Fatty Dihydropyridines With Anti-Hypertensive and Cardioprotective Potential During Ischemia and Reperfusion

In this study, three fatty dihydropyridines, were tested for their anti-hypertensive and cardioprotective properties. Dihydropyridines 2c, 8c, and 9a contain the oleic chain and the nitro unit, the oleic chain and the chlorine unit, and the palmitic chain and the chlorine unit, respectively. For the experiments, animals were treated with N(ω)-nitro-L-arginine methyl ester to induce hypertension and after treated with the new dihydropyridine compounds and the standard drug nifedipine. Then, the heart was removed and subjected to global ischemia and reperfusion. Analyses of cardiac parameters were performed to monitor cardiac functionality; lactate dehydrogenase values were quantied in perfusates. After ischemia and reperfusion were performed, analyses to check the oxidative status and lipid damage. The results of the present study suggest that the new fatty DHPs have anti-hypertensive effects offering protection against ischemia to the heart of rats, accomplished by increasing antioxidants that defend and prevent the decline in cardiac function.

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.

Pharmacological induction of hypertension and treatments
Hypertension was induced by the administration of L-NAME (a non-speci c 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.

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 transesteri cation of palmitic (C16: 0) or stearic (C18:0) fatty alcohols, respectively. Next, the compounds were employed as fatty 1,3dicarbonnyl compounds in the synthesis of fatty DHPs according to procedure: In a round bottom ask equipped with a re ux 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 re ux for 24 h. Afterwards, the crude mixture was cooled to ambient temperature, concentrated under vacuum, and puri ed by column chromatography with gradient elution of hexane: ethyl acetate to afford the hybrid fatty DHP.

Blood pressure measurement
Blood pressure was veri ed 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.

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 ltered 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.

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 ampli er and data collected in real-time through software (ANCAD).

Differential pressure (± dP/dT)
To calculate the rst 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.

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].

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.
The antioxidant capacity was expressed in terms of the uorescence area, after tting uorescence 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 uorescence with and without ABAP [28].

Lipid peroxidation (LPO)
Thiobarbituric acid reactive substances (TBARS) were measured through malondialdehyde concentration in samples by measuring uorescence 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].  [30]. During 1 min, the decomposition of H 2 O 2 by catalase was evaluated at 240 nm. Catalase activity is expressed as Δ Abs/min/milligram of protein.

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.

Statistical analysis
Data were quanti ed as the mean and standard error of mean (SEM) and statistical methods to assess the degree of signi cance 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 signi cance was set at 95%.

Results
In the rst phase of our investigation, the focus was placed on the understanding of the effects pharmacological preconditioning of new fatty dihydropyridines and its cardioprotection in I/R concerning cardiac dynamic parameters, which are indicators of heart function. During the second phase of our research was approached the effects of preconditioning with new drugs on antioxidant capacity and lipid damage, oxidative stress markers, after an I/R event.

