ARTICLE Corrected: Author correction

DOI: 10.1038/s41467-017-02210-y OPEN REST regulates the cell cycle for cardiac development and regeneration

Donghong 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

1234567890 understood. RE1 silencing (REST) is a transcriptional repressor of neu- ronal . Here we show that REST also regulates the cardiomyocyte cell cycle. REST binds and represses the cell cycle inhibitor 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.

1 Department of Genetics, Albert Einstein College of Medicine, Bronx, NY 10461, USA. 2 Department of Medical Ultrasound, The First Affiliated Hospital of Nanchang University, Nanchang 330006, China. 3 Department of Developmental and Regenerative Biology, The Black Family Stem Cell Institute, and The Mindich Child Health and Development Institute, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA. 4 Department of Medicine, Indian University School of Medicine, Indianapolis, IN 46202, USA. 5 Departments 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 Aging Research, Albert Einstein College of Medicine, Bronx, NY 10461, USA. 7 Department of Cardiology of First Affiliated Hospital and State Key Laboratory of Reproductive Medicine, Nanjing Medical University, Nanjing, Jiangsu 210029, China. Correspondence and requests for materials should be addressedto B.Z. (email: [email protected])

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

ardiomyocyte proliferation is required for generating cardiomyocytes to proliferate prevents the replenishment of lost myocardial mass and building a functional four-chamber or dysfunctional cells in a diseased heart11. Because heart diseases C 1–3 11 heart during embryonic development . After birth, car- are the number one cause of death worldwide , it is important to diomyocytes continue to proliferate in a short neonatal period, identify the regulatory factors of the cardiomyocyte cell cycle, which is crucial for the final cardiac growth surge as well as for which may be used as therapeutic targets for these devastating – regeneration of injured mouse neonatal hearts4 6. The vast conditions. majority of cardiomyocytes then exits the cell cycle and stops Several transcription factors, such as GATA412, TBX2013, – proliferating after preadolescence7 10. The inability of BRG114, YAP15,16, ERBB217, PITX218, and MEIS119 have been

a b c

100 3 Cre GT/GT mKO Tnt ;Rest Control Control Rest 18 33 12 12 19 Tnt Cre;Rest GT/+ 1.00 ± 0.18 0.20 ± 0.12* 75 44 17 GT/GT DAPI 18 Rest / E12.5 121 KDa 50 11 Rest GT/+

REST 18

REST 17 43 18 / Rest mKO 25 TnT 12 36 KDa Embryos/stage (%) 18 43 17 18

GAPDH 0 E11.5 E12.5 E14.5 E16.5 E18.5

d Control Rest mKO Control Rest mKO e Control E10.5 E12.5 Rest mKO 50 *

m) 40 µ 30 *

20

10 Wall thickness ( thickness Wall 0 E10.5 E12.5

f Ki67/TnT/DAPI EdU/TnT/DAPI PH3/TnT/DAPI Aurora B/TnT/DAPI Control

E12.5 mKO Rest

60 60 * 4 2.5 * * 50 50 * 3 2.0 40 40 1.5 30 30 2 1.0 20 20

EdU/TnT (%) 1 PH3/TnT (%) KI67/TnT (%) 10 10 0.5 Aurora B/TnT (%) Aurora 0 0 0 0 Control Rest mKO Control Rest mKO Control Rest mKO Control Rest mKO

Fig. 1 Rest is required for myocardial development and cardiomyocyte proliferation. a, b Immunostaining and western blot analyses showing the efficient deletion of Rest in cardiomyocytes in TnTCre;RestGT/GT (RestmKO) hearts. Controls were RestGT/GT mice. c A summarizing graph of the distribution and number 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 hearts indicate 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

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a TAM Rest imKO b P3(iP1) P5(iP3) P9(iP7) c 120 100 iP1 Control (n=30) REST imKO imKO imKO 80 Rest imKO (iP1) (n=22) 60 imKO Control Rest Control Rest Control Rest Rest (iP3) (n=19) iP3 REST 40 Rest imKO (iP7) (n=41)

REST 121 KDa Survival (%) 20 p<0.001 iP7 REST (P) 2 4681012 14 21 28 35 P1 P3 P7 GAPDH 36 KDa

d e fgControl Control Rest imKO (iP1) Rest imKO (iP1) 12 0.6 120 * * * * 9 90 0.4 * P3 6 60 8 * 6 0.2 3 30

4 (mm) thickness LV

2 mass corrected (mg) LV

Heart/body 0 0.0 0 weight (mg/g) weight 0 LVPW LVAW LVEF (%) LVFS (%)

h ijControl Rest imKO (iP1) 80 * 100 * 200 200 90 60 80 150 G1/G0 S G2/M 150 G1/G0 S G2/M 70 40 20 * 100 100 15 * 20 50 50 10 5 0 0 (%) Cardiomyocyte/stage 0 Cardiomyocyte/total cell (%) Cardiomyocyte/total 0 0 50 100 150 200 (K) 0 50 100 150 200 (K) G1/G0 S G2/M

k Control Rest imKO (iP1) Control Rest imKO (iP1) 15 * 2.5 * 2.0 10 DAPI DAPI 1.5 / / 1.0 TnT TnT / 5 * /

