Effect of Ischemic Postconditioning on Myocardial Function and Infarct Size Following Reperfusion Injury in Diabetic Rats Pretreated With Vildagliptin

Abstract

Background:

Cardioprotective actions of ischemic postconditioning (IPostC) against ischemia/reperfusion (I/R) injury are abolished in diabetic hearts. This study has investigated the combined effects of IPostC and vildagliptin (Vilda) on myocardial function and infarct size (IS) against I/R injury in diabetic myocardium.

Methods:

Diabetes was induced by a high-fat diet/low dose of streptozotocin (35 mg/kg; intraperitoneally) in Wistar rats (200-250 g) and lasted for 12 weeks. Vilda (6 mg/kg/d) was orally administered for 5 weeks in diabetic groups after seventh week of diabetes. At the end of the 12-week period, the hearts of rats were removed and subjected to 35-minute regional ischemia (through left anterior descending ligation) followed by 60-minute reperfusion, on Langendorff apparatus. Ischemic postconditioning was induced by 6 repetitive cycles of 10-second ischemia and 10-second reperfusion, immediately at the onset of the reperfusion. Myocardial hemodynamic was measured throughout the experiment. The IS was assessed by triphenyltetrazolium chloride staining method. The myocardial contents of troponin-I (cTnI), interleukin-6 (IL-6), and 8-isoprostane were measured in the homogenate from ischemic zone of left ventricles by enzyme-linked immunosorbent assay kit.

Results:

Pretreatment of the diabetic rats with Vilda significantly recovered the diabetes-induced reduction in left ventricular developed pressures and contractility at the baseline (P < .05 to P < .01). After I/R injury, IPostC could not significantly improve the myocardial function, cTnI content, and IS of the diabetic hearts. However, in Vilda-treated hearts, concomitant application of IPostC significantly recovered the heart functions, returned cTnI content as well as myocardial IL-6 and 8-isoprostane levels back to the control values (P < .01 to P < .001), and reduced IS more effectively (by 45%) in comparison to the diabetic group (P < .001).

Conclusion:

Besides its glycemic and lipid profile controlling effects, Vilda has a protective effect on heart function and tends to restore cardioprotective effects of IPostC on diabetic hearts.

Keywords

ischemic postconditioning cardioprotection diabetes reperfusion injury infarct size

Introduction

Metabolic disorders, including diabetes mellitus, especially type 2 diabetes and insulin resistance, are spreading at an ever-increasing rate worldwide. It is estimated that 415 million adults are engaged with diabetes and this figure will rise to 642 million by 2040, largely due to poor lifestyle habits.¹ However, diabetes tends to be further mortal when accompanied with myocardial ischemic diseases. The adverse effects of reperfusion therapy following ischemic insults have been shown to be even more severe in ischemic diabetic hearts, resulting in an increased infarct size (IS) and dysfunctional left ventricle due to changes in cardiac energy metabolism, intracellular signaling, and insulin resistance.^2,3 Therefore, an approach toward prevention of diabetic heart injury from ischemia/reperfusion (I/R) insults to improve its prognosis is one of priority of experiments.

A number of mechanical strategies and pharmacological agents have been proposed in the experiments to ameliorate reperfusion injury in nondiabetic and diabetic hearts. Ischemic postconditioning (IPostC) is defined as the very short cycles of ischemia and reperfusion applied immediately at the onset of reperfusion; it has been seen to have a significant cardioprotective effect on nondiabetic hearts, however, different settings of diabetes abrogate its positive effects.^2,3 Fortunately, some recent experimental studies have showed the cardioprotective effects of combining mechanical and pharmacologic strategies in diabetic hearts.^4,5 Novel antidiabetic drugs, like glucagon-like peptide 1 (GLP-1)–based therapies are used to control the blood glucose levels of patients with diabetes. Vildagliptin (Vilda; a new dipeptidyl peptidase 4 [DPP-4] inhibitor) is a selective oral antidiabetic agent that imposes several valuable effects including reducing hyperglycemia through increasing GLP-1 level in plasma, potentiating the secretion of insulin in the beta cells, and suppressing the glucagon release by alpha cells of the pancreas.⁶ It also possesses other positive potentials such as anti-inflammatory,^7,8 antioxidative,^9,10 and cardioprotective effects in nondiabetic experimental rats.^11
-13

The present research considers that, provided a proper antidiabetic drug (with the potential of preserving cardiac function) is chosen for controlling hyperglycemia, whether or not concomitant application of IPostC would be protective on diabetic rat heart following I/R injury. In this case, combined effects of Vilda preconditioning and IPostC in diabetic conditions are yet to be studied. Thus, this experiment is focused on this issue and investigates the concomitant effects of IPostC on myocardial function and IS and some biochemical alterations following myocardial I/R injury in diabetic rats pretreated with Vilda.

