International Journal of Cardiology 167 (2013) 451–457

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International Journal of Cardiology

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Cardioprotective effect of dipeptidyl peptidase-4 inhibitor duringischemia–reperfusion injury

Kroekkiat Chinda a,b, Siripong Palee a,b, Sirirat Surinkaew a,b, Mattabhorn Phornphutkul a,Siriporn Chattipakorn a,c, Nipon Chattipakorn a,b,⁎a Cardiac Electrophysiology Research and Training Center, Faculty of Medicine, Chiang Mai University, Chiang Mai, Thailandb Cardiac Electrophysiology Unit, Department of Physiology, Faculty of Medicine, Chiang Mai University, Chiang Mai, Thailandc Faculty of Dentistry, Chiang Mai University, Chiang Mai, Thailand

⁎ Corresponding author at: Cardiac ElectrophysiologyFaculty of Medicine, Chiang Mai University, Chiang Mai945329; fax: +66 53 945368.

E-mail address: nchattip@gmail.com (N. Chattipakor

0167-5273/$ – see front matter © 2012 Elsevier Irelanddoi:10.1016/j.ijcard.2012.01.011

a b s t r a c t

a r t i c l e i n f o

Article history:

Received 13 September 2011Received in revised form 30 December 2011Accepted 6 January 2012Available online 27 January 2012

Keywords:DPP-4 inhibitorIschemia–reperfusion injuryMitochondriaElectrophysiologyInfarct size

Background: Dipeptidyl peptidase-4 (DPP-4) inhibitor is a new anti-diabetic drug for type-2 diabetes mellituspatients. Despite its benefits on glycemic control, the effects of DPP-4 inhibitor on the heart during ischemia–reperfusion (I/R) periods are not known. We investigated the effect of DPP-4 inhibitor on cardiac electro-physiology and infarct size in a clinically relevant I/R model in swine and its underlying cardioprotectivemechanism.Methods: Fourteen pigs were randomized to receive either DPP-4 inhibitor (vildagliptin) 50 mg or normalsaline intravenously prior to a 90-min left anterior descending artery occlusion, followed by a 120-min reper-fusion period. The hemodynamic, cardiac electrophysiological and arrhythmic parameters, and the infarctsize were determined before and during I/R. Rat cardiac mitochondria were used to study the protectiveeffects of DPP-4 inhibitor on cardiac mitochondrial dysfunction caused by severe oxidative stress inducedby H2O2 to mimic the I/R condition.

Results: Compared to the saline group, DPP-4 inhibitor attenuated the shortening of the effective refractoryperiod (ERP), decreased the number of PVCs, increased the ventricular fibrillation threshold (VFT) duringthe ischemic period, and also decreased the infarct size. In cardiac mitochondria, DPP-4 inhibitor decreasedthe reactive oxygen species (ROS) production and prevented cardiac mitochondrial depolarization causedby severe oxidative stress.Conclusions: During I/R, DPP-4 inhibitor stabilized the cardiac electrophysiology by preventing the ERP short-ening, decreasing the number of PVCs, increasing the VFT, and decreasing the infarct size. This cardioprotec-tive effect could be due to its prevention of cardiac mitochondrial dysfunction caused by severe oxidativestress during I/R.

© 2012 Elsevier Ireland Ltd. All rights reserved.

1. Introduction

Diabetes mellitus has been an important health problem in mostnations with the number of patients dramatically soaring and expectedto reach 366 million by the year 2030 [1]. Patients with type-2 diabetesmellitus (T2DM) have been shown to have a 2- to 4-fold higher riskin coronary heart disease and stroke mortality [2–6], and have a worseprognosis after cardiovascular events [7–9]. Although several new anti-diabetic drugs have been discovered in the past decades, the therapieshave been limited by their adverse effects such as weight gain, hypogly-cemia, fluid retention [10], and an unexpected cardiovascular risk

Research and Training Center,50200, Thailand. Tel.: +66 53

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Ltd. All rights reserved.

