The Mechanism Of Second Window Of Myocardial Protection Induced By L-NAME

BackgroundThe invention of cardiopulmanory bypass made the cardiac surgery have a great development, so that cardiac surgeron can do operation in a bloodless surgical field. As the aorta being clampped, the heart is in the ischemic state and must be under an effective protection to avoid the heart injury. The cold cardioplegia being applied in the cardiac surgery is a milestone for myocardial protection. The blood cardioplegia which is better than cold crystalloid cardioplegia reduces myocardial injury and myocardial enzyme release. Nowadays, cardioplegia is being used widely. However, there are still limits. It is not perfect that the longer cardiopulmanory bypass time is, the more surgery risks will do. How to reduce myocardial ischemia or reperfusion injury had become an important clinical problem, so more and more cardiac surgeons pay more attention to it. For a long time, cardiac surgeon has used exogenous methods to reduce myocardial ischemia or reperfusion injury, but the result were dissatisfied. Recently, some people began to study the endogenous protective mechanism, and found that it could increase the tolerance of ischemia and hypoxia of myocardial cells.Ischemic preconditioning is the most popular endogenous myocardial protective measures, but the exact mechanisms are not clear and the clinical application is not convenience.Based on the study of ischemic preconditioning, some people tried to use drugs to simulate the anti-injury capabilities in the myocardial protection, which is known as pharmacological preconditioning. Pharmacological preconditioning has become a research hotspot, previous researches show that many drugs (such as adenosine, monophosphoryl lipid A, K-ATP channel opener, inhaled anesthetics and opioids) can mimic ischemic preconditioning. Most animal experiments confirmed that pharmacological preconditioning may not be used in clinical trial.Maybe there are many reasons, the most important one is that the pharmacological preconditioning of the exact pathway still unclear, and the lack of the large of a randomized, double-blind, controlled, multicenter clinical results. Besides, no drug that simulates the preconditioning is permited to register and access to clinical. So how to find a safe, efficient and suitable way for clinical application of these drugs remains to be further studied.As the pharmacological preconditioning model has better controllability, high repeatability, and no obvious side effects and hemodynamic minor changes, it may be a promoting drug in practical application for preconditioning. With the mechanism of the ischemic preconditioning being further studied, the pharmacological preconditioning will be widely used.Our previous studies confirmed that made pre-simulated myocardial ischemia second window of myocardial protection by intraperitoneal injection of NO synthase inhibitor L-nitro-arginine methyl ester (L-NAME) before 24 hours of cold cardioplegia in the heart subjected to ischemia will further improve a variety of cardiac function on the basis of myocardial preservation solution. However, the mechanism is not clear. Base on the experiment we found that:the glands glycoside levels of treatment group were significantly higher than the control group in fluid of coronary sinus outflow, and myocardial protection of L-NAME group is the strongest. The aim of this study is to make a deeper study of pharmacological preconditioning induced by L-NAME of second window of protection mechanism and expect to develop new theories and methods of myocardial ischemia and reperfusion injury, so as to protect myocardial function better in the operation.Objective1.Whether the second window of myocardial protection induced by L-NAME preconditioning can further enhance its role in the protective effect on the protection provided by cold cardioplegia.2.By given the Adenosine Al receptor antagonist DPCPX before the drug-induced second window of myocardial protection of L-NAME and to investigate the effects of DPCPX on myocardial protection of the second window of myocardial protection, and to clarify whether adenosine receptor activation is initiation factor for drug-induced second window of myocardial protection of L-NAME.Materials and methods1.30 male SD rats that weight of 250-320 grams were randomly divided into 3 groups (each group with 10):(1) ischemic control group:making intraperitoneal injection of lml normal saline, set up isolated rat heart Langendorff perfusion model after 