PH3/TnT (%) 0.5 Ki67 PH3 0 0.0

20 * 1.5 * DAPI

15 / DAPI

/ 1.0 TnT

10 / TnT / 0.5 5

EdU/TnT (%) KI67/TnT (%) * EdU Aurora B/TnT (%) Aurora 0 B Aurora 0.0

Fig. 2 Rest is required for neonatal cardiomyocyte proliferation and cardiac function. a A graph of experimental design for generating myocardial Rest knockout (RestimKO) at different postnatal (P) stages using the inducible TnTMerCreMer deleter mice. b Western blots showing effective deletion of Rest. c Survival 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 Rest deletion at P1. n = 5/group. Scale bar = 100 µm. e–g Echocardiography showing structure defects and decreased left ventricle function of P3 RestimKO. n = 6 for RestimKO; n = 8 for Control. h–j FACS shows reduced percentage of cardiomyocytes isolated from P3 RestimKO hearts (h) and arrested cell cycle of the RestimKO 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 shown to be essential for cardiomyocyte proliferation during binds a cis-element of 21 nucleotide base pairs, named RE1 development and for regeneration following neonatal heart motif22, to silence the expression of neuronal genes in the non- injury. Transcriptional repressor element-1 silencing transcrip- neuronal cells required for neurogenesis23,24. We have recently tion factor (REST), also known as neuron-restrictive silencer reported REST acting as a transcription repressor in mouse factor (NRSF), is widely expressed in the embryonic tissues20,21.It embryonic hearts20. In this study, we reveal that suppression of

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abE12.5 P3 c p21 50 E12.5 P3 Inter gene Control Control Repeat * * RE-1 motif 40 Coding UTR Mm9 30 Intron Hg18

/DAPI 20 0 3 kb 6 kb 9 kb

mko imko p21-RE1 TnT p21/TnT (%) / Rest Rest (iP1) 10 WT

p21 Mut1 0 Mut2 Del mKo imKo

Control Control Rest Rest 690 bp

d efE12.5 Control P1 Rest imKO (iP1) 2.5 P21 0.6 Del * * * 2.0 0.5 Mut2 * 0.4 * 1.5

-RE-1 motif Mut1 * 0.3 *

p21 * 1.0 * * 0.2 WT NS NS 0.5 NS pGL3- * 0.1 enhancer Enrichment of RE-1 motif

0.0 Relative change to 5% input 0.0 0 0.5 1.0 1.5 lgG lgG lgG lgG Luciferase activity REST REST REST REST 5% input 5% input 5% input H3K4me3H3K9me3 H3K9/14ac H3K27me3

Fig. 3 REST binds p21 to repress its expression. a, b Immunostaining (n = 4/group) showing upregulated p21 level in the Rest deleted E12.5 and P3 hearts. 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 the 3′ UTR. Bottom, The p21-RE1 motif and its mutated (Mut) or deleted (Del) counterpart. d Luciferase reporter assays with a 690-bp mouse DNA fragment containing the wild type (WT), mutated, or deleted RE1 motif reveals that the intact p21-RE1 motif is required for repressing the reporter gene transcription in primary cultured P1 mouse cardiomyocytes. e qChIP using REST antibodies indicates enrichment of p21-RE1 motif containing DNA fragments in the ventricles of E12.5 and P1, but not P21. f qChIP with P2 ventricles using the REST and histone mark antibodies showing the REST-dependent histone modification 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 in b, e, f. NS no significance the cell cycle inhibitor gene p21 by REST is critically required REST expression with cardiomyocyte proliferation by immunos- during cardiac development and regeneration to maintain car- taining. The results revealed that the majority of EdU+ diomyocyte proliferation. We show that REST binds and proliferating cells expressed REST (Supplementary Fig. 2a–c). mKO represses the cell cycle inhibitor gene p21, which is required for We next inactivated Rest in the myocardium (Rest ) using Cre GT/GT mouse cardiac development and regeneration. Rest deletion de- TnT and Rest mice23 to determine its role in cardio- represses p21 and inhibits the cardiomyocyte cell cycle and pro- myocytes and confirmed that Rest was effectively deleted in the liferation in embryonic or regenerating mouse hearts. We also myocardium by immunostaining and western blot (Fig. 1a, b). mKO show that p21 knockout rescues the cardiomyocyte cell cycle and Rest embryos were runted and >80% of them were dead by mKO proliferation defects resulting from Rest deletion. By elucidating E16.5 (Fig. 1c). Rest hearts at E10.5–12.5 had thin ventricular the REST-p21 genetic mechanism underlying the cell cycle reg- walls and defective trabeculae (Fig. 1d, e). Notably, there was mKO ulation of proliferating cardiomyocytes during cardiac develop- significantly reduced percentage of Rest cardiomyocytes that ment and regeneration, our study provides an opportunity for were expressing the cell cycle markers (Ki67 for cell cycle activity, developing cell-based therapeutics for heart disease. EdU for DNA synthesis, pH3 for mitosis, and Aurora B for cytokinesis) (Fig. 1f). Such proliferation defect was not associated with changes in myocardial differentiation and apoptosis Results (Supplementary Fig. 3a–g). These observations demonstrate that Rest is required for embryonic cardiomyocyte proliferation. Rest is essential for embryonic cardiomyocyte proliferation and Our recent studies showed that REST represses transcription in chamber development. mouse embryonic hearts20. Based upon this, we did a time course of REST expression levels at various developmental stages. By western blot, we found that REST was downregulated in the Rest is required for neonatal cardiomyocyte proliferation.To ventricles of neonatal hearts (Supplementary Fig. 1a, b). Further, determine whether Rest is also requirement for neonatal cardio- immunostaining showed that REST was expressed in the majority myocyte proliferation, we deleted Rest at P1, P3, or P7 using an MerCreMer imKO of cardiomyocytes between embryonic day (E) 11.5 and postnatal inducible TnT driver (Rest , thereafter) (Fig. 2a, b). day (P) 3, whereas the number of REST-expressing cardiomyo- Rest inactivation at P1, but not at P3 or P7, resulted in the death cytes was drastically reduced from P3 to P28 (Supplementary of ~80% of neonates within 1 week after tamoxifen injection Fig. 1c, d). The downregulation of REST protein level was not (Fig. 2c). Based upon this, we focused on inactivating Rest at P1 accompanied by a change in mRNA level (Supplementary and investigating the resultant changes in cardiac morphology imKO Fig. 1e), and was possibly due to the REST protein and function at P3. H&E staining showed that the Rest hearts degradation21,25. were underdeveloped with a reduced ratio of the heart/body Given that downregulation of REST coincided with the cell weight (Fig. 2d). Echocardiography revealed a noticeable reduc- cycle exit of cardiomyocytes5, we examined the relationship of tion in the left ventricular mass, left ventricular wall thickness,

