© 2013. Published by The Company of Biologists Ltd | Development (2013) 140, 4683-4690 doi:10.1242/dev.102798

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ABSTRACTHeart failure due to cardiomyocyte loss after ischemic heart diseaseis the leading cause of death in the United States in large partbecause heart muscle regenerates poorly. The endogenousmechanisms preventing mammalian cardiomyocyte regeneration arepoorly understood. Hippo signaling, an ancient organ size controlpathway, is a kinase cascade that inhibits developing cardiomyocyteproliferation but it has not been studied postnatally or in fully matureadult cardiomyocytes. Here, we investigated Hippo signaling in adultcardiomyocyte renewal and regeneration. We found that unstressedHippo-deficient adult mouse cardiomyocytes re-enter the cell cycleand undergo cytokinesis. Moreover, Hippo deficiency enhancescardiomyocyte regeneration with functional recovery after adultmyocardial infarction as well as after postnatal day eight (P8) cardiacapex resection and P8 myocardial infarction. In damaged hearts,Hippo mutant cardiomyocytes also have elevated proliferation. Ourfindings reveal that Hippo signaling is an endogenous repressor ofadult cardiomyocyte renewal and regeneration. Targeting the Hippopathway in human disease might be beneficial for the treatment ofheart disease.

KEY WORDS: Cell cycle, Proliferation, Regeneration, Mouse

INTRODUCTIONWhereas other organs have some regenerative capacity, heart muscleor cardiomyocytes fail to renew or regenerate sufficiently to repairthe damaged heart. Although both cardiac stem cells andendogenous cardiomyocyte renewal have been described, theseendogenous mechanisms are overwhelmed in the face of acutecardiomyocyte loss (Kikuchi and Poss, 2012). This clinical realityhas prompted multiple efforts to supplement human damagedmyocardium with exogenous cells, with some successes reported(Chugh et al., 2012). In addition to cell therapy, addition ofexogenous factors such as periostin, neuregulin 1 and microRNAshave been shown to promote cardiomyocyte renewal (Eulalio et al.,2012; Kikuchi and Poss, 2012; Boon et al., 2013; Porrello et al.,2013). However, the endogenous inhibitory mechanisms preventingcardiomyocyte renewal and regeneration are poorly understood.

The mammalian core Hippo signaling components include theSte20 kinases Mst1 and Mst2 (also known as Stk3), which areorthologous to the Drosophila kinase Hippo. Mst kinases,complexed with the salvador (Sav; also known as Salv) scaffold

RESEARCH ARTICLE STEM CELLS AND REGENERATION

1Department of Molecular Physiology and Biophysics, Baylor College ofMedicine, One Baylor Plaza, Houston, TX 77030, USA. 2Texas Heart Institute,Houston, TX 77030, USA. 3Program in Developmental Biology, Baylor College ofMedicine, One Baylor, Plaza, Houston, TX 77030, USA. 4Department ofBiochemistry and Molecular Biology, MD Anderson Cancer Center, Houston, TX 77030, USA.*These authors contributed equally to this work

‡Author for correspondence (jfmartin@bcm.edu)

Received 21 August 2013; Accepted 4 September 2013

protein, phosphorylate the large tumor suppressor homolog (Lats)kinases. Mammalian Lats1 and Lats2 are NDR family kinases andare orthologous to Drosophila Warts. Lats kinases, in turn,phosphorylate Yap and Taz, two related transcriptional co-activatorsthat are the most downstream Hippo signaling components andpartner with transcription factors, such as Tead, to regulate geneexpression. Upon phosphorylation, Yap and Taz are excluded fromthe nucleus and rendered transcriptionally inactive.

Previous cardiac loss-of-function studies in mice revealed thatHippo signaling inhibits cardiomyocyte proliferation duringdevelopment to control heart size (Heallen et al., 2011). Salv-deficient hearts develop severe cardiomegaly with a 2.5-foldincrease in heart size. Additionally, experiments investigating Yapin cardiomyocyte development support the conclusion that Yap isthe major Hippo effector molecule during cardiomyocytedevelopment (Xin et al., 2011; von Gise et al., 2012). These earlierstudies also uncovered important interactions between Hippo andcanonical Wnt and insulin-like growth factor signaling, suggestingthat Hippo may represent a regulatory hub during cardiomyocytedevelopment. Although Hippo pathway kinases have beeninvestigated using dominant-negative approaches, direct loss-of-function genetic evidence is lacking in the postnatal and adult heart(Odashima et al., 2007; Matsui et al., 2008).

