Cardiovascular pharmacology

Protective effects of sitagliptin on myocardial injuryand cardiac function in an ischemia/reperfusion rat model

Guanglei Chang, Peng Zhang, Lin Ye, Kai Lu, Ying Wang, Qin Duan, Aihua Zheng, Shu Qin n,Dongying Zhang nn

Department of Cardiology, The First Affiliated Hospital of Chongqing Medical University, No.1 Yixueyuan Road, Chongqing 400016, PR China

a r t i c l e i n f o

Article history:Received 20 March 2013Received in revised form28 August 2013Accepted 4 September 2013Available online 13 September 2013

Keywords:DPP4 inhibitorSitagliptinIschemia/reperfusionMyocardial injuryCardiomyocyte apoptosisCardiac function

a b s t r a c t

The purpose of this study is to investigate the effects and the underlying mechanisms of sitagliptinpretreatment on myocardial injury and cardiac function in myocardial ischemia/reperfusion (I/R) ratmodel. The rat model of myocardial I/R was constructed by coronary occlusion. Rats were pretreated withsitagliptin (300 mg/kg/day) for 2 weeks, and then subjected to 30 min ischemia and 2 h reperfusion.The release of lactate dehydrogenase (LDH) and creatine kinase-MB (CK-MB), cardiac function andcardiomyocyte apoptosis were evaluated. The levels of malondialdehyde (MDA), glutathione peroxidase(GSH-Px) and superoxide dismutase (SOD) in heart and glucagon-like peptide-1 (GLP-1) level in plasmawere measured. Western blot analysis was performed to detect the target proteins of sitagliptin.Our results showed that sitagliptin pretreatment decreased LDH and CK-MB release, and MDA level in I/Rrats. More importantly, we revealed for the first time that sitagliptin pretreatment decreased cardio-myocyte apoptosis while increased the levels of GSH-Px and SOD in heart. Sitagliptin also increased GLP-1 level and enhanced cardiac function in I/R rats. Furthermore, sitagliptin pretreatment up-regulatedAktserine473 and Badserine136 phosphorylation, reduced the ratio of Bax/Bcl-2, and decreased expressionlevels of cleaved caspase-3 and caspase-3. Interestingly, the above observed effects of sitagliptin were allabolished when co-administered with GLP-1 receptor antagonist exendin-(9-39) or PI3K inhibitorLY294002. Taken together, our data indicate that sitagliptin pretreatment could reduce myocardialinjury and improve cardiac function in I/R rats by reducing apoptosis and oxidative damage. Theunderlying mechanism might be the activation of PI3K/Akt signaling pathway by GLP-1/GLP-1 receptor.

Crown Copyright & 2013 Published by Elsevier B.V. All rights reserved.

1. Introduction

Myocardial infarction is a major cause of mortality and morbid-ity of patients with diabetes mellitus (Acar et al., 2011). In order toprevent the myocardium from further damage, the best therapeuticstrategy for myocardial infarction is to reestablish the blood flowas earlier as possible. Nevertheless, ischemia/reperfusion (I/R) injurysuch as cardiomyocyte apoptosis is inevitable. Cardiomyocyteapoptosis induced by I/R plays an important role in causinga gradual decline of cardiac function (Gottlieb, 2011). Therefore,the exploration of new therapeutic agents that reduce I/R injury ofmyocardial infarction patients has become very important.

Glucagon-like peptide-1 (GLP-1) is secreted by the entero-endocrine L cells of the intestinal mucosa and released in responseto nutrient ingestion (Nauck et al., 1993). It exerts insulinotropic andinsulinomimetic effects via the G-protein-coupled GLP-1 receptor

(Verge and Lopez, 2010). The therapy based on the functions of GLP-1 is currently used as a novel anti-diabetic approach (Doupis andVeves, 2008; Garber, 2012). However, GLP-1 is rapidly degradedby dipeptidyl peptidase-4 (DPP4) enzyme in the blood (Green et al.,2006). The short half life time limited its clinical use. Thus, twoclasses of drugs, including GLP-1 analogs (Garber, 2012)(i.e. exenatide) and DPP4 inhibitors (Doupis and Veves, 2008)(i.e. sitagliptin), have been recently used for treating type 2 diabetes.

Recently, growing evidences have demonstrated the beneficialeffects of GLP-1 analogs during I/R injury in both animal models andin clinical studies, such as limiting infarct, improving cardiac functionand enhancing myocardial glucose uptake (Bhashyam et al., 2010;Chinda et al., 2012a; Lorber, 2012; Mundil et al., 2012). The mechan-isms underlying the cardioprotective effects of GLP-1 analogs maybe both GLP-1 receptor dependent and independent pathways(Ban et al., 2008; Chinda et al., 2012a). Unlike GLP-1 analogs,evidences regarding the cardioprotective effects of DPP4 inhibitorsare scarce and controversial. Recently, more and more researchershave paid close attention to the cardioprotective effects of DPP4inhibitors. Chinda, et al. (2012b) reported that DPP4 inhibitor couldstabilize cardiac electrophysiology in a myocardial I/R pig model.

