【作者】 潘孝贵；

【导师】 潘珊珊；

【作者基本信息】 上海体育学院 ， 运动人体科学， 2008， 博士

【摘要】 研究目的:“运动员心脏”(athlete’s heart)是指运动员特有的高功能、高储备、大心脏,是机体对长期运动训练良好适应的结果。运动心脏的形成不仅仅是由于血流动力学超负荷导致的心肌细胞体积增大及相应亚细胞结构改变的简单过程,而且是在神经-体液调节下,产生的一系列代谢、结构、功能诸方面的重塑过程。降钙素基因相关肽(calcitonin gene related peptide,CGRP)是目前已知最强的舒血管活性物质,对心肌具有正性变力和变时作用,使心率加快,心肌收缩力增强,心输出量增加;能明显地舒张冠状血管,增加冠状动脉血流量;能有效防治心肌的缺血/再灌注损伤;是心脏重塑和保护作用的重要调节物质。经长期的耐力训练后,心脏和血液中的CGRP显著升高,提示CGRP可能参与运动诱导的心脏重塑和保护作用。目前,有关运动与CGRP的研究主要集中在心脏和血液CGRP含量的变化,这些研究结果不能全面和系统地反映运动对CGRP的影响,而且运动对CGRP合成及CGRP mRNA表达影响的研究少有报道。因此,本研究在运动心脏动物模型的基础上,探讨运动心脏重塑后CGRP的变化和耐力训练诱导的心脏保护作用机制,为科学制定运动训练方案、有效预防运动心脏损伤、提高体育人口心脏健康及制定心血管疾病的运动处方等提供新的理论和实验依据。研究方法:雄性健康SD大鼠,随机分为耐力训练组(n=33),对照组(n=33)。耐力训练组大鼠进行持续10 week的耐力跑台训练(75% VO2max)建立运动心脏动物模型。建模成功后,各组随机选取半数大鼠进行连续3 d的力竭运动,以检验大鼠的训练效果和诱导心肌微损伤。力竭运动后即刻麻醉大鼠,取血、心脏和胸腰段背根神经节。采用酶联免疫法检测血清CGRP浓度,免疫化学发光法检测血清心肌肌钙蛋白I含量;用HE染色和碱性复红苦味酸染色法观察心肌组织结构和缺血缺氧改变;用放射免疫法测定心肌组织CGRP含量;用免疫组织化学和计算机图像分析显示心肌、背根神经节CGRP分布与表达;用荧光定量聚合酶链反应检测背根神经节CGRP mRNA表达;用酶还原法、硝酸还原法、亚硝酸盐还原法和硫代巴比妥酸法分别检测血清和心肌乳酸含量、一氧化氮含量、超氧化物歧化酶活性和丙二醛含量。研究结果:(1)10 week耐力训练后,耐力训练组大鼠心脏重量与体重比显著大于对照组(P=0.01),升高了9.67%;组织学观察也发现,耐力训练组大鼠心房和心室肌细胞体积有一定程度的增大,提示耐力训练诱导了心脏形态上的肥大。(2)力竭运动测试表明,耐力训练组平均距离显著大于对照组(P<0.01),提示耐力训练诱导的心脏肥大是生理性的。(3)耐力训练组力竭运动时血乳酸较对照组显著升高(P<0.05),但心肌乳酸含量没有显著差异,提示耐力训练诱导的肥大心肌代谢表型改善。(4)与对照组比较,安静状态下耐力训练组大鼠血清CGRP浓度显著升高(P<0.05);心脏CGRP免疫反应活性明显增强,免疫反应阳性面积和平均光密度均显著增大(P<0.05),心室肌组织CGRP含量显著升高(P<0.01);背根神经节CGRP免疫反应阳性神经元数量、阳性反应面积和平均光密度上显著增加(P<0.01),但背根神经节CGRP mRNA表达量显著下降(P<0.01),表明运动心脏CGRP释放、储备、合成增加,但基因表达下调,提示耐力训练可能是在转录后水平上调节心脏CGRP。(5)力竭运动后,两组大鼠心脏CGRP免疫反应活性明显减弱,平均光密度显著下降(P<0.05),耐力训练组心室肌CGRP含量显著下降(P<0.05);背根神经节CGRP免疫反应阳性神经元数量减少,免疫阳性反应面积和平均光密度显著下降(P<0.05)。但两组大鼠血清CGRP和背根神经节CGRP表达的变化不同:耐力训练组血清CGRP显著提高(P<0.01),mRNA表达量没有显著变化,而对照组血清CGRP没有显著变化,CGRP基因表达显著下调(P<0.01)。提示力竭运动损害了CGRP基因表达,减少了CGRP的合成,降低了心脏CGRP储备,但预先的耐力训练可以保持力竭运动时CGRP正常的基因表达和释放,提高心脏对长时间运动的耐受能力。(6)安静状态下,两组大鼠血清和心肌一氧化氮含量没有显著差异,但力竭运动后对照组心肌一氧化氮含量显著下降(P<0.01),提示力竭运动可造成未经训练大鼠心脏一氧化氮合成能力下降。(7)力竭运动后,对照组大鼠血清心肌肌钙蛋白I浓度显著升高(升高11.37倍),心肌组织出现明显的缺血缺氧改变;而耐力训练组血清心肌肌钙蛋白I浓度没有显著变化(升高0.54倍),心肌组织仅出现轻微的缺血缺氧改变。提示力竭运动诱导了心肌损伤,耐力训练具有一定的心肌保护作用。(8)耐力训练后,大鼠心肌超氧化物歧化酶总活性显著上升(P<0.01),但血清超氧化物歧化酶活性以及血清、心肌丙二醛含量没有显著变化,表明耐力训练可以上调心肌超氧化物歧化酶活性。(9)力竭运动后,对照组血清超氧化物歧化酶总活性没有显著变化,心肌超氧化物歧化酶总活性显著上升(P<0.01),血清和心肌丙二醛含量均显著提高(P<0.01)。而耐力训练组血清、心肌超氧化物歧化酶总活性显著降低(P<0.01),血清丙二醛含量并没有显著变化,心肌丙二醛含量显著升高(P<0.01),提示力竭运动显著增强了血液和/或心肌脂质过氧化。研究结论:(1)10周耐力跑台训练可以诱导大鼠心系数增加、心肌细胞肥大、心脏泵血功能显著提高,是建立运动心脏动物模型的可靠方法。(2)耐力训练通过增强背根神经节CGRP的合成,提高心脏CGRP的储备,促进CGRP的释放,从而生理性重塑了运动心脏。(3)力竭运动可以降低了背根神经节CGRP基因表达和蛋白合成,减少了心脏CGRP储备,导致降钙素基因相关肽的可释放量减少,心脏保护能力下降,心肌发生微损伤。(4)耐力训练诱导的心脏保护作用可能是通过提高机体CGRP释放能力,上调超氧化物歧化酶活性,降低脂质过氧化以及促进一氧化氮的合成,从而提高冠脉血流量来实现的。更多还原

【Abstract】 Objective: The profound heart changes including morphological enlargement, functional improvement, capacity elevation that occur in athletes are a normal adaptive response to chronic athletic training, which have been recognized as the athlete’s heart. The notion has been accepted that the process of exercise-induced cardiac remodeling is not only the alternation of myocardium volume and responding ultrastructure by hemodynamic overload, but also the re-establishment of cardiac morphology, function and metabolism under the regulation of nerves and hormones. Calcitonin gene-related peptide (CGRP) is one of the most potent vasodilator substances identified to date. Biological effects of CGRP include increasing