5a). These findings were then confirmed byqRT-PCR (Supplementary Fig. 5b, c). Notably, p21 was also theonly cell cycle inhibitor gene which expression was significantlyupregulated in P2 ventricles with myocardial Rest inactivation atP1 (Supplementary Fig. 5d). Immunostaining further showed anincreased number of p21-expressing cardiomyocytes in E12.5 andP3 Rest null hearts (Fig. 3a, b). We identified one conservedREST-binding site (RE1 motif) located at the 3′ end of mouse p21gene by sequence alignment between mouse and human (Fig. 3c).

Luciferase reporter gene assays and mutational analyses revealedthat the p21-RE1 motif was required for repressing transcriptionin cultured P1 mouse cardiomyocytes (Fig. 3d). Quantitativechromatin immunoprecipitation assays (qChIP) showed RESToccupancy of the p21-RE1 motif in E12.5 and P1 ventricles, butthe occupancy was greatly reduced in the ventricles of P21 whenREST level was largely diminished (Fig. 3e). Further, qChIPshowed that Rest deletion resulted in an enrichment of H3K4me3histone modification and a reduction of H3K9me3 andH3K27me3 histone marks at the p21-RE1 motif (Fig. 3f). Thesefindings support that REST directly binds p21 and represses its

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Fig. 5 p21 deletion rescues neonatal heart defects resulting from Rest deletion. a Survival curve, no difference between control and p21 null (p21KO) or p21/Rest double knockout (DKO) group by log-rank (Mantel–Cox) test. p< 0.001 between RestimKO and each of other groups. b, c H&E-stained P3 heartsections showing that underdeveloped Rest null hearts are restored by p21 deletion (n= 5/group). Scale bar= 100 μm. d, e Echocardiograph showing p21knockout rescues cardiac function resulting from Rest deletion at P1 (n= 6–8/group). f Representative western blot revealing upregulation of p21 anddownregulation of Cyclin B, Cyclin E, and CDK1/2 in P3 Rest null hearts are restored by p21 deletion to the normal levels of control hearts. gImmunostaining for cell cycle markers indicating p21 inactivation rescues the cell cycle defect resulting from Rest deletion (n= 4/group). Scale bar= 40μm. Controls were the Tam-treated Rest+/+;TnTMerCreMer/+ mice. Mean± s.d. *p< 0.05 by one-way ANOVA followed by Tukey’s test




transcription in cardiomyocytes, possibly through histonemethylation changes26.

To determine whether such repression promoted cardiomyo-cyte proliferation during cardiac development, we deleted p21 inRestmKO mice by breeding them with p21 knockout mice27.Inactivation of p21 rescued embryonic lethality and cardiacphenotypes of RestmKO mice at E12.5 (Fig. 4a–c). Inactivation ofp21 also restored the levels of Cyclin B, Cyclin E, and CDK1/2(Fig. 4d), and re-established normal cardiomyocyte cell cycleand proliferation, as indicated by recovered expression ofKi67, PH3, and Aurora B, and EdU incorporation (Fig. 4e).Double knockout (DKO) mice were survived to birth andappeared healthy postnatally and in adulthood, with normalcardiac morphology, heart/body weight ratio, and cardiacfunction (Fig. 4f–i). Similarly, inactivation of p21 rescuedpostnatal lethality of mice caused by inactivation of myocardialRest at P1 (Fig. 5a). Cardiac phenotypes were rescued, includingthe cardiac size, heart/body ratio, cardiac function (Fig. 5b–e),