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

ab cd KO 120 Control Rest mKO Control p21 mKO KO mKO Rest DKO p21 DKO 100 ControlRest 60 REST 121 KDa Control (n=30) 80 m) 50 * * Rest mKO (n=82) µ p21 21 KDa 60 p21KO (n=20) 40 p21KO DKO Cyclin B 63 KDa 40 DKO (19) 30 53 KDa 30 Cyclin E Survival rate (%) Survival rate 20 p<0.001 10 CDK1/2 33 KDa

0 µ ( thickness Wall E12.5 100 m 0 GAPDH 36 KDa P1 P28 P63 E10.5 E16.5

KO e Control Rest mKO p21KO DKO Control p21 Rest mKO DKO 80 * 60 * TnT / 40

Ki67 20

Ki67/TnT (%) 0

60 * 50 * TnT / 40 30 EdU 20 10 EdU/TnT (%) 0 4 * 3 * TnT / 2

PH3 1

PH3/TnT (%) 0

2.5 * TnT

/ * 2.0 1.5 1.0 Aurora B Aurora 0.5 0 Aurora B/TnT (%) Aurora fghi 8 100 60 NS NS Control p21KO DKO NS NS NS NS 50 6 80 40 60 4 30 40

LVFS (%) LVFS 20 2 (%) LVEF 20 P28 10

Heart/body (mg/g) weight 0 0 0 Control p21KO DKO Control p21KO DKO Control p21KO DKO

Fig. 4 p21 deletion rescues embryonic heart defects resulting from myocardial Rest deletion. a Survival curve, no difference between control and p21 null (p21KO)orp21/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-stained E12.5 heart sections showing that thin ventricular walls of Rest deleted hearts are restored by p21 deletion (n = 3/group). Scale bar = 100 μm. d Representative western blot revealing that upregulation of p21 and downregulation of Cyclin B, Cyclin E, and CDK1/2 in E12.5 Rest null hearts are restored to the normal levels of control hearts by p21 deletion. e Immunostaining for cell cycle markers indicating that p21 inactivation rescues the cell cycle defect resulting 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), and echocardiography (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 and contractility (Fig. 2e–g). Flow cytometry assays further neonatal cardiomyocyte proliferation through maintaining cell imKO revealed a reduced percentage of cardiomyocytes in Rest cycle progression, and neonatal cardiac growth and function. hearts (Fig. 2h). The null cardiomyocytes were accumulated at the G0/G1 phase of cell cycle, while their presence at the S and G2/M phases was reduced (Fig. 2i, j). Immunostaining for Ki67 and REST binds and represses p21 to regulate cardiomyocyte pro- EdU confirmed the cell cycle defect (Fig. 2k). In contrast, Rest liferation. To identify the downstream factor(s) of REST involved deletion at P3 or P7, when cardiomyocytes were exiting the cell in the cell cycle regulation, we re-analyzed our previously pub- mKO cycle, had no effect on cardiac morphology and function (Sup- lished RNA-seq data from E12.5 control versus Rest hearts20 plementary Fig. 4a–d). Therefore, Rest is also required for and found that p21 (Cdkn1a) was the only cell cycle inhibitor

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abc120 imKO KO Control Rest Control p21 imKO 100 Rest DKO 8 80 Control (n=30) * * imKO 60 Rest (n=22) 6 KO p21 (n=25) p21KO DKO 40 4

Survival (%) DKO (n=20)

20 p<0.001 2 0

P0 2 4 6 810121416 P3 Heart/body (‰) weight 0

imKO d KO e KO f KO Control p21 Control p21 imKO DKO imKO Control Rest p21 DKO 120 Rest 90 Rest DKO REST 121 KDa * * 75 * * 90 p21 21 KDa 60 Cyclin B 63 KDa 60 45 Cyclin E 53 KDa

LVFS (%) LVFS 30 LVEF (%) LVEF 30 CDK1/2 33 KDa 15 GAPDH 36 KDa 0 0

KO g Control Rest imKO p21KO DKO Control p21 Rest imKO DKO 15 * * DAPI / 10 TnT / 5 Ki67 Ki67/TnT (%) 0 20 * * DAPI