cardiac output, elevating coronary blood flow, and significant prevention against ischemia-reperfusion injury. Consequently, CGRP is thought to play a vital role in cardiac remodeling and cardioprotection. The fact that plasma and myocardial concentration of CGRP increased after systematic endurance training has indicated that CGRP is a candidate regulator of exercise-induced cardiac remodeling and cardioprotection. At present, the focus is mostly on the changes of plasma and myocardial content of CGRP during or/ and after exercise, no comprehensive and systematic data exist on the effect of exercise on CGRP, especially for its synthesis and gene expression. The purpose of the present study was to investigate the changes and possible mechanism of CGRP involved with exercise-induced cardiac remodeling and cardioprotection following the establishment of animal model of exercise-induced cardiac hypertrophy, to provide the newer theory and experiment foundation for optimal sports program, effective prevention of cardiac injury, heart health promotion, exercise prescription for cardiovascular disease.Methods: Male SD rats were randomly divided into two groups: sedentary control group (CG) and endurance training group (EG). After the animal model establishment of exercise-induced cardiac hypertrophy by graded treadmill training lasting 10 weeks (at the intensity of 75%VO2max), half rats of each group were subjected to consecutive, exhaustive treadmill running three times, to test the effect of endurance training and induce cardiac injury. Blood, heart and dorsal root ganglions were removed immediately after exhaustion. The level of CGRP and cardiac troponin I in serum were determined by methods of enzyme immunoassay and chemiluminescence, respectively. Histological examination of Cardiac muscle was determined by hematoxylin-eosin staining and special dyeing of ischemia and hypoxia. Concentration of CGRP in left ventricular myocardium was detected by radio immunoassay. CGRP-immunoreactivity in heart and dorsal root ganglion tissue were showed by immunohistochemistry method and quantitatively analyzed by the system of computerized image analysis. The expression of CGRP mRNA in dorsal root ganglions were determined by real-time fluorescence quantitative polymerase chain reaction. Spectrophotometry were used to demonstrate the content of lactic acid, nitric oxide and malondialdehyde and activity of superoxide dismutase in serum and left ventricular myocardium.Results: (1) The ratio of cardiac weight to body weight and distance of run to exhaustion was greater in EG than in CG after 10-week endurance treadmill training (P=0.01). Meanwhile, histological evidence of heart specimen from EG demonstrated myocyte hypertrophy to some degree. Concentration of lactic acid in blood, not in myocardium, was more significantly increased in EG than in CG after exhaustive exercises. The results showed that cardiac hypertrophy by endurance training is a physiological, not pathphysiological, adaptation to exercise. (2) In EG, CGRP concentration in serum, immunoreactivity in cardiac muscle and dorsal root ganglions and content in left ventricular increased significantly (P<0.05 or 0.01, respectively), but gene expression in dorsal root ganglia decreased significantly (P<0.01), by