experimental & clinical cardiology

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Cardiac biopsies were stained for collagen and immunostained for pSmad3, collagen triple-helix repeat containing 1 (cthrc1). Collagen α1(I) was evaluated by ...
EXPERIMENTAL & CLINICAL CARDIOLOGY

Volume 20, Issue 1, 2014 Title: "Inhibition of Fibrosis and Improvement of Function of the Myopathic Hamster Cardiac Muscle by Halofuginone" Authors: Yves Fromes, Sophie Bouyon, Sadia Nagi, Veronique Roussel, Olga Genin, Oshrat Levi and Mark Pines How to reference: Inhibition of Fibrosis and Improvement of Function of the Myopathic Hamster Cardiac Muscle by Halofuginone/Yves Fromes, Sophie Bouyon, Sadia Nagi, Veronique Roussel, Olga Genin, Oshrat Levi and Mark Pines/Exp Clin Cardiol Vol 20 Issue1 pages 2351-2383 / 2014

Inhibition of Fibrosis and Improvement of Function of the Myopathic Hamster Cardiac Muscle by H...

Inhibition of Fibrosis and Improvement of Function of the Myopathic Hamster Cardiac Muscle by Halofuginone

Yves Fromes, MD, Ph.D.1 Sophie Bouyon, M.Sc 1 Sadia Nagi, M.Sc. 1 Veronique Roussel, M.Sc. 1 Olga Genin ,M.Sc.2 Oshrat Levi, M.Sc,2 Mark Pines, Ph.D. 2

1

UPMC -Institut de Myologie, Groupe Hospitalier Pitié-Salpêtrière, Paris, France,

2

Institute of Animal Sciences, Volcani Center, Bet Dagan, Israel

Running head - Fibrosis and cardiac function

Corresponding author: Mark Pines, Ph.D. Institute of Animal Sciences The Volcani Center P.O. Box 6, Bet Dagan 50250, Israel Tel- 972-8-9484408 Fax- 972-8-9475075 e-mail –[email protected]

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BACKGROUND: Heart injury, which results from various causes, leads to a common final pathway of pathological remodeling and fibrosis that causes heart failure. OBJECTIVES: To evaluated the efficacy of halofuginone in reducing fibrosis and improving cardiac function in the myopathic hamster strain CHF147 that develop a cardiomyopathy which evolves into heart failure with a major increase in fibrosis. METHODS: Echocardiography and surface ECG were assessed with and without halofuginone treatment. Cardiac biopsies were stained for collagen and immunostained for pSmad3, collagen triple-helix repeat containing 1 (cthrc1). Collagen 1(I) was evaluated by in situ hybridization. Myofibroblasts number was assessed by immunostaining for collagen cross-linking enzyme. RESULTS: Halofuginone treatment resulted in a major reduction in cardiac collagen and cthrc1 levels that was associated with prevention and deceleration of the impairment of left ventricular function. Halofuginone reduced the number of myofibroblasts and reduced the number of nuclei with pSmad3 downstream of the TGFβ signaling. CONCLUSIONS: Halofuginone that inhibited cardiac collagen synthesis and improved cardiac function meets the criteria for a potential novel antifibrotic therapy for patients with cardiac fibrosis of various etiologies.

Key Words: collagen, halofuginone, fibrosis, cthrc1, myofibroblasts

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Cardiac involvement occurs as a degenerative process with fibrosis and some fatty replacement of the myocardium. Duchenne, Becker, and limb-girdle types 2C−2F and 2I are muscular dystrophies (MDs) in which the development of a dilated cardiomyopathy is common. Arrhythmias and conduction disease occur after the development of the dilated cardiomyopathy (1). Myotonic types 1 and 2, Emery-Dreifuss, and limb-girdle type 1B, MDs present with conduction disease and associated arrhythmias and variably with a dilated cardiomyopathy (1,2). Heart injury, which results from various causes, such as hypertension-dependent pressure overload, volume overload caused by valvular lesions, a variety of toxic insults, diabetes and obesity, and MDs to mention just a few, leads to a common final pathway of pathological remodeling and fibrosis that leads to heart failure (3). Cardiac fibrosis – characterized by net accumulation of extracellular matrix (ECM) elicits both electrophysiological alterations, such as conduction defects and arrhythmogenicity, and impairs contractile properties (4,5). Transforming growth factor-β (TGFβ) is one of the main – if not the primary – profibrotic cytokines in cardiac muscle (6,7) as demonstrated in over-expression and knockout models (8,9). TGFβ induces the endothelial-tomesenchymal transition that supplies the tissue with fibroblasts (10,11), and the fibroblasts-to-myofibroblasts transition by which the fibroblasts undergo a phenotype change to proliferative and contractile myofibroblasts, which acquire migratory capabilities and produce high levels of ECM (3,12). These processes require phosphorylation of Smad3 downstream of the TGFβ pathway (13). Collagen is the major ECM protein in cardiac fibrosis, and 80-85% of it is collagen type I, which is an integral

