An Experimental Study On Rats Myocardial Injury Induced By Exhaustion Swimming Stress

BackgroundStress is a process of adaptation and response when the individual faces or detects (perceives,appraises) threats or challenges from environmental changes(stressors). Moderate stress may activate thinking, and improve people’s alertness and working efficiency. But more than the capacity of the individual stress pressure tends to induce a variety of diseases. As heart is one of the primary target organs of stress,the occurrence and development of a variety of cardiovascular disease is closely related to the activation of stress mechanisms.Exercise is a special kind of stress, appropriate exercise can improve cardiac function, such as thickening myocardial fiber, enhancing the contractility, and so on.But the exhaustive exercise or overtraining has a negative effect on the heart.It not only inhibits the improvement of cardiac function, but also causes myocardial injury and pathological changes in the heart.This has been proved by the frequent sudden death in ordinary life. Therefore, how to clarify the pathogenesis and find the methods to control overtraining-induced acute myocardial injury (OTIAMI) is an important subject in military medicine,sports medicine and clinical medicine.N-acetylcysteine(NAC) is a classic antioxidant and free radical scavenger, which may effectively improve the content of cardiac glutathione and prevent the toxic effects of high concentrations of TNF-a on the heart. Meanwhile, glutathione may inhibit the activity of NF-κB by improving myocardial redox state and make the rehabilitation of cardiac hypertrophy, improving hypertensive cardiac remodeling and enhancing the cardiac contractility.Myocardial ischemia-reperfusion experiments show NAC may significantly decrease the level of cardiac troponin I (CTnI) and Lipid Peroxidation (LPO) in serum and reduce the area of myocardial infarction. Studies have also confirmed that NAC plays a cardioprotective role by inhibiting the expression of iNOS to inhibit the activity of NF-κB which may reduce the downstream cytokines.And NAC may also reduce the apoptosis rate of myocardial cells through TLR4/NF-κB way. Moreover, NAC pretreatment can obviously reduce the cellular ROS formation, mitigate the collapse of mitochondrial membrane potential and decrease the caspase activity. But it is unclear that whether NAC has a preventive effect on OTIAMI and its mechanisms.The animal model of overtraining-induced myocardial injury was developed by repeated exhaustive swimming stress.The purpose of this study is to research the relationship among cytochrome c (CytC), programmed cell death5(PDCD5) and myocardial injury.We also observe the effects of exhaustion swimming stress on the heart and whether NAC have a preventive effect on OTIAMI or not during the experiments.Of course, the main purpose of this study is to provide a new theoretical foundation and experimental basis for the prevention and treatment on clinical OTIAMI.Methods1. The model materials:Thirty-six specific pathogen-free male wistar rats, weighed110±20g, were used in our experiment. After purchased, the rats were adapted to swimming two times once a day with20minutes for each time, then eliminated the abnormal rats. They were randomly divided into three groups before experiments:control group(group C),exhaustive swimming stress group (group S) and exhaustive swimming stress plus NAC treatment group(group N)(n=12in per group). NAC in a dose of150mg/kg/d was given by irrigation for7weeks in group N once daily.The other groups were given the same amount of double distilled water.After carrying5%of body weight heavy loads,the rats in group S and group N conducted exhaustive swimming training.Exhausted standard were as follows:the motor coordination in rats significantly decreased, the water flooded the nose of rats, the rats could not surfaced submerged10seconds for3times in a row, and the rats could not complete quickly righting reflex when they were placed on a plane after training. Swimming condition were as follows:the rats were immediately trained in a SLY-WMS water maze and forced to swim.Briefly, the water maze system was a drum with a diameter of about1.2m and its inner wall was smooth.It contained time and temperature control devices and no platform. The depth of water was set at about45cm and the temperature was maintained at (30±2)℃. During the forced swimming training, the rats could not escape or rest.2.Executing animal and taking material:After the last swimming training, All rats were anesthetized by an intraperitoneal injection of10%ketamine (0.1ml/100g) and then killed.Continuous recording HR, EKG and blood pressur for5minutes were performed by powerlab physiological recorder (AD Instrument, Australia) before rats were sacrificed.After skin preparation and disinfection were performed, the middle of neck was cut in rats, then the tracheotomy and tracheal intubation were