as well as the expression of cell cycle activators and markers(Fig. 5f, g). We also examined whether forced overexpression ofREST would promote cardiomyocyte proliferation in culturedcardiomyocytes isolated from P1 or P5 hearts. We first treated theculture with siRNAs against β-TrCP that mediates RESTdegradation via ubiquitination21,25. The results of western blotfor REST, qRT-PCR for Rest, and REST-targeted genes andimmunostaining for EdU and PH3 showed that downregulationof β-TrCP slowed down the REST degradation (SupplementaryFig. 6a), suppressed the expression of REST target genes (Hcn2,p21, Nppa, and Syn1) (Supplementary Fig. 6b), and upregulatedcardiomyocyte proliferation (Supplementary Fig. 6c). Next, weoverexpressed REST by transfection of a plasmid (pHR-NRSF-CITE-GFP) containing Rest cDNA28 and found by western blotand immunostaining for EdU and PH3 that overexpression ofREST dose-dependently repressed p21 expression and increasedcardiomyocyte proliferation (Supplementary Fig. 6d–f). Thesefindings, therefore, identify that a Rest-p21 axis underlies the

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Fig. 6 Co-upregulation of REST and cardiomyocyte proliferation in injured neonatal hearts. a, b Quantitative immunofluorescence (a Scale bar= 40 μm.)and scatter plot b showing increased expression of REST and its positive relation with the expression of cell cycle markers in cardiomyocytes. Averagecounts from six fields/section, four sections/ventricle, four ventricles/stage at P7, P14, and P28, mean± s.d. *p< 0.05 by one-way ANOVA followed byTukey’s test. APB/APR apex border/apex remote. c Co-immunofluorescence showing consistently ~60% of EdU+ cells express REST (arrow) inregenerating neonatal hearts. Scale bar= 10 μm. Data are presented as mean± s.d. (n= 4/stage)





regulation of cardiomyocyte cell cycle for fetal and neonatalcardiac development.

Rest is required for regenerative cardiomyocyte proliferation.To further investigate whether REST had a role in cardiacregeneration, we investigated Rest expression in repairing neo-natal hearts using an apical resection model4. Consistent with

previous reports4,6, immunostaining for the cell cycle markersshowed a significantly increased number of cardiomyocytes thatre-entered the cell cycle in the injured region at P7, 3 days afterablation at P4 (Fig. 6a). Notably, the number of REST-expressingcardiomyocytes was also increased in the same region and wascorrelated with the increased number of cells undergoing DNAsynthesis, mitosis, and division (Fig. 6b). The percentage of EdU+cells that also expressed REST was sustained in the regenerating

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Fig. 7 Rest is required for regeneration of injured neonatal hearts. a, b Quantitative Masson’s trichrome staining shows increased cardiac fibrosis in micewith induced myocardial Rest deletion at P3 and injured by apex ablation at P4, n= 5–8/group. Scale bar= 100 μm. c, d Echocardiography indicatingreduced cardiac function of myocardial Rest null mice after injury, n= 6–8/group. e–g Quantitative immunofluorescence (n= 4/group) showing decreasedEdU+ cardiomyocytes in regenerating hearts in mice with Rest deletion. Scale bar= 40 μm. Mean± s.d. p< 0.05 by two-way ANOVA followed by Sidak’stest in b–d; unpaired two-tailed Student’s t test in f, g




myocardium from P7 to P28 (Fig. 6c). About 90% of REST-expressing cells in the apex undergoing repair were cardiomyo-cytes; the reminder was epicardial or endocardial cells (Supple-mentary Fig. 7a–d). These findings indicated that the majority ofREST-expressing cells were proliferating cardiomyocytes inducedby injury. We deleted Rest in the myocardium at P3 and per-formed apical resection at P4. The P3 deletion bypassed the deathof neonates when Rest was deleted at P1, while apical ablation atP4 was still within the window of myocardial regeneration2. Wefound by Masson’s trichrome staining that the fibrotic scar in theapex of Rest null hearts (RestimKO) was not resolved at P14 andP28 compared to the control hearts (Fig. 7a, b). Consistent withthe persistent scaring phenotype, RestimKO hearts had reduced leftventricular function as observed by echocardiography (Fig. 7c, d).Further, immunostaining revealed that the number of EdU+proliferating cardiomyocytes in the injured apex of RestimKO

hearts was significantly reduced (Fig. 7e–g). Together, thesefindings suggest that REST re-expression is necessary for theregeneration of neonatal hearts.

