【作者】 马继政；

【导师】 张寄南；

【作者基本信息】 南京医科大学 ， 内科学， 2007， 博士

【摘要】 背景运动致心源性猝死(SCD)是某些器质性心脏病，包括肥厚型心肌病(HCM)、致心律失常性右室心肌病、先天性冠状动脉异常、预激综合症、传导系统疾病和离子通道疾病等，受到短暂、急剧的异常刺激而诱发的突然死亡。诱发因素包括急性心肌缺血，交感神经过渡兴奋，血流动力骤变等引起致死性室性心律失常。国外调查资料表明对运动员赛前进行HCM筛查可显著降低运动猝死的发生(3.6／10万降至0.4／10万)。长期大强度训练导致左室生理性肥厚与病理性左室肥厚难以鉴别，因此，制定一个积极、有效的赛前筛查策略对预防运动员SCD十分重要。HCM是一类具有不同表型的遗传性心脏疾病，可由多种基因突变引起，呈常染色体显性遗传。HCM是35岁以下运动员猝死的首要病因。已有22个突变基因，超过400个突变位点与HCM的发病相关，其中，60％是由编码肌小节蛋白的基因发生突变造成。已有资料表明β-肌球蛋白重链(Arg403Gln，Arg453Cys，Arg719Trp，Arg723Gly)，肌钙蛋白T(Arg92Gln，Arg92Trp，Ile79Asn，Arg278Cys)，α-肌凝蛋白(Val95Ala)，肌球蛋白结合蛋白C(InsG791)和肌钙蛋白I(Lys183del)等突变伴运动猝死发生率较高。建立HCM基因突变动物模型是阐述HCM发生和运动致SCD的重要途径之一。本学科梯队用HCM病人心肌肌钙蛋白I突变(cTnI R145W，小鼠为cTnI R146W)，制备转基因小鼠。本论文将观察和探讨游泳与cTnI R146W基因阳性小鼠SCD发生率及可能机制。目的本研究采用个人基本情况调查、超声心动图、心电图、血浆肌钙蛋白I检测和基因筛查等方法，对江苏省优秀运动员进行赛前心血管疾病筛查，检测高危的心血管疾病，预防运动猝死。并利用心肌肌钙蛋白I突变位点cTnI R146W转基因小鼠(人为cTnI R145W)初步探讨运动诱发心肌病猝死相关机制。内容和方法一、优秀运动员心血管疾病筛查1研究对象：运动员(训练年限＞6年)2个人基本情况调查3临床检查：超声心动图、心电图和血浆肌钙蛋白I的检测4 cTnI基因突变位点检测二、cTnI R146W转基因小鼠传代和基因型鉴定三、运动对心肌肌钙蛋白I R146W转基因小鼠的影响1实验分组：8个月小鼠(4组)和2个月小鼠(5组)2运动负荷方案：运动起始一周，10min／次，1天两次，中间间隔4h，每天递增10min直至90min，以后90min／次，1天两次，中间间隔4h，持续总时间为7周。3超声心动图4心肌组织病理学检测：HE染色、Masson染色和电镜5 Western Blotting检测Genotype(+)和Genotype(-)小鼠心肌蛋白激酶C不同亚型(PKCβⅠ、PKCβⅡ和PKCγ)的表达变化四、统计学方法：数据均测量数据以平均数加标准差((?)±s)表示。组间差异采用独立样本t检验、单因素方差分析和x~2检验(SPSS 11.0)，P＜0.05有统计学意义。结果1优秀运动员心血管疾病筛查1．1 170名男子运动员超声未见明确心血管疾病。9名男子运动员发现轻微的瓣膜返流，包括二尖瓣，三尖瓣，肺动脉瓣和主动脉瓣。19名(10.9％)运动员LVDd≥60mm，最大上限为65.5mm。3名(1.7％)IVS＞13mm；181名女子运动员，18名(9.9％)运动员LVDd≥50mm；7名(3.84％)运动员LVDd≥55mm，最大上限为62.3mm。181名女子运动员未发现室壁厚度超过12mm。1．2 351名运动员，有16名(4.5％)表现为心电图异常，其中4名明显异常，12名表现为中度异常。另外335名(95.5％)运动员中，157例心电图完全正常，另有178例表现为轻度异常。1．3 351名运动员中，3名(1.8％)男子运动员和2(1.1％)名女子运动员的血浆cTnI值＞0.5 ng／ml。1．4微阵列芯片杂交定量荧光分析结果显示351名运动员cTnI基因第7外显子Cy3荧光强度介于6340.78-47540.32之间，Cy5荧光强度接近背景值介于489.28-964.17之间，Cy5／Cy3比率介于0.01-0.11，提示351名运动员cTnI基因第7外显子为4693C，均为野生型；351名运动员cTnI基因第8外显子Cy3荧光强度介于8870.93-58481.75之间，Cy5荧光强度介于711.72-1021.56之间，Cy5／Cy3比率介于0.01-0.11，提示351名运动员cTnI第8外显子为6488G，均为野生型。2运动对心肌肌钙蛋白I-R146W转基因小鼠的影响2．1 8个月小鼠游泳训练7周后，与Genotype(-)小鼠安静组相比，Genotype(-)小鼠游泳组左室心肌重量与体重之比增加了9％(4.46±0.39 VS 4.05±0.31mg/g，P＜0.05)，表明运动可诱导心肌发生生理性肥大。Genotype(+)小鼠游泳组左室的左室心肌重量与体重之比比Genotype(+)安静组，增加了27％(4.97±0.45 VS 3.95±0.52 mg/g，P＜0.05)；与Genotype(-)小鼠游泳组相比，Genotype(+)游泳组左室左室心肌重量与体重之比显著增加(P＜0.05)。2．2 M型超声心动图结果显示运动前8个月Genotype(+)和Genotype(-)小鼠AOd、LAD、IVSd和LVPWD均无显著性差异；但7周游泳训练后，Genotype(+)游泳组的AOd和LAD显著大于Genotype(-)运动组(P＜0.05)；7周运动前后，Genotype(+)小鼠LVDd显著大于Genotype(-)小鼠(P＜0.05)，Genotype(+)组小鼠的EF显著低于Genotype(-)组小鼠(P＜0.01)。2．3 8个月小鼠脉冲多普勒结果显示7周运动前后Genotype(+)小鼠二尖瓣瓣口的舒张早期血流速度E峰峰值流速显著低于Genotype(-)小鼠，而舒张血流速度A峰峰值流速基本维持不变，从而导致Genotype(+)小鼠E／A显著降低。组织多普勒超声结果显示Genotype(+)小鼠左室长轴切面左室后壁乳头肌位置心肌舒张早期的运动速度Ea以及Ea／Aa显著小于Genotype(-)小鼠。2．4 2个月小鼠脉冲多普勒结果显示Genotype(+)左室长轴切面或心尖四腔切面二尖瓣瓣口血流变化趋势与8个月Genotype(+)小鼠一致。组织多普勒超声结果显示Genotype(+)小鼠前后叶瓣环舒张早期的运动速度Ea显著小于Genotype(-)和C57B／6组小鼠(P＜0.05)，Ea／Aa显著小于Genotype(-)和C57B／6组小鼠，表明心肌舒张功能降低。2．5 7周运动期间，8个月Genotype(+)小鼠的死亡率为54％(13／24)，8个月Genotype(-)小鼠的死亡率为18％(4／22)。x~2检验结果显示8个月Genotype(+)小鼠的死亡率显著大于8个月Genotype(-)小鼠(P＜0.01)2个月Genotype(+)小鼠的死亡率为40％(6／15)，2个月Genotype(-)小鼠的死亡率为25％(4／16)。x~2检验结果显示2个月Genotype(-)小鼠和2个月Genotype(+)小鼠的死亡率无显著差异，但2个月Genotype(+)小鼠的死亡率呈增加趋势。2．6左室心肌心尖和室间隔部位HE染色结果显示：7周运动后，8个月和2个月Genotype(+)小鼠表现出心肌细胞肥大、细胞核增大、增生、畸形、肌浆溶解和肌丝模糊。