Dipeptidyl peptidase-4 inhibitor improves cardiac function via attenuating adverse cardiac remodeling in rats with chronic myocardial infarction

Tharnwimol Inthachai 1,2, Suree Lekawanvijit 1,3, Sirinart Kumfu 1,2, Nattayaporn Apaijai 1,2, Wanpitak Pongkun 1,2, Siriporn C. Chattipakorn 1,4, Nipon Chattipakorn 1,2,*

Short title: DPP-4 inhibitor in chronic myocardial infarction Total word count: 6229

1

Cardiac Electrophysiology Research and Training Center, Faculty of Medicine,

Chiang Mai University, Chiang Mai, Thailand 2

Cardiac Electrophysiology Unit, Department of Physiology, Faculty of Medicine,

Chiang Mai University, Chiang Mai, Thailand 3

Department of Pathology, Faculty of Medicine, Chiang Mai University,

Chiang Mai, Thailand 4

Department of Oral Biology and Diagnostic Science, Faculty of Dentistry,

Chiang Mai University, Chiang Mai, Thailand

* Address for correspondence: Nipon Chattipakorn, MD, PhD Cardiac Electrophysiology Research and Training Center Faculty of Medicine, Chiang Mai University, Chiang Mai, 52000, Thailand Tel: +66-53-945329, Fax: +66-53-945368 E-mall: [email protected]

This is an Accepted Article that has been peer-reviewed and approved for publication in the Experimental Physiology, but has yet to undergo copy-editing and proof correction. Please cite this article as an Accepted Article; doi: 10.1113/EP085108. This article is protected by copyright. All rights reserved.

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New findings



What is the central question of this study?

Although cardioprotective effects of the dipeptidyl peptidase-4 (DPP-4) inhibitor are

demonstrated, it is unclear of its cardiac effects in chronic myocardial infarction (MI). Thus,

we determined the effects of DPP-4 inhibitor on cardiac function and remodeling in chronic

MI rats.



What is the main finding and its importance?

We demonstrated for the first time that DPP-4 inhibitor, but not metformin, exerted

similar efficacy in improving cardiac function and attenuating cardiac fibrosis compared to

enalapril in chronic MI rats. These findings reveal additional benefits beyond the glycemic

control of the DPP-4 inhibitor in chronic MI, and might be the new drug of choice for MI in

diabetes subjects.

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ABSTRACT Adverse cardiac remodeling after myocardial infarction (MI) leads to progressive heart failure. Dipeptidyl peptidase-4 (DPP-4) inhibitor is a new antidiabetic drug that exerts cardioprotection. However, its role on cardiac function and remodeling in chronic MI is unclear. We hypothesized that DPP-4 inhibitor (vildagliptin) reduces adverse cardiac remodeling and improves cardiac function in chronic MI rats. These effects were also compared with enalapril and metformin. Male Wistar rats (n=36) with chronic MI induced by left anterior descending coronary arterial ligation were divided into 6 groups to receive vehicle, vildagliptin (3 mg/kg/d), metformin (30 mg/kg/d), enalapril (10 mg/kg/d), combined metformin and enalapril, or combined vildagliptin and enalapril for 8 weeks. At the end of the study, plasma malondialdehyde, heart rate variability (HRV), left ventricular (LV) function, pathological and biochemical studies of cardiac remodeling were investigated. Our study demonstrated that chronic MI rats had increased oxidative stress levels, depressed HRV, and adverse cardiac remodeling indicated by cardiac fibrosis, and LV dysfunction. Treatment with vildagliptin and enalapril both significantly decreased oxidative stress, attenuated cardiac fibrosis, and improved HRV and LV function. We conclude that vildagliptin exerts similar cardioprotective effects as enalapril in attenuating oxidative stress and cardiac fibrosis and improving cardiac function in chronic MI rats. Metformin does not provide these benefits in this model. Moreover, addition of either metformin or vildagliptin to enalapril does not provide additional benefit in attenuating cardiac remodeling or improving LV function, compared to enalapril alone.

