FGF1�p38 MAP kinase inhibitor therapy inducescardiomyocyte mitosis, reduces scarring, and rescuesfunction after myocardial infarctionFelix B. Engel*†‡, Patrick C. H. Hsieh§¶, Richard T. Lee§, and Mark T. Keating†�

*Department of Pediatrics, Harvard Medical School, and Department of Cardiology, Children’s Hospital, 320 Longwood Avenue, Boston, MA 02115;§Cardiovascular Division, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA 02115; and �Novartis Institutes for BioMedical Research,Cambridge, MA 02139

Contributed by Mark T. Keating, August 24, 2006

Mammalian cardiomyocytes have limited proliferation potential,and acutely injured mammalian hearts do not regenerate ade-quately. Instead, injured myocardium develops fibrosis and scar-ring. Here we show that FGF1�p38 MAP kinase inhibitor treatmentafter acute myocardial injury in 8- to 10-week-old rats increasescardiomyocyte mitosis. At 3 months after injury, 4 weeks ofFGF1�p38 MAP kinase inhibitor therapy results in reduced scarringand wall thinning, with markedly improved cardiac function. Incontrast, p38 MAP kinase inhibition alone fails to rescue heartfunction despite increased cardiomyocyte mitosis. FGF1 improvesangiogenesis, possibly contributing to the survival of newly gen-erated cardiomyocytes. Our data indicate that FGF1 and p38 MAPkinase, proteins involved in cardiomyocyte proliferation and an-giogenesis during development, may be delivered therapeuticallyto enhance cardiac regeneration.

cardiac regeneration � fractional shortening � Cyclin D2 � Cyclin A �angiogenesis

The mammalian heart has little or no capacity to regenerate(1, 2). This is a major medical problem, because inadequate

regeneration contributes to myocardial scarring, heart failure,arrhythmia, and death (3). As a single cause of death, ischemicheart disease is one of the leading causes of mortality worldwideamong people ages 15–59. This disease results from occlusion ofcardiac vessels leading to loss of cardiomyocytes causing �7million deaths every year.

Observations from naturally occurring regeneration in ze-brafish suggest that mammalian cardiac regeneration might beachieved through cardiomyocyte proliferation. Importantly,hearts of zebrafish carrying a mutation preventing proliferationdo not regenerate; instead, they scar (4). It has been previouslysuggested that the mammalian heart induces cardiomyocyteproliferation after injury but the rate of proliferation is too lowto repair the heart (5). In addition, we have recently shown thatpostnatal mammalian cardiomyocytes can proliferate in vitro bycombining FGF1 stimulation and p38 MAP kinase (p38) inhi-bition (6). FGF1 and p38 inhibitor administration have beenshown to be protective in ischemic heart disease, decreasingcardiomyocyte apoptosis (7, 8). However, constitutive cardiac-specific FGF1 overexpression only delayed myocardial infarctformation with no difference in maximal infarct size (9). Theeffects of p38 inhibition as well as FGF1 stimulation afterpermanent coronary occlusion are not well characterized.

ResultsTo determine whether FGF1�p38 inhibitor treatment can in-crease cardiomyocyte proliferation in vivo and improve heartfunction after cardiac injury, we performed a blinded andrandomized study. We induced cardiac injury by permanentcoronary artery occlusion, or myocardial infarction (MI), andtreated animals with saline plus BSA (control), the p38 MAPkinase inhibitor SB203580 plus BSA (p38i), FGF1 plus saline

(FGF1), or FGF1 plus SB203580 (FGF1�p38i) (n � 9 pergroup). SB203580 and saline were injected i.p. once every 3 days.FGF1 and its carrier BSA were injected with self-assemblingpeptides once into the infarct border zone immediately aftercoronary artery ligation (10).