performed. The inputport of the rodent ventilator was connected to a16-gauge catheter which was connected to the lung of rats,but the other port of the rodent ventilator was not connected to the oxygen devices. Subsequently, intubation of rats was performed through incision of the tracheal and ventilation was performed at3-4ml/kg per minute with atmospheric air.The chest was opened through a lower sternal incision. Following pericardiotomy, a24-gauge catheter was delivered into the left ventricle (LV) chamber and the changes of ventricular pressure could be monitored continuously.2-3ml blood sample was collected from the piezometer tube and let it stand for1h, then centrifuged at3600r/min for10min at4℃.The serum sample was stored at-70℃.Myocardial specimens were reserved with scissors and forceps which had been soaked by DEPC water. Part of myocardial specimens myocardial specimens were fixed with4%paraformaldehyde for24h, and then were embedded in paraffin and serially cut for histologic examination. The rest was examined using transmission electron microscope (TEM).3.The detection indexes:Rats and food were weighed daily in the three groups, and the changes of behavior were also observed daily. Real-time hemodynamic data, including heart rate (HR),electrocardiogram(EKG), the left ventricular systolic pressure(LVSP), the maximum and minimum first derivative of left ventricular systolic pressure (±dp/dtmax) were recorded continuously after the last swimming training by powerlab physiological recorder(ADInstrument,Australia).When the experiment is over,the left ventricular developed pressure (LVDP),the cardiac weight index,the ventricular weight index,the maximum and minimum first derivative of LVSP(±dp/dtmax) were calculated.By hematoxylin-eosin staining (HE staining),the myocardial morphology was observed under the light microscope. And the myocardial ultrastructure was examined using transmission electron microscope (TEM). Total superoxide dismutase (T-SOD) activity were detected by the methods of xanthine oxidase. Copper-zinc superoxide dismutase (CuZn-SOD), manganese superoxide dismutase (Mn-SOD) activity were measured by the extraction method.The content of malondialdehyde (MDA) was measured by usin gthiobarbituric acid method.The content of myocardial tissue protein was determined by coomassie brilliant blue method.Real-time fluorescence quantitative PCR methods were used to detect the changes of PDCD5mRNA and westten blotting methods were used to detect the changes of cytochrome C(CytC) protein in rat myocardium. Serum level of Troponin I Type3(TNNI3) and creatine kinase MB (CK-MB) were measured using a double-antibody sandwich enzyme-linked immunosorbentassay (ELISA).4.Statistical methods:All data were expressed as means±SE unless otherwise stated and statistical analysis was performed with SPSS13.0statistics software.The differences between groups and among groups were examined by t-test and one way analysis of variance(ANOVA), respectively. A p value of less than0.05was considered statistically significant.Results1. The general condition of rats:In the fourte week,there was a rat drowned in group S.In the sixth week,there was another drowned in group S. After7weeks of swimming training, poor fur condition and corneal opacities were noticed in group S. And the rats were inactive, listless, and weakly responsive. In addition, the group S rats had twitches and jerks frequently at the later stage of the experiment. But the control group rats appeared responsive and alert, the eyes were eloquent and the furs tidy.The weight of two groups were positively growth in the first three weeks.But the weight gain in group S was slower than group C. And at4weeks,the weight of rats in group S were no longer growing (272.68±16.96g), showed a downward trend. Compared with the control group (345.98±23.11g),afore-mentioned difference was statistically significant (t=8.603,P<0.05) During the experimental period,the food intake of group C rats were also positively growth. At the end of experiment, there was a trend of food growth72.1%for group C and2.1%for group S. Compared with the maximum food intake at the3rd week(32.15±3.32g),the food intake of group S(21.32±2.75g) were significantly decreased, the difference was statistically significant (t=29.762,P<0.05)2. The rats myocardial pathological conformation and ultrastructural:The light microscope showed part of myocardial muscle fibers swelling hypertrophy, edema and vasodilation in group S. And the myocardial muscle fibers swelling hypertrophy, edema and vasodilation could be accidentally found under the light microscope in group N rats. But there were no obvious changes in group C rats. Transmission electron microscopy showed that in the control group, the myocardial ultrastructure was normal, the mitochondrias were distributed in belt and neatly arranged, the cardiac sarcomere arrangement was in order, the lines Z, M and bands I,A were clear, the