Materials and Methods

Animals

Eight-week-old male Wistar rats (200-250 g) were housed in individual cages with access to food and water ad libitum and maintained at 22°C ± 2°C on a 12-hour light/dark cycle; they were under strict surveillance until the end of the study. All experiments were approved by the ethical committee of Tabriz University of Medical Sciences and performed in accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH publication No 85-23, revised in 1996).

Induction of Type 2 Diabetes

The method of high-fat diet and low-dose streptozotocin was used to induce type 2 diabetes.^14,15 The experimental diabetic period was lasted for 12 weeks in this study. After a week of acclimatization, diabetic groups were fed, for 6 weeks ad libitum, a high-fat diet containing 35% normal pellet, 30% lard, 4% sucrose, 24% casein, 1% cholesterol (Chol), and 0.3% dl-Methionine (total caloric value was about 4.6 kcal/g, 62% of which was from fat). Nondiabetic groups of rats were fed normal diet. At the end of the sixth week, the rats were injected intraperitoneally with a low dose of streptozotocin (35 mg/kg in citrate buffer, pH 4) after 8 hours of fasting. Blood glucose levels and oral glucose tolerance test (OGTT) were measured using a glucometer at the end of the seventh week. The rats with blood glucose levels beyond 250 mg/dL and impaired OGTT were considered a transitional phase in the development of type 2 diabetes (as diabetic animals).

Experimental Preparation and Protocols

The animals were randomly divided into 5 diabetic and 2 nondiabetic subgroups (n = 10-12 in each group). Diabetic (D) groups included D-Sham, D-Control, D-IPostC, D-Vilda, and D-Vilda-IPostC, while nondiabetic healthy animals were divided into H-Sham and H-Control subgroups (Figure 1). Rats in D-Vilda and D-Vilda-IPostC groups were treated with Vilda (6 mg/kg/d; Novartis, Basel, Switzerland) through gavage in the last 5 weeks.¹⁶ The animal grouping was made in 2 series of similar conditions, one for IS measurements and another for the rest of experiments. Body weights (BWs) of the rats were recorded on a weekly basis, with the final BWs recorded prior to excision and isolation of the heart for Langendorff perfused heart preparation. The heart weight (HW)/BW ratio (%) was used as diabetic cardiomyopathy index.

Figure 1.

Langendorff experimental protocols. Hearts isolated from rats were subjected to one of the following protocols in 2 separate series: H-Sham (110-minute perfusion only), H-Control (35-minute ischemia + 60-minute reperfusion), D-Sham (110-minute perfusion only), D-Control (35-minute ischemia + 60-minute reperfusion), D-IPostC and D-Vilda-IPostC (35-minute ischemia + 6 cycles of 10-second ischemia [black]/10-second reperfusion [white] immediately at the onset of 60 minutes reperfusion). At the end of each experiment, hearts were collected for infarct size assessment or other experiments. D indicates diabetic; H, healthy; IPostC, ischemic postconditioning; Vilda, vildagliptin.

Langendorff Perfused Hearts

At the end of the treatment phase, the hearts of the animals were isolated as mentioned previously.¹⁷ They were heparinized (500 IU/kg) and anesthetized with a mixture of ketamine (60 mg/kg) and xylazine (10 mg/kg) intraperitoneally, then the hearts were rapidly excised and connected to the perfusion cannula via the aorta and mounted on a Langendorff perfusion apparatus (ML176-V; AD Instruments, New South Wales, Australia) at constant pressure (80 mm Hg), in which the isolated hearts were perfused with a Krebs-Henseleit solution containing (in mM/L) NaCl 118, KCl 4.7, CaCl₂ 2.5, MgSO₄ 1.2, NaHCO₃ 25, KH₂PO₄ 1.2, and glucose 11.1. The perfusion solution was gassed with a mixture of 95% O₂ and 5% CO₂ at 37°C and pH 7.4. A water-filled latex balloon was inserted into the left ventricle through an incision in the left atrium, for measuring the hemodynamic parameters using a Power lab data acquisition system and related Chart 7.3 software (AD Instruments). The balloon volume was adjusted to produce 5 to 10 mm Hg of left ventricular end-diastolic pressure (LVEDP) in all experimental groups. The heart rate (HR), LVEDP, left ventricular systolic pressure (LVSP), rate pressure product (RPP = [HR × left ventricular developed pressure [LVDP]]/1000), and coronary flow (CF) were recorded throughout the experiment. Figure 1 shows the experimental protocol used in this study.