[11–13]. Therefore, new anti-diabetic drugs that could control hypergly-cemia and reduce the risk of cardiovascular events are of potential ben-efits to T2DM patients.

In the past few years, a potent dipeptidyl peptidase-4 (DPP-4)inhibitor, which is a novel anti-diabetic drug, has been shown to beeffective in treating T2DM patients. Its action is to inhibit the proteo-lytic enzyme DPP-4 activity, resulting in postponing the degradationof glucagon-like peptide-1 (GLP-1), thus improving glycemic control[14,15]. Although previous studies demonstrated the cardioprotectiveactions of GLP-1 in an ischemic heart model of ex vivo isolated rodentLangendorff heart [16,17], in vivo rats, rabbits, canine, and swine[18–21], as well as in acute myocardial infarction patients [22],reports on the cardioprotective effect of DPP-4 inhibitor are scantand controversial [23–26]. Furthermore, the effect of DPP-4 inhibitoron cardiac electrophysiology during ischemia–reperfusion (I/R) hasnever been investigated.

452 K. Chinda et al. / International Journal of Cardiology 167 (2013) 451–457

The purpose of this study was to investigate the effect of vilda-gliptin, a DPP-4 inhibitor, on cardiac electrophysiology and infarctsize in a clinically relevant I/R model in swine. We hypothesizedthat vildagliptin can attenuate the occurrence of cardiac arrhyth-mias, increase the ventricular fibrillation threshold (VFT), improvedefibrillation efficacy by lowering the defibrillation threshold(DFT), and reduce the infarct size during I/R in the swine heart. Tostudy the cardioprotective mechanism, we determined the effectof vildagliptin in isolated rat's cardiac mitochondria. We tested thehypothesis that the cardioprotective mechanism of vildagliptin isvia its prevention of cardiac mitochondrial dysfunction caused bysevere oxidative stress during I/R.

2. Materials and methods

2.1. Animal preparation

All experiments were approved by the Institutional Animal Care and Use Commit-tees of the Faculty of Medicine, Chiang Mai University, Chiang Mai, Thailand. Pigs wereanesthetized by intramuscular injection of a combination of atropine (0.04 mg/kg),zoletil® (4.4 mg/kg) and xylazine (2.2 mg/kg). After endotracheal intubation, anesthe-sia was maintained by 1.5–3.0% isoflurane delivered in 100% oxygen. Surface electro-cardiogram (lead II), femoral arterial blood pressure (BP), heart rates (HR), corebody temperature as well as blood gases and electrolytes were continuously monitoredto maintain a normal physiological condition. Platinum coated titanium coil electrodes(34- and 68-mm) were advanced into the right ventricular apex (RV) and junctionbetween right atrium and superior vena cava, respectively, to deliver electrical stimu-lus during VFT and DFT determinations [27]. After a median sternotomy, two pacingelectrodes were attached to the epicardium at the right ventricular outflow tract(RVOT) and left ventricular apex (LV) to evaluate the effective refractory period(ERP) and diastolic pacing threshold (DPT) at each site. The electrode at the tip ofthe endocardial RV apex catheter was also used to determine the ERP and DPT at thissite.