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feature of most cardiac pathological conditions (14). In the healthy heart, the collagen network, which consists of thin bundles, provides the physical support that maintains myocardial structure. In the majority of heart diseases, a major increase in collagen deposition has been observed, and the extent of fibrosis differs among the various diseases and varies with time after onset of the disease (15). An inverse correlation between cardiac fibrosis and function was observed in various MDs (16-19). Upstream of TGFβ the fibrotic process is controlled by a factor called collagen triple helix repeat containing 1 (cthrc1), a secreted 30-kDa glycosylated protein that is associated with myofibroblasts at the sites of collagen matrix deposition (20). Cthrc1 has been observed in various fibrotic tissues (21), its gene expression is under the control of TGFβ and its increased expression was associated with increased cell migration, motility and invasion (22,23), and with inhibition of TGFβ-stimulated synthesis of collagen type I (24,25). Thus, it has been suggested that cthrc1 contributes to tissue matrix remodeling and repair by limiting collagen deposition and promoting cell migration (26). In the fibrotic cardiac muscles of mice exhibiting various muscular dystrophies (MDs) the collagen bundles appear smaller at sites where cthrc1 was located adjacent to collagen type I (27). Dilated cardiomyopathies (DCM) are very often associated with MDs, especially when the pathology involves mutations in the genes that code for components of the dystrophin-associated glycoprotein complex (DGC), such as the sarcoglycan family (28). Mutations in the δ-sarcoglycan (δ-SG) gene are responsible for limb-girdle muscular dystrophy (LGMD) 2F, which is associated with DCM (29). The myopathic hamster strain CHF147 is a representative model for autosomal recessive cardiomyopathy and

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LGMD. The cardiomyopathy results from a mutation in the δ-SG gene – a component of the dystrophin complex (30-32). At young age (5-6 weeks), the hamsters show a mildly enlarged left ventricular diastolic dimension (LVDD) with decreased LV fractional shortening (FS) or ejection fraction (EF). This hamster model develops a cardiomyopathy that rapidly evolves into heart failure. Histological lesions can be characterized by focal cell death, increasing degree of fibrosis that correlates inversely with LV function, and sometimes, tissue calcifications (33). Beside these characteristic features of the disease, there is development of a nonspecific interstitial fibrosis, mainly linked to the progression of the heart failure. The development of this interstitial fibrosis is related to the hemodynamic changes and contributes to the alterations of the myocardial relaxation and contractility. Furthermore, increased interstitial collagen content disrupts the coordination of electronic connectivity of adjacent myocytes that provide a key substrate for arrhythmogenesis. Halofuginone is an antifibrotic agent that inhibits Smad3 phosphorylation downstream of the TGFβ signaling pathway (34,35). In animal models, in which excess collagen is the hallmark of the disease, halofuginone prevented the increase in collagen synthesis and elicited resolution of established fibrosis. These involved mice and patients afflicted with chronic graft-versus-host disease (cGvHD) (36,37), tight skin (Tsk) mice (38), and rats with pulmonary and hepatic fibrosis (39,40). In murine models of Duchenne and Congenital MD (DMD and CMD, respectively) and in dysferlinopathy, halofuginone was shown to inhibit Smad3 phosphorylation in cardiac and skeletal muscles – an effect that was associated with decreased collagen and cthrc1 levels and with improvement of

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cardiac function and enhanced motor coordination and balance (41-43). Also, lung and cardiac muscle functions were improved in mice with established fibrosis (44). In the present study we evaluated the efficacy of halofuginone in improving cardiac histopathology and myocardial function in CHF147 hamsters.

