Comparative Analysis of the Effects of Omeprazole and Ischemic Preconditioning in Protection against Ischemia and Reperfusion Myocardial: Experimental Study in Isolated Rat Hearts

Introduction: Myocardial protection against ischemic injury has been a major focus of studies of cardiovascular sciences worldwide. In 1986, Murry et al. became pioneers by showing a special ischemic preconditioning technique in hearts of dogs which motivated researches for better understanding of endogenous protective mechanisms of the human heart. The aim of this study was to evaluate the effects of omeprazole on protection of functional recovery of isolated rat hearts subjected to ischemia-reperfusion with and without ischemic preconditioning (IP).


Introduction
The myocardial protection refers to the strategies used to mitigate or prevent post-ischemic myocardial dysfunction, a major degenerative diseases of the heart and constant concern of the objectives of all clinical and surgical cardiology since its origins.The ischemic preconditioning (IP) is an endogenous protective mechanism, first described in dog hearts [1] and subsequently demonstrated in humans [2]. Considering the therapeutic possibilities of IP, it is appropriate to expand the knowledge on the effects of drugs against it, not to damage or even enhance its protective mechanism. The pump inhibitors, H + /K + ATPase, are widely prescribed drugs in clinical medicine for ordinary people. The pioneering studies by Lindberg et al. [3] in 1986, on the action of inhibitors of proton pump H + /K + , showed a new line of research and, in 1994, Nagashima et al. [4] was able to prove the existence of active sites of proton pumps, H + /K + ATPase in the myocardium of guinea pigs [5]. In 1998, studies by Hotta et al. [6], using fluorometry and magnetic resonance imaging, in hearts of guinea-pigs, showed ionic and also pH alterations during ischemia and reperfusion. The pump inhibitors including omeprazole used in this study showed myocardial protection by blocking acute ionic changes in the cardiomyocytes. Recently, in 2008, studies by Budzynski et al. [7], showed the beneficial effects of omeprazole in the protection of angina attacks in coronary patients undergoing an exercise stress test. New experimental studies have recently shown the influence of these drugs on ischemic preconditioning of the heart [8,9]. This may cause a major impact in the clinical and surgical cardiovascular area. However, there are still no specific experimental demonstrations showing the comparative analysis of the effects of omeprazole and ischemic preconditioning in protection against ischemia-reperfusion myocardial.The aim was to analyze the effects of omeprazole in the protection of the functional recovery of isolated rat hearts subjected to ischemia-reperfusion with and without ischemic preconditioning.

Materials and Methods
The guidelines of the Brazilian College of Animal Experimentation (COBEA) [10], were respected when conducting this experimental study. The Ethics Committee of the local institution also approved all the experimental project.
Forty male albino hearts of the Wistarbreed rats were isolated and after sedation by inhalation of ethyl ether and anesthesia with an intra-abdominal injection of 10 milligrams of ketamine and 2 milligrams of xylazine, the hearts were removed and maintained in antegradeperfusion with Krebs-Henseleit solution (95% O 2 , 5% CO 2 , 37°C and 110-120 mmHg perfusion pressure and diastolic pressure of 8 mm Hg) using the modified and disposable Langendorffsystem, model FCSFA-ServCor (COMEX Ind & Com Ltda -Belo Horizonte -MG) ( Figure 1).
The 3-wire technique was used to ensure isolation of the ascending aorta [11]. The first wire pulls and lifts the aortic root to protect the aortic valve, the second wire pulls and lifts the edge (top) of the distal ascending aorta, and the third wire is passed between the first two and promotes the ligature of the aortic cannula set, immediately after cannulation of the aorta (Figure 2). A perfusion cannula was introduced cautiously and secured in the ascending aorta to prevent damage to the aortic valve leaflets ( Figure 3). Later on, an incision was made in the left atrium, and the heart was transfixed by a multiperforated cannula which used the apex of the left ventricle as outflow ( Figure 4). Another incision was made in the pulmonary artery for the purpose of draining the right ventricle. Then, the hearts were removed, 3 minutes after the procedure at most, and after 10 minutes of reperfusion, the multiperforated cannula was replaced by a balloon catheter to better evaluation of ventricular function.
The specimens were randomly divided into five groups of eight hearts each. Initially, all isolated hearts were subjected to a stabilization period of fifteen minutes. Then the specimens in Group I were perfused for thirty minutes counted from the end of the stabilization. Groups II and III were subjected to twenty minutes of ischemia followed by thirty minutes of reperfusion, and the Group III received 200 micrograms (μg) of omeprazole before ischemia. The isolated hearts of Groups IV and V were subjected to IP, one cycle consisting of a 5-minutes ischemia and a 5-minutes reperfusion, followed by an ischemia prolonged period of 20 minutes, on the other hand, the Group V received 200 μg of omeprazole immediately prior to prolonged ischemia (