Recent data indicated that overexpression of Cyclin D2 as wellas Cyclin A2 can enhance cardiac regeneration (11, 12). Todetermine whether FGF1�p38i treatment enhances Cyclin D2and�or Cyclin A2 expression, we examined heart sections 2weeks after treatment. Caveolin 3 staining was used to identifycardiomyocytes (see Fig. 5, which is published as supportinginformation on the PNAS web site). MI increased the number ofCyclin D2-positive cardiomyocytes by 11.5-fold within the infarctarea and border zone (P � 0.01, Fig. 1 A and B). This finding isin accord with the previous findings of an increase in cyclinexpression and cell cycle activation after MI and during devel-opment of hypertrophy (13–15). Neither FGF1 nor p38i treat-ment increased the number of Cyclin D2-positive cardiomyo-cytes; this finding might be explained by increased myocardialgrowth factor production after infarction (6, 16). Our in vitrodata indicated that FGF1 stimulation induces cell cycle reentryof cardiomyocytes but has only a minor effect on mitosis. Incontrast, p38 inhibition increased cyclin A2 expression andprogression through mitosis after growth factor stimulation (6).To determine whether FGF1�p38i treatment enhances Cyclin Aexpression and the progression of cardiomyocyte mitosis, weassayed for Cyclin A expression and histone H3 phosphorylation(H3P). Indeed, the number of Cyclin A- and H3P-positivecardiomyocytes within the infarct and the border zone were2-fold (P � 0.01) and 3.4-fold (P � 0.0001) increased, respec-tively, in animals treated with FGF1�p38i compared with un-treated MI (Fig. 1 C–E). p38i could, in contrast to our in vitrostudy, increase the number of Cyclin A-positive (2.2-fold, P �0.001) and H3P-positive (3.4-fold, P � 0.0001) cardiomyocytes(Fig. 1 C and D), whereas FGF1 stimulation increased H3P-positive cardiomyocytes only 2.3-fold (P � 0.001, Fig. 1D).Mitosis was not detected 3 months after MI. This is most likelydue to the fact that treatment was stopped after 1 month. Takentogether, our data support the idea that endogenous growth

Author contributions: F.B.E., P.C.H.H., R.T.L., and M.T.K. designed research; F.B.E. andP.C.H.H. performed research; P.C.H.H. and R.T.L. contributed new reagents�analytic tools;F.B.E., P.C.H.H., R.T.L., and M.T.K. analyzed data; and F.B.E. wrote the paper.

The authors declare no conflict of interest.

Abbreviations: MI, myocardial infarction; EDD, end diastolic dimension; ESD, end systolicdimension; FS, fractional shortening.

†To whom correspondence may be addressed. E-mail: mark.keating@novartis.com orengel@enders.tch.harvard.edu.

‡Present address: Max Planck Institute for Heart and Lung Research, 61231 Bad Nauheim,Germany.

¶Present address: Institute of Clinical Medicine, Department of Surgery, National Cheng-Kung University, Tainan 701, Taiwan.

© 2006 by The National Academy of Sciences of the USA

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factor signaling is present after MI, and indicate that p38inhibition facilitates FGF1 and�or growth factor-mediated car-diomyocyte mitosis after MI within the infarct and the borderzone.

Injury from MI disturbs loading conditions within the heart,causes ischemic and oxidative stresses, and activates various localand systemic neurohormonal systems (17). These alterations tothe extracellular environment trigger left ventricular (LV) re-modeling characterized by necrosis and thinning of the infarctedmyocardium, LV chamber dilation, fibrosis, and hypertrophy ofviable cardiomyocytes. Persistent remodeling contributes tofunctional decompensation and eventually leads to heart failure(18). Because p38i improves cardiomyocyte proliferation moreefficiently than FGF1, we tested whether this translates into agreater preservation of heart function. Cardiac function after MIwas assessed by percentage left ventricular fractional shortening(%FS), end diastolic dimension (EDD), and end systolic dimen-sion (ESD). Twenty-four hours after MI, %FS decreased from59.1 � 13.1% to 30.1 � 10.9% (P � 0.0001, Fig. 2A). Injectionof saline and BSA did not significantly improve %FS. In contrast,rats treated with p38i, FGF1, and FGF1�p38i significantlyincreased %FS (43.0 � 11.4%, 44.3 � 10.1%, 50.8 � 15.2%,respectively; P � 0.05 over MI, Fig. 2 A). This early improvementindicates that all treatments may have had an antiapoptotic effectas previously described (8, 19, 20). Two weeks after MI, %FS wasmaintained in animals that received FGF1 and�or p38i (P � 0.05over MI; Fig. 2B). Apoptosis rates were low and did not differbetween treated and untreated animals (data not shown). Con-