To investigate Hippo signaling in postnatal cardiomyocyterenewal and regeneration, we inactivated Salv and both Lats1 andLats2 (hereafter Lats1/2) in the postnatal heart. Hippo pathwayinactivation in the unstressed adult mouse heart inducedcardiomyocyte renewal. Moreover, Hippo deficiency promotedefficient heart regeneration in both postnatal cardiac apex resectionand adult myocardial infarction models revealing a crucial,inhibitory role for Hippo signaling in cardiomyocyte renewal andregeneration.

RESULTSHippo inhibits adult cardiomyocyte renewalTo test the role of Salv, Lats1 and Lats2 in adult cardiomyocytes, weused conditional null alleles for Hippo genes and the Myh6creERT2

transgene, which directs tamoxifen-regulated cardiomyocyte creactivity (Sohal et al., 2001). Because the heart contains multiple celltypes, we visualized cardiomyocytes using the R26mTmG (mTmG)allele, which expresses eGFP upon cre activation, to trace thecardiomyocyte lineage (Muzumdar et al., 2007).

We generated adult cardiomyocytes that were mutant for Salv andLats1/2 by injecting three-month-old mice with tamoxifen (Fig. 1A).Efficient deletion of Hippo components was determined byimmunohistochemistry with antibodies for phosphoYap (pYap) andSalv (Fig. 2D; supplementary material Fig. S2). To determinewhether Hippo deficiency results in cell cycle re-entry, we alsoinjected mice with 5-ethynyl-2′-deoxyuridine (EdU). Nuclear EdUincorporation, indicating de novo DNA synthesis, was detected inboth Salv conditional knockout (CKO) and Lats1/2 CKO mutantcardiomyocytes revealing an endogenous cardiomyocyte renewal

Hippo signaling impedes adult heart regenerationTodd Heallen1,*, Yuka Morikawa2,*, John Leach1, Ge Tao1, James T. Willerson2, Randy L. Johnson3,4 andJames F. Martin1,2,3,‡

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capacity when Hippo signaling is deleted. In contrast to Hippomutant hearts, control hearts only incorporated EdU in cardiacfibroblasts (Fig. 1A,B). Quantification of EdU-positive cells showedsignificant induction of DNA synthesis in Hippo-deficient heartswith a greater increase in Lats1/2 mutants compared with Salv CKOcardiomyocytes (Fig. 1B). Cell cycle re-entry was also quantified inisolated cardiomyocyte nuclei using fluorescence-activated cellsorting (FACS) analysis (Fig. 1C) (Bergmann et al., 2009). BothLats1/2 CKO and Salv CKO cardiomyocyte nuclei had increasednumbers of Ki-67 (Mki67)-expressing cardiomyocytes comparedwith controls (Fig. 1C; supplementary material Fig. S1A).Moreover, total cardiomyocyte number was increased with moremononuclear cardiomyocytes in Lats1/2 and Salv CKO hearts thanin controls (Fig. 1D,E). Average cardiomyocyte size in these heartswas significantly smaller than that of controls (Fig. 1F). These

results show that cardiomyocytes re-enter the cell cycle upon Hippopathway disruption and support the hypothesis that Hippo signalingis a negative regulator of adult cardiomyocyte renewal.

In addition to de novo DNA synthesis and cell counting, weevaluated whether Salv CKO and Lats1/2 CKO cardiomyocytesprogress through mitosis and cytokinesis using other methods. Weperformed ploidy analysis using FACS to sort nuclei isolated fromcontrol and Hippo-deficient cardiomyocytes (Fig. 1G;supplementary material Fig. S1B). We reasoned that if DNAduplication occurred in the absence of karyokinesis, then total DNAcontent per nuclei in Hippo-deficient hearts would be greater thanthat in controls. Total DNA content was unchanged between controland both the Salv CKO and the Lats1/2 CKO cardiomyocyte nuclei,supporting the notion that Hippo-deficient nuclei re-enter the cellcycle and progress through karyokinesis (Fig. 1G).

RESEARCH ARTICLE Development (2013) doi:10.1242/dev.102798

Fig. 1. Adult cardiomyocyte renewal via deletion of Hippo pathway genes. Lats1/2 and Salv were conditionally deleted in cardiomyocytes using theinducible Myh6-Cre/Esr line via tamoxifen injection. All analyses were performed with 3-4 month stage hearts. (A) Diagram shows tamoxifen (Tam) and EdUinjection scheme. de novo DNA synthesis was monitored by EdU incorporation (yellow) in cardiomyocytes (green) using the mTmG reporter line.(B) Percentage of EdU-positive cells for each genotype (n=3). EdU-positive cells were manually counted. (C) Percentage of cardiomyocyte nuclei in cell cycle(n=3). Isolated nuclei were stained with the cardiac marker cTnI and the cell cycle marker Ki-67. (D) Cardiomyocyte density. Numbers of cardiomyocytes werecounted in each section (n=3). (E) Cardiomyocyte nucleation. Nuclei per cardiomyocyte was calculated for each genotype (n=3). (F) Cardiomyocyte cross-sectional area was measured and averaged for multiple sections per specimen (n=3). (G) Ploidy analysis (n=3). (H) Immunohistochemical analysis: Aurkb (red)and cardiomyocytes (eGFP, green). Arrowheads indicate Aurkb staining. (I) Quantification of Aurkb-positive cardiomyocytes (n=3). Error bars represent s.e.m.