Ischemia/Reperfusion Injury and Postconditioning Algorithm

Regional ischemia (for 35 minutes) was induced by tightening the suture (5-0 silk with round/tapered needles) around the left anterior descending (LAD) coronary artery, close to its origin and holding it in place with the snare. The snare was then removed, and the hearts were reperfused for 60 minutes. Ischemic postconditioning was applied as 6 cycles of 10-second reperfusion followed by 10-second ischemia, immediately at the onset of 60-minute reperfusion (Figure 1).¹⁸

Plasma Insulin Levels and Lipid Profiles Measurement

In order to measure plasma lipid profiles, blood sampling was made before isolation of the hearts. Plasma of the collected samples was separated using a centrifuge. Thereafter, plasma insulin levels were assayed using a rat-specific enzyme-linked immunosorbent assay (ELISA) kit (Cayman Chemical, Ann Arbor, Michigan). In addition, plasma levels of triglyceride (TG), total Chol, high-density lipoprotein (HDL), and low-density lipoprotein (LDL) were measured using appropriate ELISA kits (Pars Azmoon, Tehran, Iran), according to the manufacturer’s instructions.

Infarct Size Assessment

After 60 minutes of reperfusion, the LAD coronary artery was reoccluded and a 0.25% Evans blue dye was infused via the aortic root into the coronary system. This remains the area at risk (AAR) unstained, whereas nonischemic part of the myocardium becomes blue. Then the heart was frozen, cut into thin slices (2 mm) from the apex to the base, and incubated for 15 minutes in 1% 2,3,5-triphenyltetrazolium chloride (TTC) phosphate-buffered solution (pH 7.4) at 37°C to identify the viable myocardium as red stains, while infarct (necrotic) tissue remains pale gray. Thereafter, the slices were fixed in 10% formalin to enhance the contrast of the staining. The infarct volume within the risk zone of each slice was calculated using ImageJ software. Infarct size was expressed as a percentage of AAR.¹⁷

Preparation of Tissue Homogenates and Determination of Total Protein

The ischemic zone of the left ventricle was removed and immediately frozen in liquid nitrogen. The tissue was then weighted and cut into pieces in ice-cold lysis buffer containing (mM/mL) 1 KH₂PO₄, 1 KCl, 50 Tris–HCl pH 7.4, 1 EDTA, 1 NaF, 1 Na₃VO₄, and 1% Triton 100X and protease inhibitor cocktail and then homogenized with an ice-cooled Dounce glass tissue homogenizer (Tabriz, Iran). The homogenate was centrifuged at 10 000 RCF for 10 minutes at 4°C. The supernatants were removed from the homogenates and quickly frozen at −70°C until the analysis of cardiac content of troponin-I (cTnI) and total protein.¹⁷ Protein content of supernatants was detected spectrophotometrically using Bradford method.¹⁹

Determination of Myocardial Content of Troponin-I

To evaluate the changes in cTnI intracellular content, as a tissue injury marker in diabetic hearts,^20,21 the ELISA method and a special detection kit (Life Diagnostics, Koln, Germany) were used according to the manufacturer’s instructions. Supernatant of the sample was placed on the solid phase with an ascertain amount of antigen, and the antibody was added to react with the antigen. Between each step, plates were washed with detergent solution. Before the last step of washing, plates were incubated with the enzyme substrate. Finally, the absorbance was measured in 450 nm using the ELISA reader (Lab System, Vantaa, Finland), and the cTnI contents of the hearts were reported as ng/mg of sample proteins.

Measurement of Myocardial Levels of 8-Isoprostane and Cytokine Interleukin-6

Enzyme-linked immunoassay was used to measure myocardial free 8-isoprostane levels as a marker of lipid peroxidation according to the related kit and methods provided by the manufacturer (Cayman Chemical). Fifty milliliters of standards and samples were added in duplicate to the 96-well plate provided in the kit, followed by addition of 8-isoprostane acetylcholinesterase tracer and antibody. The prepared plates were then incubated overnight at room temperature. The next day, the plates were washed 5 times with the wash buffer, followed by addition of Ellman’s reagent. After optimal development, the plates were read at 405 nm, and the optical density values were transferred to the final concentration base on protein content of each sample and expressed as pg/mg of protein.

