Adult male Wistar normotensive rats (250-300 g) were used for all the experiments. The animals were randomly housed in appropriate cages (258 cm2 base and 17,8 cm high) at a controlled temperature (23 ± 2 °C) on a 12-h light/dark cycle (6:00 a.m. to 6:00 p.m.) with free access to food (Purina®, Sao Paulo, Brazil) and tap water. In addition, all efforts were made to minimize the number of animals used and any discomfort.
Nerolidol (NRD, ≥ 98% purity), (-)-isoproterenol (ISO), hexamethonium bromide (HEXA), atropine sulphate (ATR), indomethacin (INDO), nifedipine and NG-nitro-L-arginine-methyl-ester (L-NAME) were all purchased from Sigma-Aldrich™ (St. Louis, MO, USA). Sodium chloride (NaCl) from Neon™, Brazil; Tween 80 from Oxiteno™, Brazil; and ketamine chloride and xylazine from SESPO™, SP, Brazil.
Myocardial infarction (MI) protocol
MI was induced by two doses of (-)-isoproterenol (ISO) (100 mg/kg; i.p.) with a 24h-interval between each dose. After 30 min of the second dose of ISO, the animals were treated with NRD (50 or 100 mg/kg; i.p.). ISO was diluted in saline solution (NaCl 0.9%), and NRD in saline solution + tween 80 (0.2%). The animals were divided randomly into four groups as follows: CTR (vehicle), MI – ISO (100 mg/kg of isoproterenol), ISO + NRD (50 mg/kg of nerolidol), and ISO + NRD (100 mg/kg of nerolidol) (Fig. 2).
Acquisition of the Electrocardiographic profile
After MI induction (3º day), ECG was recorded in anesthetized animals (ketamine 80 mg/kg and xylazine 10 mg/kg; i.p.) by subdermal electrodes placed in the DII derivation (negative pole placed in the right and left superior thoracic region, and positive pole in the left inguinal region) connected to a cardioscope (TEB Electronics, São Paulo, Brazil). The electrical signals were amplified and digitalized (PowerLab 4/35 ADInstrument, EUA). The ECG signals were registered for 5 minutes and the LabChart 8 program (ADInstruments) was used for analysis. Measurements of heart rate (HR) corrected QT interval (QTc), QRS complex were used in all experimental groups. The QT interval was corrected normalized for rodents by HR using Bazett’s modified formula (QTc = QT / √RR /f). Any abnormalities in the ST segment morphology for MI were also analysed (Preda and Burlacu 2010).
Determination of myocardial infarct size
After MI was induced, the animals were anesthetized with ketamine (80 mg/kg; i.p.) and xylazine (10 mg/kg; i.p.), and decapitated. The hearts were quickly removed, and the myocardial infarct size was measured using the 2,3,5-triphenyl tetrazolium tetrachloride (TTC) staining method. All hearts were sectioned in a transversal fashion in the median region to obtain a better exposure of the left ventricle. The cardiac apex region was placed in a Falcon tube with TTC 1% diluted in Krebs−Henseleit solution (composition in mM: NaCl 120, KCl 5.4, MgCl2 1.2, NaHCO3 27, CaCl2 1.25, glucose 11, NaH2PO4 2.0, pH 7.4) for 15 min at a 37 ºC water bath. Then, after staining, the area was scanned in high resolution and analysed with ImageJ software (free software ImageJ bundled with 64-bit Java 1.8.0_112, NIH, Bethesda, MD, USA).
Measurement of biochemical markers of tissue damage - Lactate Dehydrogenase (LDH) and Creatine Kinase Total and Myocardial Band (CK-TOTAL and CK-MB)
After experiments, the animals were euthanatized and decapitated, and the blood was collected. For serum obtainment, the blood was centrifuged at 3.500 rpm (Neofuge 15R, Heal Force, Shanghai, China) for 15 min at 4 ºC. The enzymatic essays were performed in triplicate using Labtest commercial dosing kit. All following sample analysis were performed on the LABMAX 240 Premium apparatus.
Oxidative stress assays
Measurement of Lipoperoxidation by the Thiobarbituric Acid (TBARS) Method
Malondialdehyde (MDA) is a known stress oxidative biomarker, one of the oxidation products of hydroperoxides of polyunsaturated fatty acids that are formed during lipoperoxidation process (Amara et al. 1995).