The increased number of REST-expressing cardiomyocytes inthe regenerating neonatal hearts was correlated with increasedREST occupancy at the p21-RE1 motif (Supplementary Fig. 8a).Concurrently, the regenerating myocardium expressed a low levelof p21 mRNA (Supplementary Fig. 8b). Immunostaining alsoshowed that the number of p21-expressing cardiomyocytes wasnegatively correlated with that of REST-expressing cells in theregenerating myocardium (Supplementary Fig. 8c, d). In contrast,p21 transcription was de-repressed in the injured RestimKO apex,

which was accompanied with downregulation of the cell cycleactivators (Supplementary Fig. 8e, f). To determine whether p21upregulation was responsible for the reduced proliferation ofcardiomyocytes and poor regeneration of myocardium after Restdeletion, we performed the apical resection on the Rest and p21DKO hearts at P4, 1 day after Rest deletion (Fig. 8a). Like controlhearts, p21−/− hearts had normal left ventricular function at P28(Fig. 8b, c). We noted that p21−/− hearts had less fibrotic scartissue as compared to controls (Fig. 8d). Importantly, p21 deletionimproved regeneration and function of RestimKO hearts andrestored the number of EdU+ cardiomyocytes in the injuredmyocardium (Fig. 4e). These results demonstrate that the Rest-p21 axis regulation of cardiomyocyte proliferation also underliesthe neonatal cardiac regeneration.

DiscussionOverall, our study identifies a molecular mechanism by whichREST repression of the cell cycle inhibitor p21 is necessary andsufficient for promoting the cardiomyocyte cell cycle and pro-liferation during cardiac development and regeneration. p21 is awell known cell cycle inhibitor through interacting with the G1/Mand G2/S CDK cyclins and inhibits their function to drive the cellcycle29. Of particular note, p21 is the only cell cycle inhibitor genesignificantly repressed by REST in the proliferating cardiomyo-cytes during cardiac development and regeneration, suggestingthat REST repression of p21 is a major mechanism underlying theregulation of cardiomyocyte cell cycle. Consistent with this

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Fig. 8 REST-dependent p21 expression is required for cardiac regeneration. a Schematic diagram showing experimental procedure of cardiac apex resection(AR) on neonatal mice. b, c Echocardiography indicates that mice with control (n= 9), single p21 knockout (p21KO, n= 8), and double knockout of Rest andp21 (DKO, n= 8) injured hearts have cardiac function comparable to that of the sham-operated control (s-Control, n= 9) at P28. Injured Rest singleknockout (RestimKO, n= 9) hearts have significantly decreased left ventricular function comparable to injured control. d, e Quantitative Masson’s trichromestaining (n= 8/group) (d Scale bar= 100 µm) and EdU assays (n= 4/group) (e Scale bar= 40 µm) showing p21 inactivation reduces the cardiac fibrosisand rescues cardiomyocyte proliferation defect in the injured apex resulting from Rest deletion. Mean± s.d. *p< 0.05 by one-way ANOVA followed byBonferroni test. NS no significance





notion, p21 deletion has been reported to enhance the regen-eration of injured appendage30 and neonatal hearts31. Interest-ingly, GATA432 and MEIS119 also repress p21 transcription inmouse embryonic and injured neonatal hearts, respectively. Sinceneither GATA4 nor MEIS1 expression is affected by RESTinactivation, REST repression of p21 is likely independent ofGATA4 and MEIS1. REST thus joins a network of transcriptionfactors or cofactors, including MEIS119, YAP15,16, GATA412,33,34,and TBX2013, as a new member essential for cardiac developmentand regeneration.