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European Journal of Pharmacology

0014-2999/$ - see front matter Crown Copyright & 2013 Published by Elsevier B.V. All rights reserved.http://dx.doi.org/10.1016/j.ejphar.2013.09.007

n Corresponding author. Tel.: 86 13101345177; fax: 86 2389011562.nn Corresponding author. Tel.: 86 13650502588; fax: 86 2368055542.E-mail addresses: Qinshu@21cn.com (S. Qin),

augustlei127@aliyun.com (D. Zhang).

European Journal of Pharmacology 718 (2013) 105–113

In addition, DPP4 inhibitor has been shown to attenuate the infarctsize and improve the left ventricular function during myocardial I/Rinjury (Chinda et al., 2012a; Jose and Inzucchi, 2012; Lenski et al., 2011;Scheen, 2012). However, the correlation between its cardioprotectiveeffect and cardiomyocyte apoptosis during myocardial I/R is unclear.

Hereby, the purpose of this study is to investigate whether thecardioprotective effects of sitagliptin, a DPP4 inhibitor, is relative toits anti-apoptotic function and to explore the underlying mechanism.We hypothesized that sitagliptin played the role of cardioprotectionin a myocardial I/R rat model by reducing cardiomyocyte apoptosis.To test this hypothesis, we pretreated rats with sitagliptin for 2 weeksbefore inducing myocardial I/R. Then the effects of sitagliptin onmyocardial injury and cardiomyocyte apoptosis were determined.Finally, we used the GLP-1 receptor antagonist exendin-(9-39) toassess the role of GLP-1 receptor-dependent pathway in the cardio-protective effects of sitagliptin.

2. Materials and methods

2.1. Experimental animals and drugs

Male Sprague–Dawley rats aged between 6 and 8 weeks werepurchased from the Laboratory Animal Center of Chongqing MedicalUniversity [certificate: SCXK (YU) 2007-0001]. Rats were housedunder optimal conditions with standard hygiene, temperature, photo-periods (12L: 12D), standard rat chow and water ad libitum. All ofthese conditions were conformed to the Guidelines for Care and Use ofLaboratory Animals. All procedures on animals were approved by theEthical Committee of the Chongqing Medical University.

The DPP4 inhibitor sitagliptin was purchased from Merck Sharp& Dohme Italia SPA. The PI3K inhibitor LY294002 was purchasedfrom Santa Cruz Biotechnology, Inc. The GLP-1 receptor antagonistexendin-(9-39) was purchased from Sigma, St. Louis, MO, USA.

2.2. Establishment of myocardial I/R injury model

Forty Male Sprague–Dawley rats were randomly divided into thefollowing five groups (n¼8): the Sham group, the I/R group,the sitagliptinþ I/R group (sitagliptin), the sitagliptinþexendin-(9-39)þ I/R group (sitagliptinþE) and the sitagliptinþLY294002þ I/Rgroup (sitagliptinþL). Sitagliptin (300 mg/kg/day) was administratedby intraperitoneal injection for 2 weeks. Exendin-(9-39) (45 μg/kg/3days) and LY294002 (0.3 mg/kg/3 days) were given by intraperitonealinjection 30min before sitagliptin injection. Sitagliptin, exendin-(9-39)and LY294002 were all dissolved in dimethyl sulfoxide (DMSO). TheSham group and the I/R group received the same volume of DMSO for2 weeks.

After pretreatment with sitagliptin for 2 weeks, all rats wereanesthetized by chloral hydrate (concentration 3.5%, 10 ml/kg).Tracheotomy was carried out for ventilation by a respirator(ALC-V8B, Shanghai Alcott Biotech Co., Ltd.) with a stroke volumeof 28 ml/kg, air pressure of 10 mmHg, respiration rate of 1:1 and ata rate of 86 strokes per minute. And the electrocardiogram of leadII was monitored. Thoracotomy was performed and the leftanterior descending coronary artery was ligated by 6-0 silk. Thenthe left anterior descending coronary artery was subjected to30 min of ischemia followed by reperfusion for 2 h. Rats in theSham group were subjected to the same surgery process withoutcoronary artery ligation.

