In this study, 56 male Wistar rats (2-month old) were provided from laboratory animal house of the Research Institute for Endocrine Sciences (RIES), Shahid Beheshti University of Medical Sciences. All animals were housed in an animal room with temperature and light controlled (23±1℃ 12/12-h dark-light cycle) conditions, and had free access to food and water at all times before initiation of surgical procedures. This study was carried out according to guidelines for the care and use of laboratory animals confirmed by the RIES ethics committee. For IR experiments, the control and hyperthyroid animals were randomly divided into 7 subgroups (n=8 each): Control (C), control-IR (C-IR), control-postconditioning (C-IPost), hyperthyroidism (HP), hyperthyroidism-IR (HP-IR), hyperthyroidism- postconditioning (HP-IPost), and hyperthyroidism-postconditioning-aminoguanidine (HP-IPost-AG). Hyperthyroidism in rats was induced by adding T₄ (Sigma; 12 mg/l) to the drinking water for a period of 21 days [17].

Serum total T₄ (thyroxine), T₃ (triiodothyronine), and TSH (thyroid stimulating hormone) levels in the control and hyperthyroid animals were determined at the end of treatment phase using commercial Elisa kits. Changes in the heart weight (HW) to body weight ratio (cardiac hypertrophy) during the treatment and citrate synthase (CS) activity (indicator for oxidative capacity) in the soleus muscle of rats were assessed for determining efficacy of treatment with T₄, the latter using the Srere procedure [18].

All animals were anesthetized with an intraperitoneal injection of combination of ketamine and xylazine (50 mg/kg and 10 mg/kg,). Perfusion experiments were carried out 48 h following the last T₄ dose. The hearts of control and hyperthyroid rats were rapidly excised and embedded in an ice-cold perfusion buffer; the aorta was then cannulated and the hearts were fixed on the Langendorff perfusion device and perfused through the aorta with a Krebs-Henseleit solution containing (mM/L): NaCl 118, NaHCO₃ 25, KCl 4.7, MgCl₂ 1.2, CaCl₂ 2.5, KH₂PO₄ 1.2, and glucose 11 at a constant pressure (75 mm Hg) and pH 7.4. The Krebs solution was gassed with a combination of 95% O₂ and 5% CO₂ at 37℃. All isolated hearts were stabilized for 20 minutes with the purpose of obtaining baseline data. After stabilization period of 20 min, in C-IR and HP-IR groups, hearts were subjected to global ischemia for 30 minutes followed by reperfusion for 120 minutes. The hearts from rats in the HP-IPost-AG group were perfused during the last 10 min of stabilization with 100 µmol/L of AG (Sigma-Aldrich), a selective iNOS-inhibitor [19]. IPost was induced by six cycles of 10 sec reperfusion and ischemia (6 cycles of 10s IR) immediately at the onset of reperfusion following 30 minutes of global ischemia. Left ventricular hemodynamical parameters were measured via a Latex balloon inserted in the left ventricle. The balloon capacity was adjusted to create 5~10 mm Hg of end diastolic pressure in all hearts by filling it with water. Hemodynamic parameters were digitized and recorded by a data acquisition system (power lab, AD instrument, Australia). Left ventricular end diastolic pressure (LVEDP), heart rate (HR), left ventricular developed pressure (LVDP), and the maximum rate of increase and decrease of left ventricular pressure (±dp/dt) were recorded. Postischemic recoveries were assessed by LVDP, LVEDP, and ±dp/dt and are represented as percent of the basal values.

Following 2-hour reperfusion, samples of left ventricle (LV) tissue were immediately frozen in liquid nitrogen and stored at -80℃. NO_x contents in serum and myocardium homogenates were determined using the Griess method [20]. Briefly, after homogenization of samples in PBS (phosphate-buffered saline) (1:5, w/v), the homogenates were centrifuged at 15,000 g for 20 min at 4℃. The supernatants were removed from the homogenates and were deproteinized by addition of zinc sulfate (15 mg/ml). Serum samples were also deproteinized using zinc sulfate (15 mg/ml) and centrifuged at 10,000 g for 10 min at 4℃. For both serum and tissue samples, a 100 µl of the supernatant was added to a microplate well and 100 µl vanadium (III) chloride (8 mg/ml) was added to each well (for reduction of nitrate to nitrite), followed by addition of 50 µl sulfanilamide (2%) and 50 µl NEDD (N-(1-naphtyl) ethylendiamine dihydrochloride) (0.1%). After 30 min incubation at 37℃, the absorbance was read at 540 nm using the ELISA reader (BioTek, Powerwave XS2). NO_x concentrations in serum and heart homogenates samples were measured from the linear standard curve established by 0-150 and 0-50 µmol/L of sodium nitrate, respectively. Heart and serum NO_x levels are presented as µmol/L. Intra-assay coefficient of variation was 5.2%.

At the end of the reperfusion period, infarct sizes (IS) were determined as described previously [21]. The frozen heart samples were cut into thin slices (2~3 mm) and were incubated for 10 min in 1% of 2, 3, 5-triphenyltetrazolium chloride (TTC) in phosphate buffer solution 20 mM/L, pH 7.4 at 37℃. The slices were immersed in 10% formalin for 24 h to identify viable myocardium as red stained, while necrotic (infracted) tissue remains unstained (pale gray). The sections were photographed using a digital camera version DV101 (Samsung, Japan). IS was measured by Photoshop CS6 software (version 13) and expressed as percentage of the total area.

All data are presented as means±SEM. LVDP, LVEDP, ±dp/dt, IS and NO_x values were evaluated by repeated measure ANOVA; a post-hoc Tukey test was applied using SPSS software for comparing means between groups, with p values <0.05 being considered significant.