Journal of Molecular and Cellular Cardiology 52 (2012) 1257–1264

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Journal of Molecular and Cellular Cardiology

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Original article

Reduction of heart failure by pharmacological inhibition or gene deletion of proteintyrosine phosphatase 1B

Elodie Gomez a,b,1, Magali Vercauteren a,b,1, Baptiste Kurtz a,b, Antoine Ouvrard-Pascaud a,b, Paul Mulder a,b,Jean-Paul Henry a,b, Marie Besnier a,b, Aurélie Waget c, Rob Hooft Van Huijsduijnen d, Michel L. Tremblay e,Remy Burcelin c, Christian Thuillez a,b, Vincent Richard a,b,⁎a Inserm U1096, Rouen, Franceb University of Rouen, Institute for Research and Innovation in Biomedicine, Rouen, Francec Inserm U858, Rangueil Institute of Molecular Medicine, Toulouse, Franced MSPRI, Geneva, Switzerlande Goodman Cancer Centre, Mc Gill University, Montreal, Canada

⁎ Corresponding author at: Inserm U1096, UFR MédBoulevard Gambetta, 76183 Rouen, France. Tel.: +33 214 83 65.

E-mail address: Vincent.Richard@univ-rouen.fr (V. R1 E Gomez and M Vercauteren contributed equally to

0022-2828/$ – see front matter © 2012 Elsevier Ltd. Alldoi:10.1016/j.yjmcc.2012.03.003

a b s t r a c t

a r t i c l e i n f o

Article history:Received 26 October 2011Received in revised form 24 February 2012Accepted 7 March 2012Available online 15 March 2012

Keywords:Heart failureEchocardiographyEndotheliumSignal transductionInsulinTyrosine

Protein tyrosine phosphatase 1B (PTP1B) regulates tyrosine kinase receptor-mediated responses, and espe-cially negatively influences insulin sensitivity, thus PTP1B inhibitors (PTP1Bi) are currently evaluated inthe context of diabetes. We recently revealed another important target for PTP1Bi, consisting in endothelialprotection. The present study was designed to test whether reduction of PTP1B activity may be beneficial inchronic heart failure (CHF).We evaluated the impact of either a 2 month pharmacological inhibition, or a gene deletion of PTP1B(PTP1B−/−) in CHF mice (2 months post-myocardial infarction). PTP1Bi and PTP1B deficiency reduced ad-verse LV remodeling, and improved LV function, as shown by the increased LV fractional shortening and car-diac output (measured by echocardiography), the increased LV end systolic pressure, and the decreased LVend diastolic pressure, at identical infarct sizes. This was accompanied by reduced cardiac fibrosis, myocytehypertrophy and cardiac expression of ANP. In vitro vascular studies performed in small mesenteric arterysegments showed a restored endothelial function (i.e. improved NO-dependent, flow-mediated dilatation, in-creased eNOS phosphorylation) after either pharmacological inhibition or gene deletion. PTP1B−/− CHF alsodisplayed an improved insulin sensitivity (assessed by euglycemic–hyperinsulinemic clamp studies), whencompared to wild-type CHF associated with an increased insulin mediated mesenteric artery dilation.Thus, chronic pharmacological inhibition or gene deletion of PTP1B improves cardiac dysfunction and cardiacremodeling in the absence of changes in infarct size. Thus this enzyme may be a new therapeutic target inCHF. Diabetic patients with cardiac complications may potentially benefit from PTP1B inhibition via two dif-ferent mechanisms, reduced diabetic complications, and reduced heart failure.

© 2012 Elsevier Ltd. All rights reserved.

1. Introduction

Protein tyrosine phosphatase 1B (PTP1B) is an important regula-tor of tyrosine kinase receptor-mediated responses, and preciselynegatively influences insulin sensitivity [1–4]. Thus PTP1B inhibitorsare currently evaluated in the treatment of insulin resistance. Otherfunctions of PTP1B have also been recently identified [5], includingregulation of leptin sensitivity and modulation of oncogenesis. We

ecine-Pharmacie de Rouen, 2235 14 83 62; fax: +33 2 35

ichard).this work.

rights reserved.

also previously revealed another important, previously unrecognized,biological role for PTP1B, consisting in a negative regulation of NOproduction in endothelial cells. Especially, we showed that in vitro in-cubation with PTP1B inhibitors acutely restored NO-mediated vasodi-latation in peripheral resistance arteries isolated from mice withchronic heart failure (CHF) [6].

