2.1 Animals
The research protocol was approved by the Animal Care and Ethical Use Committee of the Federal University of Rio Grande (P003/2017), RS, Brazil. Forty-two male Wistar rats (weighing 250 – 400 g) were obtained from the Central Animal House of the Federal University of Rio Grande do Sul, RS, Brazil. They were maintained on a 12h light / 12h dark cycle in constant temperature 22 ± 2 ° C and received commercial rodent food (25 g/animal/day) and water ad libitum.
2.2 Pharmacological induction of hypertension and treatments
Hypertension was induced by the administration of L-NAME (a non-specific nitric oxide inhibitor) (10 mg/kg/day) by gavage for eight weeks, except for the control group. The animals were divided randomly into seven groups (n = 6), during the last four weeks of treatment with L-NAME. The new fatty dihydropyridines and nifedipine were also administered by gavage. The groups were subdivided into control and group I/R that received a solution of the vehicle (DMSO 1%, 1 mL/400 g of weight) for 8 weeks; Group high blood pressure (HBP + I/R) received pretreatment with L-NAME (10 mg/kg/day); Group nifedipine and L-NAME (HBP + nifedipine + I/R) received at the same time pretreatment with L-NAME (10 mg/kg/day) and nifedipine (0.42 mg/kg/day). The remaining groups received pretreatment with a combination of L-NAME (10 mg/kg/day) and one of the new compounds: 2c (HBP + 2c + I/R), 8c (HBP + 8c + I/R), and 9a (HBP + 9a + I/R) all at a concentration of 0.42 mg/kg/day (same concentration used by Santa-Helena et al. [19]. The experiments with isolated hearts were performed 24h after the last administration of drugs using a retrograde perfusion system for all groups with exception of the control group, as described in 2.6.
2.3 Fatty 2,4-dihydropyridine synthesis
The synthesis of fatty 2,4-Dihydropyridines was realized according to previous work [18] (Figure 1). Initially the oleyl and stearic fatty β-ketoesters, were synthesized via transesterification of palmitic (C16:0) or stearic (C18:0) fatty alcohols, respectively. Next, the compounds were employed as fatty 1,3-dicarbonnyl compounds in the synthesis of fatty DHPs according to procedure: In a round bottom flask equipped with a reflux condenser were added, 2 mmol of oleyl or stearic fatty β-ketoesters, 1 mmol of respectively aromatic aldehydes (2-nitro-, 2-chloro or 4 chlorobenzaldehydes), 3mmol of ammonium acetate and 0.30 mmol of sulfamic acid as a catalyst in the presence of 5 mL methanol. The reaction mixture was stirred constantly at reflux for 24 h. Afterwards, the crude mixture was cooled to ambient temperature, concentrated under vacuum, and purified by column chromatography with gradient elution of hexane: ethyl acetate to afford the hybrid fatty DHP.
Di((Z)-octadec-9-en-1-yl)2,6-dimethyl-4-(2-nitrophenyl)-1,4-dihydropyridine-3,5-dicarboxylate (2c) Yield: 55%; M.W 818.6 gmol-1; oil; 1H NMR (400MHz, CDCl3): δ 0.90 (t, 6H, J= 6.6Hz); 1.28 (m, 44H); 1.55 (m, 4H); 2.03 (m, 8H); 2.34 (s, 6H); 4.01 (m, 4H); 5.36 (m, 4H); 5.72 (s, 1H); 5.84 (s, H); 7.23-7.74 (m,
4H); 13C NMR (100MHz, CDCl3): 14.1 (2C); 19.7 (2C); 22.7 (2C); 25.9 (2C); 27.2 (2C); 28.5-29.8 (20C);
31.9 (2C); 34.8; 64.3 (2C); 104.0 (2C); 124.0; 126.9; 129.8 (2C); 129.9 (2C); 131.3; 132.6; 142.5; 144.4 (2C);
147.9; 167.3 (2C). IR (film, νmax cm-1): 772, 1214, 1524, 1696, 2845, 2924, 3010, 3360.