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We performed immunohistochemistry with the M-phase markerAurora B kinase (Aurkb) to determine if cytokinesis occurred inHippo-deficient adult cardiomyocytes. Aurkb expression in Lats1/2and Salv CKO cardiomyocytes was clearly detectable at thecleavage furrow providing direct evidence for cytokinesis(Fig. 1H,I). In contrast to Hippo-deficient hearts, Aurkb expressionwas barely detected in control hearts (Fig. 1I).

Hippo-deficient postnatal cardiomyocytes regenerateResection of the cardiac apex in the first 6 days of life results incardiac regeneration whereas resections performed at postnatal day(P) 7 or later results in fibrosis and scarring (Porrello et al., 2011).Moreover, the cells that contribute to ventricular wall repair, asdetermined by lineage-tracing experiments, are primarily derivedfrom myosin heavy chain-expressing cardiomyocytes (Porrello etal., 2011). If Hippo signaling represses cardiomyocyte regenerativecapacity beyond P6, then Hippo activity should be higher at P7 andlater stages. Western blots indicated that Hippo activity, as measuredby pYap expression, is low in the P2 regenerative phase hearts. Bycontrast, pYap levels sharply increase at the P10 and P21 non-regenerative stages, consistent with the hypothesis that Hipposignaling represses cardiac regeneration (Fig. 2A).

To test regenerative capability, we performed apex resection ofuniform size at the normally non-regenerative P8 in control andHippo-deficient hearts. To inactivate Salv, we injected mice withfour tamoxifen doses prior to and after the resection (Fig. 2B). BothGFP fluorescence, detecting recombination in the mTmG reporter,and immunofluorescence with an anti-Salv antibody indicated

efficient deletion of Salv in mutant myocardium at 4 and 21 dayspost resection (dpr) and in three-month-old adults after tamoxifeninjection (Fig. 2C,D).

Evaluation of 21 dpr hearts by serial sectioning revealed severescarring of control hearts in all but a few cases (Fig. 2E,F; Fig. 3D-F). By contrast, resected Hippo-deficient hearts efficiently regeneratedthe myocardium with reduced scar size (Fig. 2E,F; Fig. 3D,E).Lineage tracing indicated that the regenerated cardiac apex wasderived primarily from pre-existing alpha myosin heavy chain-expressing cardiomyocytes, although this experiment does not rule outa small contribution from resident stem cells (Fig. 3A-C). In additionto the cardiomyocyte-specific Salv CKO, we also used the Nkx2.5 credriver, which inactivates Salv during development and is known toefficiently delete Salv (Heallen et al., 2011). Nkx2.5cre Salv mutantsalso robustly repaired the heart (Fig. 2F; Fig. 3E). Echocardiographyrevealed that fractional shortening (FS) of resected control hearts wassignificantly reduced whereas resected Hippo-deficient FS resembledsham levels (Fig. 2G), indicating normal contractile function in thesehearts. Histological staining revealed that some Salv CKO resectedhearts appeared slightly more dilated compared with controls(Fig. 2E), although this was not evident by echocardiography. Furtherexperiments are required to determine whether the mild dilation inSalv CKO hearts is progressive or transitory.

Hippo-deficient adult and postnatal cardiomyocytesregenerate after infarctionWe next investigated whether Hippo-deficiency enhanced heartregeneration after myocardial infarction, an experimental system

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Fig. 2. Salvador deletion promotes regenerationof ventricular myocardium. (A) Western blot (left)of postnatal wild-type heart extracts with designatedantibodies. For quantification of western blot (right)pYAP band intensity was normalized against totalYap. (B) Strategy for cardiomyocyte-specificknockout of salvador and apical resection of P8-stage hearts. (C) Cre recombinase reporter activity(eGFP) in 4 dpr control and Salv CKO hearts. (D) 4dpr control and Salv CKO coronal sections: salvador(green) and DAPI (blue). (E) High magnificationinset images of 21 dpr control and Salv CKOtrichrome-stained sections (Fig. 3D). Arrow indicatespersistent fibrotic scar. (F) Scar surface areameasurements were obtained from multiplespecimens and averages calculated. Statisticalanalysis was by unpaired Student’s t-test. *P<0.05,**P<0.01. Salvf/f (n=18), Nkx2.5cre; Salvf/f (n=6), SalvCKO (n=12), Lats1-2 CKO (n=10). (G) Left: M-modeechocardiographic image from a representative 21dpr heart. LV, left ventricle; IVS, interventricularseptum. Right: ejection fraction and fractionalshortening percentages of resected (R) and sham-operated (S) control (wt) and Salv CKO (CKO)hearts at 21-32 dpr. wt/R (n=9), CKO/R (n=6), wt/S(n=3), CKO/S (n=9). Statistical analysis was by one-way ANOVA. *P<0.05, **P<0.01, ***P<0.001. Errorbars represent s.e.m.