Knowing the central role of both impaired NO availability [7,8]and insulin resistance [9,10] in the development and aggravation ofCHF, both being targets of PTP1B inhibition, it is possible that a longterm reduction of PTP1B activity may be beneficial in CHF, but this hy-pothesis has not yet been tested.

The purpose of our study was to evaluate the impact of chronicpharmacological inhibition or gene deletion of PTP1B in chronicheart failure, and the associated changes in endothelial function andinsulin sensitivity.

1258 E. Gomez et al. / Journal of Molecular and Cellular Cardiology 52 (2012) 1257–1264

2. Methods

2.1. Animals, induction of heart failure and treatment

The investigation conforms to the Directive 2010/63/EU of the Eu-ropean Parliament. The animal protocol was approved by Rouen Uni-versity ethics review board (Authorization 76-95).

Gene deletion experiments were performed in BalbC/J mice (sincethis was the mouse strain from which PTP1B−/− mice were derived),while pharmacological experiments were performed in C57BL/6Jmice, in order to be consistent with our previous PTP1B studies [6].CHF was induced by left coronary artery ligation in eight-week-oldmale mice that were anesthetized with 3.6 mg/kg xylazine 2% i.p. fol-lowed by continuous isoflurane 2% inhalation (1.5 ml/min; Baxter)during artificial ventilation. Anesthesia and sedation were controlledby monitoring heart rate, and by performing paw pinch reflex andcorneal reflex tests.

For pharmacological treatment, CHF mice were either untreatedor treated with the PTP1B inhibitor AS279 [6] (Merck-Serono Phar-maceutical Research Institute) at the dose of 60 mg/kg/day in drink-ing water for 2 months, starting 7 days after ligation. For genedeletion, we used PTP1B−/− mice generated as described previously[1]. Early post-operative mortality (measured within the first 7 days,before the introduction of the PTP1Bi treatment that was started7 days post ligation), was around 50% in all groups. No mortalitywas observed in any groups between 7 days and 2 months post-infarction.

2.2. Cardiac echocardiographic and hemodynamic parameters

The development of heart failure was assessed by echocardiogra-phy in isoflurane-anesthetized closed-chest mice at 2 months (phar-macological study), or 1 week, 1 month and 2 months (genedeletion study) with the use of an echocardiographic system (HDI5000, ATL), equipped with a 10 MHz transducer. At 2 months, afterthis analysis, the right carotid artery was cannulated with a micro-manometer (1.2F, Millar Instruments) advanced into the aorta, to re-cord arterial pressure. The micromanometer was then advanced intothe LV to record LV pressures and dP/dt.

2.3. Studies in isolated arteries

Vascular studies were performed in mesenteric resistance arterysegments as previously described [6]. Flow-mediated vasodilatation(FMD) was evaluated at baseline or in the presence of the NOsynthase inhibitor NG-nitro-L-arginine (L-NNA, 10−4 M, Sigma) orthe PI3 Kinase inhibitor, wortmannin (10−7 M, Sigma). In some ex-periments, FMD was assessed after a 30 min infusion with TNFα(0.1 ng/ml), in the absence or in the presence of the PTP1B inhibitorAS279 (10−5 M).

Western blot analysis of small arteries was performed as de-scribed previously [6].

2.4. RNA extraction and quantitative RT-PCR

Total RNA was extracted from mesenteric arteries or from LV withTrizol (Gibco Life sciences), followed by DNAse (Sigma). Quantitativereal-time reverse transcription-polymerase chain reaction (RT-PCR)was performed with a light-cycler (Roche, Basel, Switzerland). Thefollowing primers (Proligo) were used:

MMP-2: Forward 5′-AGATCTTCTTCTTCAAGGACCGGTT-3′; Reverse5′-GGCTGGTCAGTGGCTTGGGGTA-3′. MMP-9: Forward 5′-CCCA-CATTTGACGTCCAGAGAAGAA-3′; Reverse 5′-CCCACATTTGACGTCCA-GAGAAAGAA-3′. α-MHC: Forward 5′-GGCACAGAAGATGCTGACAA-3′; Reverse 5′-CTGCCCCTTGGTGACATACT-3′. β-MHC: Forward 5′-