Result of cardiac parameters
The induction of high arterial pressure in animals was performed with the use of L-NAME (10 mg/kg/day). In Figure 3A, a signi cant increase in systolic blood pressure (occurred on the 14 th day) and an increase of diastolic blood pressure were veri ed on the 21 st day ( Figure 3B). When comparing the groups that received LNAME (all HBP) with the groups that received saline solution (Control and I/R) (p <0.05), we can verify that the treatment to induce hypertension was effective ( Figure 3). On the 29 th day, treatment was started with new fatty dihydropyridines and nifedipine simultaneously. From the 35 th day of treatment with DHPs, compounds 2c, 8c, 9a, and NIF reduced the systolic blood pressure when compared with the HBP + I/R group (p < 0.05). The diastolic blood pressure shows a reduction with treatments of 9a, NIF (day 35) and 2c (day 49) in relation to the HBP group + I/R (p < 0.05). These results indicate that the new DHPs, 2c and 9a, were able to decrease the blood pressure values, both diastolic and systolic, and the new DHP, 8c, reduce the values of systolic pressure only ( Figure 3). These ndings suggest that the effects of these new compounds seem to have maintained the characteristic blocking of calcium channels.
The heart rate during treatment is shown in gure 4. Throughout the experiment (56 days) the heart rate was veri ed weekly ( gure 4). From day 21 L-NAME treatment presents the signi cant increase (p <0.05) in the treated groups in relation to the control and I / R group.
Hemodynamic parameters were evaluated to verify the protection of new fatty DHPs. Heart rate was evaluated in isolated hearts of rats submitted to ischemia and reperfusion and BPM values were determined. All treatments show lower BPM values ( Figure 5, p < 0.05) before ischemia concerning the HBP + I/R group. During ischemia, BPM values were decreased signi cantly for all groups, except for the control group that maintained consistent values. In reperfusion, after 60 minutes, all treatments normalize BPM and show a difference from the HBP + I/R group (p < 0.05).
When left ventricle developed pressure (LVDP) was analyzed (Figure 6), pretreatment with compounds signi cantly improved LVDP values during reperfusion, while the untreated groups present lower values. At 60 minutes after ischemia, the groups HBP + 2c + I/R, and HBP + NIF + I/R are not statistically different from the control group (p > 0.05), and the groups HBP + 2c + I/R and HBP + NIF + I/R are different (p < 0.05) from the HBP + I/R group ( Figure 5).
Evaluating the left ventricular contraction speed, only treatments with compounds 8c and NIF can normalize left ventricular contraction time at the end of reperfusion (Figure 7).
When we analyze the differential pressure, meaning the ventricular contraction speed (± dP/dT), at the end of I/R, after 80 minutes of the experiment, the group HBP + 8c + I/R and HBP + NIF + I/R differ from the HBP + I/R group (p < 0.05). All other groups are different from the control (p < 0.05) at all times of reperfusion (Figure 7). Only treatments with compounds 8c and NIF can normalize the left ventricular contraction time at the end of reperfusion.
Mean arterial pressure values for all treatments (2c, 8c, 9a and NIF) are not different from the control group (p > 0.05) and they are different (p < 0.05) from the HBP + I/R group in the time 60 minutes (reperfusion) (Figure 8).
The only treatment that allowed an increase in vasodilation during the reperfusion period was 2c when compared to the HBP + I/R group (p < 0.05) (Figure 9).

Biochemical Analyses
Biochemical data demonstrate that treatment with compound 2c (HBP + 2c + I/R) showed a decrease in LDH values concerning groups I/R and HBP + I/R (p < 0.05) (Figure 10), suggesting a cardioprotective effect of this compound.
The analysis of antioxidant capacity showed that the ischemia reperfusion protocol decreased the values of the total antioxidant capacity ( Figure 11) and that the treatments with compounds 2c, 8c, 9a, and NIF, showing none differences in relation to group HBP + I/R (p > 0.05) also show no differences in relation to the control group ( p > 0.05), except for 8C group that was lower.
The lipid peroxidation (LPO) values ( Figure 13) demonstrate that treatment with compounds 2c and 9a seems to decrease lipid damage, presenting results not different (p > 0.05) to the control group, but also not different from the group HBP + I/R (p > 0.05).

