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) (Fig. 5) group may indicate a benefit of this treatment. Undoubtedly, restoring normal blood flow to the ischemic heart leads to a lower mortality rate in ischemic patients [7, 37]. However, reperfusion alone can cause a sequence of harmful events, including arrhythmias (mainly), microvascular injury, myocardial dysfunction, myocardial stunning, and finally, death [38], showing the importance of establishing reperfusion without increasing BPM.
These results follow an experimental study in isolated hearts [39], which demonstrated that previous treatment with nifedipine induces preconditioning, as shown by improved left ventricular pressure after ischemia. These findings support our results, considering that NIF and 2c present values of PDVE after 60 minutes are 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), suggesting an improvement in LVDP after ischemia. The decrease in left ventricular pressure values demonstrates an evident impairment in left ventricular function caused by pressure overload, confirming this increase in blood pressure values in untreated groups (Fig. 6).
The reduction in LVDP and ± dP/dT can be used to indicate 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 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 verapamil use in myocardial contraction and the protective effect on excessive calcium overload [42]. Thus, calcium channel blockers such as dihydropyridines 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 essential 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) (Fig. 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. Preconditioning 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-inflammatory and profibrotic pathways in the heart, leading to diastolic dysfunction, probably due to increased left ventricular diastolic stiffness.
The perfusate was collected before ischemia (time = 15 minutes), 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 significant decrease in LDH levels, which are the biomarkers of cardiac injuries.
In the early phase of reperfusion, free radicals are released. Also, I/R will decrease antioxidant activity, which renders the myocardium extremely vulnerable [48]. Previous findings support that increased lipophilicity can increase the antioxidant potential of DHP [12, 17, 18, and 19]. Tanaka et al. [49] demonstrate that amlodipine, a highly lipophilic DHP, reduces oxidative stress. Our previous results also demonstrate that DHPs that received the addition of fatty acids have more significant antioxidant potential and 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 discovering new compounds with an antioxidant effect is central 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 (Fig. 12).
In their study with rats, Alam et al. [52] 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 catalase activity observed in ischemic/reperfused untreated hearts was significantly reversed by treatment with compound 2c. This result suggests that the intervention assisted the inherent cardiac antioxidant system in combating hydrogen peroxide generated by the I/R episode. The enormous amount of H2O2 resulting from the I/R cycle could trigger the production of the highly reactive hydroxyl radicals via Fenton reaction involving transition metal ions, like Fe2+ [54]. Such hydroxyl radicals and other resulting reactive species can degrade polyunsaturated lipids, thus forming malondialdehyde. The relatively high degree of membrane peroxidation observed for I/R untreated hearts compared with that of the non-ischemic and 2c and 9a treated hearts might indicate 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 weights 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 positions C2 and C3 of the DHP ring influences its antioxidant activity [55], 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. It is also known that halogen addition 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 NO2 group also affects redox properties [57].
Manidipine is a highly lipophilic calcium antagonist, which guarantees a 24-hour action profile; 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 and 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 antihypertensive and anti-arrhythmic effect and offer protection against I/R injury in the hearts of rats, and prevent 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.