/ 15 TnT

/ 10 5 EdU EdU/TnT (%) 0 2.5 * * 2.0 DAPI / 1.5 TnT / 1.0

PH3 0.5 PH3/TnT (%) 0 1.5 DAPI

/ ** 1.0 TnT / 0.5 Aurora B Aurora

Aurora B/TnT (%) Aurora 0.0

Fig. 5 p21 deletion rescues neonatal heart defects resulting from Rest deletion. a Survival curve, no difference between control and p21 null (p21KO)orp21/ 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 heart sections showing that underdeveloped Rest null hearts are restored by p21 deletion (n = 5/group). Scale bar = 100 μm. d, e Echocardiograph showing p21 knockout rescues cardiac function resulting from Rest deletion at P1 (n = 6–8/group). f Representative western blot revealing upregulation of p21 and downregulation 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. g Immunostaining for cell cycle markers indicating p21 inactivation rescues the cell cycle defect resulting from Rest deletion (n = 4/group). Scale bar = 40 μm. Controls were the Tam-treated Rest+/+;TnTMerCreMer/+ mice. Mean ± s.d. *p < 0.05 by one-way ANOVA followed by Tukey’s test gene significantly upregulated in Rest null hearts, whereas the Luciferase reporter gene assays and mutational analyses revealed expression of several cell cycle activators was downregulated that the p21-RE1 motif was required for repressing transcription (Supplementary Fig. 5a). These findings were then confirmed by in cultured P1 mouse cardiomyocytes (Fig. 3d). Quantitative qRT-PCR (Supplementary Fig. 5b, c). Notably, p21 was also the chromatin immunoprecipitation assays (qChIP) showed REST only cell cycle inhibitor gene which expression was significantly occupancy of the p21-RE1 motif in E12.5 and P1 ventricles, but upregulated in P2 ventricles with myocardial Rest inactivation at the occupancy was greatly reduced in the ventricles of P21 when P1 (Supplementary Fig. 5d). Immunostaining further showed an REST level was largely diminished (Fig. 3e). Further, qChIP increased number of p21-expressing cardiomyocytes in E12.5 and showed that Rest deletion resulted in an enrichment of H3K4me3 P3 Rest null hearts (Fig. 3a, b). We identified one conserved histone modification and a reduction of H3K9me3 and REST-binding site (RE1 motif) located at the 3′ end of mouse p21 H3K27me3 histone marks at the p21-RE1 motif (Fig. 3f). These gene by sequence alignment between mouse and human (Fig. 3c). findings support that REST directly binds p21 and represses its

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

a REST/TnT/DAPI EdU/TnT/DAPI pH3/TnT/DAPI Aurora B/TnT/DAPI Sham-APB AR-APR AR-APB P7 2

b 2 2 2 100 * 600 600 * * 20 * 500 500 80 15 400 400 * * * 60 300 300 NS 10 200 200 40 5 100 100 20 PH3+ CMs/mm 0 EdU+ CMs/mm 0 0 0 REST+ CMs/mm Aurora B+ CMs/mm Aurora

Sham APR APB Sham APR APB Sham APR APB Sham APR APB

800 100 2 30 r=0.808 r=0.764 r=0.567 2 2 25 600 p<0.001 80 p<0.001 p<0.001 20 60 400 15 40 10 200 20 5 PH3+ CMs/mm EdU+ CMs/mm

0 0 B+ CMs/mm Aurora 0 0 100 200 300 400 500 600 0 100 200 300 400 500 600 0 100 200 300 400 500 600 REST+ CMs/mm2 REST+ CMs/mm2 REST+ CMs/mm2

c DAPI EdU REST DAPI/EdU/REST 80 60 40 20 0

P7 EdU+/REST+ (%) P7 P14 P28

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. Average counts 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 by Tukey’s test. APB/APR apex border/apex remote. c Co-immunofluorescence showing consistently ~60% of EdU+ cells express REST (arrow) in regenerating neonatal hearts. Scale bar = 10 μm. Data are presented as mean ± s.d. (n = 4/stage) transcription in cardiomyocytes, possibly through histone as well as the expression of cell cycle activators and markers methylation changes26. (Fig. 5f, g). We also examined whether forced overexpression of To determine whether such repression promoted cardiomyo- REST would promote cardiomyocyte proliferation in cultured cyte proliferation during cardiac development, we deleted p21 in cardiomyocytes isolated from P1 or P5 hearts. We first treated the mKO Rest mice by breeding them with p21 knockout mice27. culture with siRNAs against β-TrCP that mediates REST Inactivation of p21 rescued embryonic lethality and cardiac degradation via ubiquitination21,25. The results of western blot mKO phenotypes of Rest mice at E12.5 (Fig. 4a–c). Inactivation of for REST, qRT-PCR for Rest, and REST-targeted genes and p21 also restored the levels of Cyclin B, Cyclin E, and CDK1/2 immunostaining for EdU and PH3 showed that downregulation (Fig. 4d), and re-established normal cardiomyocyte cell cycle of β-TrCP slowed down the REST degradation (Supplementary and proliferation, as indicated by recovered expression of Fig. 6a), suppressed the expression of REST target genes (Hcn2, Ki67, PH3, and Aurora B, and EdU incorporation (Fig. 4e). p21, Nppa, and Syn1) (Supplementary Fig. 6b), and upregulated Double knockout (DKO) mice were survived to birth and cardiomyocyte proliferation (Supplementary Fig. 6c). Next, we appeared healthy postnatally and in adulthood, with normal overexpressed REST by transfection of a plasmid (pHR-NRSF- cardiac morphology, heart/body weight ratio, and cardiac CITE-GFP) containing Rest cDNA28 and found by western blot function (Fig. 4f–i). Similarly, inactivation of p21 rescued and immunostaining for EdU and PH3 that overexpression of postnatal lethality of mice caused by inactivation of myocardial REST dose-dependently repressed p21 expression and increased Rest at P1 (Fig. 5a). Cardiac phenotypes were rescued, including cardiomyocyte proliferation (Supplementary Fig. 6d–f). These the cardiac size, heart/body ratio, cardiac function (Fig. 5b–e), findings, therefore, identify that a Rest-p21 axis underlies the