Glucose levels were measured with a blood glucose monitor(Accu-Checks, Roche, Germany). Body weights of rats were weightedafter the establishment of the I/R model. At the end of hemodynamicmeasurement, the blood plasma samples were collected fromthe heart using the anticoagulant tube. The hearts were rapidlyexcised and arrested in diastole in cold diethyl pyrocarbonate water

after the rats were euthanized. Then the heart was transected parallelto the atrioventricular groove at the center of the ischemia area aspreviously described (Li et al., 2010). The right ventricle and atriawere rapidly removed, and the left ventricle was weighed. The leftventricular weight index was expressed as the ratio of left ventricularweight to body weight. And the blood plasma samples and hearttissue were collected immediately and stored at �80 1C.

2.3. Hemodynamic measurements

During the entire I/R period, the right common carotid arteryand left femoral artery were isolated. A polystyrene PE-20 catheterwas inserted into the left ventricle via right common carotidartery, with one end connected to MPA-2000 multichannel phy-siologic recorder. The left ventricular end-systolic pressure(LVESP), left ventricular end-diastolic pressure (LVEDP) and therates of maximum positive and negative left ventricular pressuredevelopment (7LVdp/dt max) were measured.

2.4. ELISA assay

Levels of active GLP-1 and creatine kinase-MB (CK-MB) in theplasma were detected using ELISA kits according to the instructionsprovided by the manufacturer (R&D Systems, Minneapolis, MN,USA). Briefly, plasma was centrifuged at 1600g for 10 min at 4 1C.The supernatants were collected for the detection of GLP-1 and CK-MB. Then the supernatants were incubated with the regents in kits.Finally, the absorbance values were measured using a microplatereader (Multiskan MK33, Thermolab systems, Helsinki, Finland). TheGLP-1 level was expressed as pmol/l. The CK-MB level was expressedas U/l. The experiment of CK-MB was conducted for three times.

2.5. Colorimetry

The activity of lactate dehydrogenase (LDH) in plasma and theconcentrations of malondialdehyde (MDA), glutathione peroxidase(GSH-Px) and superoxide dismutase (SOD) in heart homogenate weredetermined by colorimetry. The experiment was performed usingcommercially available kits, according to the manufacturer's instruc-tions (Jiancheng Bioengineering Institute, Nanjing, China). Briefly,plasma was collected as above described. Heart tissues were collectedand lysed by cell lysis buffer. Then cell lysates were centrifuged at1600g for 10 min at 4 1C. The supernatants of plasma and heart celllysates were collected for the detection of LDH, MDA, GSH-Px andSOD. After incubation with the reagents in kits, the absorbance valuesat 340 nm, 450 nm, 412 nm and 532 nm were measured using aspectrophotometer (721D, Pudong Shanghai Physical Optical Instru-ment Factory, Shanghai, China). The LDH level was expressed as U/ml.The SOD and GSH-Px levels were expressed as U/mg protein. The MDAlevels were expressed as nmol/mg protein. The experiment of LDHwas performed for three times.

2.6. Terminal deoxynucleotidyl transferase-mediated dUTP-biotinnick end labeling (TUNEL) staining

TUNEL staining was performed with the TUNEL staining assaykit according to the manufacturer's instructions (Boster Bio-engineering Co., Ltd., Wuhan, China). Briefly, after deparaffiniza-tion, tissue sections were first treated with hydrogen peroxide (3%)and then digested with proteinase K (20 μg/ml; pH 7.4) at 25 1C.After digestion for 10 min, tissue sections were incubated with thelabeling buffer (1:18) at 37 1C. After incubation for 120 min, tissuesections were incubated with biotinylated anti-digoxin antibody(1:100) for 30 min at 37 1C. Then incorporated fluorescein wasdetected with streptavidin–biotin-peroxidase and subsequentlytissue sections were dyed with 3,3′-diaminobenzidine (DAB).

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This assay detects apoptotic cells by labeling the 3′-OH endDNA fragments with digoxigenin–deoxyuridine triphosphate(Dig–dUTP) using terminal deoxynucleotidyl transferase. Thenuclei of apoptotic cells were stained brown and the nuclei ofnormal cells were stained blue. Apoptotic index was determinedas the ratio of brown nuclei number to the total number of nuclei.Nuclei in a total of 10 fields per tissue slice (n¼6) were included.

2.7. Flow cytometry analysis

Myocardial cells were isolated from heart homogenate byfiltration. After washing with ice-cold PBS, cells were doublestained with propidium iodide and FITC-coupled annexin V for20 min. Flow cytometry was performed with a 488 nm lasercoupled to a FacsCalibur cell sorter (BD FACSvantage SE, BeckmanCoulter, America). Cells stained with both propidium iodide andannexin V were considered necrotic and cells stained only withannexin V were considered apoptotic.