Keywords: DPP-4 inhibitor; Incretins; Metformin; Vildagliptin; Enalapril; Chronic MI; Cardiac remodeling; Cardiac function.

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Abbreviations: +dp/dt, Maximum dp/dt; -dp/dt, Minimum dp/dt; ACE, Angiotensin converting enzyme; BW, Body weight; ECG, Electrocardiogram; EDP, End diastolic pressure; EDP, End systolic pressure; FS, Fractional shortening; GIP, Gastric inhibitory polypeptide, GLP-1, Glucagon like peptide 1; H&E, Hematoxylin and Eosin; HF, Heart failure; HR, Heart rate; HRV, Heart rate variability; HPLC, High performance liquid chromatography; LAD, Left anterior descending coronary; LVPWd, Left ventricular posterior wall diameter during diastole; IVSd, Left ventricular septum during diastole; LVIDd, Left ventricular internal diameter during diastole; LVPWs, Left ventricular posterior wall diameter during systole; IVSs, Left ventricular septum during systole; LVIds, Left ventricular internal diameter during systole; LF, Low frequency; LSD, Least significant difference; MDA, Malondialdehyde; MI, Myocardial infarction; NADPH, Nicotinamide adenine dinucleotide phosphate; p-p38, Phosphorylated-p38; p-p44/42, Phosphorylatedp44/42; SEM, Standard error of mean; TBA, Thiobarbituric acid; TGF-β, Transforming growth factor-beta; VLF, Very low frequency.

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Introduction Heart failure is the most common complication and the major cause of morbidity and mortality in post-myocardial infarction (MI) survivors (Minicucci et al., 2011). Post-MI cardiac structural changes, the so-called ‘cardiac remodeling’ which comprises cardiac hypertrophy, interstitial fibrosis, progressive wall thinning and ventricular chamber dilatation (Sutton et al., 2000; Gajarsa et al., 2011), contribute to progressive cardiac dysfunction and heart failure. These processes are associated with persistent activation of neurohormonal systems, such as sympathetic nervous system and renin-angiotensin-aldosterone system, and increased oxidative stress. Generally, post-MI cardiac remodeling begins rapidly usually within the first-few hours and continues to progress from weeks to months (Cohn et al., 2000). Patients with major cardiac remodeling are at high risk of heart failure progression and sudden cardiac death after MI (Cohn et al., 2000; Bunch et al., 2007). Thus, a reversal in cardiac remodeling is potentially a key therapeutic strategy in these patients to prevent sudden cardiac death and to slow the heart failure progression. An angiotensin converting enzyme (ACE) inhibitor such as enalapril has been shown to exert cardioprotective effect in MI (Wang et al., 2004; Ocaranza et al., 2006). Growing evidence demonstrates that several antidiabetic drugs exert cardioprotective effects in addition to their glycemic controlling effect (Apaijai et al., 2012; Myat et al., 2014), including a first-line antidiabetic drug metformin (Tahrani et al., 2007; Scarpello et al., 2008), and a new class of antidiabetic drug gliptin (Plutzky, 2011; Hocher et al., 2012). Previous studies demonstrated that both metformin and vildagliptin exerted beneficial effects on the cardiac autonomic regulation and cardiac function by attenuating the cardiac sympathovagal imbalance and improved cardiac contractile function in obese-insulin resistant models (Apaijai et al., 2012). In cardiac ischemia-reperfusion model, it has been shown that metformin and vildagliptin also improved cardiac function impaired by ischemia-reperfusion