2.2. Experimental protocols

Fourteen domestic pigs (25 to 30 kg) were randomly divided into 2 groups (n=7/group). The first group was assigned to receive 30 ml of normal saline solution and thesecond group received vildagliptin (prepared by dissolving 50-mg vildagliptin in 30-mlsaline solution). Both normal saline solution and vildagliptin (2 mg/Kg) were adminis-tered intravenously at a rate of 1.0 ml/min prior to the left anterior descending artery(LAD) occlusion. Hemodynamic and cardiac electrophysiological parameters includingHR, systolic (SBP) and diastolic blood pressure (DBP), DPT, ERP, corrected QT interval(QTC), VFT and DFT were determined at the beginning of the study as a baseline. Myo-cardial ischemia was induced by LAD occlusion at 5 cm above the distal end [28]. Dur-ing the first 60 min of occlusion, if spontaneous ventricular fibrillation (VF) occurred,the defibrillation shock was delivered to determine the DFT. On the other hand, if VFdid not occur, it was electrically induced with 50-Hz alternating current. Both VFT andDFTwere determinedusing a three-reversal up/downprotocol [28]. After 90 min of occlu-sion, LAD ligation was released to promote reperfusion for 120 min. All parameters weredetermined again at the end of the reperfusion. Ventricular arrhythmia, e.g. ventricularpremature contractions (PVCs), ventricular tachycardia (VT) and spontaneous VF, wasrecorded throughout the experiment.

2.3. Diastolic pacing threshold (DPT) determination

A train of 10 S1 stimuli was delivered via the electrode at the tip of RV catheter.Current strength was begun with 0.1 mA and was increased in 0.1-mA steps until allstimuli in a train elicited a ventricular response (capture) [28]. The minimum currentstrength which captures ventricular response was defined as the DPT.

2.4. Effective refractory period (ERP) determination

An S2 stimulus (2× DPT strength) was introduced in late diastole of the lastS1 beat of a train of 10 S1 to elicit a capture. S1–S2 coupling interval was decreasedin 10-ms steps until S2 failed to elicit a capture. ERP was defined as the longest S1–S2 interval which S2 stimulus failed to capture [28].

2.5. Ventricular fibrillation threshold (VFT) determination

The interval between the last S1 and the mid T-wave was determined for 3 times.An average was used as a coupling interval between the last S1 and S2 shock. VFTwas performed by delivering S2 shocks starting at 100 V. If this shock induced VF,the decrement of 10-V step was used for each successive shock until VF was no longerinduced. If the 100-V S2 shock did not induce VF, the increment of 10-V step was usedfor each successive shock until VF was induced. VFT was defined as the lowest shockstrength that successfully induced VF [28].

2.6. Defibrillation threshold (DFT) determination

Defibrillation shock was delivered after 10 s of VF to determine the DFT using athree-reversal up/down protocol [28]. However, if the tested shock failed to defibril-late, a rescue shock (600–700 V) was delivered to successfully defibrillate the heart.The DFT was defined as the lowest shock strength required for successful defibrillation.A 4-minute interval was allowed between each VF induction episode to set the heartback to physiologic condition [28].

2.7. Infarct size determination

The infarct size was assessed with 0.5% Evans Blue and 1.0% TriphenyltetrazoliumChloride (TTC) staining as previously described [28]. In brief, at the end of the study,the LAD was re-occluded at the exact same location as during ischemia. Evans Bluewas infused into the left and right coronary arteries to evaluate the area at risk(AAR). After being frozen overnight, the heart was cut into 5-mm thick slices perpen-dicular to the LAD from apex to the occlusion site. Each slice was incubated in TTC for15 min to discriminate the infarct tissues from the viable myocardium. After overnightfixation with 4% paraformaldehyde, each slice was photographed with a digital camera.An area measurement was performed using Image Tool software version 3.0.

2.8. Histological analysis

Both infarct and normal myocardium were fixed with 4% neural buffered formal-dehyde for 24 h at room temperature, followed by embedding in paraffin wax, andslicing into 5-μm slices for subsequent Hematoxylin–Eosin staining [29,30]. The infarcttissues were evaluated for microscopic changes using the Lodge–Patch classification[31–33].