2.8. Western blot analysis

Protein samples were isolated from the left ventricular myocar-dium of I/R rats. Left ventricular myocardium lysates were prepared byhomogenization in cell lysis buffer (Beyotime Institute of Biotechnol-ogy, China). Lysates were kept on ice for 45 min and total cardiacproteins were isolated by centrifugation at 14,000g for 10 min at 4 1C.Proteins were separated by SDS-PAGE and transferred to membranes.The membranes were blocked in 5% nonfat milk and incubated withprimary antibodies. The primary antibodies included anti-AKT anti-body (1:1000, Cell Signaling Technology, Inc.), anti-phospho-AKTserine473 antibody (1:1000, Cell Signaling Technology, Inc.), anti-cleaved caspase-3 antibody (1:1000, Cell Signaling Technology, Inc.),anti-caspase-3 antibody (1:1000, Cell Signaling Technology, Inc.), anti-phospho-Badserine136 antibody (1:500, Santa Cruz Biotechnology, Inc.),anti-Bcl-2 antibody (1:1000, Cell Signaling Technology, Inc.), anti-Baxantibody (1:1000, Cell Signaling Technology, Inc.) and anti-GAPDHantibody (1:1000, Beyotime Institute of Biotechnology, China). Thenthe membranes were incubated with secondary antibodies (BeyotimeInstitute of Biotechnology, China). The signals were detected with theECL system (Beyotime Institute of Biotechnology, China). Blots werescanned using Bio-Rad gel imaging system (Bio-Rad Company, USA)and bands were quantified with the Quantity One software.

2.9. Statistical analysis

The SPSS 17.0 software was used for statistical analysis. Datawere presented as mean7standard deviation (S.D.). Grouped datawere analyzed using a one-way analysis of variance (ANOVA)followed by the Student–Newman–Keuls (SNK) test. When theequal variance test failed, a Mann–Whitney Rank Sum test wasused. A P value of less than 0.05 was considered statisticallysignificant.

3. Results

3.1. Sitagliptin does not affect the glucose level, body weight, leftventricular weight and left ventricular weight index

In order to roll out the possible side effects of sitagliptin, wemeasured the basic clinical features of rats after sitagliptin treatment.The basic clinical features included the blood glucose level, bodyweight, left ventricular weight and left ventricular weight index.The blood glucose levels were measured at 2 weeks before inducingI/R. Statistically, the differences in the blood glucose levels among thefive groups were not significant (data not shown). Body weight, left

ventricular weight and left ventricular weight index were measuredafter the inducement of I/R model. Similarly there were no significantdifferences among the five groups (data not shown).

3.2. Sitagliptin upregulates the plasma GLP-1 level after myocardialinjury in I/R rats

To examine the effect of sitagliptin on plasma GLP-1, wemeasuredthe plasma level of GLP-1 after inducing I/R by ELISA assay. Ourresults (data not shown) were consistent with previous reports. Wefound that the level of GLP-1 was significantly decreased in I/R group,when compared with that in Sham group (P¼0.006). In contrast,the level of GLP-1 was significantly increased in sitagliptin groupcompared with that in I/R group (Po0.001). Meanwhile, the levels ofGLP-1 in sitagliptinþE group and sitagliptinþL group were notsignificantly different from the level of GLP-1 in sitagliptin group(P40.05).

3.3. Sitagliptin reduces the LDH and CK-MB release after myocardialinjury in I/R rats

LDH and CK-MB are the diagnostic markers of myocardial tissuedamage. Thus, we examined the effects of sitagliptin on LDH and CK-MB levels in plasma. The changes in LDH and CK-MB levels in thisstudy (data not shown) were also consistent with previous reports.Statistically, the levels of LDH (Po0.001) and CK-MB (Po0.001) inI/R group were significantly higher than those in Sham group.Compared with those in I/R group, the levels of LDH (Po0.001)and CK-MB (Po0.001) in sitagliptin group were significantly lower.In addition, the LDH levels in sitagliptinþE group (Po0.001) andsitagliptinþL group (Po0.001) were significantly higher than thosein sitagliptin group. Meanwhile, the CK-MB levels in sitagliptinþE group (Po0.001) and sitagliptinþL group (Po0.001) were alsosignificantly higher than those in sitagliptin group.

3.4. Sitagliptin increases SOD and GSH-Px and decreases MDA aftermyocardial injury in I/R rats

GSH-Px, SOD and catalase are important enzymes of the firstline cellular defense against oxidative injury. Therefore, we exam-ined the effects of sitagliptin on levels of SOD, GSH-Px and MDA inmyocardial tissue. As shown in Fig. 1A, the concentrations of SODin Sham group were significantly higher than those in I/R group(Po0.001). Compared with those in I/R group, the concentrationsof SOD in sitagliptin group were significantly increased (Po0.001).However, the concentrations of SOD in sitagliptinþE group(Po0.001) and sitagliptinþL group (Po0.001) were significantlylower than those in sitagliptin group. The effects of sitagliptinon concentrations of GSH-Px are shown in Fig. 1B. Statistically,the concentrations of GSH-Px in I/R group were significantlydecreased than those in Sham group (Po0.001). And comparedwith those in I/R group, the concentrations of GSH-Px in sitagliptingroup were significantly increased (Po0.001). However, com-pared with those in sitagliptin group, the concentrations of GSH-Px in sitagliptinþE group (Po0.001) and sitagliptinþL group(Po0.001) were significantly decreased.