24h; (2)L-NAME preconditioning group:making intraperitoneal injection of L-NAME (10mg/kg), set up isolated rat heart Langendorff perfusion model after 24h; (3)antagonist group:making intraperitoneal injection of DPCPX (O.lmg/kg), making the injection L-NAME (10mg/kg) after 30mins, setting up isolated rat heart Langendorff perfusion model after 24h.2. After weighing the rats, pentobarbital sodium (60mg/kg) was given by intraperitoneal injection of anesthesia, heparin (1000U/kg) was given by sublingual intravenous in order to anti-coagulate, rapidly opened the chest and got heart, immediately placed it in 4℃K-H solution, insert the aortic cannulation, moved quickly to the Langendorff perfusion device, perfusing with 37℃K-H solution (95% O2 and 5% CO2 continuous balance), maintaining perfusion pressure at 100cm H2O. Cut out the pulmonary root so as to fully return flow drainage of coronary. Cut the left atrial appendage after perfusion 1min later through the left atrium, mitral valve, delivering the ventricular balloon with a manometry catheter into the left ventricle, test the other end manometry catheter pressure transducer with Pc-labue Biomedical Signal collection. When the suitable amount of distilled water was slowly injected into ventricular balloon, so that left ventricular end diastolic pressure (LVEDP) keep at 10mmHg. K-H perfusion stable after 15min by aortic root infusion 10ml 4℃St Thomas'Ⅱcardioplegia, the heart of all the beating at 20℃60mins(beating heart surgery simulation process), then reuse K-H perfusion for 120mins. Only late in the stability of the following conditions:HR 240-360 beats/min, LVDP 60-130mmHg, CF 8-16 ml/min before entering the test.3. We made records of HR, LVDP at four time by Pc-labue biomedical signal acquisition and processing system:15mins when stable in the perfusion experiments, lmin after reperfusion,60mins after reperfusion and 120mins after reperfusion, calculating value of CF through the amount of coronary flow and timing, meanwhile collecting crown vein effluent and discharge in-20℃refrigerator, detecting cTnI on Siemens automatic chemiluminescence immunoassay analyzer. At the end of experiment, observing changes of myocardial ultrastructure in the left ventricular free wall myocardial after treating with electron microscopy fixative in PHILIPS CM 10 transmission electron microscope.Results1. Hemodynamics:there is no difference of overall heart rate (HR) among all the group before ischemia and after ischemic. Antagonist group is more slowly than L-NAME group, and they have significant differences (P<0.05). However the antagonist group shows no difference with ischemic control group; left ventricular developed pressure (LVDP) is the discrepancy between cardiac systolic and diastolic blood pressure, suggesting the myocardial systolic and diastolic function, LVDP of antagonist group is lower than L-NAME group, and there were significant differences (P<0.05), but show no difference with ischemic control group; coronary flow (CF) reflects coronary vascular resistance and myocardial oxygen supply of the antagonist group, CF of L-NAME group is less significant, and there were significant differences P<0.05), but show no difference with the ischemia control group.2. Enzyme:cTnl is cardiac-specific enzyme, ithas higher sensitivity and specificity to myocardial injury than other enzymes, antagonist group of cTnI was significantly higher compared with L-NAME preconditioning (P<0.05), they have no difference with ischemic control group.3. Myocardial ultramicro-structure:Under the electron microscope, ischemic control group and antagonist group show myocardial edema, irregular nucleus, chromosome margination, mitochondrial swelling, with ridges fuzzy, vacuolation of myofilament dissolution and SR expansion; L-NAME preconditioning group show slight edema of myocardial cells and mild mesenchymal, muscle fibers were arranged in order with clear filaments, mitochondria were slightly swollen, with ridges slightly fuzzy and the uniform nuclear chromosomes. The ultrastructure of myocardial injury antagonist and ischemia group is more serious than L-NAME pretreatment group.Conclusion1.L-NAME induced a second window of myocardial protection, and further strengthened its protective effect on the original cardioplegia protection.2.Adenosine A1 receptor antagonist DPCPX blocked the L-NAME preconditioning induced second window of myocardial protection, so adenosine A1 receptors may be the initiation factor caused by L-NAME preconditioning of the second window of myocardial protection.