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injury (Apaijai et al., 2014). Moreover, both metformin and gliptin, which have been used to treat type-2 diabetic patients, have been demonstrated to decrease cardiac remodeling and improve left ventricular (LV) function in both animal (Liu et al., 2010; Yin et al., 2011; Takahashi et al., 2013), and clinical studies in non-diabetic and diabetic patients with MI (Sokos et al., 2006; Hausenloy et al., 2008; Lexis et al., 2014). However, comparing cardiac effects between these two drugs in chronic MI as well as investigating on potential additional cardioprotective effects of using either metformin or gliptin in combination with an angiotensin converting enzyme (ACE) inhibitor, a conventional pharmacological treatment of chronic MI, have not been studied. In the present study, we determined the effects of metformin and vildagliptin on cardiac remodeling in chronic MI rats induced by left anterior descending (LAD) coronary arterial ligation. We hypothesized that 1) metformin and vildagliptin decrease oxidative stress and cardiac remodeling, and improve heart rate variability (HRV) and left ventricular (LV) function in chronic MI rats, and 2) a combination of either metformin or vildagliptin with enalapril exerts synergistic effects on cardioprotection in chronic MI rats.

Materials and methods Ethical Approval All of the experiment procedures were conducted in accordance with an approved protocol from the Institution Animal Care and Use Committee at the Faculty of Medicine, Chiang Mai University, Chiang Mai, Thailand, and complied with the ARRIVE guidelines.

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Animal preparation Male Wistar rats weighing 400 to 450 g were obtained from the National Animal Center, Salaya Campus, Mahidol University, Thailand. All animals were fed with normal rat chow and ad libitum water, and housed in a temperature-controlled environment with a 12:12 light-dark cycle. All rats were induced for chronic MI by LAD ligation, as previously described (Lekawanvijit et al., 2012). Briefly, animals were intubated and mechanically ventilated with 2% isoflurane with 1.0 L/min oxygen. After the animal was deeply anesthetized, the left-side thoracotomy was performed, and the LAD branch of left coronary artery was exposed and ligated at approximately 4 mm from its origin. Subsequently, positive pressure was applied to the lungs via the ventilator before closing the chest wall. At 3 days after LAD ligation, all rats were randomly divided into six groups to receive one of the following treatments: vehicle (normal saline), enalapril (10 mg/kg, Berlin Pharmaceutical Industry, Bangkok, Thailand), metformin (15 mg/kg, twice a day, Glucophage, Merck Serono, Bangkok, Thailand), vildagliptin (3 mg/kg, once a day, Galvus, Novartis, Bangkok, Thailand), combined metformin and enalapril, and combined vildagliptin and enalapril, respectively, for 8 weeks by intragastric gavage (n= 5-7/group). HRV was assessed and blood samples were collected at baseline and at the end of treatment. Echocardiography was performed at baseline, at 4 weeks post-MI, and at the end of treatment. At the end of treatment, all rats were anesthetized and LV function was assessed by the intraventricular pressure recording system using the pressure catheter (Scisense, Ontario, Canada). Then, rats were sacrificed and the heart was removed for determining the infarct size and cardiac remodeling including cardiomyocyte hypertrophy and cardiac fibrosis. Heart tissue was also used for western blot analysis.