Statistical methods
The statistical methods to analyze the effect of group was the Kruskall-Wallis test followed by Mann-Whitney was applied in (2) (12) (7)

Results
Groups I, III, IV and V showed a pattern of similar behavior (p=0.05) regarding HR. In each group the corresponding hemodynamic variables were measured at equal time intervalst0, t15' and t30' respectively ( Table 1, Chart 1). The CF analysis showed that the Groups II, III, IV and V, had a similar behavior analyzed over time (p=0.316). Group I showed a pattern different from the others, with statistical significance for all the analyzed time (p<0.001) ( Table 2, Chart 2).
In the study of SP,the groups I, III, IV and V, showed the same behavior at t0, t15' and t30' (p=0.345). Group II showed significant alteration of SP throughout the observed stages (p<0.001) ( Table 3, Chart 3).
The corresponding hemodynamic variable statistic +dP/dt max , concluded that there was the same pattern of behavior (p=0.511) between Groups I, III, IV and V. The Group II differs from other groups and presents significant change in +dP/dt max evaluated over time (p<0.001) ( Table 4, Chart 4).
The evaluation of−dP/dt max , showed similarity in behavior (p = 0.634) in Groups II and III. Groups II andIII showed significant alteration of −dP/dt max throughout the evaluations (p<0.001). Groups I, IV and V showed significant differences in analyzed mean times (p=0.005) ( Table 5, Chart 5).

Discussion
Myocardial ischemia can cause serious damage to cardiomyocytes and sometimes the damage can be irreversible. The myocardial reperfusion also causes damage, which may result in fatal arrhythmias. The magnitude of reperfusion injury is directly proportional to the prior ischemic injury [12]. In 1986, Murry et al. [1] showed an important technique for myocardial protection from ischemic events, which he called ischemic preconditioning. The pump inhibitors H + /K + pioneer studies used in Lindberg et al. [3], opened a new line of research, and Nagashima's et al. [4] work, in 1994, showed the presence of proton pump H + /K + ATPase in human heart cells. Studies by Moffat and Karmazyn [13] were able to show good results in myocardial protection against ischemia and reperfusion through drugs inhibiting the Na + / H + ATPase. This research resulted in multicenter studies involving the so-called drugs amiloride and cariporida (channel blocking Na + /    H + ), which confirmed good protection of cardiomyocytes. These good results can be associated with studies using ischemic preconditioning and also the benefits from the use of omeprazole, an inhibitor pump H + /K + ATPase initially used for reduction of gastric hyperacidity [8, [17][18][19][20]. The explanation for the action of omeprazole in the myocardium may be related with the changes of transmembrane H + /K + and flags of ischemic electrocardiographic tracings which action is expressed in the morphological changes of the T wave [21].
Recent experimental study demonstrated the protective pump inhibitor for H + /K + ATPase against ischemia and reperfusion [8]. Subsequently, a new experimental study with pump inhibitor H + / K + ATPase pantoprazole conferred preconditioning characteristics to isolated rat hearts subjected to ischemic injury [9]. There are no studies on the comparative analysis of omeprazole and ischemic preconditioning in the literature consulted.

Conclusion
Inconclusion, omeprazole conferred preconditioning characteristics to isolated rat hearts subjected to ischemic injury. There was no greater efficacy of protection shown in relation to existing methods ofischemic preconditioning. There was no synergism in the use of omeprazole in conjunction with the methods of IP.