Elucidating the Rest-p21 axis underlying the regulation ofcardiomyocyte cell cycle may have clinical implications by pro-viding an opportunity for better understanding of cardiomyocyteproliferation in cardiac regeneration. It is well known that the cellcycle arrest of cardiomyocytes is a normal physiological feature ofadult hearts5. This, however, prevents the endogenous regenera-tion of infarcted adult hearts from the proliferation of pre-existing cardiomyocytes11,35. Our studies indicate that REST israpidly diminished in the postnatal heart, but rebounded afterinjury. These observations suggest that REST downregulationmight be one of the limiting factors for sustaining proliferation ofpostnatal cardiomyocytes. Future investigations are needed todetermine whether REST is reactivated in adult hearts by injuryand if the reactivation promotes adult cardiac regeneration bymodulation of the activity of cardiomyocyte cell cycle.

MethodsMouse strains. Floxed Restmice (RestGT/GT), which carries a conditional gene trap(GT) cassette23, were bred to TnTCre36 or TnTMerCreMer37 mice to obtain Restdeletion in myocardium. Intraperitoneal tamoxifen (TAM) injection at 40 mg/kgwas used for inducible inactivation. For rescue experiments, TnTCre; RestGT/GT

(RestmKO), or TnT MerCreMer;RestGT/GT (RestimKO) mice were crossed to p21−/−

(Cdkn1a−/−) mice27. All mouse stains were in the C57B6 background. Embryoswere isolated and inspected according to expected developmental ages. Age-matched embryos and neonatal mice of both sexes were grouped according togenotypes during experiments. Noontime on the day of detecting vaginal plugs wasdesignated as E0.5, and morning on the day of observing newborns was designatedas P0. The yolk sac or tail of mice was used for genotyping by PCR using Cre andallele-specific primers (Supplementary Table 1). Mouse housing and experimentswere according to the protocols approved by the Institutional Animal Care and UseCommittee (IACUC) of Albert Einstein College of Medicine.

Neonatal heart apical resection. Neonatal mouse heart apical resection (AR) wasperformed on postnatal day (P) 4 mice of both sexes as previously described4.Briefly, we induced deletion of Rest in the myocardium at P3 by tamoxifen andperformed apical resection at P4. P4 neonates were anesthetized by inhalation of2% isoflurane and 100% oxygen on ice and then subjected to intercostal incisions toseparate the pericardium and expose the apex. The apex was amputated withmicrosurgical scissors under a dissecting microscope. About 10% of the heart wassurgically removed to produce reproducible regeneration. The chest wall was thenclosed with sutures and the skin incision was closed using skin adhesive. After theoperation, the neonates were placed back with the feeding female mice. Sham-operated mice underwent the same procedure without apical resection.

Histology. Embryos or isolated hearts were fixed in 4% paraformaldehyde (PFA),dehydration, embedded in paraffin, and sectioned frontally at 6-μm thickness. H&Eand Masson’s trichrome staining were performed for detecting cardiac structuredefects and fibrosis, respectively. The fibrotic area was quantified using theMetaMorph software (Molecular Devices).

Primary cardiomyocyte cultures. Cardiomyocytes (CMs) were isolated asdescribed in previous studies20. Ventricles were dissected out from P1 or P5neonates of both sexes, minced and dissociated by 0.045% collagenase II and 0.08%trypsin-EDTA at 37 °C. Non-CMs were removed through adhesive plating. PurifiedCMs were cultured in 12-well plates for 24 h at 37 °C with 5% CO2 in Dulbecco’smodified Eagle’s minimum essential medium (DMEM) supplemented with 10%fetal calf serum.

siRNA and cDNA transfection. siRNA against β-TrCP (si-β-TrCP, Santa CruzBiotechnology) or plasmids containing mouse Rest cDNA (pHR-NRSF-CITE-GFP,Addgene) was transfected into cultured neonatal cardiomyocytes isolated from P1or P5 neonatal hearts, at 70% confluency. Lipofectamine 3000 Regent (Invitrogen)and si-β-TrCP (40 nM) or REST-cDNA (0.5 or 2 µg/ml) were incubated in

OPTIMEM (Invitrogen) for 15 min at RT, and then added to cells and incubatedfor indicated time. qRT-PCR and western blot were used to confirm the efficiencyand effect of transfection.