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Plasma malondialdehyde (MDA) level determination Plasma MDA levels were measured by high performance liquid chromatography (HPLC) based assay (Thermo Scientific, Bangkok, Thailand) (Apaijai et al., 2012). Plasma MDA was mixed with phosphoric acid (H3PO4) and thiobarbituric acid (TBA) to create TBA reactive substance. Plasma TBA reactive substance concentration was reported as MDA equivalent concentration from a standard curve of HPLC based assay (Mateos et al., 2005). Heart rate variability (HRV) analysis HRV was used to determine the cardiac sympathovagal balance (Incharoen et al., 2007; Apaijai et al., 2012). The electrocardiogram (ECG) lead II was recorded in all rats using Powerlab (AD Instrument, Sydney, Australia). Data of RR interval variability from the ECG were analyzed using the MATLAB program (Incharoen et al., 2007; Apaijai et al., 2013). In the present study, frequency domain method was used to represent the HRV (Apaijai et al., 2012; Supakul et al., 2014). Frequency domain has three major oscillatory components, including high frequency (HF; 0.6-3 Hz) band representing cardiac parasympathetic activity, low frequency (LF; 0.2-0.6 Hz) band representing cardiac sympathetic and parasympathetic activity, and very low frequency (VLF; below 0.2 Hz) band. The LF/HF ratio was used to indicate cardiac sympathovagal balance (Apaijai et al., 2012). Depressed HRV or increased LF/HF ratio represents cardiac sympathovagal imbalance. Echocardiography The M-Mode echocardiography (HP/Agilent Philips Sonos 4500 (Agilent Technologies, Santa Clara, CA, USA) was used to determine LV posterior wall diameter during diastole (LVPWd), interventricular septum during diastole (IVSd) and LV internal diameter during diastole (LVIDd), and LV posterior wall diameter during systole (LVPWs), interventricular septum during systole (IVSs) and LV internal diameter during systole (LVIds). Percentage of fractional shortening (%FS) was calculated to indicate LV systolic performance by using the following equation: %FS = [(LVIDd-LVIds)]/LVIDd]x100 (McDonald, 1976).

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LV pressure measurement Rats were anesthetized using zoletil (50 mg/kg, Virbac, Laboratories, Carros, France) and xylazine (0.15 mg/kg, Laboratory Carlier, SA, Barcelona, Spain) via intramuscular injection (Apaijai et al., 2012; Supakul et al., 2014). Rats then were ventilated with room air via a tracheostomy tube. The right carotid artery was identified and canulated with a pressure catheter (Scisense, Ontario, Canada) for measuring LV pressure for 20 minutes (Supakul et al., 2014). Parameters including heart rate, end-systolic (ESP), end-diastolic pressure (EDP), and dP/dtmax and dP/dtmin were analyzed using the analytical software program (Labscribe, Dover, NH) (Apaijai et al., 2012; Chinda et al., 2012). Myocardial infarct size assessment Tissue harvesting was performed at the end of the LV pressure assessment while animals were still under deep anesthesia. The left ventricle was divided horizontally into 3 sections. Mid-LV cross sections were stained with Picrosirius red and scanned for infarct size analysis. Assessment was performed in animals with transmural infarction where the full thickness of LV wall from endocardium to epicardium was replaced by fibrotic scar (Goldman et al., 1995). Infarct size was reported as an average of the proportions of LV endocardial and epicardial circumferences occupied by the infarct as previously described (Goldman et al., 1995). Cardiac remodeling determination Mid-LV cross sections were stained with Hematoxylin and Eosin (H&E) and scanned with Aperio ScanScope (Aperio Technologies, Inc). Fifty LV cardiomyocytes with equalsized nuclei were randomly selected for analysis of cellular cross-sectional area using Aperio ImageScope version 11.1.2.760 (Lekawanvijit et al., 2012). Measurements from each animal were averaged and data were expressed as mean±SEM for each group. For determination of cardiac fibrosis, cardiac sections stained with Picrosirius red were scanned (Aperio Technologies, Inc). The proportional area of matrix deposition in the peri- and non-infarct areas were separately analyzed using positive pixel count v9 algorithm for picrosirius red stain intensity (Lekawanvijit et al., 2012).