Discussion
The study of the isolated heart is used in several types of tests being extremely useful in the translational evaluation of these pharmacological tests [32,33]. According to Tong et al. [34], heart retrograde perfusion models are ex vivo methods of clinical relevance for the study of myocardial infarction to evaluate function and injury.
The ability of DHPs to block calcium channels is linked to the heterocyclic ring [35,36] and this structure has not been altered in the new compounds studied here, which strengthens our suggestion that these compounds continue to bind and inhibit calcium channels. The fact that DHPs prevent the increase of BPM when compared to the HBP + I/R (baseline) ( Figure 5) group may indicate a bene t of this treatment. Undoubtedly, the restoration of normal blood ow to the ischemic heart leads to a lower mortality rate of ischemic patients [7,37], however, reperfusion alone can cause a sequence of harmful events, including arrhythmias (particularly), microvascular injury, myocardial dysfunction, myocardial stunning, and nally death [38], showing once again the importance of establishing reperfusion without increasing BPM.
These results are in accordance with an experimental study in isolated hearts [39], which demonstrated that previous treatment with nifedipine induces preconditioning, as shown by the improvement in left ventricular pressure after ischemia. These ndings support our results, considering that NIF and 2c present values of PDVE, after 60 minutes, not different from the control group (p > 0.05). In addition, compounds 8c and 9a are different from group HBP + I/R (p < 0.05), which already suggests an improvement in LVDP after ischemia. The decrease in left ventricular pressure values demonstrates a clear impairment in left ventricular function, caused by pressure overload con rming this increase in blood pressure values in untreated groups ( Figure 6).
The reduction in LVDP and ±dP/dT can be used as indicative of I/R injury [40], the lowest values of LVDP were observed in the groups I/R and HBP + I/R. These results suggest that all compounds demonstrate the ability to reduce I/R injury. Calcium channel antagonists, which can reduce the calcium overload induced by myocardial ischemia, can protect cardiac tissue from changes in its contractility and ejection strength [41]. To the values of +dP/dT, however, only treatments with compound 8c and NIF are different from HBP + I/R at the end of reperfusion. Previous studies have emphasized the inhibition of the calcium channels by the use of verapamil in myocardial contraction and the protective effect on excessive calcium overload [42] thus, calcium channel blockers such as dihydropiridines can reduce the size of the ischemic lesion of the myocardium through inhibition of calcium channels, improving the ejection strength and contractility [43].
The impact of endothelial dysfunction is important in understanding myocardial I/R injury [44] the values of vasodilation and vasoconstriction were analyzed. The only treatment that allowed an increase in vasodilation during the reperfusion period was 2c when compared to the HBP + I/R group (p < 0.05) ( Figure 9). It is important to note that, in Wistar-Kyoto rats, L-NAME has been shown to completely block the relaxation induced by acetylcholine in blood vessels [45], which suggests once again that compound 2c can block calcium channels of smooth muscle, and cause vasodilation. Simonovic and Jeremic [46] suggest that preconditioning with calcium channel blockers (verapamil, amlodipine) improved the recovery of contractile function during reperfusion. Pre-conditioning may be involved in this study because it prevents changes in the redox state of cardiac tissue, caused by increased blood pressure. The loss of redox homeostasis results in the activation of pro-in ammatory and pro brotic pathways in the heart, which leads to diastolic dysfunction, probably a result of increased left ventricular diastolic stiffness.
Before ischemia (time = 15 minutes) perfusate was collected, but the LDH values were undetectable. In cardiac studies, they found that nifedipine reduces leakage of LDH and accumulation of neutrophils after myocardial I/R injury [47]. In this study, we found that only compound 2c led to a statistically signi cant decrease in LDH levels, which is the biomarkers of cardiac injuries.
At the early phase of reperfusion free radicals are released. Also, I/R will result in a decrease in the antioxidant activity which renders the myocardium extremely vulnerable [48]. Previous ndings support that increased lipophilicity can increase the antioxidant potential of DHP [12,17, 18 and 19]. Tanaka et al. [49] demonstrates that amlodipine, a highly lipophilic DHP, reduces oxidative stress. Our previous results also demonstrate that DHPs that received the addition of fatty acids have greater antioxidant potential, in addition to attenuating reactive species in cardioblasts undergoing I/R [12,17,18].
Currently, therapies to mitigate the damage caused by I/R are based on reducing the formation of reactive species [50], so the discovery of new compounds that have an antioxidant effect is important to treat this pathology. The antioxidant protection under the conditions of oxidative injury is a complex system in which separate antioxidant elements co-operate with each other. The function of one antioxidant often potentiates the effects of another element in the system [51], as observed in the treatment with compound 2c on catalase activity ( Figure 12).