2.9. Isolated cardiac mitochondria study protocol

Male Wistar rats (300–350 g) were used for cardiac mitochondrial isolation as de-scribed previously [34]. H2O2 (2 mM, incubated for 5 min) was used to induce oxidativestress in cardiac mitochondria to mimic I/R condition [34]. Isolated cardiac mitochon-dria were divided into 6 groups (n=5/group): 1) Control, 2) Mitochondria treatedwith H2O2, 3) Mitochondria treated with vildagliptin for 30 min, 4) Mitochondriapretreated with vildagliptin for 5 min followed by H2O2 treatment, 5) Mitochondriapretreated with vildagliptin for 15 min followed by H2O2 treatment, 6) Mitochondriapretreated with vildagliptin for 30 min followed by H2O2 treatment. Vildagliptin atdoses of 0.33 mM, and 3.30 mM were used in this study.

The measurement of cardiac mitochondrial reactive oxygen species (ROS) produc-tion and mitochondrial membrane potential changes (ΔΨM) was determined in allgroups as previously described [34]. In short, dichlorohydro-fluorescein diacetate dyewas used to determine the level of ROS production in cardiac mitochondria. The ROSlevel was expressed as arbitrary units of fluorescence intensity determined at λexcitation

485 nm and λemission 530 nm [34]. JC-1 was used to determine the change of cardiacmitochondrial membrane potential at λexcitation 485 nm and λemission 530 nm forgreen and λemission 590 nm for red [34]. Cardiac mitochondrial depolarization wasindicated by a decrease in the red/green fluorescence intensity ratio.

2.10. Statistical analysis

Data were expressed as mean±SEM. Statistical comparison of cardiac electro-physiological, hemodynamic, and arrhythmic parameters as well as cardiac mitochon-dria results was performed with the Student's t test. Chi-square test was performed tocompare VT/VF incidence, and comparison of the infarct size was analyzed usingMann–Whitney's U test. All statistical analysis was performed with SPSS version10.0. A p-value less than 0.05 was considered significant.

3. Results

The hemodynamic parameters including HR, SBP, DBP and cardiacelectrophysiological parameters including QTC, QRS complex, and DPTwere not significantly different between the saline-treated group andthe vildagliptin-treated group during baseline, ischemia, and reperfu-sion periods (Table 1). Pretreatment with vildagliptin significantlyincreased both VFT energy and VFT voltage at ischemic period com-pared with the saline-treated group (Fig. 1). However, there was nodifference between the DFT of the vildagliptin-treated group andthe saline-treated group at any time period (Fig. 2). For the ERP, nodifferences were found among ERPs recorded at three sites at baselineand reperfusion periods in both saline and vildagliptin treated groups(Fig. 3A and 3C). However, during ischemia the ERP at the LV epicar-dium (i.e. ischemic site) was significantly shortened, compared tothe other two sites in the saline-treated group (Fig. 3B), thus creatingthe dispersion of the ERP in the heart during this period. In the

Table 1Basic electrophysiological and hemodynamic parameters.

Parameters Baseline Ischemia 60 min Reperfusion

NSS Vil NSS Vil NSS Vil

HR (beat/min) 88.4±5.2 98.0±5.7 91.3±3.6 95.3±4.6 102.7±8.5 107.6±4.2SBP (mm Hg) 101.1±7.4 102.9±5.1 87.1±6.1 84.6±6.1 86.1±3.4 85.6±4.7DBP (mm Hg) 63.3±5.4 64.0±3.9 55.6±4.1 52.0±4.6 54.4±2.3 53.9±2.9QTC (ms) 466.7±7.1 466.1±14.0 451.2±17.2 459.2±18.5 441.7±15.8 435.2±20.3QRS (ms) 64.4±2.0 58.0±5.0 59.6±2.0 57.6±4.5 60.8±3.2 58.0±2.2DPT RV (mA) 0.20±0.02 0.21±0.01 0.29±0.03 0.31±0.05 0.33±0.05 0.36±0.04DPT RVOT (mA) 0.19±0.04 0.17±0.02 0.14±0.02 0.16±0.03 0.40±0.13 0.23±0.07DPT LV (mA) 0.11±0.01 0.14±0.02 0.22±0.08 0.18±0.04 0.42±0.20 0.21±0.04

NSS = Normal saline solution; Vil = Vildagliptin; HR = Heart rate; SBP = Systolic blood pressure; DBP = Diastolic blood pressure; QTC = Corrected QT interval; QRS = QRScomplex; DPT = Diastolic pacing threshold; RV = Right ventricle; RVOT = Right ventricular outflow tract; LV = Left ventricle.