CTTCAACCACCACATGTTC-3′; Reverse 5′-TCTCGATGAGGTCAATG-CAG-3′. ANP: Forward 5′-GGGGGTAGGATTGACAGGAT-3′; Reverse5′-ACACACCACAAGGGCTTAGG-3′. BNP: Forward 5′-GCCAGTCTCCA-GAGCAATTC-3′; Reverse 5′-CCTTGGTCCTTCAAGAGCTG-3′. ICAM-1:Forward: 5′-ACTGGCAGTGGTTCTCTGCT-3′; Reverse 5′-AAAGTAGGT-GGGGAGGTGCT-3′. TNF-α: Forward 5′-ACTGGCAGTGGTTCTCTGCT-3′; Reverse 5′-AAAGTAGGTGGGGAGGTGCT-3′. eNOS: Forward:5′-GACCCTCACCGCTACAACAT; Reverse 5′-CACAGGGATGAGGTTGTCCT;iNOS: Forward: 5′-TGGTGGTGACAAGCACATTT; Reverse 5′-AAGGCCAAA-CACAGCATACC; nNOS: Forward: 5′-CTAGCTGGGTGTTGGGTGTT; Reverse5′-CACCCCTAGGCTAACCCTTC; 18S Forward 5′-GTGGAGCGATTT-GTCTGGTT-3′; Reverse 5′-CGCTGAGCCAGTCAGTGTAG-3′.:

2.5. Insulin sensitivity

Insulin sensitivity was assessed by euglycemic hyperinsulinemicclamp studies, performed in mice as previously described [11,12].Vascular insulin sensitivity was tested in isolated perfused mesentericresistance artery segments by measuring the changes in diameter in-duced by increasing concentrations of insulin (10−9–10−6 M).

2.6. Statistical analysis

All data are presented as mean±SEM. For vascular functionalstudies, differences between groups were analyzed by 2-factor re-peated measures ANOVA, using SigmaStat V3.5. All other differenceswere analyzed by ANOVA followed by a Tukey test for multiple com-parisons. A p value b0.05 was considered statistically significant.

3. Results

3.1. Cardiac dysfunction and remodeling

In the pharmacological inhibition study (Table 1, Fig. 1), untreatedCHF mice displayed increased LV diameters and LV end-diastolicpressure, and decreased LV fractional shortening, cardiac output, LVend-systolic pressure, LV dP/dt max, LV dP/dt min and arterial pres-sures. CHF also induce a small, non-significant decrease in heartrate. Compared to untreated CHF, PTP1B inhibition significantly re-duced LV end-systolic diameters and LV end-diastolic pressure, andincreased LV fractional shortening, LV end-systolic pressure, LV dP/dt max, and LV dP/dt min. PTP1B inhibition also increased cardiacoutput, as well as systolic and diastolic blood pressures, but thesechanges were not statistically significant.

The cardiac effects of PTP1B gene deletion are shown in Table 1 andFig. 1. In normal mice, we found no difference in cardiac echocardio-graphic and hemodynamic parameters between WT and PTP1B−/−.In WT mice, CHF increased LV diameters and LV end-diastolic pres-sure, and decreased LV fractional shortening, cardiac output, LV end-systolic pressure, LV dP/dt max, LV dP/dt min and arterial pressures.Serial echographic evaluation showed that the decreased fractionalshorteningwas already present 1 week after ligation, and progressive-ly aggravated at 1 and 2 months. Compared toWT CHF, PTP1B−/− CHFmice showed significantly decreased LV end-diastolic diameter, end-systolic diameter and LV end-diastolic pressure, and a significantly in-creased LV fractional shortening, whichwas observed both at 1 monthand 2 months. PTP1B−/−mice also displayed a significant increased inLV dP/dt and LV end-systolic pressure, as well as cardiac output, with-out changes in arterial pressure or heart rate.