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Western blot analysis At the end of the study, heart tissues were collected and quickly frozen in liquid nitrogen and stored at -80oC until utilization as described previously (Kumphune et al., 2011). Heart tissue was homogenized with 1 ml of tissue lysis buffer, then subsequently homogenized at 4oC and incubated on ice for 30 minutes followed by centrifugation at 13,000 rpm for 10 minutes at 4oC after which the supernatant was collected as 100-µl aliquots. Equal amount (34 µg) of the total protein was mixed with the loading buffer (1 mg/ml) (10% mercaptoethanol, 0.05 % bromophenol blue, 75 mM Tris-HCl, pH 6.8, 2% sodium dodecyl sulphate (SDS) and 10% glycerol), boiled for 5 minutes and loaded onto 10% gradient SDSpolyacrylamide gel. Then, the protein was transferred to a nitrocellulose membrane in the presence of the glycine/methanol transfer buffer (20 mM Tris base, 0.15 M glycine, 20% methanol) in a transfer system (Bio-Rad, CA, USA). Nitrocellulose membrane was blocked with 5% skim milk in 1xTBS-T buffer (20 mM Tris-HCl pH 7.6, 137 mM NaCl, 0.05% tween-20) for one hour at room temperature. The membrane was subsequently exposed to anti-Bax (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti–Bcl 2, anti-active caspase 3, anti-phosphorylated connexin (Cx) 43, anti-transforming growth factor beta (TGFβ), antiphosphorylated-p38 (p-p38) and p38 mitogen‐activated protein kinase (MAPK) kinase and anti-phosphorylated p44/42 (p-p44/42) and p44/42 MAPK (Cell Signaling Technology, Danvers, MA, USA) overnight at 4oC. The signals were developed by incubating with enhanced chemiluminescence reagent and subjected to autoradiography.

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Statistical analysis All data were expressed as mean ± SEM. Two-way analysis of variance (ANOVA) with repeated measures followed by least significant difference (LSD) post‐hoc test was used to determine variables measured repeatedly at multiple time-points including %fs, lf/hf ratio, and plasma mda. One-way ANOVA followed by LSD post‐hoc test was used to determine variables measured at a single time-point. P value < 0.05 was considered statistically significant.

Results Metabolic parameters and organ weight index in chronic MI rats among various treatment groups At 8 weeks post-MI, the effects of pharmacological interventions on the body weight (BW), food intake, LV/BW, lung/BW, right ventricle/BW and atrium/BW are shown in Table 1. Our results demonstrated that these parameters were not significantly different among different treatment groups, compared with the vehicle-treated group. Infarct size in chronic MI rats among various treatment groups LAD ligation resulted in the infarct size of 36±3%, 36±5%, 37±4%, 37±4%, 31±4%, 29±3% in the vehicle group (MI+V), enalapril-treated group (MI+E), metformin-treated group (MI+M), vildagliptin-treated group (MI+Vil), combined metformin and vildagliptin treated group (MI+M&E), and combined vildagliptin and enalapril treated group (MI+Vil&E), respectively. There was no significant difference of the infarct size among different treatment groups (Table 1). These results indicated the similar degree of infarct size induction caused by LAD occlusion in all treatment groups.

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LV function in chronic MI rats among various treatment groups At 4 weeks post-MI, %FS was decreased in all groups of chronic MI rats. All pharmacological treatments in the present study did not significantly improve %FS when compared with MI+V group after 4 weeks of treatment (Fig. 1A). However, %FS was significantly improved after 8 weeks of treatment in MI+E, MI+Vil, MI+M&E and MI+Vil&E groups, but not in the MI+M group. Moreover, rats in the MI+E group had significantly higher %FS than those in the MI+Vil group. No significant difference was observed when compared %FS of MI+E group with MI+M&E and MI+Vil&E groups (Fig. 1A). Results from the LV pressure study showed that all treatment groups, except MI+M, showed a significant increase in ESP and dP/dtmax, and a significant decrease in EDP and dP/dtmin at 8 weeks after treatment, compared with the MI+V group (Table 2). In addition, MI+E group had significantly higher ESP and +dP/dt than MI+Vil group, whilst no difference was observed when compared with MI+M&E and MI+Vil&E groups (Table 2). There was no significant difference of EDP and -dP/dt among MI+E, MI+Vil, MI+M&E and MI+Vil&E groups. HRV in chronic MI rats among various treatment groups At baseline, the LF/HF ratio was not different among different treatment groups (Fig. 1B). At 8 weeks after treatment, all groups had an increased LF/HF ratio when compared to the baseline. However, all treatment groups, except the MI+M group, showed a significant reduction in the LF/HF ratio when compared with the MI+V group. There was no significant difference of HRV among MI+E, MI+Vil, MI+M&E and MI+Vil&E groups.