Di((Z)-octadec-9-en-1-yl) 4-(2-chlorophenyl)-2,6-dimethyl-1,4-dihydropyridine-3,5-dicarboxylate (8c) Yield: 71%; M.W 807.6gmol-1; oil; 1H NMR (400MHz, CDCl3): δ 0.90 (t, 6H, J= 6.5Hz); 1.29 (m, 44H); 1.60 (m, 4H); 2.04 (m, 8H); 2.33 (s, 6H); 4.04 (t, 4H, J=6.75Hz); 5.37 (m, 4H); 5.40 (s, 1H); 5.66 (s, H); 7.03-7.41(m, 4H); 13C NMR (100MHz, CDCl3): 14.1 (2C); 19.6 (2C); 22.7 (2C); 26.0 (2C); 27.2-29.8 (20C); 31.9(2C); 32.6 (2C); 37.6; 64.1 (2C); 103.9 (2C) ; 126.6; 127.2; 129.4; 129.8 (2C); 129.9 (2C); 131.5; 132.6; 143.8 (2C); 145.5; 167.7 (2C); IV (film, νmax cm-1): 796, 1115, 1465, 1676, 2845, 2918, 3003, 3333.
Dihexadecyl4-(4-chlorophenyl)-2,6-dimethyl-1,4-dihydropyridine-3,5-dicarboxylate (9a) Yield: 89%; M.W 755.6gmol-1; M.P 57-58ºC; solid; 1H NMR (400MHz, CDCl3): δ 0.90 (t, 6H, J= 6.7Hz); 1.28 (m, 52H); 1.60 (m, 4H); 2.35 (s, 6H); 4.04 (m, 4H); 4.98 (s, 1H); 5.67 (s, H); 7.17-7.24 (m, 4H); 13C NMR (100MHz,
CDCl3): 14.1 (2C); 19.6 (2C); 22.7 (2C); 26.0 (2C); 28.7-29.7 (22C); 31.9 (2C); 39.2; 64.1 (2C); 103.9 (2C);
127.9 (2C); 129.4 (2C); 131.7; 144.0 (2C); 146.2; 167.5 (2C); IR (film, νmax cm-1): 745, 1267, 1465, 1696,
2851, 2918, 3069, 3340.
2.4 Blood pressure measurement
Blood pressure was verified by tail-cuff plethysmography using a Non-Invasive Blood Pressure System (LE5001, Panlab Harvard Apparatus). The rats were placed in a restrainer of the appropriate size to balance for a few minutes before checking systolic and diastolic blood pressure, and heart rate. Each animal was adapted to blood pressure and heart rate measurements for three weeks before the start of the experiment. These parameters were measured for each animal before the start of treatment and weekly thereafter. Three consecutive checks were performed, and the averages were calculated for each parameter.
2.5 Ischemia and reperfusion protocol
The animals were heparinized (1000 IU) intraperitoneally and after 10 minutes euthanized. After thoracotomy, the inferior cava vena was sectioned to minimize venous return, and the pulmonary veins and aorta were then cut, and the heart was removed and placed in a cold solution (Krebs-Henseleit). Shortly after cannulation (18G cannula), the heart was perfused with cooled Krebs-Henseleit with the constant pressure of 90 mmHg at 37 ± 1 °C. The heart-nourishing solution was prepared following the concentrations proposed by [60]. Thus producing non-recirculating Krebs-Henseleit solution with pH 7.4 (120 mM NaCl, 5.9 mM KCl, 1.2 mM MgSO4, 1.75 mM CaCl2, 25 NaHCO3, and 11 mM glucose) was filtered at 0.25 µm and posteriorly saturated with carbogen (95% oxygen and 5% carbon dioxide).
After cannulation, each heart underwent the same procedure, except for the control group. The hearts were stabilized for 10 minutes. After the baseline time (10 minutes), the global ischemia was induced for 20 minutes and the reperfusion time was reestablished for 40 minutes (Figure 2). During the experiments, 2 mL of perfusate were collected at 15 and 80 minutes, in addition to photographing the heart every 10 minutes.