左室心肌心尖和室间隔部位HE染色结果显示：7周运动后，8个月Genotype(+)小鼠心肌和室间隔部位出现许多纤维母细胞。左室心肌心尖和室间隔部位Masson染色结果显示：7周运动后，8个月Genotype(+)小鼠心尖和室间隔部均出现明显细胞间质纤维化。2．7 7周运动期间发生死亡8个月和2个月Genotype(+)小鼠左室心尖部见心肌排列紊乱。2．8 2个月Genotype(+)和Genotype(-)小鼠安静对照组心肌总蛋白、胞浆和结合蛋白PKCβⅠ、PKCβⅡ和PKCγ无显著差异，但8个月Genotype(+)未运动组心肌胞浆蛋白和结合蛋白PKCβⅠ，心肌胞总蛋白PKCβⅡ表达增加；7周运动后，2个月运动组Genotype(+)心肌总蛋白PKCβⅠ表达增加，而8个月运动组Genotype(+)小鼠心肌总蛋白和胞浆蛋白PKCβⅠ表达增加，心肌结合蛋白PKCβⅡ表达增加。结论1 351名运动员赛前筛查未检测到易发运动猝死的高危心血管疾病，如HCM、Marfan’s综合征、AVRC等。其原因可能是1)这些高危心血管疾病在本次入选运动员中的发病率较低。2)长期运动训练可导致左室发生重构现象，肥厚类似于HCM，本次所采用的筛查手段很难区分运动性心肌重构和HCM。2单基因芯片筛查有助于对HCM运动员进行早期诊断，对运动员心肌肥厚与HCM可进行鉴别诊断。3 Genotype(+)小鼠表现异常的心血管反应，死亡率比Genotype (-)小鼠高36％。舒张功能进一步损害。4 Genetype(+)小鼠运动后心肌细胞间质纤维化增加，心肌排列紊乱，部分心肌细胞断裂，心肌细胞核增大。以上病变可能是cTnI R146W阳性小鼠SCD的基础。5游泳后Genotype(+)小鼠心肌PKCβs表达发生变化，对于SCD发生通路和影响因素需进一步研究和证实。更多还原

【Abstract】 BackgroudThe causes of sudden cardiac death associated with exercise includeHypertrophic cardiomyopathy (HCM), Arrhythmogenic right ventricularcardiomyopathy, Congenital anomalies of coronary arteries, Pre-excitationsyndromes, Conduction diseases, Ion channel disease and so on. Suddencardiac death is usually the result of an interaction between structural lesions ofthe heart (’substrate’) and transient acute abnormalities (’trigger’). Themechanisms of exercise related sudden death in young competitive athletesinclude a number of triggers such as acute myocardial ischaemia, sympatheticstimulation and abrupt haemodynamic changes leading to lifethreateningventricular arrhythmias. HCM is the first cause in athletes under 35 years. Theforeign data showed that the annual incidence of sudden cardiovascular deathamong competitive athletes decreased from 3.6 to 0.4 deaths per 100,000. Thisreduction in mortality during organized training and competition was probablyattributable to the power of the screening program to identify athletes withcardiomyopathies. There are very difficult to distinguish LV physiologicalhypertrophy induced by long high intensity training from HCM. So it isimportant to establish efficacy of pre-participation screening program foridentification of cardiovascular diseases in competitive athletes. HCM is agenetically heterogeneous autosomal dominant inherited heart disease and hasbeen linked to at least 22genes (more than 400 mutations). Among thesemutations, 60% HCM was caused by sarcomeric gene mutation. Severalgenetic mutations have recently been associated with a high risk of suddendeath with the phenotype of normal or moderate hypertrophy, in particular,some mutations of theβ-myosin heavy chain gene (Arg403Gln, Arg453Cys,Arg719Trp, Arg723Gly), of the troponin T gene (Arg92Gln, Arg92Trp,Ile79Asn, Arg278Cys), ofα-Tropomyosin gene (Val95Ala), of the Cardiacmyosin binding protein C gene (InsG791)and of the troponin Igene(Lys183del). So it is an important research fields to develop the transgenicHCM animal models to shed light on this mechanisms. A novel mutation, cTnI Arginine145→tryptophan linking withhypertrophic cardiomyopathy among Chinese people was found in our institute.And the transgenic mice (cTnI R146W in the mouse sequence) animal modelwas established successfully. So this animal model was used to shed light onunderlying mechanisms