endurance training. It suggested that CGRP release, reserve and synthesis in exercise-induced hypertrophied heart increased, but gene expression decreased, which demonstrates the fact that endurance training may regulate cardiac CGRP at the post transcription level. (3) After exhaustive exercises, CGRP immunoreactivity in cardiac muscle and dorsal root ganglia was markedly weakened in CG and EG (P<0.05), and CGRP content in left ventricular myocardium of EG decreased (P<0.05). Meanwhile, the CGRP release in EG increased and gene expression of CGRP in EG down-regulated significantly (P<0.01), but results of serum CGRP in CG and CGRP mRNA in EG showed no changes. It was reasonable to conclude that exhaustive exercises do harm to CGRP gene expression, protein synthesis and local store, but pretraining can maintain the normal expression and release of CGRP to improve the tolerance to prolonged exercise. (4) Nitric oxide content in serum and cardiac muscle did not differ between CG and EG after 10-week endurance training. However, lower nitric oxide content in cardiac muscle from CG was found immediately after exhaustive exercises (P<0.01).It showed that exhaustive exercise attenuates the nitric oxide synthesis in cardiac muscle of untrained rats. (4) Serum cTnI concentration ratio of after exhaustive exercise to before was greater in CG than in EG, and evident changes demonstrated by special dyeing of ischemia and hypoxia was found in some rats, so minor cardiac injury by exhaustive exercises occurred. (5) Although cardiac activity of superoxide dismutase in EG increased, serum superoxide dismutase activity and malonaldehyde contents in serum and cardiac muscle did not so, in CG and EG after endurance training, which suggested that endurance training can elevate the activity of superoxide dismutase in heart to reduce lipid peroxidation. (6) In CG, exhaustive exercise could elevate malonaldehyde content in cardiac muscle and serum, superoxide dismutase activity in cardiac muscle, but did not affect superoxide dismutase activity in serum. On the other hand, exhaustive exercise could elevate malonaldehyde content in cardiac muscle, depress superoxide dismutase activity in cardiac muscle and serum, but did not affect malonaldehyde content in serum, and lipid peroxidation in blood and heart was increased by exhaustive exercise.Conclusion: (1) 10 week endurance treadmill training results in greater ratio of cardiac weight to body weight, hypertrophied myocyte, elevated cardiac function, and can be proposed as a reliable animal model of exercise-induced cardiac hypertrophy. (2) By endurance training, CGRP synthesis in dorsal root ganglia is enhanced, content in cardiac muscle raised, level in serum elevated, thereby cardiac hypertrophy is physiologically remodeled. (3) Exhaustive exercise stress impaires gene expression and synthesis of CGRP in dorsal root ganglia, decreases CGRP reserve in heart, and as the result, available release of CGRP reduces, minor cardiac injury occurs due to lowered ability of cardioprotection. (4) Cardiaoprotection by endurance training may be mediated by elevated release of CGRP that up-regulates the activity of superoxide dismutase to lower lipid peroxidation, and promotes nitric oxide synthesis to increase coronary blood flow.更多还原