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Oxidative stress in chronic MI rats among various treatment groups At baseline, plasma MDA levels were not significantly different among different treatment groups (Fig. 2). At 8 weeks after treatment, all groups had increased plasma MDA levels compared to the baseline. However, rats in the MI+E, MI+Vil, MI+M&E and MI+Vil&E groups had significantly lower plasma MDA levels, compared with the MI+V group. There was no significant difference for the plasma MDA among MI+E, MI+Vil, MI+M&E and MI+Vil&E groups. The plasma MDA in the MI+M group was not different from the MI+V group. Cardiac remodeling among various treatment groups At 8 weeks after treatment, cardiac fibrosis in the peri-infarct area was not significantly different among different treatment groups (Fig. 3B). Interestingly, a significant reduction in cardiac fibrosis in the non-infarct area was observed in MI+E, MI+Vil, MI+M&E and MI+Vil&E groups, but not in the MI+M group, when compared with MI+V group (Fig. 3A). Our results also demonstrated that cardiomyocyte cross sectional area was not significant different among different treatment groups, compared to MI+V group (Fig. 3C). Protein expression in chronic MI rats among various treatment groups Western blot analysis showed a decreased Bax/Bcl-2 ratio in MI+E, MI+M&E and MI+Vil&E groups, but not MI+M and MI+Vil groups, compared with MI+V group (Fig. 4). In all drug treatment groups, the p-p44/42 protein expression was significantly decreased, compared to the MI+V group (Fig. 5). However, no significant difference in p-p44/42 protein expression was found among these drug-treated groups. The expression levels of active caspase3, TGF-β, p-p38 and p-connexin43 (serine368 residue) were also not significantly different among all groups (Fig. 6).

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Discussion The major findings of the present study in the setting of chronic MI are as follows. Chronic MI rats induced by LAD ligation developed a cardiac symphathovagal imbalance (i.e. depressed HRV), increased oxidative stress level, cardiac fibrosis, and LV dysfunction. Treatment with enalapril and vildagliptin, but not metformin, improved HRV, reduced oxidative stress, attenuated cardiac fibrosis, and improved LV function. In the present study, chronic MI rats developed cardiac sympathovagal imbalance at 8 week-post MI as indicated by an increased LF/HF ratio. Our finding is consistent with previous clinical studies which demonstrated increased sympathetic and decreased parasympathetic activity in chronic MI patients (Chattipakorn et al., 2007; Oliveira et al., 2013). Moreover, the depressed HRV has been shown to be associated with increased oxidative stress level (Pavithran et al., 2008). Our findings suggested that increased plasma MDA level in chronic MI rats could be linked to the increased LF/HF ratio observed in this study. In the present study, we demonstrated that both enalapril and vildagliptin, but not metformin, could reduce oxidative stress level as well as improve HRV in chronic MI rats at similar degree, suggesting a strong association between the reduced oxidative stress level and improved cardiac autonomic balance under this condition. It has been shown that the increased sympathetic activity in chronic MI could be aggravated under the stress condition, and that this increase sympatho-excitation in turn could significantly impact the progression of heart failure (Triposkiadis et al., 2009; Kishi, 2012). It is also possible that the drugs could affect various elements of the cardiac nervous system, and that the potential cardioprotective effects of this pharmacological therapy could be related to their effects on cardiac-related neurons rather than direct effects on cardiomyocytes. Future study is needed to test this hypothesis. The fact that these drugs could improve cardiac sympathovagal