sistent with the improvement of %FS after MI, injection of FGF1and�or p38i prevented cardiac dilation as measured by EDD andESD (P � 0.05, see Table 1, which is published as supportinginformation on the PNAS web site). Trichrome stain at sevenlevels of the heart from apex to base (1.2 mm apart) revealed thatscar size at 2 weeks was reduced after all treatments by over 44%(P � 0.05, Fig. 2 C and F, see also Fig. 6, which is published assupporting information on the PNAS web site). In addition, wecalculated the thinning index (ratio of minimal ventricular wallthickness to maximal thickness of the septum, using the top fourlevels from base). The thinning index decreased as expectedfrom 0.96 � 0.05 at 2 weeks in the sham group to 0.35 � 0.08 inthe MI group (P � 0.0001, Fig. 2D). Whereas the control groupdid not reduce thinning significantly, all other treatments re-duced thinning by �34% (P � 0.05 for MI versus FGF1, P � 0.01for p38i and FGF1�p38i versus MI; Fig. 2D). Because the leftventricular wall thins after injury, the amount of scar tissue doesnot reflect the amount of muscle loss. To determine the amountof muscle loss, we divided the circumference of left ventricularwall containing at least 75% scar by the circumference of leftventricular wall using all sections. This analysis revealed that theloss of left ventricular muscle at two weeks was reduced after alltreatments by �56% (P � 0.01, Fig. 2E). There was no differ-ence between p38i, FGF1, and FGF1�p38i therapy regarding%FS, thinning, scarring, and loss of left ventricular muscle.Taken together, these data indicate that FGF1 stimulation aswell as p38 inhibition rescue cardiac structure and function afterinjury.

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Fig. 1. FGF1�p38 inhibitor treatment induces cardiomyocyte mitosis in vivo. Rats were treated after myocardial infarction (MI) with saline plus BSA (control),SB203580 plus BSA (p38i), saline plus FGF1 (FGF1), or SB203580 plus FGF1 (FGF1�p38i). Two weeks after treatment, heart sections were analyzed for cell cycleactivation (Cylin D2, Cyclin A) and mitosis (H3P) of cardiomyocytes within the scar area and the infarct border zone. (A) FGF1 and�or p38i significantly increasedthe number of Cyclin D2-positive cardiomyocytes. (B) Examples of heart section from sham or FGF1�p38i-treated animals stained for Caveolin 3 (green) and CyclinD2 (red). (C) FGF1 and�or p38i significantly increased the number of Cyclin A-positive cardiomyocytes. (D) FGF1 and�or p38i significantly increased the numberof H3P-positive cardiomyocytes. (E) Six examples of heart section after FGF1�p38i treatment stained for Caveolin 3 (green) and H3P (red). Data are �SEM; n �

9 in each group; NS, not significant; **, P � 0.01.

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To determine whether FGF1�p38i treatment has a long-termeffect, we performed an additional and separate blinded andrandomized study (n � 7 per group). Because it has been shownthat long-term exposure of humans to p38 inhibitors is toxic (21),we treated animals for 1 month and analyzed the effect of ourtreatment regimen after 3 months. Again, %FS 24 h after MI wassignificantly improved after injection of FGF1 and�or p38i (P �0.05, Fig. 3A). At 3 months after injury, 4 weeks of FGF1 andFGF1�p38i therapy increased %FS by 25% and 30%, respec-tively (P � 0.05 over MI; Fig. 3B). Importantly, there was nosignificant difference in %FS to uninjured animals. In contrast,%FS of hearts treated with p38i were not significantly differentfrom the MI group. Scar size was reduced in all rats treatedwith FGF1 and�or p38i (reduction of: p38i: 32.8%, FGF1:43.7%, FGF1�p38i: 52.3%; P � 0.05 for p38i, FGF1, and P �0.01 for FGF1�p38i over MI; Fig. 3C and F, see also Fig. 7, whichis published as supporting information on the PNAS web site).

However, ventricular wall thinning and left ventricular muscleloss were only significantly improved in rats treated with FGF1(reduction of 34.0% and 51.0% respectively; P � 0.05) orFGF1�p38i (reduction of 54.6%, P � 0.01 and 61.7%, P � 0.05respectively, Fig. 3 D and E). Treatment with p38i had no effectat this later stage. Taken together, these data indicate that FGF1and FGF1�p38i treatment preserve wall thickness, reduce scar-ing, and improve function, whereas the effect of p38i is transient.