METHODS Materials Halofuginone bromhydrate was obtained from Halo Therapeutics, LLC (Boston, MA, USA); monoclonal prolyl 4-hydroxylase β (P4Hβ) from Acris (Hiddenhausen, Germany); cthrc1 monoclonal and phosphorylated Smad3 (P-Smad3) antibodies were from Abcam (Cambridge, UK); rabbit anti-human collagen type I antibody was purchased from Novotec (Lyon, France); and 4′,6-Diamidino-2-phenylindole dihydrochloride (DAPI) from Sigma Chemicals Co. (St Louis, MO, USA). Animals Male Syrian hamsters (4-5 months old) were used for the experiments. The wild-type (wt) hamsters (Rj:AURA backcrossed on Aura Syrian hamster strain) were purchased from Elevage Janvier, France. CHF147 hamsters (n = 11) were bred at the local animal facility of the Institut de Myologie. The protocol conforms to the Guide for the Care and Use of Laboratory Animals, published by the US National Institutes of Health (NIH Publication No. 85-23, Revised 1996). Our protocol was submitted to and approved by the local committee, and we have a formal authorization issued by INSERM. Animals were assigned randomly to the different groups and were identified by implanted tags without any external sign allowed to identify individuals, which remained blinded along

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the experiments. Halofuginone (25 µg/hamster) was injected intraperitoneally (IP) three times per week for 6 weeks and control animals were injected with saline. Body weights were measured weekly and weight changes between the end of the experiment and the date of inclusion were subjected to ANOVA (unpaired t-test). All animals were sacrificed 7 weeks after termination of halofuginone treatment. ECG Surface ECG on restrained non-sedated animals was performed with an especially adapted ECG tunnel device (EMKA Technology, Paris, France) and the ECG trace was analyzed with the ECGauto specific software (EMKA Technology). Briefly: cardiac cycle duration as indicated by the RR interval duration was analyzed quantitatively according to standard criteria of heart rate variability (HRV). Specific parameters were mean RR interval duration and standard deviation from normal-to-normal R intervals (SDNN) (45). Echocardiography Echocardiographic assessments were realized with a VEVO770 echocardiograph (VisualSonics, Canada), equipped with scan heads with high-frame-rate electronics (broadband frequency 37.5 MHz and axial resolution 70 μm). All echocardiographic examinations were performed under light gas anesthesia (isoflurane 2%). 2D and Mmode imaging were used to obtain measurements of LV dimensions (LVEDD) and of LV function (FS, EF). All animals were evaluated for ECG and echocardiography parameters prior to treatment initiation 0, 5, 9, and 13 weeks later.

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Preparation of sections, collagen staining and immunohistochemistry and in situ hybridization At the end of the experiments, cardiac muscles were collected and fixed, and sections were prepared and stained for collagen with Sirius Red, as previously described (41). The collagen level was calculated by using ImagePro software (Media Cybernetics, Silver Spring, MD, USA). Photographs of the apex and base of the biopsy from each animal (five sections from each biopsy) were taken for analysis. The results were calculated as red area divided by the total (red + green) area, and presented as the proportion of fibrotic muscle area (mean ± SE). Cardiac muscles were immunostained with P4Hβ (1:25), cthrc1 (1:50), or pSmad3 (1:50) antibodies, or double-immunostained with collagen type I (1:100) and cthrc1 (1:50) antibodies. Nuclei were detected with DAPI. Phospho-Smad3positive nuclei were counted in sections from the wt, CHF147, and halofuginone-treated CHF147 hamsters (10 slides from each hamster), and presented as percentages of total nuclei. The levels of P4Hβ were determined in 10 slides from each hamster by image analysis of the ratio of the area covered by the enzyme-positive cells to the total area and expressed as arbitrary units. In situ hybridization with a digoxigenin-labeled collagen 1(I)

probe was performed as described (40).