The effects of sitagliptin on concentrations of MDA are shownin Fig. 1C. Statistically, the concentrations of MDA in I/R groupwere significantly higher than those in Sham group (Po0.001).And compared with those in I/R group, the concentrations of MDAin sitagliptin group were significantly decreased (Po0.001).However, compared with sitagliptin group, the concentrations ofMDA in sitagliptinþE group (Po0.001) and sitagliptinþL group(Po0.001) were significantly increased.

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3.5. Sitagliptin enhances left ventricular function after myocardialinjury in I/R rats

To determine the effects of sitagliptin on cardiac function in I/Rrats, hemodynamic measurements were performed during the entireI/R period. As shown in Fig. 2, compared with those in Sham group,I/R treatment significantly decreased the þLVdp/dt max (Po0.001),�LVdp/dt max (Po0.001), and LVESP (Po0.001) and significantlyincreased the LVEDP (Po0.001). Compared with those in I/R group,sitagliptin significantly enhanced the þLVdp/dt max (Po0.001),�LVdp/dt max (Po0.001), and LVESP (P¼0.014) and significantlyreduced the LVEDP (P¼0.003). However, the GLP-1 receptor antago-nist exendin-(9-39) and the PI3K inhibitor LY294002 abolished theeffects of sitagliptin on þLVdp/dt max (Po0.001, Po0.001), �LVdp/dt max (Po0.001, Po0.001), LVESP (P¼0.012, P¼0.03) and LVEDP(P¼0.017, P¼0.009).

3.6. Sitagliptin inhibits cardiomyocyte apoptosis after myocardialinjury in I/R rats

We examined the effects of sitagliptin on cell apoptosis inmyocardial tissue by TUNEL assay and flow cytometry analysis. Therepresentative graphs of TUNEL assay and flow cytometry analysisare shown in Fig. 3A and C, respectively. The apoptotic index ofTUNEL assay is shown in Fig. 3B and the apoptosis ratio of flowcytometry analysis is shown in Fig. 3D. Representative

photomicrograph showed that TUNEL staining positive apoptoticcells were more frequently observed in I/R group, sitagliptinþEgroup and sitagliptinþL group as compared with Sham group andsitagliptin group (Fig. 3A). Statistically, the apoptotic index insitagliptin group was significantly lower than those in I/R group(Po0.001), sitagliptinþE group (Po0.001) and sitagliptinþL group(Po0.001).

As analyzed by flow cytometry, apoptotic cell ratio in I/R groupwas significantly increased compared with that in Sham group(Po0.001). However, compared with that in I/R group, apoptosisratio was significantly decreased in sitagliptin group (Po0.001).Also apoptosis ratios in sitagliptinþE group (Po0.001) andsitagliptinþL group (Po0.001) were significantly increased com-pared with those in sitagliptin group.

3.7. Sitagliptin increases expression of anti-apoptotic proteins andinhibits expression of pro-apoptotic proteins after myocardial injuryin I/R rats

The effects of sitagliptin on pho-Aktserine473, pho-Badserine136,caspase-3, cleaved caspase-3, Bax and Bcl-2 in myocardial tissuewere analyzed by western blot (Fig. 4). The representative westernblot results are shown in Fig. 4A and the quantitative results areshown in Figs. 4B–E, G and H. And the ratio of Bax/Bcl-2 is shown inFig. 4F. Compared with those in Sham group, I/R treatment signifi-cantly decreased levels of pho-Aktserine473 (P¼0.006), pho-Badserine136

Fig. 1. Effects of sitagliptin on levels of SOD, GSH-Px and MDA in heart homogenate of I/R rats. Levels of SOD, GSH-Px and MDA in heart homogenate of I/R rats weremeasured by colorimetry. (A) Levels of SOD in heart homogenate of I/R rats. Significance was determined by ANOVA followed by the SNK test, F¼63.255. (B) Levels of GSH-Pxin heart homogenate of I/R rats. Significance was determined by ANOVA followed by the SNK test, F¼53.189. (C) Levels of MDA in heart homogenate of I/R rats. Significancewas determined by ANOVA followed by the SNK test, F¼74.287. SOD: superoxide dismutase; GSH-Px: glutathione peroxidase; and MDA: malondialdehyde. Values wereexpressed as mean7S.D. Sham: Sham group; I/R: ischemia/reperfusion group; sitagliptin: sitagliptin 300 mg/kg/day pretreatment group; sitagliptinþE: sitagliptin 300 mg/kg/day and exendin-(9-39) 45 μg/kg/3 days pretreatment group; sitagliptinþL: sitagliptin 300 mg/kg/day and LY294002 0.3 mg/kg/3 days pretreatment group; N¼8 in eachgroup; nPo0.05 vs. Sham group; #Po0.05 vs. I/R group; and ▲Po0.05 vs. sitagliptin group.