453K. Chinda et al. / International Journal of Cardiology 167 (2013) 451–457

vildagliptin-treated group, the ERP from all 3 sites were not differentduring the ischemic period, indicating less dispersion of the ERPduring ischemia in this group.

Regarding the occurrence of arrhythmias, the number of PVCswas markedly decreased in pigs treated with vildagliptin during90 min of ischemia, compared to the saline-treated group (Fig. 4A).The number of PVCs during reperfusion was also smaller in the vilda-gliptin group, but it did not reach statistical significance (Fig. 4A). TheVT/VF incidence (Fig. 4B) and the number of VT/VF episodes (Fig. 4C)were also smaller in the vildagliptin-treated group, but were notstatistically significant. The time to the first VT/VF onset was notdifferent between the vildagliptin and the saline treated groups(Fig. 4D). The area at risk (AAR) was not different between the saline(36.8±2.7%) and the vildagliptin treated groups (33.4±3.2%). How-ever, the infarct size in the vildagliptin-treated groupwas significantlysmaller than that in the saline-treated group (Fig. 5A), accounting for a

Fig. 1. Effect of vildagliptin on the VFT. During the ischemic period, vildagliptin signif-icantly increased both VFT energy (panel A) and voltage (panel B) compared to normalsaline group. * pb0.05 vs. NSS at ischemic period; † pb0.05 vs. vildagliptin at baseline.

17% reduction in the infarct size. To determine stages of myocardialdamage caused by I/R injury, myocardial tissues were obtained andevaluated based on microscopic changes. In the infarct area, thinningand waviness of myocardial fibers with distinct nuclei were observed(Fig. 5B).

In cardiac mitochondria, vildagliptin alone at doses 0.1 mg/ml(0.33 mM) and 1.0 mg/ml (3.30 mM) did not affect the mitochondrialROS production and the mitochondrial membrane potential change(Fig. 6). H2O2 caused a markedly increased ROS level and decreasedthe red/green fluorescent intensity ratio (i.e. an indication ofmitochon-drial depolarization). Vildagliptin at 0.1 mg/ml neither attenuatedcardiac mitochondrial ROS production nor prevented mitochondrialdepolarization caused by H2O2 (Fig. 6A and 6B). However, treatmentwith 1.0 mg/ml of vildagliptin for 5, 15, and 30 min ameliorated cardiacmitochondrial dysfunction caused by H2O2, as indicated by lower ROSproduction (Fig. 6C) and lessmitochondrialmembrane potential changes(Fig. 6D), compared to those in H2O2-treated cardiac mitochondria.

Fig. 2. Effect of vildagliptin on the DFT. Both vildagliptin and normal saline did notchange the DFT energy (panel A) and DFT voltage (panel B).

Fig. 3. Effect of vildagliptin on the ERP. ERP at baseline was not different among all 3 re-cording sites in both normal saline and vildagliptin groups (panel A). During ischemia,LV ERP was decreased compared to the other two recorded sites in the normal salinegroup (panel B), whereas the LV ERP was not different from the other two recordedsites in the vildagliptin treated group (panel B), indicating that vildagliptin preventedthe ERP dispersion among all recording sites at ischemic period. At reperfusion,the ERPs were not different among all three recorded sites in both normal saline andvildagliptin treated groups (panel C). * pb0.05 vs. RV; † pb0.05 vs. RVOT. RV = Rightventricle; RVOT = Right ventricular outflow tract; LV = Left ventricle.