Organ regeneration is a highly complex process. Studies inliver regeneration have shown that the process of regenerationdepends on the injury and involves the cooperative effort ofseveral cell types and secreted factors to rebuild a functionalcytoarchitecture (22). Although our data indicate that p38inhibition improves the proliferative potential of cardiomyo-cytes, newly generated cardiomyocytes can only survive if theyare supplied with oxygen and nutrients in functioning heartmuscle. Therefore, we speculated that FGF1 contributes to

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Fig. 2. FGF1�p38 inhibitor treatment improves heart function. Rats were treated after myocardial infarction (MI) with saline plus BSA (control), SB203580 plusBSA (p38i), saline plus FGF1 (FGF1), or SB203580 plus FGF1 (FGF1�p38i). Hearts were analyzed by using echocardiography and trichrome stain of transverse heartsections. (A and B) FGF1 and�or p38i significantly improved left ventricular %FS 1 and 14 days after MI. %FS was calculated as (EDD � ESD)�EDD � 100%, whereEDD is end-diastolic dimension and ESD is end-systolic dimension. (C) FGF1 and�or p38i significantly decreased left ventricular scarring. Percentage of scar volumewas determined as fibrotic area�(fibrotic � nonfibrotic area) using seven sections from apex to base, 1.2 mm apart. (D) FGF1 and�or p38i significantly decreasedventricular wall thinning. The thinning index is the ratio of minimal infarct wall thickness and maximal septal wall thickness using sections 1–4 from base. (E)FGF1 and�or p38i significantly decreased left ventricular muscle loss. Percentage of left ventricular muscle loss was determined as circumference of the leftventricular wall containing at least 75% scar area�circumference of the left ventricular wall based on all sections. (F) Examples of heart sections stained for scartissue (blue) and muscle tissue (brown) from base to apex in an interval of 2.4 mm 2 weeks after treatment. Data are �SEM; n � 9 in each group; NS, not significant;

*, P � 0.05; **, P � 0.01.

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long-term improved heart function not only by inducing cardi-omyocyte mitosis but also by improving angiogenesis in the heart(23, 24). Indeed, FGF1 and FGF1�p38i increased the capillarydensity in the scar by �40% at 3 months after injury (P � 0.05,Fig. 4, see also Fig. 5). In contrast, although contributing to theactivation of cardiomyocyte mitosis, p38i had no effect oncapillary density. This observation may explain why the overalleffect of p38i treatment is transient. Taken together, our dataindicate that FGF1 treatment increases angiogenesis in the scararea.

DiscussionRecent evidence suggests that a population of extracardiac orintracardiac stem cells may be feasible for cardiac repair (25–27),but this approach has been controversial and requires isolationof autologous stem cells or the use of donor cells along withimmunosuppresion. The evidence presented here suggests that

FGF1 and p38 MAP kinase, proteins involved in cardiomyocyteproliferation and angiogenesis during cardiac development, maybe redeployed therapeutically to enhance cardiac regenerationafter myocardial infarction. It has been suggested that themammalian heart induces cardiomyocyte proliferation afterinjury, but the rate of proliferation is too low to repair the heart(5). Recent work had indicated that induction of cardiomyocyteproliferation by Cyclin D2 or Cyclin A2 overexpression has thepotential to improve heart function after injury (11, 12). How-ever, this work was based on transgenic technology requiringgenetic engineering in the fetus or gene therapy. We havedeveloped a pharmacological approach to treat cardiac injury inadults. To constrain adverse effects of systemic FGF1 therapy,we used a local delivery system (10, 28). This system is advan-tageous over prolonged overexpression by gene therapy vectorsthat can lead to excessive protein production and undesiredeffects. Local and pulsed delivery also is optimal for the p38