ChIP-qPCR. ChIP-qPCR assays were performed as previously reported38. Ven-tricles (30–40 mg) from indicated stages were treated with 1% formaldehyde atroom temperature for 10 min to crosslink protein and DNA complexes beforequenched with addition of glycine. After three washes with PBS, tissues werehomogenized, lysed, and sonicated to shear DNAs into 100–500 bp fragments. TheDNA fragments were incubated with 5 µg antibodies against REST (EMD Milli-pore, 17–641, 1:100), Tri-Methyl-Histone H3 (Lys9) (CST mAb #13969, 1:100),Tri-Methyl-Histone H3 (Lys27) (CST mAb #9733, 1:100), Tri-Methyl-Histone H3(Lys4) (CST mAb #9751, 1:100), or Acetyl-Histone H3 (Lys9) (CST mAb #9649,1:100) overnight at 4 °C using Dynabead Protein G in a final volume of 500 µl andMagnet Starter Pack (Invitrogen, 10004D). Five microgram IgGs (Abcam,ab171870) were used as control. Immunoprecipitated DNAs were extracted andused for qPCR analysis to determine the enrichment of DNA fragments containingthe p21-RE1 using specific primers (Supplementary Table 1).

Western blot. Ventricles were collected at the indicated stages, homogenized inradioimmunoprecipitation assay buffer at 4 °C and quantified by bicinchoninicacid assay. Equal total proteins (20 μg) were resolved in SDS-PAGE gels andtransferred onto polyvinylidene fluoride membranes (Immobilon, Millipore). Themembranes were blocked and incubated with primary antibodies at 4 °C overnightbefore incubated with HRP-conjugated secondary antibodies. Protein bands weredetected by enhanced chemiluminescence. Antibodies, including REST (Anti-bodies-online, ABIN747683, 1:1000), P21 (Abcam, ab109199, 1:500), Cyclin E(Santa Cruz, sc-377100, 1:1000), Cyclin B (Santa Cruz, sc-166210, 1:1000), CDK1/CDK2 (Santa Cruz, sc-53219, 1:1000), and GAPDH (Thermo Fisher, MA5-15738,1:5000), were used in western blot analysis. Full scans of western blots are shown inSupplementary Figs. 9–14.

Echocardiography. Echocardiography was performed using a Vevo 770 ultrasoundsystem (Visual Sonics, Toronto, Canada). Investigators were blinded to genotypeand treatment groups. Mice were anesthetized with 1.5% isoflurane and mousehearts were viewed on two-dimensional short-axis planes and measured using M-mode. Heart rate, left ventricular internal diameter, interventricular septal, andposterior wall (PW) dimensions were measured at end ystole and end diastole forat least three cardiac cycles. Fractional shortening (FS) were calculated by theaverage of FS measurements of the left ventricular contraction for five cardiaccycles.

Quantitative reverse transcription PCR (qRT-PCR). Total RNAs were extractedfrom ventricles or cultured CMs using TRIzol (Life Technologies). One microgramof total RNAs was reverse transcribed to cDNA using a Superscript II reversetranscriptase Kit (Life Technologies). qRT-PCR was performed using Power SYBRGreen PCR Master Mix (Life Technologies) and carried out on an Applied Bio-systems 7900HT Fast Real-Time PCR System. Specific primers were listed inSupplementary Table 1. The relative expression of each gene was normalized to theexpression of Gapdh and calculated using the 2−ΔΔCT method. Biological replicateswere performed using three individual samples of each genotype and technicaltriplicates were carried out for each run of qPCR.