Confocal microscopy Microscopic observation and image acquisition were performed with an Olympus IX 81 inverted confocal laser-scanning microscope (Fluoview 500; Olympus, Tokyo, Japan) equipped with a 405-nm diode laser, a 488-nm argon-ion laser, a 543-nm helium-neon laser, and a 60 × 1.0 NA PlanApo water-immersion objective. DAPI was excited at 405 nm and the emission was collected through a BA 430-460 filter; "GREEN" was excited at

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488 nm and the emission was collected through a BA 505-525 filter; "RED" was excited at 543 nm and the emission was collected through a BA 560 IF filter. The transmittedlight images were obtained by Nomarski differential interference contrast. Goat antimouse IgG antibodies with Alexa Fluor dye (Molecular Probes, Carlsbad, CA, USA) were used. Statistical analysis For collagen analysis the Tukey-Kramer HSD test was applied at p < 0.05, with JMP software, version 6 (SAS Institute, Cary, NC, USA). Analysis of variance (ANOVA) with an unpaired t test was applied to the ECG and echocardiography results. All parameters were expressed as mean ± SEM.

RESULTS In vivo evaluations of cardiac functions Halufuginone treatment did not affect the hamster body weight, which indicated good general tolerance of the IP administration. Surface ECG did not reveal significant changes in the mean RR interval duration or SDNN and no significant changes in arrhythmogenicity.

Left

ventricular

dimensions

(LVEDD),

as

measured

by

echocardiography, did not exhibit significant changes among groups (data not shown). In the CHF147 hamsters left ventricular function, as indicated by ejection fraction (EF), declined with age (Fig. 1). Halofuginone treatment already resulted in a significant improvement in EF (p < 0.001) after 5 weeks, and it persisted for an additional 7 weeks after termination of the treatment. In all time points the difference between the control and the halofuginone-treated hamsters differ significantly by Analysis of variance.

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Effect of halofuginone on cardiac fibrosis At the end of the experiment cross-sections from the apex and basis were taken from the wt, CHF147, and halofuginone-treated CHF147 hamsters, and stained with Sirius Red for collagen (Fig. 2). The biopsies of the CHF147 hamsters – apex and basis – were eight times richer in collagen than those of the wt, and this collagen consisted of long, thick fibers (Fig. 2C) that were mostly interstitial, but with some perivascular collagen, as revealed by the light and confocal microscopy. Halofuginone treatment caused a reduction in collagen levels by a factor of 2.7 (Fig. 2B), and only small numbers of thin collagen fibers remained 7 weeks after termination of the treatment (Fig. 2C). Increases in various collagen types were demonstrated in both human and mouse models of cardiac fibrosis therefore we evaluated the effect of halofuginone on collagen type I gene expression, which is the major collagen in tissue fibrosis. Hardly any cells expressing the 1(I) collagen gene were observed in the cardiac muscle of the wild-type hamsters as demonstrated by in situ hybridization (data not shown). In the CHF147 hamsters an increase in the number of cells expressing the gene were observed. Halofuginone treatment caused a complete reduction in the number of cells expressing the collagen 1(I) gene (Fig. 3). Almost no cthrc1 or collagen was observed in the wt hamsters (Fig. 4). In the CHF147 cardiac muscle a high level of cthrc1 was observed, which was largely reduced by halofuginone treatment (Fig. 4, upper panel). Moreover, double-staining of apical cardiac biopsies with collagen type I and cthrc1 antibodies (Fig. 3, lower panel) revealed that cthrc1 expression overlapped considerably with interstitial collagens, which confirmed our previous results in cardiac muscles of MD mice (41,43), suggesting a general phenomenon. Halofuginone