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Fig. 3. FGF1�p38 inhibitor improved heart function 3 months after myocardial infarction. Rats were treated after myocardial infarction (MI) with saline plusBSA (control), SB203580 plus BSA (p38i), saline plus FGF1 (FGF1), or SB203580 plus FGF1 (FGF1�p38i). Hearts were analyzed by using echocardiography andtrichrome stain of transverse heart sections 3 months after treatment. Note that treatment was stopped after 1 month. (A and B) FGF1�p38i and FGF1 significantlyimproved %FS 1 day and 3 months after MI. In contrast, %FS after p38i treatment was only improved at day 1. (C) FGF1 and�or p38i significantly decreased scarformation. The percentage of scar volume was determined as fibrotic area�(fibrotic � nonfibrotic area) using nine sections from apex to base, 1.3 mm apart.(D) FGF1�p38i and FGF1 significantly decreased ventricular wall thinning but not p38i. The thinning index was determined by using sections 1–6 from base. (E)FGF1�p38i and FGF1 significantly decreased left ventricular muscle loss but not p38i. The percentage of left ventricular muscle loss was determined ascircumference of the left ventricular wall containing at least 75% scar area�circumference of the left ventricular wall based on all sections. (F) Examples of heartsections stained for scar tissue (blue) and muscle tissue (brown) from base to apex in an interval of 2.6 mm 3 months after treatment. Data are �SEM; n � 9 ineach group; NS, not significant; *, P � 0.05; **, P � 0.01.

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inhibitor SB203580; our previous data revealed that continuedinhibition of p38 in vitro results in cell death of adult cardiomy-ocytes (F.B.E., unpublished observation).

Although our previous in vitro data suggest that FGF1�p38itreatment induces cardiomyocyte proliferation, it is also possiblethat FGF1�p38i treatment in vivo might increase proliferation ofresident stem cells in the heart or peripheral stem cells recruitedto the heart. In our proliferation assays, it is not 100% certainthat the Cyclin D2-, Cyclin A, or H3P-positive cells are allproliferating adult cardiomyocytes, because it can be argued thatthey can also be proliferating stem cells that have alreadyexpressed cardiac markers. However, it has been reported that,20 days after MI, cardiomyocytes derived from injected cardiacstem cells are much smaller (3,400 � 560 �m3) than survivingcardiomyocytes (20,000–25,000 �m3) (29). In our study, �90%of Cyclin D2-, Cyclin A, or H3P-positive cells show no differencein size compared with surviving cardiomyocytes. Future studieswill determine whether FGF1�p38i therapy has an effect on stemcells in the heart.

FGF1�p38i therapy consistently shows the best outcomeregarding preservation of cardiac structure and function. Ourdata suggest that FGF1 improves heart function throughenhancement of angiogenesis and cardiomyocyte mitosis. p38inhibition further increases the effect on cardiomyocyte mi-tosis. Thus, FGF1�p38i therapy combines the effects of p38inhibition on apoptosis and proliferation with the effects ofFGF1 on apoptosis, proliferation, and angiogenesis to enhancecardiac regeneration. Given our findings, FGF1�p38 inhibitortreatment may prove useful for protecting patients fromcardiac injury and therefore warrants further preclinicalinvestigation.

Materials and MethodsMI and Delivery of FGF1 and p38 Inhibitor. Animal experiments wereperformed in accordance with guidelines of Children’s Hospital(Boston, MA) and were approved by the Harvard Medical SchoolStanding Committee on Animals. MI was produced in �250-g maleSprague–Dawley rats [�8–10 weeks old; Charles River (Wilming-ton, MA) and Harlan (Indianapolis, IN)] as described (10). Briefly,rats were anesthetized by pentobarbital and, after tracheal intuba-tion, the hearts were exposed via left thoracotomy. The leftcoronary artery was identified after pericardiotomy and was ligatedby suturing with 6-0 prolene at the location �3 mm below the leftatrial appendix. For the sham operation, suturing was performed

without ligation. Peptide nanofibers (peptide sequence AcN-RARADADARARADADA-CNH2 from Synpep, Dublin, CA)with BSA (0.1% in PBS) or 400 ng�ml bovine FGF1 (R & DSystems, Minneapolis, MN; diluted in 0.1% BSA�PBS) were dis-solved in 295 mM sucrose and sonicated to produce 1% solution forinjection. Eighty microliters of peptide nanofibers (NF) was in-jected into the infarcted border zone through three directionsimmediately after coronary artery ligation estimated to deliver anFGF1 concentration of �50–100 ng�ml to the cardiomyocytes (10).Subsequently, SB203580HCl (Tocris, Ellisville, MO, 2 mg�kg bodyweight) or saline was injected i.p., the chest was closed, and animalswere allowed to recover under a heating pad.