The concentrations of pro-inflammatory cytokine interleukin-6 (IL-6) in the hearts were measured using a rat-specific ELISA kit according to the kit instructions (eBioscience, Bender Medsystems, Vienna, Austria). The polyclonal antibody was added to all wells of a plate containing 50 mL standards or samples and their surface was covered and incubated at room temperature for 2 hours. After washing, streptavidin-horseradish peroxidase was added to all of the wells, and they were incubated at room temperature for an hour. Then, tetramethylbenzidine (TMB) substrate solution was added to all wells to form a colored product parallel to the amount of inflammatory cytokine in the sample. At the end, the stop solution was added into the each well to hinder the enzyme reaction and the relative absorbance was read on a spectrophotometer at 450 nm, and the final concentration was normalized and expressed as pg/mg of protein for each sample.

Statistical Analysis

All values were expressed as means ± standard error of the mean. For hemodynamic variables, the groups were compared at multiple time points using repeated analysis of variance (ANOVA) measurements, with LSD correction for multiple comparisons (mixed model) and comparison of the differences of other parameters between groups were analyzed through 1-way ANOVA followed by Tukey post hoc test utilizing SPSS v16. The differences were considered statistically significant when P < .05.

Results

Blood Glucose and OGTT, Plasma Insulin and Lipid Profiles, and HW to BW Ratio

At the end of the seventh week, impaired OGTT was observed in diabetic rats so that higher levels of blood glucose were seen in diabetic rats as compared with nondiabetics (P < .001 for all times; Figure 2). After 12 weeks of diabetes, the ANOVA showed that the diabetic rats had a significantly higher blood glucose levels, despite augmented insulin production, and higher HW and the ratio of HW to BW (P < .001, for all), TG, Chol, and LDL levels (P < .001, for all), as compared to those of the rats in the nondiabetic group (Table 1). Pretreatment of diabetic rats with Vilda significantly restored hyperglycemia (P < .001), decreased Chol, TG, and LDL levels (P < .01 to P < .001), increased insulin levels and HDL levels (P < .01), and decreased HWs and BWs (P < .01), as compared to the rats in diabetic group (Table 1).

Figure 2.

Oral glucose tolerance test (OGTT) in nondiabetic and diabetic rats. The data were expressed as mean ± standard error of the mean (SEM). n = 12 for each group. ***P < .001 as compared with nondiabetic group. Vilda indicates vildagliptin.

Table 1.

Metabolic Information of Experimental Groups.^a

Groups	Nondiabetic	Diabetic	Diabetic + Vilda
FBS, mg/dL	95 ± 3.22	514.80 ± 8.98^b	361.20 ± 4.62^b,c
Insulin, µU/mL	4.64 ± 0.27	7.33 ± 0.14^b	9.52 ± 0.20^b,d
BW, g	297.75 ± 1.67	302.92 ± 4.47	268.92 ± 4.51^d
HW, g	1.32 ± 0.03	1.63 ± 0.04^b	1.44 ± 0.04^d
HW/BW, %	0.44 ± 0.01	0.54 ± 0.00^b	0.53 ± 0.01
Chol, mg/dL	84.00 ± 3.34	443.66 ± 4.82^b	150.14 ± 2.19^b,c
TG, mg/dL	44.66 ± 3.25	231.00 ± 4.89^b	89.00 ± 1.69^b,c
HDL, mg/dL	36.66 ± 0.84	35.66 ± 1.16	49.57 ± 1.02^b,d
LDL, mg/dL	0.53 ± 0.08	35.40 ± 0.53^b	2.31 ± 0.14^c,e

Abbreviations: BW, body weight; Chol, cholesterol; FBS, fasting blood glucose; HDL, high-density lipoprotein; HW, heart weight; LDL, low-density lipoprotein; TG, triglyceride; Vilda, vildagliptin.

^aThe data were expressed as mean ± standard error of the mean (SEM). n = 12 per each group.

^bP < .001 as compared to nondiabetic group.

^cP < .001 as compared to diabetic group.

^dP < .01 as compared to diabetic group.

^eP < .01 as compared to nondiabetic group.

Hemodynamic Parameters

Baseline values

At the baseline (before ischemia), there were no significant differences in HR, CF, and LVEDP values between all experimental groups (Table 2). However, the LVDP and RPP values were significantly lower in diabetic rats rather than healthy ones (P < .001). In addition, preadministration of Vilda to diabetic rats significantly increased the LVDP and RPP levels, as compared to those of the diabetic controls (P < .05 to P < .01).

Table 2.