vascular bureaucratic was smooth and the basement membrane was normal. After7weeks of swimming training, transmission electron microscopy showed the myocardial cell and the chromatin of nuclear were swollen, the organelles decreased, mitochondrion was partly swollen, the protruding of mitochondrion was not in order, the protruding and membrane of mitochondrion were partly blended and disappeared. There were not obvious morphological changes in myocardial tissue and the changes of myocardial tissue ultrastructure were slight in group N.3.Comparison of the serum CK-MB and TNNI3contents in rats:In this study, the serum CK-MB and TNNI3contents in rats were compared using ANOVA and LSD methods, and the differences were statistically significant (CK-MB:F=429.183, df=2, P<0.05; TNNI3:F=257.953, df=2, P<0.05). Compared with group C, CK-MB and TNNI3contents in the serum were significantly increased in group S,(CK-MB:19.343±0.944pg/ml vs8.866±0.950pg/ml,TNNI3:198.107±12.060pg/ml vs84.854±11.574pg/ml), and the differences were statistically significant(P<0.05). After being interfered with NAC, serum CK-MB (11.998±0.634pg/ml) and serum TNNI3(128.265±11.489pg/ml) were significantly decreased in group N (P<0.05). But compared with group C, serum CK-MB and serum TNNI3in group N were still higher, and the differences were statistically significant(P<0.05).4.Comparison of the activity of T-SOD, CuZn-SOD, Mn-SOD and MDA contents in the myocardium of the rats:The activity of T-SOD, CuZn-SOD, Mn-SOD and MDA contents in the myocardium between three groups of rats were statistically significant (T-SOD:F=130.739, df=2, P<0.05; CuZn-SOD:F=65.098, df=2,P<0.05; Mn-SOD:F=43.234, df=2, P<0.05; MDA:F=43.703, df=2, P<0.05), and LSD method was using for pairwise comparisons. Compared with group C(T-SOD:160.638±9.419U/mgprot,CuZn-SOD:118.724±7.187U/mgprot,Mn-SOD:41.915±7.508U/mgprot, MDA:1.343±0.104nmol/mgprot), the activity of various SOD was significantly decreased and the content of MDA was obviously increased in group S (T-SOD:126.435±6.368U/mgprot, CuZn-SOD:96.809±4.998U/mgprot,Mn-SOD:29.626±8.595U/mgprot, MDA:1.913±0.206nmol/mgprot), and the differences were statistically significant (P<0.05). After being interfered with NAC, the activity of various SOD was significantly increased and the content of MDA was obviously decreased (T-SOD:197.619±13.334U/mgprot,CuZn-SOD:137.345±11.042U/mgprot,Mn-SOD:60.274±7.426U/mgprot,MDA:1.407±0.144nmol/mgprot)versusgroup S (P<0.05).Although the content of MDA in group N was higher than in group C, but the differences were not statistically significant(P>0.05).5.The results of myocardial tissue CytC protein expression and PDCD5mRNA expression in rats:The myocardial tissue CytC protein expression and PDCD5mRNA expression were compared using ANOVA and LSD methods, and the differences were statistically significant (CytC:F=79.497, df=2, P<0.05; PDCD5mRNA:F=73.290, df=2, P<0.05).The westten blotting methods were used to detect changes of cytochrome C(CytC) protein in rat myocardium, and the ratio of CytC protein expression and GAPDH was compared. Compared with group C, the expression of CytC protein in rat myocardium was significantly increased in group S (CytC:0.485±0.161vs0.327±0.120), and the difference was statistically significant (P<0.05).But after being interfered with NAC, the expression of CytC protein in rat myocardium was significantly decreased in group N versus group S (CytC:0.381±0.326VS0.485±0.161)(P<0.05) and higher than group C (CytC:0.327±0.120; TNNI3:84.854±11.574), and the difference was statistically significant (P<0.05).The dissolutioncurve of all the testing purpose gene dissolution curve were unimodal, and the specificity of experimental products was good. The assessment of PDCD5mRNA expression in rats was using2-△△CT method. Compared with group C, the expression of PDCD5mRNA in rat myocardium was significantly increased in group S and group N (PDCD5mRNA:2.297±0.098;1.739±0.065VS1.060±0.065)(P<0.05).But after being interfered with NAC, the expression of PDCD5mRNA were significantly lowered versus group S (PDCD5:1.739±0.065VS2.297±0.098),and the difference was statistically significant (P<0.05).Conclusions1.After training, the typical overtraining pathophysiological changes such as appetite decreases, weight loss, and weakly responsive were observed. Therefore, the experimental model of "forced swimming plus freeweight" was established successfully, and it can be used for the OTIAMI study.2.Exhausting swimming stress will make the heart produce a series pathological and functional changes, destroy the myocardial organizational structure,and cause myocardial injury.3.NAC has a preventive effect on OTIAMI, may prevent myocardial Myocardial peroxidation damage induced by overtraining through its antioxidant effect. 4.NAC may prevent myocardial injury induced by overtraining via down-regulating the PDCD5mRNA expression and inhibiting cytochrome C released from mitochondria.