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(P¼0.002) and Bcl-2 (Po0.001) expression. Meanwhile, comparedwith those in Sham group, I/R treatment significantly increasedthe expression levels of caspase-3 (Po0.001), cleaved caspase-3(Po0.001) and Bax (Po0.001). Compared with those in I/R group,sitagliptin significantly enhanced the levels of pho-Aktserine473

(P¼0.01), pho-Badserine136 (Po0.001) and Bcl-2 (Po0.001). At thesame time, compared with those in I/R group, sitagliptin significantlyreduced levels of caspase-3 (Po0.001), cleaved caspase-3 (P¼0.02)and Bax (Po0.001). Also the Bax/Bcl-2 ratio was significantlydecreased in sitagliptin group than that in I/R group (Po0.001).However, the GLP-1 receptor antagonist exendin-(9-39) and the PI3Kinhibitor LY294002 attenuated the effects of sitagliptin on anti-apoptotic and pro-apoptotic proteins. Statistically, compared withsitagliptin group, exendin-(9-39) and LY294002 administration sig-nificantly reduced the levels of pho-Aktserine473 (P¼0.011, P¼0.007),pho-Badserine136 (Po0.001, Po0.001) and Bcl-2 (Po0.001, Po0.001).And compared with sitagliptin group, exendin-(9-39) and LY294002administration significantly increased the expression levels of caspase-3 (Po0.001, Po0.001), cleaved caspase-3 (P¼0.001, P¼0.001) andBax (Po0.001, Po0.001).

4. Discussion

The major finding of our study was that sitagliptin pretreatmentcould reduce myocardial injury and improve cardiac function by

inhibiting cardiomyocyte apoptosis in an I/R rat model. Though DPP4inhibitors were reported to have cardioprotective effects during I/R,this is the first study to examine the effects of sitagliptin oncardiomyocyte apoptosis and oxidative stress induced by myocardialI/R. In addition, our data indicate that sitagliptin could decrease thelevels of pro-apoptotic proteins and increase the levels ofanti-apoptotic proteins. However, the above observed effects ofsitagliptin were all abolished when co-administered with GLP-1receptor antagonist exendin-(9-39) or PI3K inhibitor LY294002. Thuswe speculate that sitagliptin protected the heart in I/R rats frominjury through the decrease of apoptosis and oxidative damage andthe activation of PI3K/Akt signaling pathway.

As reported by previous studies (Khalil et al., 2005; Sun et al.,2012), myocardial I/R impaired cardiac function, increased therelease of LDH and CK-MB and the apoptotic rate of cardiomyo-cyte. We found similar results in this study. Importantly, we foundthat sitagliptin reduced LDH and CK-MB release in I/R rats. Wespeculate that this effect might be ascribed to its potential to resistagainst oxidative stress. Interestingly, we demonstrated thatsitagliptin significantly increased the levels of SOD and GSH-Px and decreased the level of MDA in myocardial tissues in I/Rrats. As we all know, SOD and GSH-Px are key antioxidantenzymes, which constitute first line cellular defense againstoxidative injury. MDA is one of the products of oxidative stress,which reflects the damage of cell caused by oxidative stress. To ourknowledge, our study demonstrated for the first time that

Fig. 2. Effects of sitagliptin on left ventricular function in I/R rats. The cardiac functions of þLVdp/dt max, �LVdp/dt max, LVESP and LVEDP were measured by multichannelphysiologic recorder. (A) Values of þLVdp/dt max (rates of maximum positive left ventricular pressure development). Significance was determined by ANOVA followed bythe SNK test, F¼66.053. (B) Values of �LVdp/dt max (rates of maximum negative left ventricular pressure development). Significance was determined by ANOVA followedby a Mann–Whitney Rank Sum test, F¼28.161. (C) Values of left ventricular end-systolic pressure (LVESP). Significance was determined by ANOVA followed by the SNK test,F¼25.952. (D) Values of left ventricular end-diastolic pressure (LVEDP). Significance was determined by ANOVA followed by the SNK test, F¼24.460. Values were expressedas mean7S.D. Sham: Sham group; I/R: ischemia/reperfusion group; sitagliptin: sitagliptin 300 mg/kg/day pretreatment group; sitagliptinþE: sitagliptin 300 mg/kg/day andexendin-(9-39) 45 μg/kg/3 days pretreatment group; sitagliptinþL: sitagliptin 300 mg/kg/day and LY294002 0.3 mg/kg/3 days pretreatment group; N¼8 in each group;nPo0.05 vs. Sham group; #Po0.05 vs. I/R group; and ▲Po0.05 vs. sitagliptin group.