of exercise induced sudden cardiac death.Object:To identify individuals at risk for cardiovascular diseases resulting frompre-participation screening of young competitive athletes to prevent the suddencardiac death. A prospective screening evaluation of elite athletes wasundertaken by including personal and family history, physical examination, 12lead electrocardiogram, echocardiography, the plasma cTnI levels and genetictesting. Furthermore, the cTnI R146W transgenic mice (cTnI R145W in thehuman sequence) were used to shed light on underlying mechanisms ofexercise induced sudden cardiac death.Materials and methods1. Cardiovascular pre-participation screening of young competitive athletes1) Selection of subjects: Athletes (the training time＞6 years)2) Personal and family history3) Clinical examination: 12 lead electrocardiogram, echocardiography andthe Plasma cTnI levels4) Genetic testing in cTnI mutations2. The cTnI R146W Genotype (+) and Genotype (-) mice were identified byPCR.3. The cardiac response to exercise in cTnI R146W transgenic mice1) Transgenic AnimalsThe 8-month-old mice were separated into four groups and the 2-month-oldmice were separated into five groups.2) Exercise protocol:Mice were initially swum for 10 min twice daily separated by 4 h and the duration increased by 10-min increments daily. Upon reaching 90 mintwice daily, this duration was maintained for the remainder of the 7-weekexercise period.3) Echocardiography4) Cardiac tissue was treated for histological examination. Sections werefixed, embedded and tissues stained with either hematoxylin and eosin orMasson’s trichrome.5) Protein expression of protein kinase C (PKCβⅠ、PKCβⅡ、PKCγ) inGenotype (+) and Genotype (-) mice was evaluated by Western blotting inthe total, cytosolic and particulate fractions, which were isolated from leftventricle.4. Statistics: All data are presented as mean±SD. The independent samples Ttest, One way ANOVA and chi-square test were used to analyze differencesand statistical tests were performed with the use of SPSS 13.0 statisticalsoftware. Statistical significance was accepted at P＜0.05.Results1 The results of cardiovascular pre-participation screening of youngcompetitive athletes1.1 A cardiovascular abnormality was not identified by echocardiography, ninemale athletes had echocardiographic evidence of relatively mild valveregurgitation, including mitral, tricuspid, pulmonary and aortic valve. Of the170 male athletes, nineteen (10.9%) athletes presented with an LVDd≥60mm with an upper limit of 65.5mm. Only three male athletes presentedwith wall thickness values＞13mm. The systolic (FS) and diastolic (E:A)function were within normal limits for all male athletes. A cardiovascularabnormality was not identified by echocardiography, four female athleteshad echocardiographic evidence of relatively mild valve regurgitation,including mitral, tricuspid, and aortic valve. Of the 181 female athletes, 18(9.9%) athletes presented with an LVDd≥50mm, and 7 (3.84%) athletespresented with an LVDd≥55mm, with an upper limit of 62.3mm. Nonewere found to have a maximum wall thickness greater than 12mm. The systolic (FS) and diastolic (E:A) function were within normal