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treatment resulted in reduction of both collagen type I and cthrc1 levels; the collagen fibers that remained 7 weeks after the treatment were much reduced in thickness (Fig. 4, lower panel). Halofuginone and cardiac myofibroblasts Cardiac fibrosis is associated with the appearance of myofibroblasts, which are the source of collagen, and that contribute to arrhythmogenic slow and discontinuous conduction (46,47). In addition to collagen, these cells express P4Hβ – a major collagen cross-linking enzyme. Almost no P4Hβ-expressing cells were observed in the basis and apex of the wt hamsters’ hearts (Fig. 5, upper panel), but many cells expressing P4Hβ were observed in the CHF147 cardiac muscle (Fig. 5, middle panel). A major reduction in the number of P4Hβ-expressing cells was observed after halofuginone treatment (Fig. 5, lower panel). Image analysis revealed a 4 × reduction in the number of myofibroblasts (P 4Hβ-positive cells) after halofuginone treatment, i.e., from 0.08 ± 0.01 to 0.02 ± 0.0010 arbitrary units; p < 0.01). Hardly any cells expressing P4Hβ were detected in the wt animals (0.002 ± 0.0001). Halofuginone and TGFβ signaling The synthesis of collagen and cthrc1 is under the control of TGFthat is mediated by Smad3 phosphorylation. Low number of nuclei was positive for pSmad3 in the Wt hamsters. Only 19±3 and 21 ± 3% of the nuclei were pSmad3-positive in the apex and basis, respectively (Fig 6, upper panel). High and similar percentages of pSmad3 nuclei were observed in biopsies taken from the apex and basis of the CHF147 hamster hearts (41 ± 4 and 42 ± 7%, in apex and basis, respectively). As much as 85-90% of the pSmad3 was located in the nuclei suggesting full activation of the TGFβ pathway (inner panel)

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(48). Halofuginone caused reductions in the numbers of nuclei exhibiting pSmad3 in the hamster cardiac muscle, which reached 26 ± 3% in both the apex and the base (Fig. 6, lower panel).

DISCUSSION The amount of fibrosis in cardiac disease is associated with retarded conduction and increase vulnerability to arrhythmias (49,50). The adverse increase in fibrillar collagen deposition that accompanies fibrosis occurred irrespective of the etiological origins of cardiomyocyte necrosis and heart disease. This excessive collagen deposition is an integral feature of both ischemic and non-ischemic cardiomyopathies, such as hypertensive heart disease, hypertrophic and dilated cardiomyopathies, and also valvular heart disease (47). In light of the immense impact that fibrosis has on the heart structure and function, antifibrotic therapies are greatly needed. Halofuginone inhibited collagen synthesis and deposition in the cardiac muscle of CHF147 hamsters (Figs. 2 & 3) as previously observed in mice representing DMD (41,44) and dysferlinopathy (43), and in each case the inhibition was associated with cardiac function improvement. In all the reported cases halofuginone did not reduce collagen levels completely; and in the present study some interstitial collagen remained in the hamsters' cardiac tissue (Fig. 2). These findings confirm our previous observations that halofuginone had minimal effects on collagen content in non-fibrotic or minimally fibrotic tissues, whereas its profound inhibitory effect on pathological fibrosis was found in organs that exhibited TGFβ –dependent fibrosis (35,39,41). These results suggest differential regulation of collagen synthesis: low in normal tissue; aggressive and rapid in

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case of chronic tissue fibrosis. Thus, systemic administration of halofuginone will primarily reduce excess cardiac collagen formation without affecting the collagen network that is of utmost important for cardiac muscle integrity, that contributes to the contractility of the heart, and that provides cardiac strength (51-54). Nor will it affect collagen synthesis in other locations. Moreover, halofuginone that inhibited collagen 1(I)

gene expression (Fig. 3) significantly attenuated the anastomotic intimal thickening

that was associated with inhibition of collagen type I but not of type III (55). Although cardiac muscle contains collagen of both types – I and III – changes in collagen I may make the larger contribution to diastolic dysfunction and heart failure and a retained ejection fraction, because it has the higher stiffness properties, occurs in higher proportions, and increases in response to pressure overload. The improvements in the hamsters' cardiac function after halofuginone treatment included prevention of the decrease in LV function that was observed in the untreated CHF147 hamsters. Most importantly, this improvement persisted even weeks after termination of the treatment (Fig. 1). In the MD mice, the halofuginone-dependent decrease in fibrosis resulted in reduction of LV hypertrophy and wall-motion abnormalities (WMAs), in both young mice that developed cardiac fibrosis and old mice with established fibrosis (41,44). Improvements in cardiac function not only rely on diameter changes, but also on lusitropic properties of the myocardium, where interstitial collagen content is a major determinant. Moreover, halofuginone can dissolve existing fibrosis and improve tissue functions as observed in older mdx mice (44), in rats with established liver fibrosis (40), in the tight skin (Tsk+) mice (38) and in graft versus-host disease patients (37). The ability of halofuginone to resolve pre-existing fibrosis is probably due its regulating