To perform TBARS experiments, the hearts were weighted and homogenized at the ration of 100 mg of tissue/mL of phosphate buffer (PBS 0.1 mol/L, pH 7.4). Next, the homogenates were incubated for 45 min at 90 ºC with a solution containing thiobarbituric acid (TBA 0.37%) in acid solution (15% trichloroacetic acid and 0.25 N hydrochloric acid). Samples were centrifuged at 14.000 rpm (Neofuge 15R, Heal Force) for 30 min at 4 °C, and the supernatant was mixed with n-butanol and saturated NaCl solution. The mixture was mixed in a vortex for 30 s and again centrifuged at 14.000 rpm for 2 min. Aliquots of the supernatant were pipetted into 96-well plates to read absorbance on a microplate reader (Biotek, ELx800 absorbance microplate reader, VT, USA) at 535 nm, correcting for absorbance values at 572 nm. The amount of MDA was expressed in nanomoles per grams of tissue (nmol/g) and was interpreted as a marker of lipid peroxidation formed by the reaction with TBARS (Esterbauer and Cheeseman 1990).
Measurement of Measurement of Protein Carbonylation
To measure the possible oxidative damage to proteins carbonylation was used, which is the formation of carbonyl groups in proteins by the reaction with 2,4-dinitrophenylhydrazine (DNPH) with reactive ketones or aldehydes to form hydrazones, which are formed from protein oxidation. The total carbonyl group concentration was expressed as nmol/mg protein (Levine et al. 1990).
Cardiac tissues were weighted and homogenized. Initially, 2 microtubes were prepared with DNPH (experimental) and control (white), 1 mg of supernatant protein was added in both microtubes and completed to 200 μl with PBS. Next, 200 μl of trichloroacetic acid (TCA) 20% was added and homogenized for 5 min, after was centrifuged at 4.000 rpm for 5 min. The supernatant was discarded, and the pellet was resuspended in 100 μl NaOH 0,2 mol/L.
Next was added 100 μl of hydrochloric acid (HCl) 2 M in the white tube and 100 μl of DNPH 10 mM in the sample following 100 μl of TCA 20 %, the tubes were centrifuged at 16.000 rpm for 5 min in triplicate. Thereafter, was added 500 μl of ethanol/ethyl acetate (1:1). The pellet was resuspended with 1 mL of urea 8 M (pH 2.3), centrifuged again at 16.000 rpm for 3 min. Finally, 200 μl was pipetted into 96-well ELISA plate to read at an absorbance from 360 to 385 nm.
Endogenous Antioxidant Enzyme Activities
Superoxide Dismutase Activity (SOD)
SOD activity was measured as previously described (Madesh and Balasubramanian 1998). Cardiac tissue was homogenized in phosphate buffer (PBS, 50 mmol/L, pH 7.4) and centrifuged at 14.000 rpm for 30 min at 4 °C. The supernatant, PBS, tetrazolium (1.25 mmol), and adrenaline (60 mmol) were transferred to a microplate and shaken for 5 min. Thereafter, DMSO was added to the mixture, and read in a spectrophotometer (ELx 800, Biotek Instruments, VT, USA) was measured at 570 nm absorbance. SOD activity was expressed as U/μg protein.
Catalase Activity (CAT)
CAT activity was measured following a protocol previously described (Nelson and Kiesow 1972). The reaction was started with the addition of H2O2 (0.3 mol/L) to the supernatant samples, which were equalized in phosphate buffer (50 mmol/L, pH 7.0) and centrifuged at 12.000 rpm for 30 min at 4 °C. The test was performed in a quartz cuvette without illumination. The measurements were made in a spectrophotometer (ELx 800, Biotek Instruments), with a periodicity of 15 s at 25 °C and a wavelength of 240 nm. The reaction was quantified by measuring the consumption of hydrogen peroxide (H2O2, 0.3 mol/L). Decomposition of H2O2 by catalase was monitored for 1 min at 25 °C. The activity of the enzyme was expressed by the difference in absorbance variation (ΔE)/min/μg protein.
Data were expressed as mean ± standard error (SEM). Statistical comparisons were performed using GraphPad Prism 6.1 (San Diego, CA, USA). The normality and equality of variance were verified by the Shapiro−Wilk and Levene tests, respectively (Santana et al. 2018; Souza et al. 2019). For the statistical decision, one-way ANOVA was used, followed by the Tukey post hoc test or chi-squared test depending on the case. Values of probability of p < 0.05 were considered statistically significant (Supplemental data).