Immunofluorescence Staining. Hearts were embedded in tissue-freezing medium (Fisher, Hampton, NH) without fixation, fro-zen in 2-methylbutane (cooled in liquid nitrogen), stored at�80°C, and finally sectioned [20 �m; Leica, Bannockburn, IL)3050S]. Staining was performed as described (see also Table 2,which is published as supporting information on the PNAS website) (30). Immune complexes were detected with Alexa Fluor488- or Alexa Fluor 594-conjugated secondary antibodies (1:400;Molecular Probes, Eugene, OR). DNA was visualized with 0.5�g�ml DAPI (Sigma, St. Louis, MO).

Trichrome Stain. Through each heart, seven to nine sections (1.2-or 1.3-mm interval) from apex to base were subjected to AFOGstaining (4). Frozen sections were fixed at room temperature(RT) with 10% neutral buffered formalin (10–15 min). Sectionswere permeabilized (0.5% Triton X-100�PBS, 10 min), incu-bated in preheated Bouins fixative (2.5 h at 56°C, 1 h at roomtemperature), washed in tap water, incubated in 1% phospho-molybdic acid (5 min), rinsed with distilled water, and stainedwith AFOG staining solution (3 g of acid fuchsin, 2 g of orangeG, 1 g of anilin blue dissolved in 200 ml of acidified distilledwater, and pH 1.1 HCl, for 5 min). Stained sections were rinsedwith distilled water, dehydrated with EtOH, cleared in Citrosolv,and mounted. This staining results in a blue coloration of thescar, and muscle tissue appears orange�brown. Images weretaken for each section to calculate the fibrotic and nonfibroticareas as well as ventricular and septal wall thickness.

Scarring, Thinning, and Muscle Loss. Scarring was determined asfibrotic area�(fibrotic � nonfibrotic area), and muscle loss wasdetermined as circumference of the left ventricular wall con-

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Fig. 4. FGF1�p38 inhibitor treatment increases angiogenesis. (A) FGF1�p38i and FGF1, but not p38i, significantly increased angiogenesis of the scar area at 3months. (B) Examples of vessel staining 3 months after MI using vessel markers smooth muscle actin (SMA, smooth muscle cells, fluorescent green) and vonWillebrand factor (vWF, endothelial cells, red) and cardiomyocyte-specific marker Caveolin 3 (pale green). (C) Examples of vessel staining of heart sections fromrats 3 months after coronary ligation treated with FGF1�p38i. Data are �SEM; n � 7 in each group; NS, not significant; *, P � 0.05; **, P � 0.01.

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taining at least 75% scar area�circumference of the left ventric-ular wall based on all sections. The thinning index is a ratio ofthe amount of wall thinning in the infarct normalized to thethickness of the septum and is calculated by dividing the minimalinfarct wall thickness with maximal septal wall thickness (2weeks, sections 1–4; 3 months, sections 1–6 from base).

Echocardiography. Echocardiographic acquisition and analysiswere performed as previously described (31). Left ventricularfractional shortening was calculated as (EDD � ESD)�EDD �100%.

Statistics. Data are shown as mean � SEM. Multiple groupcomparison was performed by one-way ANOVA followed by theBonferroni procedure for comparisons of means. Comparison

between two groups was analyzed by the one-tailed Student’s ttest because we hypothesized, based on our in vitro study, apositive effect of FGF1�p38 inhibitor treatment on heart func-tion. Values of P � 0.05 were considered statistically significant.For infarct size, wall thickness, muscle loss measurements, andproliferation analysis, the observer was blinded to treatmentgroup. Proliferation analysis was performed at two levels in eachheart, and all cells inside the scar and in the border zone wereanalyzed.

We thank David Clapham, Yibin Wang, and Kyu-Ho Lee for critique ofthe manuscript and Keating laboratory members for helpful discussions.This work was supported by a grant from the Charles H. HoodFoundation (Boston, MA; Child Health Research grant to F.B.E.) anda fellowship from the American Heart Association (to P.C.H.H.).

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