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that more accurately models human cardiomyocyte loss secondaryto coronary artery disease. We performed left anterior descending(LAD) coronary artery occlusion at both P8 and two months of age.In P8 hearts, following LAD occlusion we found that there wasfunctional recovery and reduced scar size when analyzed at 21 daysafter occlusion (Fig. 4A-D). Histology also confirmed the recoveryof myocardium with less scar tissue after LAD occlusion (Fig. 4E).In adult hearts, we found similarly strong functional (Fig. 4F-H) andhistological evidence for cardiomyocyte regeneration (Fig. 4I,J) afterLAD occlusion. FS and ejection fraction (EF) evaluated byechocardiography indicated that by three weeks post LADocclusion, adult Hippo-deficient hearts had recovered function to alevel comparable to that of sham-operated animals, suggesting thatHippo-deficient cardiomyocytes have increased survival and/orproliferation after ischemic damage (Fig. 4F,G).

Hippo-deficient cardiomyocytes are more proliferative afterinjuryTo investigate the reparative process in more depth, we evaluated 4dpr (P12) Hippo-deficient hearts after apex resection. Four hoursprior to harvest, hearts were pulsed with EdU to visualize cells thathad entered the cell cycle. In control hearts, EdU-positive cells wereprimarily found in the GFP-negative, non-cardiomyocyte lineagenear the resected zone and are likely to be infiltrating inflammatorycells and proliferating cardiac fibroblasts (Fig. 5A,C). Similarproliferating GFP-negative cells were also observed in Salv CKOhearts (Fig. 5B,D). In contrast to controls, both Salv CKO resectedand sham-operated hearts had EdU/GFP double-positive

cardiomyocytes within both the border zone, or a border zoneequivalent region in shams, and distal heart regions (Fig. 5B,D-G).To determine whether cardiomyocytes were progressing throughcytokinesis after apex resection, we evaluated Aurkb activity. In theHippo-deficient hearts, there was significantly more Aurkb staining,indicating that Hippo-deficient cardiomyocytes were progressingthrough cytokinesis (Fig. 5H). We also determined whether cellcycle progression genes that have been previously shown to beupregulated in developing Hippo-deficient hearts were required forelevated cardiomyocyte proliferation in postnatal Hippo-deficientcardiomyocytes. Small interfering RNA (siRNA) knockdown ofSalv in neonatal cardiomyocytes resulted in enhanced cell cycleentry that was repressed by knocking down Aurkb, Birc5 and Ccne2(Fig. 5I). We conclude that postnatal Hippo deficiency enhances theability of cardiomyocytes to re-enter the cell cycle and progressthrough cytokinesis after apex resection.

To gain more insight into the timing of Hippo-deficientcardiomyocyte proliferation, we evaluated EdU incorporation atmultiple stages. In P8 and P12 unstressed hearts, EdU incorporationwas significantly elevated with increased numbers of cardiomyocytes(Fig. 5J,K). In addition, similar to what was observed in the adultheart, there were increased numbers of mononucleatedcardiomyocytes in Hippo-deficient hearts (Fig. 5J,K).

DISCUSSIONOur findings indicate that Hippo is an endogenous inhibitor of adultcardiomyocyte renewal and regeneration. By inactivating Hippopathway components in postnatal cardiomyocytes, we found that

RESEARCH ARTICLE Development (2013) doi:10.1242/dev.102798

Fig. 3. Lineage tracing of 21 dprhearts. Immunohistochemical analysiswas performed on resected hearts ofcontrol and Salv CKO mice at 21 dpr.(A-B′) Representative images of eGFP(cardiomyocyte-lineage cells, green)and cTnt (cardiomyocytes, red) stainingof control and Salv CKO hearts at theresection site. Dotted lines indicateplane of resection. Salv CKO heartsdisplay repair with cTnt/eGFP-positivecells. B′ shows higher magnification ofthe boxed region in B. (C) Quantificationof eGFP+ cardiomyocytes comparingborder-zone and distal regions of theheart. No significant difference (n.s.)between repaired area and distal areawere found (n=3). (D) Trichrome-stained21 dpr control and Salv CKO serialsections from anterior (left) to posterior(right). (E) Bar graph representingqualitative differences betweengenotypes in scar severity. Control(n=18), Salv CKO (n=12), Nkx2.5cre;Salvf/f (n=6). Statistical analysis was byχ2 distribution. (F) Distribution ofresected apices measured by surfacearea between groups. No significantdifference was found indicatingequivalent resected area betweengroups. Error bars represent s.e.m.