Alam et al. [52] in their study with rats, showed that another dihydropyridine, amlodipine, decreased the levels of LPO in a dose-dependent manner. Literature data suggest that DHPs can inhibit lipid peroxidation accumulation in cardiac membranes by blocking calcium channels [53] but only compounds 2c and 9a do not show a statistical difference from the control group..
In the present investigation, the reduced levels of catalase activity observed in ischemic/reperfused untreated hearts were signi cantly reversed by treatment with compound 2c. This suggest that the intervention assisted the inherent cardiac antioxidant system in combating hydrogen peroxide generated by the I/R episode. The enormous amount of H 2 O 2 resulting from the I/R cycle could trigger the production of the highly reactive hydroxyl radicals via Fenton reaction involving transition metal ions, like Fe 2+ [54]. Such hydroxyl radicals and other resulting reactive species are capable of degrading polyunsaturated lipids, thus forming malondialdehyde. The relatively high degree of membrane peroxidation observed for I/R untreated hearts when compared with that of the non-ischemic and 2c and 9a treated hearts might be indicative of the protection afforded by the treatment.It is also important to note that the dose administered for all DHPs was 0.42 mg/kg/day. As the molecular weight of the compounds are different (NIF 346.3 g/mol; 2c 818.6 g/mol; 8c 807.6 g/mol and 9a to 755.6 g/mol), this suggests a greater potential for new fatty DHPs.
The literature describes that a substituent at position C2 and C3 of the DHP ring in uences its antioxidant activity [55], which is one of the replacement sites for the fatty acids used. The greater lipophilicity of the new DHPs, and their possible binding to a mitochondrial channel, could explain the promising results of the observed ROS reduction seen during the induced I/R. In addition, it is already known that the addition of a halogen, such as chlorine, increases the electron-donating effect of some molecules [56], this addition was carried out in compound 9c, already in compound 2c and the addition of a NO 2 group also affects redox properties [57].
Manidipine is a highly lipophilic calcium antagonist, which guarantees a 24-hour action pro le, despite a relatively short plasma half-life due to its high liposolubility, the compound is quickly removed from circulation and binds to plasma membranes continuously binding to calcium channels [58]. By adding lipid chains and observing the results obtained in this work, we can suggest that the new DHPs behave similarly to manidipine, being able to bind the membrane more easily, thus blocking calcium channels more effectively and preventing the accumulation of calcium ions inside the cell. The following mechanism may explain the better performance of the new DHPs when compared to nifedipine. When under conditions of cytosolic calcium overload, calcium can cause mitochondrial damage by triggering the opening of the mitochondrial permeability transition pore, leading to mitochondrial membrane permeabilization and generating reactive oxygen species, resulting in mitochondrial dysfunction and cell death [59] and thus generating a change in cardiac functionality.
In conclusion, the present study suggests that new fatty DHPs have an anti-hypertensive and antiarrhythmic effect, in addition to offering protection against I/R injury in the hearts of rats and preventing a decline in cardiac function. The new fatty DHPs, mainly 2c, which had the addition of oleic acid and nitrogen dioxide, could serve as sources of user agents to combat the complications associated with myocardial I/R injury. However, more studies are still needed to elucidate the pharmacodynamics and pharmacokinetics of these new dihydropyridines.

Figure 1
Schematic images of fatty dihydropyridines tested in this study [12,18,19].  symbol represents the statistical difference between High blood pressure treated with nifedipine after the ischemia and reperfusion group (HBP + NIF + I/R) versus all other groups. Different letters represent statistically signi cant differences among the times of the same group.

Figure 4
The heart rate was monitored throughout of the experiment. For 28 days, a high blood pressure induction was performed, from day 28 to day 56 of animals that received treatments or saline solution (Control, I/R and HBP + I/R). Data are shown as mean and SEM. The * symbol represents the statistical difference between the group High blood pressure and ischemia and reperfusion (HBP + I/R) vs all other groups. The # symbol represents the statistical difference between the Control group versus all other groups. The & symbol represents the statistical difference between High blood pressure treated with nifedipine after the ischemia and reperfusion group (HBP + NIF + I/R) versus all other groups. Different letters represent statistically signi cant differences among times of the same group.         Catalase activity analyzed in cardiac tissue after ischemia and reperfusion. Values are mean ± SEM and the different letters indicate statistical difference (p < 0.05) between the groups.