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pretreatment with sitagliptin could increase the concentrations ofantioxidant defense enzymes including GSH-Px and SOD, anddecrease the production of MDA in I/R rats.

We also found that sitagliptin administration significantlyimproved the cardiac function via increasing 7LVdp/dt max,LVESP and limiting the increase of LVEDP. Similarly, Sauvé et al.(2010) observed that the left ventricular function was improved in

mice pretreated with sitagliptin for 12 h prior to aortic occlusionand in DPP4 deleted mice after I/R injury. Ku et al. (2011) alsoreported that DPP4 deficiency could preserve cardiac function inrats subjected to myocardial I/R. In a clinical study, single dosesitagliptin treatment could improve the regional and global leftventricular function in patients with coronary artery disease (Readet al., 2010). However, a recent study demonstrated that in rats

Fig. 3. Effects of sitagliptin on cardiomyocyte apoptosis in I/R rats. Apoptosis were analyzed by TUNEL assay and flow cytometry analysis, respectively. (A) Representativegraphics of TUNEL staining. (B) Quantitative results of TUNEL staining (400� ). Apoptotic index was determined as the ratio of brown nuclei number to the total number ofnuclei. Nuclei in a total of 10 fields per tissue slice (N¼6) were included. Significance was determined by ANOVA followed by the SNK test, F¼69.639. (C) Representative flowcytometry results. (D) Quantitative results of flow cytometry results, N¼8. Apoptosis ratio was determined as the ratio of Annexin V positive and propidium iodide negativecells to total cells analyzed. Significance was determined by ANOVA followed by the SNK test, F¼70.412. Values were expressed as mean7S.D. Sham: Sham group; I/R:ischemia/reperfusion group; sitagliptin: sitagliptin 300 mg/kg/day pretreatment group; sitagliptinþE: sitagliptin 300 mg/kg/day and exendin-(9-39) 45 μg/kg/3 dayspretreatment group; sitagliptinþL: sitagliptin 300 mg/kg/day and LY294002 0.3 mg/kg/3 days pretreatment group; nPo0.05 vs. Sham group; #Po0.05 vs. I/R group; and▲Po0.05 vs. sitagliptin group.

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with ischemic heart failure, early or late treatment with vildaglip-tin had no beneficial effect on ventricular performance (Yin et al.,2011). Meanwhile, Sauvé et al. (2010) also reported that acutetreatment with sitagliptin for 20 min prior to I/R injury failed toimprove ventricular function. The discrepancy in these studiesmight be attributed to the differences in the duration of treatmenttime and differences in types of drug administration as well as thedifferences in study models.

Cardiomyocyte apoptosis is one of the critical reasons of heartfailure after myocardial infarction. Many studies have confirmedthat blocking the apoptosis process could reduce the loss ofcardiomyocyte, minimize myocardial injury and improve ventri-cular performance induced by I/R (Mughal et al., 2012). Given thesignificantly improved recovery of cardiac function, we examinedthe effects of sitagliptin on preventing cardiomyocyte apoptosis inI/R rats. We demonstrated that sitagliptin administration

Fig. 4. Effects of sitagliptin on expression levels of anti-apoptotic proteins and pro-apoptotic proteins in I/R rats. Expression levels of apoptosis related proteins were analyzed bywestern blot. (A) Representative western blot results. (B) Ratios of phospho-AKTserine473 to total AKT. Significance was determined by ANOVA followed by a Mann–Whitney Rank Sumtest, F¼20.546. (C) Ratios of phospho-Badserine136 to GAPDH. Significance was determined by ANOVA followed by the SNK test, F¼10.407. (D) Ratios of Bax to GAPDH. Significance wasdetermined by ANOVA followed by a Mann–Whitney Rank Sum test, F¼212.404. (E) Ratios of Bcl-2 to GAPDH. Significance was determined by ANOVA followed by a Mann–WhitneyRank Sum test, F¼75.357 (F) Ratios of Bax to Bcl-2. Significance was determined by ANOVA followed by a Mann–Whitney Rank Sum test, F¼84.528. (G) Ratios of Caspase-3 to GAPDH.Significance was determined by ANOVA followed by the SNK test, F¼70.092. (H) Ratios of cleaved caspase-3 to GAPDH. Significance was determined by ANOVA followed by the SNKtest, F¼17.488. Values were expressed as mean7S.D. Sham: Sham group; I/R: ischemia/reperfusion group; sitagliptin: sitagliptin 300 mg/kg/day pretreatment group; sitagliptinþE:sitagliptin 300 mg/kg/day and exendin-(9-39) 45 μg/kg/3 days pretreatment group; sitagliptinþL: sitagliptin 300 mg/kg/day and LY294002 0.3 mg/kg/3 days pretreatment group;N¼8 in every group; nPo0.05 vs. Sham group; #Po0.05 vs. I/R group; and ▲Po0.05 vs. sitagliptin group.