The effects of pharmacological inhibition and of gene deletion ofPTP1B on cardiac hypertrophy and fibrosis are shown in Fig. 2. Atidentical infarct sizes, chronic PTP1B inhibition reduced LV weightand LV weight/tibia length ratio, suggesting reduced LV hypertrophy,and also significantly prevented CHF-induced increased collagen

Table 1Cardiac and hemodynamic parameters.

Pharmacological inhibition Gene deletion

Normal CHF CHF+PTP1Bi WT PTP1B−/− CHF WT CHF PTP1B−/−

CO (ml/min) 25.5±1 18.2±1.1** 21.9±1.3 27±1.8 28.2±1.4 16.3±1⁎⁎ 22.2±1.1†

HR (bts/min) 527±8 493±23 532±18 494±14 521±19 509±16 503±11SABP (mm Hg) 105±3 87±2⁎ 92±4 109±4 114±4 88±3⁎ 94±5DABP (mm Hg) 69±2 57±2⁎ 64±4 78±4 87±4 66±3⁎ 73±3LVESP (mm Hg) 108±9 83±2⁎ 92±3† 92±3 106±5 64±8⁎ 89±6†

LVEDP (mm Hg) 0.5±0.5 4.0±1.3⁎ 0.8±0.5† 0.4±0.4 0.3±0.3 11.0±6.6⁎ 0.8±0.6†

⁎ pb0.05.⁎⁎ pb0.01 vs WT.† pb0.05 vs CHF WT.

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density, suggesting that it also reduced fibrosis. In normal mice, com-pared to WT, PTP1B deficiency was not associated with changes in LVweight or collagen density. At identical infarct sizes, PTP1B−/− CHFmice displayed no changes in LV weight and LV weight/tibia length

Normal CHF

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sec

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Fig. 1. a) Echocardiographic and hemodynamic parameters in normal (n=13), or 2-montAS279 (n=15). * pb0.05 and ** pb0.01 vs. normal mice; † pb0.05 and †† pb0.01 vs. untrCHF mice, either WT (normal n=11, CHF n=12) or PTP1B−/− (normal n=11, CHF n=9)*

ratio, compared to WT. However, cardiac collagen density was re-duced in PTP1B−/− CHF mice, indicating reduced fibrosis.

The effect of CHF and PTP1B deletion on the expression of variouscardiac genes is shown in Table 2. In normal mice, no differences were

CHF + chronic PTP1Bi

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h CHF mice, either untreated (n=13) or chronically treated with the PTP1B inhibitoreated CHFb) Echocardiographic and hemodynamic parameters in normal or 2-monthpb0.05 and ** pb0.01 vs. WT normal mice; † pb0.05 and †† pb0.01 vs. WT CHF mice.

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Fig. 2. a) LVweight, LVweight/tibia length, infarct size and LV collagen density obtained normal (n=13) or CHFmice, either untreated (n=11) or chronically treated with the PTP1Binhibitor AS279 (n=15) (top)b) LV weight, LV weight/tibia length, infarct size and LV collagen density obtained in WT (normal n=17, CHF n=15) or PTP1B−/− mice (normaln=20, CHF n=16). Representative images of collagen density in the four groups are also presented.** pb0.01 vs. normalmice; † pb0.05 and †† pb0.01 vs. untreated orWT CHFmice.

Table 2LV gene expression.

WT PTP1B−/− CHF WT CHF PTP1B−/−

α-MHC/18S (%) 1.04±0.05 0.94±0.04 0.91±0.1 0.73±0.05β-MHC/18S (%) 1.27±0.06 1.24±0.04 1.16±0.17 0.92±0.07ANP/18S (%) 1.41±0.42 0.86±0.18 7.09±2.05⁎⁎ 3.23±0.93†

BNP/18S (%) 0.72±0.08 0.86±0.13 1.87±0.34⁎⁎ 1.48±0.24MMP-2/18S (%) 1.07±0.1 0.88±0.04 1.24±0.04⁎ 1.12±0.11MMP-9/18S (%) 0.85±0.14 1.27±0.33 0.64±0.12 0.66±0.15TNFα/18S (%) 0.93±0.45 1.17±0.45 0.42±0.30 1.02±0.23ICAM-1/18S (%) 0.60±0.14 0.78±0.21 0.53±0.10 0.71±0.10

⁎ pb0.05.⁎⁎ pb0.01 vs WT.† pb0.05 vs CHF WT.