2.6 Measurement of cardiac parameters
Pressure apparatus mounting
This procedure was performed according to Jia et al. [26], which consists in the measurement and monitoring of left ventricular developed pressure (LVDP) through a latex balloon containing ultrapure water (pressure of 10 mmHg) and connected to a pressure transducer and an amplifier and data collected in real-time through software (ANCAD).
2.7 Differential pressure (± dP/dT)
To calculate the first derivative of ventricular pressure over time, ±dP/dT was obtained following the calculations of Migliore [24], which considers the rate of change of inclination of a signal over a period , associated with the method of Sarazan et al. [25] for the up-to-date integration of data collection software for converting the signal into mathematical data.
2.8 Mean arterial pressure (MAP)
Digitized systolic pressure (SP) and diastolic pressure (DP) data were used with the following calculation MAP = DP + 0.333(∆P), with ∆P = DP-SP and 0.333 is 1/3 of the cardiac cycle, assuming that systole occupies 2/3 according to Razminia et al. [26].
2.9 Caliber image in situ evaluation
The left anterior descending coronary artery was constantly monitored in situ using a portable digital microscope (GL1600x), placed next to the heart, with a focus plane on the artery that nourishes the interventricular septum and the cardiac apex. With ambient light, 40-50X lens, 1024x768 pixel image, and 30,000 frame rates were obtained intact and functioning heart surface images by adapting the method of Chen et al. [27]. Images were recorded every 10 minutes and then analyzed using Image J software.
2.10 Biochemical Analyses
Total antioxidant capacity against peroxyl radical (ACAP)
After experimental procedures with the isolated heart were completed, the heart was homogenized and centrifuged (10,000 x g) at 4 °C for 20 minutes. Aliquots of 15 µL of each sample (three replicates) were placed into a plate with 120 µL of buffer (Hepes, KCl, MgCl2), 10 μl of ABAP ([2,2’-Azobis (2 methylpropionamidine) dihydrochloride]), and 20 μl of H2DCF-DA (2',7'-dichlorodihydrofluorescein diacetate). The fluorescence intensity was determined over 60 minutes at 37 °C using a fluorometer (FilterMax F5, molecular device) at excitation and emission wavelengths of 450 and 535 nm, respectively. The antioxidant capacity was expressed in terms of the fluorescence area, after fitting fluorescence data to a second order polynomial and integrating between 0 and 60 min to obtain its area, being the inverse of the area difference in fluorescence with and without ABAP [28].
2.11 Lipid peroxidation (LPO)
Thiobarbituric acid reactive substances (TBARS) were measured through malondialdehyde concentration in samples by measuring fluorescence at 520 and 595 nm for excitation and emission wavelengths, respectively. TBARS levels were expressed as malondialdehyde/mg of tissue, using tetramethoxypropane (TMP, Acros Organics) as a standard [29].
2.12 Catalase Activity
Catalase activity occurred by analyzing the decomposition of H2O2 as a function of time. The reaction was performed in triplicate adding H2O2 (0.3 M), 2 mL potassium phosphate buffer (KPB, 50 mM) in pH 7.0 at 25 °C, according to Nelson and Kiesow [30]. During 1 min, the decomposition of H2O2 by catalase was evaluated at 240 nm. Catalase activity is expressed as Δ Abs/min/milligram of protein.
2.13 Lactate dehydrogenase (LDH) activity
LDH activity was measured in heart perfusate samples collected from the coronary, during the I/R protocol according to the manufacturer's recommendations (LDH Liquiform – Labtest, Brazil). This method was performed using a spectrophotometer (800 ELX Universal Microplate Reader, Bio-TEK) and evaluated the decreasing absorption of NADH at 340 nm for 3 min at 37 °C.
2.14 Statistical analysis
Data were quantified as the mean and standard error of mean (SEM) and statistical methods to assess the degree of significance were utilized [31]. For cardiac contractility and arterial caliber data (HR, LVDP, ± dP/dT, MAP and vasoconstriction) two-way ANOVA analysis of variance followed by the Newmann Kells posttest was performed. In the biochemical tests, one-way ANOVA followed by the Newmann Kells posttest was completed. The level of statistical significance was set at 95%.