454 K. Chinda et al. / International Journal of Cardiology 167 (2013) 451–457

4. Discussion

In this study, we assessed the cardioprotective effects of vildaglip-tin in a clinically relevant swine I/R model. The major findings of thisstudy were as follows. Vildagliptin demonstrated its cardioprotectiveeffects by 1) attenuating ERP shortening caused by myocardial ische-mia, thus decreasing the ERP dispersion in the heart during ischemia,2) increasing the VFT, 3) reducing the number of PVCs, 4) decreasing

the infarct size, and 5) attenuating cardiac mitochondrial dysfunctiondue to oxidative stress caused by H2O2.

In the present study, we demonstrated for the first time thecardiac electrophysiological effects of DPP-4 inhibitor, vildagliptin,in the I/R heart. Our study demonstrated that vildagliptin could atten-uate the ERP shortening in the ischemic myocardium, thus decreasingthe degree of the dispersion of refractoriness caused by cardiac ische-mia. Since dispersion of refractoriness plays an important role infacilitating arrhythmias [35], prevention of the ERP dispersion in theheart during ischemia could attenuate the occurrence of arrhythmiain this study. Our study also demonstrated the decreased number ofPVCs and the reduced VFT in the vildagliptin treated pigs, suggestingthat vildagliptin could stabilize cardiac electrophysiology duringischemia, thus attenuating myocardial vulnerability to arrhythmiaduring ischemia.

In the present study, the AAR in both saline and vildagliptin trea-ted groups was not different, indicating the similar extent of ischemicmyocardium caused by an LAD occlusion in both groups. However,vildagliptin significantly decreased the infarct size, compared withthat in the saline-treated group. The histological appearance of theinfarct tissues represented an early myocardial infarction (stage 1)according to Lodge–Patch classification [31,33], which was consistentwith the duration of I/R in this study. The beneficial effect on the in-farct size reduction of vildagliptin in the swine I/R model as shownin our study was consistent with previous reports in I/R model inmice using DPP-4 inhibitor sitagliptin [24], and in pre-diabetic insulinresistance rat using vildagliptin [23]. Since a reduced infarct size hasbeen shown to be associated with a decreased myocardial vulnerabil-ity to arrhythmias during I/R, the reduction of infarct size and de-creased ERP dispersion caused by vildagliptin could help attenuatearrhythmia and reduce the VFT as found in this study. However, notall reports on the effects of DPP-4 inhibitors on the infarct size areconsistent. In ischemic study of DPP-4 gene deleted mice and DPP-4inhibitor treated mice, no infarct size reduction was observed inthat study [26]. Furthermore, treatment with vildagliptin or valinepyrrolidide did not reduce the infarct size in rat I/R model [18,23].This discrepancy in results could be due to the difference in animalmodels. Nevertheless, our study is the first study to demonstrate theinfarct limiting effect of DPP-4 inhibitor in the swine I/R model. Thisfinding suggests that the effect of DPP-4 inhibitor on the infarct sizereduction could differ in different animal species. In the clinicallyrelevant swine I/R model, the augmentation of GLP-1 level by GLP-1analog could also reduce infarct size [21]. However, Kavianipouret al. [36] and Kristensen et al. [37] failed to show infarct size limitingprofit. This inconsistent finding could be due to different type of drug,routes, and the timing of drug administration. In a swine study thatshowed infarct limiting benefit, exendin-4 (GLP-1 analog) was givenat 10 μg subcutaneously and intravenously prior to reperfusion and10 μg subcutaneously twice daily until day 3 of reperfusion [21].However, in two swine studies that failed to show infarct limitingbenefit, liraglutide (GLP-1 analog) was given at 10 μg/kg/day subcuta-neously for 3 days prior to the I/R conduction [37], and recombinantGLP-1 was given at 3 pmol/kg/min intravenously starting at 15 minbefore ischemia to the end of reperfusion [36]. All of these findings in-dicate the importance of species, type of drug, routes, and timing ofdrug administration in I/R models.