1260 E. Gomez et al. / Journal of Molecular and Cellular Cardiology 52 (2012) 1257–1264

1261E. Gomez et al. / Journal of Molecular and Cellular Cardiology 52 (2012) 1257–1264

detected between WT and PTP1B−/−. In WT, CHF induced significantincreases in LV expression of ANP, BNP and MMP-2, but did not affectα-MHC, β-MHC, MMP-9, TNFα or ICAM-1 expression. In CHF mice,PTP1B deletion significantly decreased ANP and also decreased (al-though non significantly) BNP and MMP-2.

3.2. In vitro vascular studies

3.2.1. Pharmacological inhibitionCHF abolished FMD of mice mesenteric arteries (Fig. 3a), without

affecting the responses to acetylcholine or SNP (Fig. 3b). PTP1B inhi-bition restored FMD to levels similar to those of normal mice(Fig. 3a), suggesting that this chronic treatment restored endothelialfunction ex vivo. Chronic PTP1B inhibition did not affect the re-sponses to acetylcholine or sodium nitroprusside (Fig. 3b).

In arteries obtained from CHF mice chronically treated with thePTP1B inhibitor, FMD was reduced by the PI3 kinase inhibitor wort-mannin and abolished by the NOS inhibitor L-NNA (Fig. 3c), suggest-ing that the improved endothelial function involved a restoration ofPI3K-mediated activation of eNOS. Indeed, Fig. 3d shows that CHFmarkedly reduced eNOS phosphorylation on Serine 1177 in arteriessubmitted to intraluminal flow, and this was prevented by chronicPTP1B inhibition. These changes in phosphorylation status were notassociated with any changes in eNOS levels.

Normal

CHF CHF + chronic PTP1Bi

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Pharmacologica

Fig. 3. a) Effect of CHF and chronic pharmacological PTP1B inhibition on flow-mediated vANOVA revealed statistically significant differences (pb0.01) between normal and CHF andcological PTP1B inhibition on the vasodilatory responses of mesenteric arteries to acetylchocubation with the NOS inhibitor L-NNA and the PI3K inhibitor wortmannin of arteries isolatand 11 for wortmannin). Repeated measures ANOVA revealed statistically significant d(pb0.05).d) Representative Western blot data of total eNOS or P Ser1177-eNOS , and meafrom normal or CHF mice, either untreated or chronically treated with the PTP1B inhibitor

Since previous data showed that TNFα induces endothelial dys-function [13] and is increased in heart failure, we also tested whetherthe endothelial effects of TNFαwere sensitive to PTP1B inhibition.Weconfirmed that incubation with TNFα (0.1 ng/ml) markedly reducedFMD (from 14±4 to 5±3%, n=3), and this was fully prevented byincubation with the PTP1B inhibitor (20±6, n=3).

3.2.2. PTP1B gene deletionThe effects of gene deletion of PTP1B on vascular function are

shown in Fig. 4. Wild type BalbC/J mice displayed a vascular profilesimilar to that of C57BL/6j mice, with a progressive increase in diam-eter in response to stepwise increases in intraluminal flow (FMD,Fig. 4a), and this response was virtually abolished after 2 monthsCHF (Fig. 4b). In contrast, no changes in FMD were observed after ashorter (2 weeks) period of CHF (maximal dilatation normal 18±4;CHF 15±5, n=6; data not shown).

In normal mice (without CHF), PTP1B deficiency reduced FMD(Fig. 4a). In contrast, PTP1B−/− CHF mice displayed no alteration ofFMD (Fig. 4b), demonstrating that these mice were protected againstCHF-induced endothelial dysfunction.

Fig. 4c shows that acute in vitro incubation with the pharmaco-logical PTP1B inhibitor AS279 failed to improve FMD in arteries iso-lated from PTP1B−/− CHF mice (in contrast to its effect describedbefore by us in wild-type mice [6]), suggesting that PTP1B is the

Responses to acetylcholine and sodium nitroprusside

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asodilatation of mesenteric arteries (n=8–9 animals per group). Repeated measuresbetween CHF and CHF+chronic PTP1B inhibition.b) Effect of CHF and chronic pharma-line or sodium nitroprusside (SNP) (n=8–9 animals per group).c) Effect of in vitro in-ed from CHF mice chronically treated with the PTP1B inhibitor AS279 (n=7 for L-NNAifferences between base and L-NNA (pb0.01) and between base and wortmanninn±SEM P Ser1177-eNOS/eNOS ratio and eNOS (n=6) in mesenteric arteries isolatedAS279, and exposed to flow at 200 μl/min for 2 min. and 30 sec.