Baseline Values of Hemodynamic Parameters in Hearts of Experimental Groups.^a

Groups	HR, bpm	CF, mL/min	LVEDP, mm Hg	LVDP, mm Hg	RPP (1/1000), mm Hg × bpm
H-Sham	276.00 ± 3.87	12.66 ± 1.02	8.00 ± 0.36	97.83 ± 4.25	26.95 ± 1.04
H-Control	273.68 ± 4.30	13.00 ± 1.03	8.83 ± 0.40	92.15 ± 2.34	25.23 ± 0.79
D-Sham	265.50 ± 1.20	13.33 ± 0.79	8.33 ± 0.61	58.50 ± 3.85	15.54 ± 1.04
D-Control	268.83 ± 3.94	13.83 ± 0.75	9.17 ± 0.31	61.83 ± 2.66^b	16.62 ± 0.77^b
D-IPostC	264.50 ± 1.40	12.67 ±1.28	9.00 ± 0.51	58.33 ± 1.41	15.43 ± 0.37
D-Vilda	265.20 ± 0.86	13.20 ± 1.39	8.40 ± 0.51	77.20 ± 2.31^c	20.47 ± 0.87^c
D-Vilda-IPostC	264.00 ± 1.30	11.20 ± 1.16	8.20 ± 0.37	76.00 ± 2.46^c	19.34 ± 0.51^d

Abbreviations: bpm, beat per minute; CF, coronary flow; D, diabetic; H, healthy; HR, heart rate; IPostC, ischemic postconditioning; LVEDP, left ventricular end diastolic pressure; LVSP, LV systolic pressure; RPP: rate pressure product; Vilda, vildagliptin.

^aThe data was expressed as mean ± standard error of the mean (SEM). n = 6 for each groups.

^bP < .001 as compared to H-Control group.

^cP < .01 as compared to D-Control group.

^dP < .05 as compared to D-Control group.

Ischemia–reperfusion values

A 2-way ANOVA was performed to analyze the main influences of the time and group conditions and their interactive effects on the hemodynamic parameters in the setting of ischemia–reperfusion. According to the analysis outputs, the main effects of group conditions and time, as well as their interaction effects on all hemodynamic variables, were statistically significant (P < .01 to P < .001).

Heart Rates

The post hoc test showed that, at the ischemic phase, the HRs were lower in H-Control group versus H-Sham group (P < .01), in D-Control group versus H-Control group (P < .05 at 15 minutes of ischemia), and in D-Vilda and D-Vilda-IPostC groups (P < .01 and P < .001 at 15 and 30 minutes of ischemia, respectively) versus D-Control group. At reperfusion phase, the HRs increased compared to the corresponding values in the ischemic phase but were still lower than the baseline (Figure 3A). Heart rates in D-IPostC, D-Vilda, and D-Vilda-IPostC groups were not statistically different with that of D-Control group. The HRs in D-IPostC and D-Vilda groups were higher than those in the D-Vilda-IPostC group (P < .01 and P < .001, at different minutes of reperfusion).

Figure 3.

Cardiac function assessment in nondiabetic and diabetic hearts subjected to 35-minute ischemia and 60-minute reperfusion. A, Heart rate (HR; beat per minutes [bpm]). B, Coronary flow (CF, mL/min). C, Left ventricular end diastolic pressure (LVEDP, mm Hg). D, Left ventricular developed pressure (LVDP, percentage changes of baseline), E, Rate pressure product (RPP, percentage changes of baseline). Left ventricular pressures were monitored via an inserted left ventricular latex balloon, and RPP was calculated as HR × LVDP as a measure of cardiac contractility. Data are shown as mean ± standard error of the mean (SEM), n = 6. +P < .05, ++P < .01, and +++P < .001 as compared to D-Control group. ΨP < .05, ΨΨP < .01, and ΨΨΨP < .001 as compared to D-Vilda or D-IPostC group. D indicates diabetic; H, healthy; IPostC, ischemic postconditioning; Vilda, vildagliptin.

Coronary Flow

The CFs in ischemic phase were lower in all of those groups experiencing I/R injury, as compared to their baseline values and respective sham groups (Figure 3B). Reperfusion of ischemic hearts restored the CF in all groups, yet no significant difference was seen among D-Control, H-Control, D-Vilda, and D-Vilda-IPostC groups.