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balance and decrease oxidative stress may also provide a long-term benefit on the heart failure progression in this chronic MI model. In chronic MI rats, cardiac systolic and diastolic dysfunction is commonly found (Bauersachs et al., 2001; Izutani et al., 2002). Since the infarct size of our chronic MI rats was not different among different groups of treatments, the improved LV function observed in both enalapril and vildagliptin, but not metformin, treatments could contribute to the improved cardiac remodeling by these drugs. In a pressure overload-induced heart failure model, vildagliptin was shown to decrease cardiac interstitial fibrosis and increase %FS and survival rate (Takahashi et al., 2013). In the present study, vildagliptin reduced cardiac fibrosis significantly, consistent with a previous report in a different model. However, the effect of vildagliptin on LV function has been inconsistently reported. In a study of Yin et al., vildagliptin given at 3 weeks after LAD ligation provided no protective effects on cardiac function and remodeling in this long-term chronic MI model (Yin et al., 2011). This could be due to the late administration of the drug after MI onset. In post-MI, it has been shown that ventricular remodeling could continue for weeks in a subacute stage to months in a chronic stage of remodeling (Gajarsa et al., 2011). Therefore, in Yin et al. study it is possible that the remodeling process was already completed at the time vildagliptin was administered, leading to failed protection of cardiac dysfunction and adverse remodeling. Unlike Yin et al. study, the treatment was started on the third day after LAD ligation in the present study, when the remodeling process was still in an acute stage, thus exerting the cardioprotection in our chronic MI rats. These findings suggest that early administration of vildagliptin may attenuate adverse cardiac remodeling in the chronic MI model. Previous studies demonstrated that increased cardiac fibrosis can lead to diastolic dysfunction (Moreo et al., 2009; Konstam et al., 2011). Interestingly, we found that treatment with vildagliptin, but not metformin, could decrease cardiac fibrosis, similar to that

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found in the enalapril group in chronic MI rats. Therefore, improved cardiac fibrosis by enalapril and vildagliptin observed in this study could be responsible for the improved LV function. Moreover, oxidative stress and cardiac sympathovagal imbalance has been demonstrated to be associated with cardiac dysfunction (Ferrara et al., 2002; Grieve et al., 2003). This notion is supported by our findings that both enalapril and vildagliptin, but not metformin, attenuated these impairments and might play an important role in improved LV function in chronic MI rats observed in our study. In addition to fibrosis and oxidative stress, increased cellular apoptosis has also been shown to involve in LV dysfunction (Sam et al., 2000; Grieve et al., 2003). Bax and Bcl-2 are proteins involved in the apoptotic process (Krijnen et al., 2002). The activation of Bax leads to its translocation into the mitochondria which triggers mitochondrial membrane permeability, resulting in the release of cytochrome c and activation of caspase-9 and -3, leading to apoptotic cell death (Crow et al., 2004). Previous studies demonstrated that increased Bax/Bcl-2 ratio indicates increased apoptosis (Yang et al., 2001). In the present study, only enalapril, but neither metformin nor vildagliptin, could decrease the Bax/Bcl-2 ratio, suggesting that only enalapril exerted anti-apoptotic effect in chronic MI condition. In chronic MI, inflammatory process also plays an important role in cardiac remodeling (Nian et al., 2004; Anzai, 2013). TGF-β, i.e. a pro-inflammatory and pro-fibrotic cytokine, has been shown to involve in cardiac remodeling (Dobaczewski et al., 2011). Both phospho-p38 and phospho-p44/42 MAPKs, which are downstream signaling mediator of TGF-β, have been reported to be involved in the fibroblast proliferation and extracellular matrix deposition (Bujak et al., 2007). In the present study, both vildagliptin and enalapril significantly decreased phospho-p44/42 MAPK expression, which could be responsible for decreased cardiac fibrosis in the non-infarcted area in chronic MI rat hearts. In this regard, although metformin decreased phospho-p44/42 MAPK protein expression, it did not exert