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ab5 P14 * 4 P7 P28 3 Control NS * 2

1 Fibrosis area (%)

imKO 0 Rest imKO imKO imKO t Control Control Control P7 P14 P28 Rest Rest Res

cdP7 P14 P28 P7 P14 P28 100 70 * * * * * ** * * * NS * 90 * * 60 NS * * 80 50 *

70 40 LVEF (%) LVEF LVFS (%) LVFS

60 30

50 20 AR AR AR AR AR AR AR AR AR AR AR AR Sham Sham Sham Sham Sham Sham Sham Sham Sham Sham Sham Sham

Control Rest imKO ControlRest imKO Control Rest imKO Control Rest imKO ControlRest imKO Control Rest imKO

e P7 P14 P28 Control Rest imKO ControlRest imKO Control Rest imKO TnT / REST TnT / EdU

f g 600 600 Control * Control imKO * Rest imKO Rest 500 500 2 2 400 400

300 300

200 * 200

* EdU+ CMs/mm * REST+ CMs/mm 100 100 *

0 0 P7 P14 P28 P7 P14 P28

Fig. 7 Rest is required for regeneration of injured neonatal hearts. a, b Quantitative Masson’s trichrome staining shows increased cardiac fibrosis in mice with induced myocardial Rest deletion at P3 and injured by apex ablation at P4, n = 5–8/group. Scale bar = 100 μm. c, d Echocardiography indicating reduced cardiac function of myocardial Rest null mice after injury, n = 6–8/group. e–g Quantitative immunofluorescence (n = 4/group) showing decreased EdU+ 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’s test in b–d; unpaired two-tailed Student’s t test in f, g regulation of cardiomyocyte cell cycle for fetal and neonatal previous reports4,6, immunostaining for the cell cycle markers cardiac development. showed a significantly increased number of cardiomyocytes that re-entered the cell cycle in the injured region at P7, 3 days after ablation at P4 (Fig. 6a). Notably, the number of REST-expressing Rest is required for regenerative cardiomyocyte proliferation. cardiomyocytes was also increased in the same region and was To further investigate whether REST had a role in cardiac correlated with the increased number of cells undergoing DNA regeneration, we investigated Rest expression in repairing neo- synthesis, mitosis, and division (Fig. 6b). The percentage of EdU+ natal hearts using an apical resection model4. Consistent with cells that also expressed REST was sustained in the regenerating

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

abP28 P28 100 c 70 Schematic model of heart regeneration * * 90 60 ** s-Control TAM 80 60 Control 70 P3 P4 P7 P14 P28 40 imKO 60 Rest (%) LVEF LVFS (%) LVFS 30 p21KO 50 Apical & DKO 40 20 resection functional assays KO KO imKO DKO imKO DKO Control p21 Control p21 s-Control Rest s-Control Rest

d 2.5 Masson staining (P28) * * 2.0 imKO KO s-Control Control Rest p21 DKO 1.5 1.0 * 0.5

Fibrosis area (%) 0.0

KO imKO DKO Control p21 s-Control Rest e EdU/TnT/DAPI (P28) ) 50 2 * 40 s-Control Control Rest imKO p21KO DKO 30 * * 20 10

