Influence of apocynin on cardiac remodeling in rats with streptozotocin-induced diabetes mellitus

Six-month-old male Wistar rats were divided into four groups: control (CTL, n = 15); control treated with apocynin (CTL + APO, n = 20); diabetes (DM, n = 20); and diabetes treated with apocynin (DM + APO, n = 20). All animals were housed in a room under temperature control at 23 °C and kept on a 12-h light/dark cycle. Commercial chow and water were supplied ad libitum.

Diabetes was induced by intraperitoneal injection of streptozotocin (Sigma, St. Louis, MO, USA) at 50 mg/kg body weight diluted in 0.01 M citrate buffer pH 4.5 [26, 27]. Control groups received an intraperitoneal injection of vehicle only. Seven days after streptozotocin administration, blood glucose was measured by glucometer (Advantage®). Only rats with glycemia > 220 mg/dL were considered diabetic and included in the study [28]. Apocynin (Sigma, St. Louis, MO, USA) was added to drinking water at a dosage of 16 mg/kg/day for 8 weeks [20, 29]. Systolic blood pressure was measured at the end of experiment by the tail-cuff method using an electro-sphygmomanometer (Narco Bio-System^®, International Biomedical Inc., USA) [30]. All rats underwent environment adaption before tail-cuff measurements.

Echocardiographic evaluation was performed at the end of the experimental period. We used a commercially available echocardiograph (General Electric Medical Systems, Vivid S6, Tirat Carmel, Israel) equipped with a 5–11.5 MHz multifrequency probe, as previously described [31‐34]. Rats were anesthetized by intraperitoneal injection of a mixture of ketamine (50 mg/kg) and xylazine (0.5 mg/kg). Two-dimensionally guided M-mode images were obtained from short-axis views of the left ventricle (LV) just below the tip of the mitral-valve leaflets, and at the level of the aortic valve and left atrium. All LV structures were measured by the same observer (KO) and according to the leading-edge method of the American Society of Echocardiography [35]. Values obtained were the mean of at least five cardiac cycles on M-mode tracings. The following structural variables were measured: left atrial diameter (LA), LV diastolic (LVDD) and systolic (LVSD) diameter, and LV diastolic posterior wall thickness (PWT). LV mass was calculated using the formula [(LVDD + PWT + SWT)³ − (LVDD)³] × 1.04. LV function was assessed by the following parameters: endocardial fractional shortening (EFS), ejection fraction (EF), posterior wall shortening velocity (PWSV), tissue Doppler imaging (TDI) of mitral annulus systolic velocity (S′), early and late diastolic mitral inflow velocities (E and A waves), E/A ratio, isovolumic relaxation time (IVRT), TDI of early mitral annulus diastolic velocity (E′), and E/E′ ratio. Tissue Doppler variables (S′ and E′) were measured at the septal and lateral walls, and average values presented [36, 37].

At the end of the experimental period, myocardial contractile performance was evaluated in isolated LV papillary muscle preparation as previously described [38‐40]. Rats were anesthetized (pentobarbital sodium, 50 mg/kg, intraperitoneally) and decapitated. Hearts were quickly removed and placed in oxygenated Krebs–Henseleit solution at 28 °C. LV anterior or posterior papillary muscle was dissected free, mounted between two spring clips, and placed vertically in a chamber containing Krebs–Henseleit solution at 28 °C and oxygenated with a mixture of 95% O₂ and 5% CO₂ (pH 7.38). The composition of the Krebs–Henseleit solution in mM was as follows: 118.5 NaCl, 4.69 KCl, 1.25 CaCl₂, 1.16 MgSO₄, 1.18 KH₂PO₄, 5.50 glucose, and 25.88 NaHCO₃. The spring clips were attached to a Kyowa model 120T-20B force transducer and a lever system which allowed for muscle length adjustment. Preparations were stimulated 12 times/min at a voltage 10% above threshold.

After a 60 min period, during which the preparations were permitted to shorten while carrying light loads, muscles were loaded to contract isometrically and stretched to the point where active tension development reached its maximum. After a 5 min period, during which preparations performed isotonic contractions, muscles were again placed under isometric conditions, and the peak of the length-tension curve (L_max) was determined. A 15 min period of stable isometric contraction was imposed prior to the experimental period. One isometric contraction was then recorded for later analysis.