Left Ventricular End-Diastolic Pressure

Following the induction of ischemia, LVEDPs were increased in I/R hearts in relation to respective baselines (Figure 3C). The post hoc test showed that LVEDP in D-Vila-IPostC group was significantly lower than those of D-Control (P < .001 at the beginning and P < .05 at other times of reperfusion). The LVEDP levels in D-IPostC and D-Vilda groups (P < .01, for both) were significantly higher than those of D-Vila-IPostC group at the beginning of reperfusion.

Left Ventricular Developed Pressure

Figure 3D shows the alterations in LVDP during ischemia and reperfusion periods among all groups. Left ventricular developed pressure in D-Vilda-IPostC group was recovered to 92.00% of baseline value, compared to only 69.33% recovery in D-Control group (P < .05), 55.32% in D-IPostC group (P < .001), and 80.80% in D-Vilda group at 5 minutes of reperfusion phase (P < .05). Although the percentages of LVDP recovery throughout reperfusion in Vilda-treated groups were higher than that of D-Control group, these changes were not statistically significant (Figure 3D).

Rate Pressure Product

Changes in RPP, as an index of cardiac contractility, were expressed as percentage of baseline. Cardiac contractility was increased in Vilda-treated groups, as compared to D-Control group, at reperfusion phase (Figure 3E). The post hoc test showed that the RPP percentage was significantly increased in D-Vilda-IPostC group at any time points of reperfusion phase when compared to D-IPostC group (P < .05 to P < .01) and at 5 minutes of reperfusion when compared to D-control group (P < .05).

Infarct Size

The AAR assessment by Evan’s blue staining showed that AAR was approximately consistent among all of the hearts subjected to 35-minute regional ischemia and 60-minute reperfusion (Figure 4A). Infarct size assessments by TTC solution showed that there was no significant difference between D-Control group (38% ± 2%) and H-Control group (42% ± 2%; P = .76). The reduction in IS by IPostC alone (31% ± 3%; P = .19) or Vilda alone (28% ± 2%; P = .08) was not statistically significant compared to those of nontreated diabetic hearts. However, the combination of Vilda and postconditioning reduced the IS more effectively and significantly (approximately by 45%) in comparison to D-Control group (17% ± 5% vs 38% ± 2%; P < .001; Figure 4B).

Figure 4.

Infarct sizes (IS; A) and area at risk (AAR; B) percentages in nondiabetic and diabetic hearts subjected to 35-minute ischemia and 60-minute reperfusion. The data were expressed as mean ± standard error of the mean (SEM). n = 5 for each group. +++P < .001 as compared to D-Control group and ΨP < .05 as compared to D-IPostC group. D indicates diabetic; H, healthy; IPostC, ischemic postconditioning; Vilda, vildagliptin.

Cardiac Content of Troponin-I

The cTnI (intracellular) in AAR of myocardium was measured and expressed as ng/mg of total protein. The changes in cTnI content among different groups were statistically significant (P < .001; Figure 5). The post hoc test showed that cTnI content was significantly higher in the hearts of sham groups rather than control groups, both for nondiabetic and diabetic hearts (P < .01 for both). Cardiac content of troponin-I content in D-Control hearts was higher than H-Controls (P < .01). In the hearts of D-Vilda-IPostC group, the content of cTnI was higher than those of D-Control, D-IPostC, and D-Vilda groups (all P < .001). These results showed that the decrease in cTnI concentration of the tissue was associated with injury in D-Control group and that the increase in cTnI by the combination of Vilda with IPostC prevented diabetic hearts against tissue injury.

Figure 5.

Cardiac tissue (intracellular) troponin-I (cTnI) content (ng/mg of total protein) in nondiabetic and diabetic hearts subjected to 35-minute ischemia and 60-minute reperfusion. The data were expressed as mean ± standard error of the mean (SEM). n = 6 for each group.**P < .01 as compared to H-Sham group. ##P < .01 as compared to D-Sham group. $$P < .01 as compared to H-Control group. +++P < .001 as compared to D-Control group. ΨΨΨP < .001 as compared to D-Vilda and D-IPostC groups. D indicates diabetic; H, healthy; IPostC, ischemic postconditioning; Vilda, vildagliptin.

Myocardial 8-Isoprostane and IL-6 Levels

Induction of I/R injury in both diabetic and nondiabetic hearts significantly increased the levels of both 8-isoprostane and IL-6 as compared with corresponding sham groups (Figure 6A and B, respectively). The increment of 8-isoprostane but not IL-6 levels in diabetic control group was greater than that of healthy control group (P < .01). Application of IPostC could not reduce the concentrations of both parameters in comparison to diabetic control group. Vildagliptin alone significantly reduced only the levels of IL-6 compared to D-Control hearts (P < .05; Figure 6B). However, concomitant administration of both IPostC and Vilda to diabetic groups significantly and more potently reduced the myocardial levels of both 8-isoprostane (P < .05) and IL-6 (P < .001) as compared with D-Control hearts (Figure 6).