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Hippo-deficient cardiomyocytes regain proliferative andregenerative capacity. Our data provide new insight into the cellularmechanisms underlying cardiac regeneration and indicate that Hipposignaling is a viable target for therapeutic approaches to heartdisease.

Data from human and mouse studies indicate that cardiomyocytesregenerate inefficiently at ~1% per year in young hearts (Bergmannet al., 2009; Kajstura et al., 2012; Mollova et al., 2013; Senyo et al.,

2013). Our data, showing that Hippo-deficient adult cardiomyocytesre-enter the cell cycle and undergo cytokinesis, indicate that Hipposignaling is a major endogenous repressor for cardiomyocyteproliferation. Because cardiomyocyte renewal diminishes with age,it will be important to determine whether Hippo signaling is alsoinvolved in loss of renewal capacity during cardiac aging. Indeed,our preliminary results indicate that Hippo inactivation in eight-month-old hearts leads to cardiomyocyte cell cycle re-entry.

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Fig. 4. Cardiac regeneration and functionalrecovery following myocardial infarction. (A) B-mode echocardiographic image from representative21 day post-occlusion heart. LV, left ventricle; P,papillary muscle. (B,C) Ejection fraction (B) andfractional shortening (C) percentages of P8 LAD-Ohearts. Occluded (O) and sham-operated (S)control (ctrl) and Salv CKO (CKO) hearts at 21-32days post myocardial infarction (dpmi). ctrl/O (n=7),CKO/O (n=9), ctrl/S (n=6), CKO/S (n=9). Statisticalanalysis was by one-way ANOVA with Bonferroni’smultiple comparison test. (D) Infarct surface areameasurements were obtained from multiplespecimens and averages calculated. Statisticalanalysis was by unpaired Student’s t-test. Control(n=6), Salv CKO (n=8). (E) Serial transversesections of 4-week post-occlusion hearts.(F,G) Ejection fraction (F) and fractional shortening(G) percentages of adult LAD-O hearts. 1 week:Ctrl Sham (n=4), Ctrl LAD-O (n=6), CKO Sham(n=3), CKO LAD-O (n=3); 2 weeks: Ctrl Sham(n=3), Ctrl LAD-O (n=5), CKO Sham (n=3), CKOLAD-O (n=2); 3 weeks: Ctrl Sham (n=7), Ctrl LAD-O (n=7), CKO Sham (n=3), CKO LAD-O (n=5).(H) Adult LAD-O infarct size 3 weeks post-occlusion measured as percentage fibrosis of LVmyocardium (total fibrotic area/total myocardialarea×100). Ctrl LAD-O (n=6), CKO LAD-O (n=3).Statistical analysis was by two-way ANOVA withBonferroni’s multiple comparison test. (I) Illustrationof transverse section planes presented in J. Crossindicates plane of occlusion. IVS, interventricularseptum; LV, left ventricle; RV, right ventricle.(J) Serial transverse sections of representative 3-week post-occlusion adult hearts. Allechocardiographic measurements were collectedblind from genotype. Scale bars: 1 mm. *P<0.05,**P<0.01, ***P<0.001.

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It is notable that cell cycle re-entry occurs as an organ-wideresponse in Hippo-deficient hearts at the 4 dpr stage we examinedhere. This is similar to zebrafish and neonatal heart regenerationin which apex resection induces cell cycle re-entry throughout theheart (Kikuchi and Poss, 2012). The timing of activated cell cyclere-entry varies depending on the heart region examined. Inneonatal mouse hearts, cardiomyocyte cell cycle re-entry in thedistal area peaks at 1 dpr but persists until 7 dpr whereas borderzone cardiomyocyte cell cycle re-entry peaks at 7 dpr (Porrello etal., 2011). It will be important in the future to examine Hippo-deficient hearts carefully to determine the kinetics of cell cycle re-

entry and perhaps to uncover the mechanisms underlying organ-wide cell cycle response.