EdU+ CMs (mm 0

KO imKO t DKO Control p21 s-Control Res

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 and p21 (DKO, n = 8) injured hearts have cardiac function comparable to that of the sham-operated control (s-Control, n = 9) at P28. Injured Rest single knockout (RestimKO, n = 9) hearts have significantly decreased left ventricular function comparable to injured control. d, e Quantitative Masson’s trichrome staining (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 fibrosis and rescues cardiomyocyte proliferation defect in the injured apex resulting from Rest deletion. Mean ± s.d. *p < 0.05 by one-way ANOVA followed by Bonferroni test. NS no significance myocardium from P7 to P28 (Fig. 6c). About 90% of REST- which was accompanied with downregulation of the cell cycle expressing cells in the apex undergoing repair were cardiomyo- activators (Supplementary Fig. 8e, f). To determine whether p21 cytes; the reminder was epicardial or endocardial cells (Supple- upregulation was responsible for the reduced proliferation of mentary Fig. 7a–d). These findings indicated that the majority of cardiomyocytes and poor regeneration of myocardium after Rest REST-expressing cells were proliferating cardiomyocytes induced deletion, we performed the apical resection on the Rest and p21 by injury. We deleted Rest in the myocardium at P3 and per- DKO hearts at P4, 1 day after Rest deletion (Fig. 8a). Like control −/− formed apical resection at P4. The P3 deletion bypassed the death hearts, p21 hearts had normal left ventricular function at P28 −/− of neonates when Rest was deleted at P1, while apical ablation at (Fig. 8b, c). We noted that p21 hearts had less fibrotic scar P4 was still within the window of myocardial regeneration2.We tissue as compared to controls (Fig. 8d). Importantly, p21 deletion imKO found by Masson’s trichrome staining that the fibrotic scar in the improved regeneration and function of Rest hearts and imKO apex of Rest null hearts (Rest ) was not resolved at P14 and restored the number of EdU+ cardiomyocytes in the injured P28 compared to the control hearts (Fig. 7a, b). Consistent with myocardium (Fig. 4e). These results demonstrate that the Rest- imKO the persistent scaring phenotype, Rest hearts had reduced left p21 axis regulation of cardiomyocyte proliferation also underlies ventricular function as observed by echocardiography (Fig. 7c, d). the neonatal cardiac regeneration. Further, immunostaining revealed that the number of EdU+ imKO proliferating cardiomyocytes in the injured apex of Rest hearts was significantly reduced (Fig. 7e–g). Together, these Discussion findings suggest that REST re-expression is necessary for the Overall, our study identifies a molecular mechanism by which regeneration of neonatal hearts. REST repression of the cell cycle inhibitor p21 is necessary and The increased number of REST-expressing cardiomyocytes in sufficient for promoting the cardiomyocyte cell cycle and pro- the regenerating neonatal hearts was correlated with increased liferation during cardiac development and regeneration. p21 is a REST occupancy at the p21-RE1 motif (Supplementary Fig. 8a). well known cell cycle inhibitor through interacting with the G1/M Concurrently, the regenerating myocardium expressed a low level and G2/S CDK cyclins and inhibits their function to drive the cell of p21 mRNA (Supplementary Fig. 8b). Immunostaining also cycle29. Of particular note, p21 is the only cell cycle inhibitor gene showed that the number of p21-expressing cardiomyocytes was significantly repressed by REST in the proliferating cardiomyo- negatively correlated with that of REST-expressing cells in the cytes during cardiac development and regeneration, suggesting regenerating myocardium (Supplementary Fig. 8c, d). In contrast, p21 imKO that REST repression of is a major mechanism underlying the p21 transcription was de-repressed in the injured Rest apex, regulation of cardiomyocyte cell cycle. Consistent with this