Figure 6.

Myocardial levels of 8-Isoprostane (8-Isop; A) and interleukin-6 (IL-6; B, in pg/mg of protein) in nondiabetic and diabetic hearts subjected to 35-minute ischemia and 60-minute reperfusion. The data were expressed as mean ± standard error of the mean (SEM). n = 5 for each group. *P < .05 and ***P < .001 as compared to H-Sham group. ##P < .01 as compared to D-Sham group. $$$P < .001 as compared to H-Control group. +++P < .001 as compared to D-Control group. D indicates diabetic; H, healthy; IPostC, ischemic postconditioning; Vilda, vildagliptin.

Discussion

This study showed that IPostC provided better cardioprotection against I/R injury in type 2 diabetic rats, only when the rats were pretreated with Vilda, a selective oral antidiabetic agent. Co-application of Vilda and IPostC could significantly reduce the inflammatory and oxidative markers in diabetic hearts, even though the effects of individual treatments were not significant. In addition, Vilda improved metabolic parameters and lipid profiles while attenuating the cardiac dysfunction induced by chronic diabetes.

In this study, high fat diet/streptozotocin (HFD/STZ) method in rats (as a reliable model to induce diabetes in experimental animals) induced dyslipidemia, hyperinsulinemia, and hyperglycemia and increased HW/BW ratio of rats. The hearts from diabetic rats showed lower baseline LVDP and RPP levels compared to those of the healthy rats. Hyperglycemia and hyperlipidemia lead to excess fat deposition, increased inflammatory mediators, and increased imbalance between oxidant and antioxidant mediators. These changes can ultimately lead to the development of myocardial lipotoxiciy, adverse cardiac remodeling, hypertrophy, and fibrosis, all of which contribute to the progression of diabetic cardiomyopathy and heart dysfunction.^22

-25 Therefore, according to the results, lower baseline cardiac function and increased HW/BW ratio in the diabetic rats can be attributed to the higher levels of blood glucose, 8-isoprostane, Chol, TG, and LDL and lower level of HDL in these rats.

Vildagliptin represents a new classes of antidiabetic drugs that inhibit the inactivation of GLP-1 and GIP by DPP-4, allowing them to potentiate the secretion of insulin in the pancreatic β cells. It has been showed that this drug can decrease glucose and glucagon levels in plasma⁶ and possess anti-inflammatory^7,8 and antioxidative effects.^9,10 In the present study, Vilda restored blood glucose level and lipid profile to mostly normal value, while reducing cardiac dysfunction in diabetic rats. This is consistent with a previous study showing that Vilda may have beneficial effects on lipid profiles and cardiac contractile function in diabetes participants.²⁶ In addition, it has improved left ventricular function and mitochondrial biogenesis^11,16 and decreased Chol and oxidative stress levels in obese rats.^27,28

Following myocardial I/R injury, diabetic hearts had lower recovery of cardiac function in comparison to nondiabetic ones. In addition, the hearts from diabetic rats developed the myocardial contractile dysfunction, as manifested by increased LVEDP and decreased LVDP and RPP, which were associated with decreased cardiac content of cTnI. Clinical studies and most animal experiments have indicated that diabetes tends to increase vulnerability of the heart to I/R injury and exaggerates the IS.^3,29,30 In contrary, our findings showed that, although the AAR of the hearts were similar for all groups, chronic diabetes could not significantly increase the IS. The effects of diabetes on myocardial I/R injury and IS in animal studies are controversial.^31
-33 These inconsistent results can be explained by the levels of metabolic changes following the induction of diabetes, duration of diabetes, plasma level of insulin, and the degree of I/R injury.^34,35 Moreover, it has been also reported that preceding diabetes, per se, may increase the resistance of the myocardium to I/R injury. This has been attributed to the possible preconditioning effects of diabetes, due to the production of many humoral mediators and factors during diabetic circumstances.^35
-37