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Gene Deletion

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Liver Mes. artery

-/- +/+ -/- +/+ -/- +/+ -/- +/+ -/- +/+

Lung LV RV Neg RT

18S

Fig. 4. a) Flow-mediated vasodilatation of mesenteric arteries obtained from WT (n=16) or PTP1B−/− (n=8) normal mice (in the absence of CHF). Repeated measures ANOVArevealed statistically significant differences (pb0.01) between WT and PTP1B−/−.b) Flow-mediated vasodilatation of mesenteric arteries obtained from WT (n=10) or PTP1B−/−

mice (n=12) with CHF. Repeated measures ANOVA revealed statistically significant differences (pb0.01) betweenWT and PTP1B−/− CHF mice.c) Effect of acute in vitro incubationwith the PTP1B inhibitor AS179 on flow-mediated vasodilatation ofmesenteric arteries isolated from PTP1B−/− CHFmice (n=7 ).d) Representative PCR experiments showing PTP1Bexpression in the liver, lung, RV, LV, and small mesenteric artery (MA) from WT or PTP1B−/− normal mice.

1262 E. Gomez et al. / Journal of Molecular and Cellular Cardiology 52 (2012) 1257–1264

molecular target involved in the endothelial protection exerted bythis compound.

Finally, we verified the absence in PTP1B−/− mice of PTP1B mRNAexpression in the liver, lung, RV and LV, as well as in the mesentericartery segments used in functional studies (Fig. 4d).

3.3. Arterial and cardiac NOS gene expression

The effects of CHF or gene deletion on eNOS, iNOS and nNOSmRNA expression in mesenteric arteries and in the LV are shown in

Normal WT PTP1B-/-

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Fig. 5. mRNA expression of NOS isoforms (normalized to 18S) in mesenteric arteries (left)

Fig. 5. Neither CHF, not PTP1B gene deletion affected the expressionof any of the NOS isoforms, whether in the mesenteric artery and inthe heart.

3.4. Insulin sensitivity

Fig. 6a shows glucose turnover rate in euglycemic hyperinsuline-mic clamp studies. In normal mice, PTP1B deletion did not affect glu-cose turnover rate. Compared to normal, CHF was not associatedwith changes in insulin sensitivity. However, in CHF mice, PTP1B

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or hearts (right) from WT or PTP1B−/− mice, either normal or CHF (n=6 per group).

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Fig. 6. a) Glucose turnover rate (ml/kg·min) assessed in euglycemic hyperinsulinemicclamp studies performed in normal mice (n=16), or CHF mice, either WT (n=9) orPTP1B−/− (n=17). * pb0.05 vs. CHF WT.b) Vasodilatory responses to insulin of mes-enteric arteries obtained from normal (n=6) or CHF mice, either WT (n=7) orPTP1B−/− (n=5). Repeated measures ANOVA revealed statistically significant differ-ences between normal and CHF mice and between WT CHF and PTP1B−/− CHF mice.

1263E. Gomez et al. / Journal of Molecular and Cellular Cardiology 52 (2012) 1257–1264

deficiency induced a significant increase in glucose turnover rate, sug-gesting an increased insulin sensitivity in this disease, similar to whathas been previously described in diabetes or obesity.

Fig. 6b shows the vasodilatory responses of mesenteric artery seg-ments to increasing concentrations of insulin. Compared to normal,CHF markedly reduced the vascular response to insulin. This impair-ment was partly prevented in PTP1B−/− mice.

4. Discussion

The main result of our study is that long term pharmacological in-hibition or gene deletion of PTP1B improved heart failure, as shownby the increased LV function and decreased adverse LV remodeling.