Our findings provide new insight into mammalian heartregeneration and lend support to the idea that endogenouscardiomyocytes can be manipulated in vivo to repair the heart.Recent work has shown that the Hippo effector Yap also promotescardiomyocyte regeneration when overexpressed in late fetal andneonatal cardiomyocytes (Xin et al., 2013). Moreover, deleting Yapat the same immature cardiomyocyte stage results in reducedcardiomyocyte proliferation (Del Re et al., 2013). Our data supportand extend those findings by revealing that, in addition to immature

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Fig. 5. Increased proliferation ofcardiomyocytes in resectedsalvador mutant hearts.(A-F) Control and Salv CKO 4 dprresected hearts. Cardiomyocytelineage (eGFP positive). Borderzone (A,B,E) and distal area (C,D,F)showing EdU-positive nuclei(arrowheads, yellow) after apexresection. E and F show high-magnification representative imagesof control and Salv CKO,respectively. (G) Proliferation ratewas quantified by percentage of EdUincorporation in cardiomyocytes(n=3). Salv CKO hearts showsignificant increase of proliferation inboth border zone and distal area.(H) Quantification of Aurora Bkinase-positive cardiomyocytes(n=3). *P<0.001. (I) Quantification ofEdU+ cardiomyocytes followingsiRNA knockdown of indicatedgenes in wild-type neonatalcardiomyocytes (n=3).(J,K) Cardiomyocyte proliferation,density and nucleation fromunstressed control and Salv CKOmice at multiple stages werecalculated. Cnt (n=3), Salv CKO(n=3). Error bars represent s.e.m.

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cardiomyocytes, Hippo deficiency enhances regeneration in maturecardiomyocytes. It will be important to investigate Yap target genesin the context of regeneration in order to understand the regenerativeprocess in more depth.

Typically, a stressed cardiomyocyte re-enters the cell cycle butfails to proceed through cytokinesis, perhaps owing to an unyieldingsarcomere structure or anillin localization defect (Engel et al., 2006;Kikuchi and Poss, 2012). Recently reported innovative approacheswith therapeutic promise include in vivo reprogramming of cardiacfibroblasts to cardiomyocytes and delivering microRNAs directly todamaged heart (Efe et al., 2011; Bruneau, 2012; Jayawardena et al.,2012). It is conceivable that combining these different methods withmolecules that can transiently reduce Hippo signaling in the heartmight prove to be an effective method of regenerating adult humancardiomyocytes.

MATERIALS AND METHODSMouse alleles and transgenic linesThe Nkx2.5cre transgenic line and floxed alleles for ww45/Salvador and lats1-2/Warts have been described previously (Heallen et al., 2011). The Myh6-cre/Esr1 transgenic line (The Jackson Laboratory) directsexpression of a tamoxifen-inducible Cre in cardiomyocytes. TheGt(ROSA)26Sortm4(ACTB-tdTomato,-EGFP)Luo/J (abbreviated to mTmG)Cre reporter line (The Jackson Laboratory) expresses red fluorescence in theabsence of Cre recombinase in all cell types. In the presence of Cre, the loxP-flanked mT cassette is deleted, red fluorescence is abolished, and downstreamexpression of eGFP (mG) green fluorescence is activated. Myh6-cre/Esr1 micewere mated to mTmG mice to generate progeny that express cardiomyocyte-specific Cre that is tamoxifen-inducible and traceable via immunofluorescenceimaging. DNA was extracted from tail biopsies for genotyping. Genotypingprimers for Myh6-cre/Esr1 and mTmG are as follows: Myh6-cre/Esr1 5′-AGGTGGACCTGATCATGGAG-3′, 5′-ATACCGGAGATCATGCAAGC-3′;mTmG 5′-TCAATGGGCGGGGGT CGTT-3′, 5′-CTCTGCTGCCTCCTGGC -TTCT-3′, 5′-CGAGGCGGATCA CAAGCAATA-3′.

For Figs 1, 4 and 5, control was Mhccre-Ert; mTmG and Salv CKO wasMhccre-Ert; mTmG; Salvf/f. For Figs 2, 3 and supplementary material Figs S1and S2, control was Salvf/f and Salv CKO was Mhccre-Ert; Salvf/f. For Figs 1,2 and supplementary material Fig. S1, Lats1/2 CKO was Mhccre-Ert; mTmG;Lats1f/f; Lats2f/f.

DNA incorporation in the adult heartFor adult cardiomyocyte renewal studies, 3- to 4-month-old animals ofcontrol or conditional knockout lines of Salv and Lats1/2 were used. Allmice were crossed with Myh6-cre/Esr1 driver and the mTmG reporter linesfor lineage tracing. For Cre activation, tamoxifen (1 mg) was injected for 2consecutive days. EdU (0.5 mg) was injected into each mouse daily fromday 2 to day 5 and hearts were harvested 6 hours after EdU injection on day5. Following dissection, hearts were fixed with 10% neutral bufferedformalin, dehydrated, embedded in paraffin, then sectioned into 7-μm-thickslices. EdU incorporation was detected using the Click-it EdU imaging kit(Life Technologies).