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effect on metalloproteinases (MMPs) activity and subsequent fine-tuning of the ECM turnover (56). Taken together, these results suggest a general mechanism by which halofuginone reduces cardiac fibrosis and improves cardiac function, regardless of the species involved or the cause of the fibrosis. TGFthat derives from macrophages that accumulated in close proximity to matrix producing myofibroblasts in the injured heart is the major cytokine driving fibroblasts-to myofibroblasts transition and initiate fibrosis (3,5,57,58). Thus, inhibition of the TGF signaling pathway can be an important cardioprotective strategy. Halofuginone inhibited Smad3 phosphorylation in the CHF147 hamsters’ cardiac muscle (Fig. 5) as it did in other fibrotic muscles representing various MDs without affecting TGF or its receptor levels (39-41). Because of the extremely pleiotropic nature of TGF therapies that target its expression or its receptors are likely to be associated with an array of adverse effects, including abnormal cell proliferation, inflammation and autoimmunity (59). Therefore, drugs that selectively target downstream events are expected to be more successful, because they elicit fewer side effects. Moreover, since halofuginone reduced fibrosis irrespective of the cause or muscle type it can be used to treat various disorders that result in increased fibrosis. The source of collagen type I and cthrc1 is the myofibroblasts (27). These cells are derived from the resident cardiac fibroblasts during myocardial injury, under the control of TGFβ (60,61) or from fibrocytes recruited from the circulation (62,63). The myofibroblasts that synthesize high levels of collagen also synthesize the enzymes responsible for post-translational modifications of collagen, such as P4Hβ – a major 14

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collagen cross-linking enzyme. Halofuginone reduced the number of P4Hβ-expressing cells (Fig. 4), as well as collagen and cthrc1 levels (Fig. 3) in the hamsters' hearts, suggesting a halofuginone-dependent inhibition of the fibroblasts-to-myofibroblasts transition. The observations that cthrc1 and collagen type I occupied different but adjacent areas and that collagen fibers became smaller after halofuginone treatment (27) (Fig. 3) support the notion that cthrc1 is involved in inhibition of collagen synthesis, or even in collagen degradation. Thus, cthrc1 is probably part of the physiological machinery that reverses the fibrotic reaction of the TGF stimulus regardless of the stimulus. Another feature of the myofibroblasts is their contractile and migratory capabilities: cthrc1 has been implicated in cell migration because: A. it was found at high levels in various metastatic and invasive cancers (26, 64,65); B. knockdown of cthrc1 inhibited cell motility (22); and C. fibroblasts and smooth muscle cells over-expressing cthrc1 gene were able to migrate into the wound area significantly faster than control cells (16). Thus, the antifibrotic properties of halofuginone partially spring from its inhibition both of myofibroblast migration and of myofibroblasts' ability to synthesize collagen. In summary, irrespective of whether the necrotic cell death of the cardiomyocyte occurs in the infarcted heart or in non-ischemic heart disease, the myofibroblasts, through their continuous production of signaling molecules, promote fibrogenesis in an autocrine manner. This vicious circle results in formation of scar tissue and interstitial fibrosis and thereby affects the electrical behavior and contractile properties of the myocardium (47, 66, 67). Halofuginone, which has significant impacts on cardiac fibrosis at various levels

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and demonstrates good safety profile, meets the criteria for a potential novel antifibrotic medication for patients with cardiac fibrosis of various etiologies.

Acknowledgments This study was supported in part by the Association Française contre les Myopathies (AFM 14105). We thank Edouard Belauso for his help with the confocal microscopy. This paper is contribution from the Agricultural Research Organization, the Volcani Center, P.O. Box 6, Bet Dagan 50250, Israel

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Legends to figures

Figure 1– Effect of halofuginone on left ventricular ejection fraction (EF). Hamsters (n = 11) were treated with halofuginone (25 g/hamster, 3 times a week) for 6 weeks. During this period and 7 weeks later EF was measured by echocardiography. In all time points the EF was higher (p