The beneficial cardiac effects of PTP1B blockade were observed ona large series of parameters of cardiac remodeling and dysfunction.This includes 1) increased cardiac contractile function and hemody-namics, with increased LV fractional shortening, cardiac output, LVend systolic pressure, LV dP/dt max, LV dP/dt min, and decreased LVend diastolic pressure, and 2) reduced LV remodeling, with decreasedLV diameters, cardiac hypertrophy and fibrosis, reduced ANP and BNPgene expression. Since these beneficial were observed in the absenceof changes in infarct size (as expected in this permanent ligationmodel), they are most likely due to the improvement of cardiac remo-deling at the remote area. We could not however assess the effects ofthis approach on other important parameters such as cardiac inflam-mation or long-term mortality, since the present mouse model didnot appear to be associated with any detectable changes in cardiacinflammatory markers or any mortality in the absence of treatment.The beneficial effects obtained in a series of clinically relevantparameters point toward a new important target for PTP1B inhibitors,as promising treatments of heart failure. Whether this new approach

provides additional protectionwhen combinedwith standard therapyof heart failure (and especially with treatments known to induceendothelial protection in this setting, such as angiotensin convertingenzyme inhibitors [14]) is unknown and was not addressed in thepresent study.

There are different possible mechanisms for this beneficial cardiacimpact in CHF, including restoration of NO production. Indeed, the va-soconstriction associated with impaired NO production leads to an el-evation of peripheral resistance and thus of cardiac afterload. Thischange in afterload results in an aggravation of CHF, via an increasedcardiac work. Conversely, restored peripheral NO production may perse decrease cardiac work and reduce the severity of CHF. In support ofthis hypothesis, eNOS overexpression was shown to reduce heart fail-ure in mice [8], while the beneficial effects of statins or exercise in thisdisease are reduced in eNOS-deficient mice [15–17], although notonly endothelial, but also cardiac eNOS, may contribute to these ef-fects. At this point, we can only postulate on the role of endothelialprotection in the reduction of heart failure. Demonstration of such arole would require assessing the development of heart failure inmice with endothelial specific PTP1B deficiency; however this ap-proach is beyond the scope of the present study. In addition, thefact that cardiac dysfunction precedes endothelial dysfunction in thepresent model (as evidenced by the absence of impaired FMD at2 weeks while fractional shortening is already markedly reduced at1 week) would argue against a deleterious role of endothelial dys-function. This however does not fully rule out the hypothesis that en-dothelial dysfunction participates to the aggravation of heart failure,especially since cardiac dysfunction progressively aggravated overtime, as shown by the progressive decrease in fractional shorteningand 1 and 2 months, compared with 1 week.

Another mechanism which may contribute to the improvement ofheart failure is the increased insulin sensitivity. Indeed, although wedid not observe a change in glucose turnover rate in CHF mice whencompared to normal, suggesting that this disease is not associatedwith a clear development of insulin resistance in our model, wefound that glucose turnover rate was higher in PTP1B−/− CHF mice,compared to WT. This suggests that PTP1B deficiency is associatedwith increased insulin sensitivity in CHF. In parallel, PTP1B deficiencypartially prevented the reduced vasodilatory response to insulin in-duced by CHF, and this effect may participate both to the increased in-sulin sensitivity and to the reduced heart failure.

There are several mechanisms by which increased insulin sensitiv-ity may be beneficial in CHF, including improved myocardial energymetabolism [18–20], but also increased tissue perfusion, decreasedoxidative stress, or reduced sympathetic activation. However whetherthese effects actually contribute to the reduction of heart failure is cur-rently unknown. In any case, since it is likely that the improved LVfunction and reduced adverse LV remodeling may per se be secondar-ily responsible for the increased insulin sensitivity, it seems extremelydifficult to separate whether this mechanism is a cause or conse-quence of the reduced heart failure.