Luciferase reporter gene assay. Transcriptional suppression of p21 via the p21-RE1-pGL3 enhancer by REST was evaluated using luciferase reporter gene assay asdescribed previously20. DNA fragment of p21 gene with or without mutation ordeletion of the p21-RE1 was PCR cloned into a PGL3 enhancer luciferase reportervector (Supplementary Table 1). All constructs were confirmed by Sangersequencing. 0.8 µg of each p21-RE1-pGL3 enhancer construct was co-transfectedwith 0.2 µg of pRL-SV40 vector (Promega, E2231) into cultured P1 CMs in 96-wellplates using Lipofectamine 3000 Reagents (Invitrogen). After 24 h transfection, celllysates were processed for luciferase activity using the luciferase reporter system(Promega, E1980). Luciferase reporter activities were calculated as firefly luciferasenormalized to Renilla luciferase luminescence. Biological triplicates were per-formed in technical triplicates for each vector.

Immunostaining. Immunostaining was performed on sections of paraffin-embedded embryos or hearts, as previously described39. Embryos or hearts werecollected at the indicated stages and fixed in 4% PFA for 2 h, rinsed in PBS,dehydrated in 15 and 30% sucrose sequentially, embedded in optimal cuttingtemperature (OCT) compound and sectioned at 8-μm thickness. Heart sectionswere blocked with 5% horse serum for 1 h at room temperature before beingincubated overnight at 4 temperature with the single or double staining with fol-lowing antibodies diluted in blocking solution: TnT (Fisher Scientific, MS-295-P0,1:400), PECAM1 (Santa, sc-1506, 1:400), REST (Antibodies-online, ABIN747683,1:400 dilution), Ki67 (Abcam, ab66155, 1:200), P21 (Abcam, ab109199, 1:100dilution), cleaved caspase-3 (CST, mAb #9664, 1:200), phosphorylated-histone3(pH3) (Abcam, ab32107, 1:200), or aurora B (Abcam, ab2254,1:100). After three




washes with PBS for 5 min each, samples were stained for 1 h at room temperaturewith fluorescent secondary antibodies followed by DAPI (4′,6-diamidino-2-phe-nylindole dihydrochloride) staining for nuclei visualization. Images were acquiredand analyzed using a Zeiss fluorescence microscope with Axiovision image analysissoftware. For paraffin sections, 6-µm tissue sections were de-waxed in xylene,rehydrated in descending grades of alcohol and antigen-retried by incubating insodium citrate for 10 min at 95 °C. After blocked with 5% horse serum for 60 min,sections were incubated with antibodies against NKX2.5 (Santa, sc-14033, 1:500)overnight at 4 °C, followed by incubation with biotinylated secondary antibodies(Vectastain, PK-6100) for 1 h and stained with DAB (Vector, SK-4100). Coun-terstaining with hematoxylin was used for visualization of nuclei. All immunos-taining were performed on four sections per sample, and total four samples of eachgenotype and treatment group were experimented. Images were taken from sixfields per section, processed using ImageJ software and counted blindly to genotypeand treatment groups.

EdU incorporation. Proliferative cells were pulse labeled for 2 h by intraperitonealinjection of mice with 5-ethynyl-2′-deoxyuridine (EdU, 100 mg/kg). Hearts werethen collected, fixed in 4% PFA, rinsed in PBS, soaked in 15 and 30% sucrosesequentially, embedded in OCT, and sectioned at 8-μm thickness. Sections werestained with antibodies against TnT (Fisher Scientific, MS-295-P0, 1:400) or REST(Antibodies-online, ABIN747683, 1:400), followed by EdU staining (Click-iT® EdUAlexa Fluor® 488 Imaging Kit, Life Technologies) and DAPI counterstaining(Vector lab, H1200). Stained sections were photographed under a Zeiss micro-scope, and images were processed for quantitative analysis as described above.