Nuclei isolation and flow analysisNuclei isolation was performed as described previously (Bergmann et al.,2009). Three mouse hearts of each genotype for each experiment were used.Isolated nuclei were stained with FITC-conjugated anti-TnI (Bioss)overnight. Nuclei were stained with Alexa700-conjugated anti-Ki-67 (BDBiosciences, 581277; 1:100 dilution) and/or 7-AAD (BD Biosciences, 51-68981E). Nuclei were analyzed using a BD FACSAria cell sorter. Threeindependent experiments were performed for each analysis.

Cardiomyocyte proliferation studiesTo assess cardiomyocyte proliferation rates, density and nucleationfollowing Hippo disruption, control or Salv and Lats1/2 conditionalknockout mice (as described above) were used. Cre activation and EdUincorporation were performed as follows: for P7 samples, tamoxifen (0.5

mg) was administered at P5 and P6 (0.25 mg EdU 4 hours before harvest);for P12 samples, tamoxifen (0.5 mg) was administered at P8, P9, P10 andP11 (0.25 mg EdU 4 hours before harvest).

Apex resectionSurgical resection of the heart apex was performed on P8 mice as describedby Porrello et al. (Porrello et al., 2011) using modified procedures. Vicrylsutures (6-0 absorbable) were used to close the thoracic cavity, and the entireprocedure required ~12 minutes from the onset of hypothermia to recovery.Sham procedures excluded apex amputation. To increase survival rate andprevent maternal neglect and cannibalization, pups were fostered to nursingICR mothers of litters approximately the same age. Mice recovered up to 21dpr, then were euthanized and dissected hearts were processed for histologyand immunocytochemistry. Surface area measurements of resected apex tissuewere calculated using a Zeiss SteREO Discovery.V12 stereoscope equippedwith an AxioCam HRc digital camera to assay surgical reproducibility anddetermine amount of resected tissue of hearts categorized by scar severity. Allmeasurements and functions were controlled by the Carl Zeiss Axiovisionsoftware program (Carl Zeiss Microimaging). Similarly, fibrotic scar size wasmeasured using the procedures described above. Scar severity was categorizedas follows: severe (transmural fibrosis of the apex), mild (trace fibrosis at theapex) or absent (fibrosis not detected). Lineage tracing of cardiomyocytes afterheart apex resection was performed by crossing the Salvador floxed allele withMyh6-cre/Esr1; mTmG mice. Cre activity was induced with four consecutiveintraperitoneal or subcutaneous injections of tamoxifen from P7 to P10.Following apex resection at P8, pups were injected with EdU (0.5 mg) 4 hoursprior to heart excision. Hearts were excised at 4 and 21 dpr. For EdU pulsechase experiments, pups recovered for 4 dpr and EdU (0.25 mg/animal) wasinjected to label replicating DNA. Four hours after EdU injection, pups wereeuthanized and hearts excised. Fixation and tissue processing were performedas described below.

LAD occlusionFor P8 samples, surgical permanent occlusion of the left anterior descendingcoronary artery (LAD-O) was performed on P8 mice (Mahmoud et al.,2013). Nylon sutures (8-0 non-absorbable) were used to occlude the LAD.Proper occlusion was noted by blanching of the myocardium and also duringdissection 3-4 weeks post occlusion via visual inspection. Vicryl sutures (6-0 absorbable) were used to close the thoracic cavity, and the entire procedurerequired ~12 minutes from the onset of hypothermia to recovery. Shamprocedures excluded placement of a suture around the LAD. To increasesurvival rate and prevent maternal neglect and cannibalization, pups werefostered to nursing ICR mothers of litters approximately the same age. Micerecovered up to 3-4 weeks post-occlusion, were euthanized and dissectedhearts were processed for histology and immunocytochemistry. Automatedfibrotic scar size was measured using image segmentation MIQuant, opensource code for Matlab (Nascimento et al., 2011).

For adult samples, LAD-O was performed as described for P8 with minormodifications. Tamoxifen (1.5 mg) was administered at three time points: 7and 6 days pre-LAD-O and within 2 hours post LAD-O. At 1, 2 and 3 weekspost LAD-O, echocardiography was performed in the Baylor College ofMedicine Mouse Phenotyping core using a VisualSonics 770 systemequipped with a 30 MHz scanhead (RMV7007B). Mice recovered up to 3weeks post-occlusion, were euthanized and dissected hearts were processedfor histology and immunocytochemistry. Automated fibrotic scar size wasmeasured as described for P8 LAD-O.