Both long term administration of a PTP1B inhibitor and gene dele-tion of this phosphatase induced a complete prevention of endotheli-al dysfunction. Several mechanisms may contribute to this beneficialendothelial effect; however the most likely hypothesis is that it re-flects a direct impact of PTP1B on the endothelium and especiallyon eNOS activation and NO production, via increased tyrosine phos-phorylation of endothelial proteins, possibly upstream of PI3K/Akt.This improvement was observed in the absence of changes in eNOSexpression, or in other NOS isoforms (iNOS and nNOS). In the presentwork we were not able to address the specific target proteins, as ourstudies were performed in very small arteries, which present the ad-vantage of a strong pathophysiological relevance (through their con-tribution to the control of peripheral resistance), but from which wecan extract a very small amount of proteins, with just a portionbeing from endothelial origin. The exact endothelial target(s) of

1264 E. Gomez et al. / Journal of Molecular and Cellular Cardiology 52 (2012) 1257–1264

PTP1B remain to be determined, probably in other models such ascultured endothelial cells, since this is currently practically unfeasiblein complex chronic in vivo models such as the one used in our study.

Apart from the direct effects on eNOS transduction pathways, onecannot rule out that at least part of the endothelial protective effectmay be mediated by indirect mechanisms, secondary to the improvedhemodynamic status. This includes chronic increase in vascular shearstress, or decrease in the neurohumoral and/or inflammatory media-tors such as angiotensin II or TNFα which are increased in CHF andare triggers of endothelial dysfunction. In agreement with this latterhypothesis, we found that PTP1B inhibition prevented the impairedFMD induced by in vitro incubation with TNFα. These changes in neu-rohumoral activation would most likely result in altered oxidativestress, known to be a potent regulator of NO bioavailability. However,due to the small size of the vascular samples of the study, we havebeen so far unable to detect either reactive oxygen species , or varia-tions of NO levels, by electron spin resonance in small segments ofisolated mesenteric arteries such as the one used in the presentstudy. Thus, at present, the exact role of the NO/reactive oxygen spe-cies balance in the observed results is still unknown. In any case, thefact that endothelial function of CHF mice is also improved by in vitroincubation of isolated arteries with a PTP1B inhibitor [6] strongly sug-gests that part of the effects is direct and does not occur secondary tothe improved hemodynamic status.

Another possibility is that the improved endothelial function ispartly a consequence of the increased insulin sensitivity after block-ade of PTP1B, again as suggested by the improved vasodilatory re-sponse to this peptide in arteries from CHF mice. Surprisingly, wefound that while in CHF mice, PTP1B deficiency was associated witha markedly improved endothelial function, shown by the increasedFMD, in normal mice (i.e. in the absence of CHF) the absence ofPTP1B was associated with a reduced FMD, when compared to WT.This may represent chronic in vivo adaptation to PTP1B deficiency,e.g. expression of other phosphatases [21]. However, the fact that asimilar reduction was observed after acute in vitro PTP1B inhibition[6] would suggest that tyrosine dephosphorylation by PTP1B is im-portant for normal regulation of shear stress-induced NO productionin the absence of any disease.

PTP1B inhibitors are currently evaluated in the treatment of diabe-tes [22], and diabetic patients have an increased risk of heart failureeven after adjusting for concomitant risk of micro- andmacrovasculardiseases [23]. Importantly, diabetes is an independent predictor ofcardiovascular mortality in patients with ischemic heart failure [24].Thus, our study suggests that diabetic patients with heart failuremay benefit from PTP1B inhibition via two different mechanisms,i.e. reduced diabetic complications, and reduced heart failure.

In conclusion, our study performed in a mouse model of heart fail-ure demonstrates that long-term pharmacological inhibition or genedeletion of PTP1B reduces LV dysfunction and adverse LV remodeling.These data suggest for the first time that PTP1B inhibition, currentlyconsidered for the treatment of diabetes, may also be a new therapeu-tic approach in heart failure.

Funding

This work was supported by a grant from the Fondation deFrance. Magali Vercauteren was supported by fellowships from theConseil Régional de Haute Normandie and the Société Françaised'Hypertension. Elodie Gomez was supported by fellowships fromthe Société Française de Pharmacologie et de Thérapeutique andfrom the Groupe de Réflexion sur la Recherche Cardiovasculaire.Baptiste Kurtz was supported by a fellowship from the FédérationFrançaise de Cardiologie. Vincent Richard was supported by a Con-trat d'interface Inserm.

Disclosures

Dr Rob Hooft Van Huijsduijnen is a full time employee ofMerck-Serono.

Acknowledgments

We thank Christophe Arnoult for his help in genotyping the mice,and Brigitte Dautreaux, Sylvanie Renet and Cathy Vendeville for ex-pert technical assistance.

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