Flow cytometry. P3 ventricles of Rest knockout (Restimko) mice and littermatecontrols of both sexes were digested into single cell suspension, fixed with 70%ethanol. CMs were stained with TnT antibodies (Fisher Scientific, MS-295-P0,1:400) for 2 h at room temperature and then incubated with secondary antibodies(Alexa Fluor™ 488 Tyramide SuperBoost™ Kit, goat anti-mouse IgG). Cell pelletswere re-suspended in FxCycle™ PI/RNase Staining Solution (Fisher Scientific,F10797) and analyzed on a FACStar Plus flow cytometer (Becton Dickinson,Franklin Lakes, NJ). An average of 104 CMs (FITC+) were collected in the gatefrom total cells per ventricle using forward and side scatter. We used pulse widthversus pulse area as first gate on the single cell population and then applied thisgate to the scatter plot to gate out cell debris. The combined gates were applied tothe PI histogram plot to obtain the percentage of cells in each cell cycle phase.

Apoptosis assay. Apoptotic cells were visualized by TUNEL assay. Frozen sectionswere stained with TnT antibodies (Fisher Scientific, MS-295-P0, 1:400), followed byTUNEL staining using a DeadEndTM Fluorometric TUNEL kit (Promega). AfterDAPI counterstaining, sections were photographed under a Leica SP5 confocalmicroscope. Stained sections were photographed, and images were processed forquantitative analysis as described above.

Statistics. No statistical methods were used to predetermine sample size. Student’st test (two-tailed) or one-way ANOVA analysis followed by Tukey’s, Sidak’s, orBonferroni test was used for statistical difference between or among groups. Therelationship between REST expression and cell proliferation during cardiacregeneration was analyzed by Pearson correlation coefficient. Survival rate of miceat various stages was calculated using log-rank (Mantel–Cox) test. Normality wasassumed and variance was compared between or among groups. Sample size wasdetermined based upon previous experience in the assessment of experimentalvariability. The investigators were not blinded to the group allocation duringexperiments and outcome assessment, unless stated otherwise. We chose theadequate statistic tests according to the data distribution to fulfill test assumptions.Statistical comparison for all quantitative assays was performed in at least threeindependent experiments. All numerical data were presented as mean± s.d. and pvalue of <0.05 was considered as significant. Statistical analyses were performedusing the SPSS v16.0 software (SPSS Inc., Chicago, IL).

Data availability. Data supporting the findings of this study are available withinthe article and its Supplementary Information files. All relevant data are availablefrom the authors on reasonable request. The RNA-Seq data have been downloadedfrom the NIH GEO database with accession code GSE80378.

Received: 4 June 2017 Accepted: 13 November 2017

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AcknowledgementsWe thank B. Morrow, R.N. Kitsis, and N.G. Frangogiannis for critically reading themanuscript. We also thank T. Floss and T. Nechiporuk for RestGT/GT mice. This workwas supported by the National Institutes of Health/National Heart, Lung, and BloodInstitute grants (R01HL116997 to B.Z.; R01HL133120 to B.Z., and De.Z.; R01HL131735to C.C.), and an American Heart Association grants (16POST30480021 to Do.Z.;15GRNT25710153 to C.C.).

Author contributionsDo.Z., Y.W., P.L., X.Y., and B.W. performed most of the experiments. P.W. and De.Z.helped RNA-sequence data analysis. J.Y. and C.C. generated TnTMerCreMer mice. Do.Z. andB.Z. conceptualized the project. B.W. and B.Z. supervised the experiments. C.-P.C. offeredhelp in data analysis and manuscript revision. Do.Z. and B.Z. wrote the manuscript.

Additional informationSupplementary Information accompanies this paper at https://doi.org/10.1038/s41467-017-02210-y.

Competing interests: The authors declare no competing financial interests.

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