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cardioprotection. This could be due to the fact that metformin did not exert antioxidative effect and did not attenuate cardiac sympathovagal imbalance as the other two drugs did. These findings suggest that the antioxidative effect might play a crucial role in preventing adverse cardiac remodeling and cardiac dysfunction. However, several studies demonstrated beneficial effects of metformin in chronic MI model with higher dose and longer duration than that used in our study (Yin et al., 2011; Cittadini et al., 2012). In addition, a previous study showed that administration of metformin at the same dose used in our study could improve diastolic function in insulin resistance rats (Apaijai et al., 2012). This cardioprotective benefit of metformin in this model could be due to the fact that metformin is an antidiabetic drug which exerts its beneficial effects on the cardiovascular system through its reduction of insulin resistance. Thus, metformin could be more beneficial in diabetic or insulin resistant conditions than in normal subjects. Nevertheless, further investigation is needed to verify the effect of metformin on cardiac function under non-diabetic and diabetic with superimposed chronic MI. Although enalapril and vildalgliptin attenuated oxidative stress, cardiac sympathovagal imbalance, cardiac fibrosis and cardiac dysfunction, combined vildagliptin and enalapril did not show synergistic effect on cardioprotection. This could be due to the fact that enalapril exerted maximal effects on the reduction of oxidative stress and phospho p44/42 expression, compared to vildagliptin alone. Interestingly, vildagliptin which is an antidiabetic drug could decrease cardiac fibrosis similar to enalapril which is a first line drug for treatment of chronic MI. This finding suggests that DPP-4 inhibitor may be the drug of choice for chronic MI patients who have insulin resistance or type 2 diabetes. Further investigation is needed to warrant its clinical application. In conclusion, we demonstrated that chronic MI rats induced by LAD ligation developed a cardiac sympathovagal imbalance, increased oxidative stress, cardiac fibrosis

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and cardiac contractile dysfunction. In addition, vildagliptin, but not metformin, attenuated these impairments by improving HRV, reducing oxidative stress and cardiac fibrosis, and improving LV function similar to that observed in enalapril, suggesting cardioprotective benefits of vildagliptin in addition to its glycemic control in chronic MI model.

Conflict of interest The authors declare that they have no conflict of interest.

Acknowledgements TI, SL, SK, NP, WP: Data collection, analysis, interpretation, drafting the work, and final approval of the manuscript; SC and NC: Study concept and design, data analysis and interpretation, drafting the work, and final approval of the manuscript. This work was supported by the NSTDA Research Chair Grant from the National Science and Technology Development Agency (NC), Thailand Research Fund RTA5580006 (NC), BRG5780016 (SC), MRG5680079 (SL), TRG5780002 (SK) and Chiang Mai University Center of Excellence Award (NC). Vildagliptin was kindly provided by Novartis (Thailand).

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Figure 1. Effects of pharmacological interventions on percentage of fractional shortening (%FS) and low frequency/high frequency (LF/HF) ratio in chronic myocardial infarction (MI) rats (n=5-7/group). A, All pharmacological interventions, except metformin (MI+M) group improved %FS at 8 weeks post‐MI. B, All pharmacological interventions, except MI+M group, decreased LF/HF ratio at 8 weeks post‐MI. Two-way ANOVA; %FS, time course effect (F=426.608, p = 0.000), treatment effect (F=2.634, p = 0.044, time course & treatment effects (F=2.888, p = 0.005). LF/HF ratio; time course effect (F=113.640, p =0.000), treatment effect (F=3.098, p = 0.002), time course & treatment effects (F=3.670, p = 0.011). V, vehicle; E, enalapril; M, metformin; Vil; vildagliptin; M&E, combined metformin and enalapril; Vil&E, combined vildagliptin and enalapril. *p

Dipeptidyl peptidase-4 inhibitor improves cardiac function by attenuating adverse cardiac remodelling in rats with chronic myocardial infarction.

What is the central question of this study? Although cardioprotective effects of dipeptidyl peptidase-4 (DPP-4) inhibitors have been demonstrated, the...
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