ABC | Volume 112, Nº2, February 2019

Original Article Badavi et al Trimetazidine effects on diabetic hearts Arq Bras Cardiol. 2019; 112(2):173-178 Methods Chemical Trimetazidine (TMZ), heparin and alloxan were obtained from Sigma Chemical Co. (St. Louis, MO, U.S.A.) and Ketamine and xylazine purchased from Alfasan Co (Woderen- Holland). Animal Twenty-four adult male Sprague-Dawley rats (250 ± 20 g) were housed under standard conditions (20 ± 5°C, 12-hour light/dark cycle, and free available to water and food) during the study period. All the experimental protocols followed the Consensus Author Guidelines on Animal Ethics and Welfare and the national guidelines for conducting animal studies (Ethics Committee permission No. APRC-94-25 Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran). 15 The sample size of each group was computed to be eight by the formula: 16 (Z 1– α /2 + Z 1– β ) 2 x (S 1 2 + S 2 2 ) d 2 (89–70) 2 n = = (1.96 x 1.29) 2 x (13.52 2 + 9.07 2 ) = 7.75~8 (Z 1– α /2 + Z 1– β ) 2 x (S 1 2 + S 2 2 ) d 2 (89–70) 2 = (1.96 x 1.29) 2 x (13.52 2 + 9.07 2 ) = 7.75~8 where S 1 2 and S 2 2 are means. The animals were randomly divided into three groups (n=8) by simple random sampling method. Control (C), diabetic (D) and diabetic administrated with TMZ at 10 mg/kg (T10). 17 TMZ was treated orally by gavage once daily for 8 weeks. Diabetic model Diabetes was induced by intraperitoneal administration of alloxan at 120 mg/kg. After 6 h, the animals were orally treated with 10% glucose solution (10 mL). They were further kept for 24h on 5% glucose solution to reduce fatal hypoglycemic resulted from alloxan. The rats, indicating fasting blood glucose ≥ 250 mg/dL, reduced body weight, dyslipidemia, increased hepatic enzymes and clear signs of polyuria, polyphagia and polydipsia after 4 days were regarded as diabetic animals and used for the experiment. 18 Electrocardiography The animals were anesthetized by heparin, ketamine and xylazine (1000 U/kg, 50, and 5 mg/kg, respectively), lead II was recorded by Bio Amp and controlled using a Power Lab system (AD Instruments, Australia). QT interval and heart rate were measured. Corrected QT interval (QTc) was calculated by Bazett formula normalized as QTc = QT/(RR/ f ) 1/2 , where RR is R–R interval and f = 150 ms. 19,20 Isolation of hearts After echocardiogram (ECG) recording, the cannulation and ventilation of trachea were performed using an animal ventilator (UGO BASILE, model: 7025). The cannulation of aorta was carried out by a central incision in the aorta. The hearts were conveyed to the Langendorff system. The perfusion of heart was carried out by Krebs-Henseleit solution (5% carbon dioxide and 95% oxygen, 37°C, pH = 7.4, 8 ml/min). A latex balloon was inserted in the left ventricle for the measurement of left ventricular pressure (LVP) by Power Lab system (AD Instruments, Australia). Left ventricular end diastolic pressure (LVEDP) was approximately regulated 5-10 mmHg by the alteration of balloon volume. Left ventricular systolic pressure (LVSP), Max dp/dt, and min dp/dt (±dp/dt max) were measure. 21 Left ventricular diastolic pressure (LVDP) and rate pressure product (RPP) were calculated by following formula: LVDP = LVSP – LVEDP RPP = LVDP x heart rate Measurement of hypertrophy After assessment of hemodynamic parameters using the Langendorff system, the hearts were removed and put in saline, then on a paper for assessment of the heart weight. Cardiac hypertrophy index (mg/g) was calculated from the total heart weight (mg) relative to total body weight (g) of the rat. 22 Measurement of antioxidant enzymes After measurement of hypertrophy, 100 mg of heart tissue was frozen in liquid nitrogen and stored at -70°C. The tissue samples were homogenized in phosphate buffered saline (PBS; 50 mM at pH of 7.4) using a Homogenizer (Heidolph Silenterosher M, Germany), and centrifuged at 14000 g for 15 minutes. The assessment of enzyme levels including glutathione peroxidase (GPx), catalase (CAT) and superoxide dismutase (SOD) was performed on supernatant. GPx and SOD were measured using Randox kits (Randox Lab, UK) and CAT activity was evaluated using Zellbio kit (Zellbio Lab, Ulm, Germany). Statistical analysis The results were indicated as mean and standard deviation (SD). In the present study, the normal distribution of the results was carried out by Kolmogorov-Smirnov analysis. One-way ANOVA and Least Significant Difference (LSD) test were used for comparison between the various groups. The comparison of pre and post metabolic in each group was performed by paired t-test using SPSS (version 16). A p < 0.05 was regarded statistically significant. Results Electrocardiographic parameters The QT and QTc intervals significantly increased in the diabetic animals in comparison with the control group (100 ± 13.80 vs. 70 ± 8.34, 82.52 ± 13.03 vs. 58.4 ± 7.33, p = 0.007 and p=0.009, respectively). TMZ treatment was associated with a significant reduction in the QT and QTc intervals in comparison with the untreated diabetic rats (80 ± 10.69 vs. 100 ± 13.80, 63.11 ± 7.05 vs. 82.52 ± 13.03, p = 0.043 and p = 0.040, respectively, Figure 1). As shown in 174