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significantly decreased apoptosis ratio of cardiomyocytes asrevealed by TUNEL staining and flow cytometry analysis.And sitagliptin administration significantly reduced expressionlevels of caspase-3 and cleaved caspase-3 in rats subjected to I/Rinjury.

It has been reported that PI3K/Akt signaling pathway activationcould inhibit cardiomyocyte apoptosis after I/R injury (Fujio et al.,2000; Matsui et al., 2001; Mullonkal and Toledo-Pereyra, 2007).The mechanisms of the anti-apoptotic effect are acted throughvarious means, such as inhibiting caspase activation, inhibitingdeath genes expression and regulating the activity of Bcl-2 family.Bcl-2 family, the key regulators of apoptosis, consists of both celldeath promoters such as Bax and Bad, and cell death inhibitorsincluding Bcl-2, Bcl-x, etc. It is reported that the high ratio of Bax/Bcl-2 was associated with great possibility to apoptotic activation(García-Sáez, 2012). In our study, we demonstrated that sitagliptincould increase phosphorylation levels of Akt and Bad and decreaseexpression levels of caspase-3, cleaved caspase-3 and Bax. Mean-while, we also found that sitagliptin could up-regulate Bcl-2expression, resulting in decreased Bax/Bcl-2 ratio in I/R rats. Theseeffects of sitagliptin were correlated with cardiomyocyte apoptosisattenuation. Hence, we next examined whether the sitagliptinexerted its anti-apoptotic action through activation of PI3K/Aktpathway in rats subjected to I/R injury. The PI3K inhibitorLY294002 was employed. We found that co-administrationof LY294002 and sitagliptin decreased phosphorylation levels ofAkt and Bad and increased expression levels of caspase-3, cleavedcaspase-3, Bax, and Bax/Bcl-2 ratio. These findings suggested thatLY294002 could abolish the anti-apoptotic effects of sitagliptin.The anti-apoptotic effects of sitagliptin were related to, at least inpart, activation of PI3K/Akt signaling pathway.

The function of DPP4 inhibitor is to inhibit the proteolyticactivity of DPP4 enzyme, postponing the myocardial degradationof GLP-1 (Barnett, 2006). Our data showed that sitagliptinadministration resulted in significant accumulation of plasmaGLP-1 in I/R rats. This result was similar to previous data reportedby Ku et al. (2011) and Ye et al. (2010). Moreover, our resultsshowed that higher levels of plasma GLP-1 were associated withlower levels of cardiac injury markers, higher levels of antiox-idant enzymes, lower ratio of cardiomyocyte apoptosis and betterventricular performance in I/R rats. However, these effects wereattenuated by the GLP-1 receptor antagonist exendin-(9-39).Exendin-(9-39) has been widely used to estimate the role ofreceptor-dependent pathway. Thus we speculated that the car-dioprotective effects of sitagliptin might be attributed to, at leastin part, the GLP-1 receptor-dependent pathway. In summary,sitagliptin exerted its action via up-regulating the level of GLP-1,which activated the PI3K/Akt signaling pathway via binding GLP-1 receptor.

The prominent finding of this study was that sitagliptin pretreat-ment could reduce myocardial injury and improve cardiac function inan I/R rat model. The possible mechanisms might be relative todecrease of apoptosis and oxidative damage, up-regulation of GLP-1level (which activated the PI3K/Akt signaling pathway via bindingGLP-1 receptor), increase of Bad phosphorylation and decrease ofBax/Bcl-2 ratio. However, exact cardioprotective mechanisms of DPP4inhibitors need a further study. To sum up, we conclude that sitagliptinadministration has protective effects on myocardial I/R injury. Ourfindings could provide deeper insights into the treatment of heartdisease.

Acknowledgments

This work was supported by the National Natural Science Fundsfor Youths (Grant no. 81100196), Natural Science Foundation

Project of CQ CSTC (Grant no. CSTC, 2011BB5133). Foundationproject of Traditional Chinese Medicine of Chongqing MunicipalHealth Bureau (Grant no. 2012-2-125) and Pfizer pharmaceuticallimited competition grants (Grant no. ws1790576). We greatlyappreciate Jianyong Wu and Dezhang Zhao (Institute of LifeSciences, Chongqing Medical University) for their excellent tech-nical support for the flow cytometry analysis.

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