NATURE COMMUNICATIONS | 8: 1979 | DOI: 10.1038/s41467-017-02210-y | www.nature.com/naturecommunications 9 ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/s41467-017-02210-y notion, p21 deletion has been reported to enhance the regen- OPTIMEM (Invitrogen) for 15 min at RT, and then added to cells and incubated eration of injured appendage30 and neonatal hearts31. Interest- for indicated time. qRT-PCR and western blot were used to confirm the efficiency ingly, GATA432 and MEIS119 also repress p21 transcription in and effect of transfection. mouse embryonic and injured neonatal hearts, respectively. Since 38 neither GATA4 nor MEIS1 expression is affected by REST ChIP-qPCR. ChIP-qPCR assays were performed as previously reported . Ven- p21 tricles (30–40 mg) from indicated stages were treated with 1% formaldehyde at inactivation, REST repression of is likely independent of room temperature for 10 min to crosslink protein and DNA complexes before GATA4 and MEIS1. REST thus joins a network of transcription quenched with addition of glycine. After three washes with PBS, tissues were factors or cofactors, including MEIS119, YAP15,16, GATA412,33,34, homogenized, lysed, and sonicated to shear DNAs into 100–500 bp fragments. The and TBX2013, as a new member essential for cardiac development DNA 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), and regeneration. Tri-Methyl-Histone H3 (Lys27) (CST mAb #9733, 1:100), Tri-Methyl-Histone H3 Elucidating the Rest-p21 axis underlying the regulation of (Lys4) (CST mAb #9751, 1:100), or Acetyl-Histone H3 (Lys9) (CST mAb #9649, cardiomyocyte cell cycle may have clinical implications by pro- 1:100) overnight at 4 °C using Dynabead Protein G in a final volume of 500 µl and viding an opportunity for better understanding of cardiomyocyte Magnet Starter Pack (Invitrogen, 10004D). Five microgram IgGs (Abcam, ab171870) were used as control. Immunoprecipitated DNAs were extracted and proliferation in cardiac regeneration. It is well known that the cell used for qPCR analysis to determine the enrichment of DNA fragments containing cycle arrest of cardiomyocytes is a normal physiological feature of the p21-RE1 using specific primers (Supplementary Table 1). adult hearts5. This, however, prevents the endogenous regenera- tion of infarcted adult hearts from the proliferation of pre- Western blot. Ventricles were collected at the indicated stages, homogenized in 11,35 existing cardiomyocytes . Our studies indicate that REST is radioimmunoprecipitation assay buffer at 4 °C and quantified by bicinchoninic rapidly diminished in the postnatal heart, but rebounded after acid assay. Equal total (20 μg) were resolved in SDS-PAGE gels and injury. These observations suggest that REST downregulation transferred onto polyvinylidene fluoride membranes (Immobilon, Millipore). The membranes were blocked and incubated with primary antibodies at 4 °C overnight might be one of the limiting factors for sustaining proliferation of before incubated with HRP-conjugated secondary antibodies. Protein bands were postnatal cardiomyocytes. Future investigations are needed to detected by enhanced chemiluminescence. Antibodies, including REST (Anti- determine whether REST is reactivated in adult hearts by injury bodies-online, ABIN747683, 1:1000), P21 (Abcam, ab109199, 1:500), Cyclin E and if the reactivation promotes adult cardiac regeneration by (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, modulation of the activity of cardiomyocyte cell cycle. 1:5000), were used in western blot analysis. Full scans of western blots are shown in Supplementary Figs. 9–14. Methods Rest RestGT/GT Mouse strains. Floxed mice ( ), which carries a conditional gene trap Echocardiography. Echocardiography was performed using a Vevo 770 ultrasound 23 TnTCre36 TnTMerCreMer37 Rest (GT) cassette , were bred to or mice to obtain system (Visual Sonics, Toronto, Canada). Investigators were blinded to genotype deletion in myocardium. Intraperitoneal tamoxifen (TAM) injection at 40 mg/kg and treatment groups. Mice were anesthetized with 1.5% isoflurane and mouse TnTCre RestGT/GT was used for inducible inactivation. For rescue experiments, ; hearts were viewed on two-dimensional short-axis planes and measured using M- RestmKO TnT MerCreMer;RestGT/GT RestimKO p21−/− ( ), or ( ) mice were crossed to mode. Heart rate, left ventricular internal diameter, interventricular septal, and Cdkn1a−/− 27 ( ) mice . All mouse stains were in the C57B6 background. Embryos posterior wall (PW) dimensions were measured at end ystole and end diastole for were isolated and inspected according to expected developmental ages. Age- at least three cardiac cycles. Fractional shortening (FS) were calculated by the matched embryos and neonatal mice of both sexes were grouped according to average of FS measurements of the left ventricular contraction for five cardiac genotypes during experiments. Noontime on the day of detecting vaginal plugs was cycles. designated as E0.5, and morning on the day of observing newborns was designated as P0. The yolk sac or tail of mice was used for genotyping by PCR using Cre and allele-specific primers (Supplementary Table 1). Mouse housing and experiments Quantitative reverse transcription PCR (qRT-PCR). Total RNAs were extracted were according to the protocols approved by the Institutional Animal Care and Use from ventricles or cultured CMs using TRIzol (Life Technologies). One microgram Committee (IACUC) of Albert Einstein College of Medicine. of total RNAs was reverse transcribed to cDNA using a Superscript II reverse transcriptase Kit (Life Technologies). qRT-PCR was performed using Power SYBR Green PCR Master Mix (Life Technologies) and carried out on an Applied Bio- Neonatal heart apical resection. Neonatal mouse heart apical resection (AR) was fi 4 systems 7900HT Fast Real-Time PCR System. Speci c primers were listed in performed on postnatal day (P) 4 mice of both sexes as previously described . Supplementary Table 1. The relative expression of each gene was normalized to the fl Rest −ΔΔ Brie y, we induced deletion of in the myocardium at P3 by tamoxifen and expression of Gapdh and calculated using the 2 CT method. Biological replicates performed apical resection at P4. P4 neonates were anesthetized by inhalation of fl were performed using three individual samples of each genotype and technical 2% iso urane and 100% oxygen on ice and then subjected to intercostal incisions to triplicates were carried out for each run of qPCR. separate the pericardium and expose the apex. The apex was amputated with microsurgical scissors under a dissecting microscope. About 10% of the heart was surgically removed to produce reproducible regeneration. The chest wall was then Luciferase reporter gene assay. Transcriptional suppression of p21 via the p21- closed with sutures and the skin incision was closed using skin adhesive. After the RE1-pGL3 enhancer by REST was evaluated using luciferase reporter gene assay as operation, the neonates were placed back with the feeding female mice. Sham- described previously20. DNA fragment of p21 gene with or without mutation or operated mice underwent the same procedure without apical resection. deletion of the p21-RE1 was PCR cloned into a PGL3 enhancer luciferase reporter vector (Supplementary Table 1). All constructs were confirmed by Sanger sequencing. 0.8 µg of each p21-RE1-pGL3 enhancer construct was co-transfected Histology. Embryos or isolated hearts were fixed in 4% paraformaldehyde (PFA), µ fi μ with 0.2 g of pRL-SV40 vector (Promega, E2231) into cultured P1 CMs in 96-well dehydration, embedded in paraf n, and sectioned frontally at 6- m thickness. H&E plates using Lipofectamine 3000 Reagents (Invitrogen). After 24 h transfection, cell and Masson’s trichrome staining were performed for detecting cardiac structure fi fi fi lysates were processed for luciferase activity using the luciferase reporter system defects and brosis, respectively. The brotic area was quanti ed using the (Promega, E1980). Luciferase reporter activities were calculated as firefly luciferase MetaMorph software (Molecular Devices). normalized to Renilla luciferase luminescence. Biological triplicates were per- formed in technical triplicates for each vector. Primary cardiomyocyte cultures. Cardiomyocytes (CMs) were isolated as described in previous studies20. Ventricles were dissected out from P1 or P5 Immunostaining. Immunostaining was performed on sections of paraffin- neonates of both sexes, minced and dissociated by 0.045% collagenase II and 0.08% embedded embryos or hearts, as previously described39. Embryos or hearts were trypsin-EDTA at 37 °C. Non-CMs were removed through adhesive plating. Purified collected at the indicated stages and fixed in 4% PFA for 2 h, rinsed in PBS, CMs were cultured in 12-well plates for 24 h at 37 °C with 5% CO in Dulbecco’s 2 dehydrated in 15 and 30% sucrose sequentially, embedded in optimal cutting modified Eagle’s minimum essential medium (DMEM) supplemented with 10% temperature (OCT) compound and sectioned at 8-μm thickness. Heart sections fetal calf serum. were blocked with 5% horse serum for 1 h at room temperature before being incubated overnight at 4 temperature with the single or double staining with fol- siRNA and cDNA transfection. siRNA against β-TrCP (si-β-TrCP, Santa Cruz lowing antibodies diluted in blocking solution: TnT (Fisher Scientific, MS-295-P0, Biotechnology) or plasmids containing mouse Rest cDNA (pHR-NRSF-CITE-GFP, 1:400), PECAM1 (Santa, sc-1506, 1:400), REST (Antibodies-online, ABIN747683, Addgene) was transfected into cultured neonatal cardiomyocytes isolated from P1 1:400 dilution), Ki67 (Abcam, ab66155, 1:200), P21 (Abcam, ab109199, 1:100 or P5 neonatal hearts, at 70% confluency. Lipofectamine 3000 Regent (Invitrogen) dilution), cleaved caspase-3 (CST, mAb #9664, 1:200), phosphorylated-histone3 and si-β-TrCP (40 nM) or REST-cDNA (0.5 or 2 µg/ml) were incubated in (pH3) (Abcam, ab32107, 1:200), or aurora B (Abcam, ab2254,1:100). After three

