Apocynin influence on oxidative stress and cardiac remodeling of spontaneously hypertensive rats with diabetes mellitus

Seven-month-old male spontaneously hypertensive rats (SHR) were purchased from the Multidisciplinary Center for Biological Investigation in Laboratory Animals Science, State University of Campinas, SP, Brazil. 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. The rats were assigned into four groups: control SHR (n = 16); SHR treated with apocynin (SHR-APO, n = 16); diabetic SHR (SHR-DM, n = 18); and diabetic SHR treated with apocynin (SHR-DM-APO, n = 19).

Diabetes was induced by intraperitoneal injection of streptozotocin (Sigma, St. Louis, MO, USA) at 40 mg/kg body weight diluted in 0.01 M citrate buffer pH 4.5 [36]. Control groups received an intraperitoneal injection of vehicle only. As the rats received only one moderate dose of streptozotocin, sucrose administration was not necessary to avoid hypoglycemia caused by sudden release of insulin due to massive islet β-cells necrosis [37]. 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 [5, 36]. Apocynin (Sigma, St. Louis, MO, USA) was added to drinking water at a dosage of 16 mg/kg/day for 8 weeks [38]. Water consumption was measured daily and body weight weekly. Systolic arterial pressure was measured before streptozotocin injection and at the end of experiment by tail-cuff method using a model 709-0610 electro-sphygmomanometer (Narco Bio-System ^®, International Biomedical Inc., USA).

Echocardiographic evaluation was performed before diabetes induction and 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 [39‐41]. Rats were anesthetized by intramuscular 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. M-mode images were printed on a thermal printer (Sony UP-890MD) at a sweep speed of 100 mm/s. All LV structures were manually measured by the same observer (KO). Values obtained were the mean of at least five cardiac cycles on M-mode tracings. The following structural variables were measured: left atrium diameter (LA), LV diastolic diameter (LVDD), and LV diastolic and systolic posterior wall thickness (PWT and SWT, respectively). LV relative wall thickness (RWT) was calculated using the formula 2 × PWT/LVDD. LV mass was calculated using the formula [(LVDD + PWT + SWT)³ − (LVDD)³] × 1.04. LV function was assessed by the following parameters: midwall fractional shortening (MFS), tissue Doppler imaging (TDI) of mitral annulus systolic velocity (S’), myocardial performance index (Tei index), early and late diastolic mitral inflow velocities (E and A waves), E/A ratio, isovolumetric 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 [42].

At the end of the experimental period, myocardial contractile performance was evaluated in isolated LV papillary muscle preparation as previously described [43, 44]. 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 apices of their length-tension curves. After a 5 min period, during which preparations performed isotonic contractions, muscles were again placed under isometric conditions, and the apex 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 developed tension (DT), resting tension (RT), time to peak tension (TPT), maximum tension development rate (+dT/dt), and maximum tension decline rate (−dT/dt). To evaluate myocardial contractile reserve, papillary muscle mechanical performance was evaluated after the following positive inotropic stimulation: 30 s post-rest contraction, extracellular Ca²⁺ concentration increase to 2.5 mM, and β-adrenergic agonist isoproterenol (10⁻⁶ M) addition to the nutrient solution [45]. 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) [46]. In at least 50 myocytes from each LV, where the nucleus could clearly be identified, the smallest transverse diameters were measured [47]. On average, 20 microscopic fields were used to quantify interstitial collagen fraction. Perivascular collagen was excluded from this analysis [48]. 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).

Hydroxyproline concentration was measured in LV to estimate myocardial collagen content [49]. Tissue was dried using a Speedvac Concentrator (SC 100) attached to a refrigerated condensation trap (TRL 100) and vacuum pump (VP 100, Savant Instruments, Inc., Farmingdale, NY, USA). Dry tissue weight was measured and samples were hydrolyzed overnight at 100 °C with 6 N HCl (1 mL/10 mg dry tissue). An aliquot of the hydrolysate (50 μL) was transferred to an eppendorf tube and dried in the speedvac concentrator. One milliliter of deionized water was added and the sample transferred to a tube with a teflon screw cap. One milliliter of potassium borate buffer (pH 8.7) was added to maintain constant pH and the sample was oxidized with 0.3 mL of chloramine T solution at room temperature for 20 min. The addition of 1 mL of 3.6 M/L sodium thiosulfate with thorough mixing for 10 s stopped the oxidative process. The solution was saturated with 1.5 g KCl. The tubes were heated in boiling water for 20 min. After cooling to room temperature, the aqueous layer was extracted with 2.5 mL of toluene. One and a half milliliters of toluene extract were transferred to a 12 × 75 mm test tube. Then 0.6 mL of Ehrlich’s reagent was added and color allowed to develop for 30 min. Absorbance was read at 565 nm against a reagent blank. Deionized water and 20 μg/mL hydroxyproline were used as the blank and standard, respectively.