HistologyFor trichrome staining, dissected hearts were immediately fixed with 10%formalin overnight at room temperature, dehydrated in an ethanol series, andparaffin embedded. Coronally sectioned tissues (7 μm) were deparaffinizedin xylene, rehydrated and fixed in Bouin’s Fluid (EMS) at 56°C for15 minutes. Following washes in deionized water, sections were sequentiallystained with Weigerts’ Iron Hematoxylin, Beibrich Scarlet-Acid Fuchsinsolution, phosphotungstic/phosphomolybdic solution and Aniline Bluesolution (Sigma). Sections were dehydrated via ethanol, cleared in Xyleneand slides were mounted.

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ImmunofluorescenceFixation, tissue processing, antigen retrieval and blocking for non-specificstaining have been described previously (Heallen et al., 2011). Samples wereincubated in primary antibody at 4°C overnight. After washing in PBST(0.05% Tween in PBS), sections were incubated in the appropriatefluorescence-labeled secondary antibodies, followed by counterstaining withDAPI (Sigma) then mounted in Aqua-Poly/Mount (Polysciences). Primaryantibodies used were as follows: mouse monoclonal anti-eGFP (1:500;Clontech, 632569), rabbit anti-phosphoYap (1:200; Cell Signaling, 4911),rabbit anti-Aurora B kinase (1:200; Abcam, ab2254), rabbit anti-eGFP (1:400;Abcam, ab290), mouse monoclonal anti-cTnt (1:200; Thermo Scientific, ms-295) and mouse monoclonal anti-Sav1 (1:200; Santa Cruz, sc-101205).Secondary antibodies used were as follows: Alexa Fluor 488 goat anti-mouseIgG, Alexa Fluor 488 goat anti-rabbit IgG, Alexa Fluor 594 goat anti-rabbitIgG and Alexa Fluor 546 donkey anti-rabbit IgG (1:200-1:1000; MolecularProbes). Immunofluorescence images were captured on (1) a Leica TCS SP5confocal microscope (all functions controlled via Leica LAS AF software) or(2) a Zeiss LSM 510 META laser scanning confocal microscope (all functionscontrolled via Ziess LSM Image Browser software). All manuscript figureswere prepared using Adobe Photoshop CS5 (Adobe Systems).

siRNA knockdownsiRNA knockdown was performed on cultured mouse neonatalcardiomyocytes in vitro in 4-well plate format. At 80% confluency, cells weretransfected with 1.5 μl Lipofectamine RNAiMax Transfection Reagent (LifeTechnologies) and 1.5 μl of predesigned siRNAs (10 μM; IDT, Coralville,Iowa) diluted in 50 μl of OPTIMEM. Cells were stained for EdU following a24-hour incubation at normal growth conditions. siRNA duplex names: AurkB(MMC.RNAI.N011496.12.7), Birc5 (MMC.RNAI.N001012273.12.2), Ccne2(MMC.RNAI.N001037134.12.1), Salv (MMC.RNAI.N022028.12.1), siRNAneg control (NC1 Negative Control Sequence).

StatisticsDifferences between groups were examined for statistical significance usingunpaired Student’s t-tests, ANOVA or χ2 distribution. All error bars represents.e.m. P<0.05 was regarded as significant.

AcknowledgementsWe gratefully acknowledge Hesham Sadek and Ahmed Mahmoud for instruction ofthe cardiac apex resection procedure. All echocardiographic measurements wereperformed by the Mouse Phenotyping Core at Baylor College of Medicine.

Competing interestsThe authors declare no competing financial interests.

Author contributionsT.H. and Y.M. designed and performed experiments and analyzed data; J.L. andG.T. performed experiments and analyzed data; J.T.W. and R.L.J. providedreagents; J.F.M. designed and supervised experiments and analyzed data; T.H.,Y.M. and J.F.M. wrote the manuscript.

FundingThis work was supported by grants from the National Institutes of Health (NIH)[R56DK094865 to R.L.J.; 1U01HL087365 to J.T.W.; 5T32HL007676-23 to J.L.], theVivian L. Smith Foundation [J.F.M.], the Cancer Prevention Research Institute ofTexas (CPRIT) [P120138 IIRA to R.L.J.], and the American Heart Association(AHA) [AHA10POST4140029 and AHA12POST11760019 to T.H.; AHA NCRPSDG 0930240N to Y.M.]. Deposited in PMC for release after 12 months.

Supplementary materialSupplementary material available online athttp://dev.biologists.org/lookup/suppl/doi:10.1242/dev.102798/-/DC1

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RESEARCH ARTICLE Development (2013) doi:10.1242/dev.102798

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