10 NATURE COMMUNICATIONS | 8: 1979 | DOI: 10.1038/s41467-017-02210-y | www.nature.com/naturecommunications NATURE COMMUNICATIONS | DOI: 10.1038/s41467-017-02210-y ARTICLE washes with PBS for 5 min each, samples were stained for 1 h at room temperature 2. von Gise, A. et al. YAP1, the nuclear target of Hippo signaling, stimulates heart with fluorescent secondary antibodies followed by DAPI (4′,6-diamidino-2-phe- growth through cardiomyocyte proliferation but not hypertrophy. Proc. Natl nylindole dihydrochloride) staining for nuclei visualization. Images were acquired Acad. Sci. USA 109, 2394–2399 (2012). and analyzed using a Zeiss fluorescence microscope with Axiovision image analysis 3. Pasumarthi, K. B. & Field, L. J. Cardiomyocyte cell cycle regulation. Circ. Res. software. For paraffin sections, 6-µm tissue sections were de-waxed in xylene, 90, 1044–1054 (2002). rehydrated in descending grades of alcohol and antigen-retried by incubating in 4. Porrello, E. R. et al. 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Images were taken from six (2013). fields per section, processed using ImageJ software and counted blindly to genotype Dev. Cell 36 and treatment groups. 7. Uygur, A. & Lee, R. T. Mechanisms of cardiac regeneration. , 362–374 (2016). 8. Bergmann, O. et al. Dynamics of cell generation and turnover in the human EdU incorporation. Proliferative cells were pulse labeled for 2 h by intraperitoneal heart. Cell 161, 1566–1575 (2015). injection of mice with 5-ethynyl-2′-deoxyuridine (EdU, 100 mg/kg). Hearts were 9. Soonpaa, M. H. et al. Cardiomyocyte cell-cycle activity during Preadolescence. then collected, fixed in 4% PFA, rinsed in PBS, soaked in 15 and 30% sucrose Cell 163, 781–782 (2015). μ sequentially, embedded in OCT, and sectioned at 8- m thickness. Sections were 10. Walsh, S., Ponten, A., Fleischmann, B. K. & Jovinge, S. 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Acknowledgements Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, We thank B. Morrow, R.N. Kitsis, and N.G. Frangogiannis for critically reading the GT/GT adaptation, distribution and reproduction in any medium or format, as long as you give manuscript. We also thank T. Floss and T. Nechiporuk for Rest mice. This work appropriate credit to the original author(s) and the source, provide a link to the Creative was supported by the National Institutes of Health/National Heart, Lung, and Blood Commons license, and indicate if changes were made. The images or other third party Institute grants (R01HL116997 to B.Z.; R01HL133120 to B.Z., and De.Z.; R01HL131735 material in this article are included in the article’s Creative Commons license, unless to C.C.), and an American Heart Association grants (16POST30480021 to Do.Z.; indicated otherwise in a credit line to the material. If material is not included in the 15GRNT25710153 to C.C.). article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from Author contributions the copyright holder. To view a copy of this license, visit http://creativecommons.org/ Do.Z., Y.W., P.L., X.Y., and B.W. performed most of the experiments. P.W. and De.Z. licenses/by/4.0/. MerCreMer helped RNA-sequence data analysis. J.Y. and C.C. generated TnT mice. Do.Z. and B.Z. conceptualized the project. B.W. and B.Z. supervised the experiments. C.-P.C. offered help in data analysis and manuscript revision. Do.Z. and B.Z. wrote the manuscript. © The Author(s) 2017

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