The following parameters were measured from the isometric contraction: peak of developed tension (DT), resting tension (RT), time to peak tension (TPT), maximum rate of tension development (+ dT/dt), and maximum rate of tension decline (− dT/dt). To evaluate myocardial contractile reserve, papillary muscle mechanical performance was evaluated after the following positive inotropic stimuli: 30 s post-rest contraction, extracellular Ca²⁺ concentration increase to 2.5 mM, and β-adrenergic agonist isoproterenol (10⁻⁶ M) added to the nutrient solution [41, 42]. Papillary muscle cross-sectional area (CSA) was calculated from muscle weight and length by assuming cylindrical uniformity and a specific gravity of 1.0. All force data were normalized for muscle CSA.

Transverse LV sections were fixed in 10% buffered formalin and embedded in paraffin. Sections (5 µm thick) were stained with hematoxylin–eosin and collagen-specific stain picrosirius red (Sirius red F3BA in aqueous saturated picric acid) [43]. The smallest transverse diameters were measured in at least 50 myocytes from each LV, where the nucleus could be clearly identified [44]. On average, 20 microscopic fields were used to quantify interstitial collagen fraction. Perivascular collagen was excluded from this analysis [45]. Measurements were performed using a Leica microscope (magnification 40×) attached to a video camera and connected to a computer equipped with image analysis software (Image-Pro Plus 3.0, Media Cybernetics, Silver Spring, MD, USA).

After anesthesia and before heart removal from the chest, rats were decapitated and blood collected. Plasma was separated by centrifugation and used for quantification of glucose, glutathione peroxidase (GSH-Px), superoxide dismutase (SOD), and catalase.

Glutathione peroxidase (GSH-Px, E.C.1.11.1.9) activity was assayed in 15 μL of serum using 0.15 M phosphate buffer, pH 7.0, containing 5 mM EDTA, 0.1 mL of 0.0084 M NADPH, 4 μg of GSSG-reductase, 1.125 M sodium azide, and 0.15 M glutathione reduced form in a total volume of 0.3 mL [32]. Superoxide dismutase (SOD, E.C.1.15.1.1.) activity was determined in serum aliquots of 50 μL using its ability to inhibit reduction of nitroblue tetrazolium, in medium containing 50 mM phosphate buffer pH 7.4, 0.1 mM EDTA, 50 μM NBT, 78 μM NADH, and 3.3 μM phenazine methosulfate. One unit of superoxide dismutase was defined as the amount of protein needed to decrease the reference rate to 50% of maximum inhibition [28]. Catalase (E.C.1.11.1.6.) activity was evaluated in 50 mM phosphate buffer, pH 7.0, with 10 mM hydrogen peroxide. One unit of catalase was defined as the amount of enzyme needed to degrade 1 μmol H₂O₂ over 60 s, at 240 nm. Enzyme activity was analyzed at 25 °C using a microplate reader system (μQuant-MQX 200 with KC Junior software, Bio-TeK Instruments, Winooski, VT, USA). All reagents were purchased from Sigma (St. Louis, MO, USA).

NADPH oxidase activity was evaluated in membrane-enriched cellular fraction by quantifying two dihydroethidium (DHE) oxidation-derived fluorescent compounds, 2-hydroxyethidium (EOH) and ethidium, by HPLC according to a previously described method [46, 47]. After washing cardiac tissue in phosphate buffered saline, muscle fragments (∼ 200 mg) were homogenized in 1 mL of ice-cold lysis buffer containing 50 mM Tris (pH 7.4), 100 mM DTPA, 0.1% β-mercaptoethanol, and protease inhibitors. The samples were sonicated (3 cycles of 10 s at 8 W) and centrifuged at 1000g for 3 min, at 4 °C. The supernatant was transferred to another microtube and centrifuged at 18,000g for 10 min at 4 °C. The supernatant was then centrifuged at 100,000g for 45 min, at 4 °C. The pellet was resuspended in 100 µL of lysis buffer. Total protein content was quantified by the Bradford method. Subsequently, 20 µg of membrane-enriched cellular fraction was incubated in phosphate buffer (50 mM, pH 7.4, with 0.1 mM DTPA) containing DHE (50 µM) and NADPH (300 µM) to a final volume of 100 µL, for 30 min at 37 °C in the dark. After adding 40 µL of 10% trichloroacetic acid, the samples were ice-cooled for 10 min in the dark, and centrifuged at 12,000g for 10 min at 4 °C. The supernatant was analyzed by HPLC and the fluorescent DHE-derived products were quantified.

Results are expressed as mean and standard deviation or median and 25th and 75th percentiles according to normal or non-normal distribution, respectively. Variables were compared by analysis of variance (ANOVA) for a 2 × 2 factorial design followed by the Bonferroni post hoc test. Comparisons of interest: CTL + APO vs CTL, DM vs CTL, and DM + APO vs both CTL + APO and DM. Statistical significance was accepted at p < 0.05.