The present study further demonstrated the protective effect ofvildagliptin on cardiac mitochondria. We demonstrated for the firsttime that DPP-4 inhibitor could effectively attenuate cardiac mito-chondrial dysfunction caused by severe oxidative stress as found inthe heart during I/R, suggesting that this mitochondrial protectioncould play a crucial role in the cardioprotective effect of vildagliptin.During I/R, it has been shown that the level of ROS could be marked-ly increased [38], resulting in the deterioration of the electron trans-port chain [39] and activation of the apoptotic pathway [40], andeventually myocardial death. In addition, the elevation of ROS level

Fig. 4. Vildagliptin and the occurrence of cardiac arrhythmia. PVCs were markedly decreased in the vildagliptin treated group at the ischemic period and tended to decrease atreperfusion period, compared with normal saline group (panel A). Ventricular tachycardia (VT) and ventricular fibrillation (VF) incidence (panel B), VT/VF episode (panel C)and time to the first VT/VF onset (panel D) were not significantly different between vildagliptin and normal saline groups. * pb0.05 vs. baseline.

Fig. 5. Effect of vildagliptin on the infarct size. Panel A: The AAR was not differentbetween the vildagliptin and normal saline treated groups. Vildagliptin could attenuatethe infarct size by 17%, compared with normal saline treated group. Panel B: a micro-scopic appearance of the myocardium from the remote area (left) and infarct area(right). Thinning and waviness of myocardial fibers with distinct nuclei were foundin the infarct area. * pb0.05 vs. NSS. AAR = Area at risk.

455K. Chinda et al. / International Journal of Cardiology 167 (2013) 451–457

could cause cardiac mitochondrial membrane depolarization, a con-dition that has been shown to be responsible for cardiac arrhythmias[41]. In our study, administration of vildagliptin at 3.30 mM to cardi-ac mitochondria could attenuate cardiac mitochondrial dysfunctioncaused by H2O2, as indicated by the reduction of ROS productionand attenuation of mitochondrial membrane potential changes.These effects could be a mechanism responsible for reduced myocar-dial vulnerability to arrhythmia and decreased infarct size found inthe present study [42].

5. Conclusions

A DPP-4 inhibitor vildagliptin stabilized cardiac electrophysiologyby attenuating ERP shortening and reducing the infarct size, resultingin decreasing myocardial vulnerability to cardiac arrhythmia duringI/R. The cardioprotective effect of vildagliptin could be due to itsability to prevent cardiac mitochondrial dysfunction caused by severeoxidative stress during I/R.

Conflict of interest

None declared.

Acknowledgments

This study is supported by grants from the Thailand ResearchFund RTA5280006 (NC), BRG5480003 (SC), and the Thailand Re-search Fund's Royal Golden Jubilee PhD program (NC and KC).

The authors of this manuscript have certified that they complywith the Principles of Ethical Publishing in the International Journalof Cardiology.

Fig. 6. Effect of vildagliptin on cardiac mitochondria. H2O2 caused markedly increased ROS level and decreased red/green florescent ratio. Vildagliptin at 0.1 mg/ml could not reduceROS level (panel A) or prevent the red/green florescent ratio reduction (panel B) caused by H2O2. However, 1.0-mg/ml vildagliptin treated for 5, 15, and 30 min could significantlydecrease ROS level (panel C) and attenuate mitochondrial depolarization (panel D) caused by H2O2. * pb0.05 vs. M; † pb0.05 vs. MH. M = Mitochondria; MV = Mitochondria+Vildagliptin; MH = Mitochondria+H2O2; MV5H = Mitochondria+Vildagliptin5 min+H2O2; MV15H = Mitochondria+Vildagliptin15 min+H2O2; MV30H = Mitochondria+Vildagliptin30 min+H2O2.

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