A previous study showed a reduction in IS of obese rats after pretreatment with Vilda.¹¹ The researchers have concluded that the Vilda-induced protection of nondiabetic hearts against ischemia might be due to the decreased cardiac apoptosis and mitochondrial dysfunction. In the present study, compared to nontreated rats, diabetic rats pretreated with Vilda exhibited some lower myocardial injury as evaluated by the alterations in IS and cardiac hemodynamics. The IS in Vilda-treated group was decreased from that of nontreated diabetic hearts, but this difference was not statistically significant. On the other hand, the effect of combination therapy on myocardial IS, hemodynamics, and cTnI was more potent and significant. This indicates that Vilda has a cardioprotective potential, but its potency is more likely reduced during diabetic conditions. In other words, this finding shows that chronic diabetes most likely interferes with the cardioprotective effects of Vilda. This is in accordance with the previous reports, indicating that diabetes attenuates the cardioprotective power of agents that are protective in nondiabetic healthy hearts.^4,36,37 Interestingly, the similar alterations were seen somewhat in myocardial IL-6 and 8-isoprostane levels.

Similarly, IPostC also failed to significantly reduce the IS and failed to protect the heart from I/R injury in diabetic rats, indicating the ineffectiveness of this therapeutic strategy in diabetic circumstances. In this case, previous reports have also demonstrated that ischemic preconditioning as well as postconditioning cannot protect the diabetic hearts against I/R insults.^36
-38 However, our results showed that, provided that diabetic rats are pretreated with Vilda prior to myocardial I/R insult, application of IPostC can induce more potent cardioprotective effect and tends to significantly reduce the IS and recover the contractile response of I/R hearts in diabetic setting. In support of the findings of the combination therapy in the present research, some recent studies have shown successful cardioprotective effects of combined mechanical and pharmacological protective strategies in diabetic hearts.⁴ In our previous study, we showed that IPostC could provide cardioprotection against I/R injury in type 1 diabetic rats, not alone but in the presence of the inhibition of mitochondrial permeability transition pores (mPTP) by cyclosporine-A.⁴ It seems that the individual treatments are not powerful to overcome the diabetes-induced alterations in I/R, but, by increasing the potency of each treatments (eg, through co-application of them), the significant full cardioprotection is achieved (combination therapy restored more potently the loss of protection in diabetes). Therefore, the combination therapy would be considered as a promising approach to correct the diabetes-induced loss of cardioprotection.

The potent cardioprotective effects of the combined therapy can be achieved through different ways. For example, both IPostC and Vilda may decrease intracellular cytokines and reactive oxygen species generation via various mechanisms, trigger the opening of mitochondrial ATP-sensitive potassium channels, and/or prevent the opening of mPTP during early reperfusion.^4,37 Also, they can activate, either independently or synergistically, the cell survival mechanisms such as PI3K/Akt/GSK-3β and JAK/STAT pathways, by which their final cardioprotection is enhanced and the normal physiology of the hearts is somewhat restored in diabetes.^17,38 In the mechanistic insights, we showed that the cardioprotective influences of combination therapy were associated with the reducing effects of this treatment on IL-6 and 8-isoprostane levels. Although Vilda alone also tended to reduce pro-inflammatory cytokine significantly, the combination effect was more potent. Thus, the anti-inflammatory and antioxidative mechanisms can explain the positive combination effects in our study. However, further studies are needed to better explain the involvements of other contributors.

In conclusion, our findings indicated that IPostC per se or even Vilda per se could not provide significant cardioprotection against myocardial I/R injury in rats with chronic diabetes. However, this loss of cardioprotection can be restored by the combination of IPostC with Vilda. This combination effect was achieved somewhat by the potentiation of antioxidative and anti-inflammatory routes. Therefore, once the diabetic participants are pretreated with effective hypoglycemic drugs with potentials of preserving normal heart function, the risk of myocardial dysfunction is reduced and the efficiency of the application of cardioprotective postconditioning strategies during I/R injury is improved.

Footnotes

Author Contributions

Goltaj Bayrami contributed to conception and design; contributed to acquisition, analysis, and interpretation; drafted the manuscript; critically revised the manuscript; gave final approval; and agrees to be accountable for all aspects of work ensuring integrity and accuracy. Pouran Karimi and Saeid Feyzizadeh contributed to design; contributed to acquisition and analysis; drafted the manuscript; and gave final approval. Fariba Agha-Hosseini contributed to design; contributed to acquisition; and gave final approval. Reza Badalzadeh contributed to conception and design; contributed to acquisition and interpretation; drafted the manuscript; critically revised manuscript the manuscript; gave final approval; and agrees to be accountable for all aspects of work ensuring integrity and accuracy.

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by a grant (93.5-4.8) from Drug Applied Research Center, Tabriz University of Medical Sciences, Tabriz-Iran.

ORCID iD

Reza Badalzadeh, PhD

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