Protein expression of type I (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA, col1a1, sc-8784-r) and type III collagen (Abcam, Cambridge, UK, col3a1, ab6310) and lysil oxidase (Abcam, LOX1, ab60178) was analyzed by Western blot as previously described [50, 51]. Samples were separated on polyacrylamide gel and then transferred to a nitrocellulose membrane. After blockage, the membrane was incubated with the primary antibodies overnight at 4 °C. The membrane was then washed with PBS and Tween 20 and incubated with secondary peroxidase-conjugated antibodies (Santa Cuz Biotechnology, anti-mouse, sc-2005, and anti-rabbit, sc-2004) for 90 min at room temperature. ECL western blotting substrate (Pierce Protein Research Products, Rockford, USA) was used to detect bound antibodies. The membrane was then stripped (Restore Western Blot Stripping Buffer, Pierce Protein Research Products, Rockford, USA) to remove antibodies. After blockage, membrane was incubated with anti-GAPDH antibody (Santa Cruz Biotechnology, GAPDH 6C5 sc-32233). Protein levels were normalized to GAPDH.

Frozen left ventricle myocardium (∼200 mg) was homogenized in phosphate buffer (0.1 M) pH 7.4 and centrifuged at 12,000g for 15 min at 4 °C. The supernatant was assayed for total protein, lipid hydroperoxide, and anti-oxidant enzyme activity [52]. Lipid hydroperoxide concentration was determined in a medium containing methanol 90 % (v/v), 250 μM ammonium ferrous sulfate, 100 μM xylenol orange, 25 mM sulfuric acid, and 4 mM butylated hydroxytoluene. The solution was incubated for 30 min at room temperature and measurement was performed at 560 nm. Glutathione peroxidase (GSH-Px, E.C.1.11.1.9) was assayed 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 (GSH) in a total volume of 0.3 mL. Superoxide dismutase (SOD, E.C.1.15.1.1.) activity was determined based on its ability to inhibit reduction of nitroblue tetrazolium, in a medium containing 50 mM phosphate buffer pH 7.4, 0.1 mM EDTA, 50 μM nitroblue tetrazolium, 78 μM NADH, and 3.3 μM phenazine methosulfate. One unit of SOD was defined as the amount of protein needed to decrease the reference rate to 50 % of maximum inhibition. 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).

Paraffin-embedded myocardial tissue was cut, mounted on slides for immunohistochemical analysis, and deparaffinized with EDTA for antigen retrieval [53, 54]. Blockade was performed with hydrogen peroxide, goat serum, and avidin/biotin. Tissue sections were incubated with primary antibody (rabbit polyclonal antibody to AGE; Ab23722, Abcam) for 1 h, then with a secondary antibody (goat polyclonal secondary antibody to rabbit IgG HL—Biotin; Ab6720, Abcam) for 2 h, at room temperature, and for 15 min with streptavidin. Sections were then stained with 3,3′-diaminobenzidine and counterstained with hematoxylin [53]. Analysis was performed using a video camera coupled to a microscope connected to a computer with an image analyzes program (Image Pro Plus 6.0, Media Cybernetics, Silver Spring, Maryland, USA). Cardiac tissue components were identified according to color enhancement. AGEs yield a brown color while myocytes are seen in purple. Areas containing blood vessels were excluded from this analysis.

NADPH oxidase activity was evaluated in membrane-enriched cellular fraction by quantifying dihydroethidium (DHE) oxidation-derived fluorescent compounds, 2-hydroxyethidium (EOH) and ethidium, by HPLC according to a previously described method [55, 56]. Cardiac muscle was washed in PBS to remove blood. 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 then 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 supernatant was discarded and the pellet resuspended in 100 µL of lysis buffer [56]. 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, as previously described [55, 56].

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 two-factor ANOVA followed by the Tukey test for normal distribution data or the Dunn test for non-normal distribution data. All rats were subjected to functional studies as their parameters usually present an elevated variability. In papillary muscle preparations, some animals were discarded due to technical problems. For all morphological and molecular analyzes, samples were randomly chosen. Statistical significance was accepted at p < 0.05.