limits for allfemale athletes.1.2 Abnormal ECGs were identified in 16 athletes (4.5%); these included 4with distinctly abnormal and 12 with mildly abnormal patterns. Of the other335 athletes (95.5%), ECGs were completely normal in 157 athletes andshowed only minor alterations in 178 athletes.1.3 The plasma cTnI of most athletes were normal, only three of men (1.8%)and two of women (1.1%) have gone beyond the limits of cut off (cTnI＞0.5ng/ml).1.4 Image scanning and data processing showed that 351 samples yieldingstrongly fluorescing Cy3 spots gave fluorescence scores between 6340.78and 47540.32 fluorescent units. The same spots yielding very low Cy5signals and near background signals gave fluorescencescores between489.28 and 964.17 fluorescent units. Cy5/Cy3 ratios were between 0.01 and0.11. The results indicated that DNA from these samples only completelymatched with detector 4693CC. They showed homozygous wild types. Astrongly fluorescing Cy5 spots and a strongly ’yellow’ fluorescence were notfound in all samples. The same results were found for G6488A locus inathletes. 351 samples yielding strongly fluorescing Cy3 spots gavefluorescence scores between 8870.93 and 58481.75 fluorescent units. Thesame spots yielding very low Cy5 signals and near background signals gavefluorescence scores between 711.72 and 1021.56 fluorescent units. Cy5/Cy3ratios were between 0.01 and 0.11. They also showed homozygous wildtypes.2 The results of the cardiac response to exercise in cTnI R146W transgenicmice2.1 At the end of the 7 weeks exercise training, compared with the Genotype (-)sedentary group (8-month-old), while Genotype (-) mice had an average of a9% increase the ratio of LV to body weight in response to exercise (Genotype (-) sedentary group: 4.05±0.31VS swim group: 4.46±0.39.P＜0.05), which showed that exercise could induce cardiac physiologicalhypertrophy. However, compared with the Genotype (+) sedentary group(8-month-old), the Genotype (+) exercise mice had an average of a 27%increase (Genotype (+) sedentary group: 3.95±0.52VS Genotype (+)exercise group: 4.97±0.45. P＜0.05). And the ratio of LV to body weight inthe Genotype (+) exercise group increased significantly than Genotype (-)exercise group.2.2 M-mode echocardiographic data showed that before exercise training, nosignificant differences were found in AOd、LAD、IVSd and LVPWD inGenotype (+) and Genotype (-) mice. However, after 7 weeks of swimmingtraining, the Genotype (+) mice significantly increased AOd and LAD(P＜0.05). Before and after 7 weeks exercise training, the LVDd in Genotype(+) mice increased significantly than Genotype (-) mice (P＜0.05), while theEF and FS in Genotype (+) mice decreased significantly than Genotype (-)mice (P＜0.05).2.3 Before and after 7 weeks exercise training, transmitral pulsed Dopplermeasurements showed that compared with the Genotype (-)mice(8-month-old), there was a significant decrease in Peak E in Genotype(+) mice (P＜0.05), and no significant differences were found the in Peak Ain Genotype (-) and Genotype (+) mice, which led to significant decrease theE/A Genotype (+) mice(P＜0.01). Doppler tissue Imaging (DTI) data showedthat compared with the Ea in Genotype (-) mice (8-month-old) within the LVposterior myocardial wall at the level of the papillary muscles, there weresignificant decrease Ea and Ea/Aa in Genotype (+) mice (P＜0.01).2.4 Similarly, these changes were found transmitral pulsed Dopplermeasurements acquired in the parasternal long-axis view and four chamberview in Genotype (+) mice (2-month-old). DTI data showed that comparedwith the Ea in Genotype (-) group MA Lateral TDI, there was a significantdecrease in Ea and Ea/Aa in Genotype (+) mice (P＜0.001). Similarly, thesechanges were found in MA septal TDI in Genotype (+) mice. 2.5 At the end of the 7-wk follow-up period, the rate of death in Genotype (+)mice swim group (8-month -old) was 54% (13/24), and the rate of death inGenotype (-) mice swim group (8-month -old) was 18% (4/22). Comparedwith the Genotype (-) mice swim group, the Genotype (+) mice swim groupdisplayed a significantly increased rate of death by x~2 analysis. The rate ofdeath in Genotype (+) mice swim group (2-month -old) was 40% (6/15), andthe rate of death in Genotype (-) mice swim group (2-month-old) was25%(4/16). Compared with the Genotype (-) mice swim group, theGenotype (+) mice swim group displayed no significantly increased rate ofdeath by x~2 analysis. But the rate of death in Genotype (+) mice swim groupshowed a tendency of increase.2.6 The histological analysis from hematoxylin and eosin in the cardiac apexand interventricular septum showed that at the end of exercise, Genotype (+)mice(8-month and 2-month-old) exhibited hypertrophied myocytes withlarge hyperchromatic nuclei, nuclei hyperplasy, nuclei anisotrophy, seriousdropsy, carcoplasm solution and cardiac muscle break. The cardiachistological results from hematoxylin and eosin also showed that at the endof exercise Genotype (+) mice (8-month) exhibited a lot of fibroblast. Andhistological results from Masson’s Trichrome in the cardiac apex andinterventricular septum showed that at the end of exercise Genotype (+)mice (8-month) exhibited serious interstitial fibrosis.2.7 During the 7-wk exercise, the Genotype (+) mice (8-month and 2 month)death in exercise showed cardiomyocyte disarray.2.8 Before 7 weeks training, there were no significant differences in threeisozymes PKC (PKCβⅠ, PKCβⅡand PKCγ) in the total, cytosolic andparticulate fractions between Genotype (-) mice (2-month-old) andGenotype (+) mice (2-month-old). However, at 8 months mice, theexpression of PKC-βⅠ,βⅡwere associated preferentially with the cytosolicor particulate fraction in left ventricle in Genotype (+)mice. After 7 weeks swimming, the expression of PKC-βⅠin total fractions was upregulated in 2months Genotype (+) mice, and the expression of PKC-βⅠin total fractionsandβⅡin particulate fractions were upregulated in 2 months or 8 monthsGenotype (+) mice.Conclusions1 This screening protocol identified no athletes (351) with definite evidence ofhypertrophic cardiomyopathy, Marfan’s syndrome or other cardiovasculardiseases that convey a significant potential risk for sudden death or diseaseprogression during athletic activity. This is largely due to the relative lowprevalence of conditions resulting in sudden cardiac death in young athletesand high false positive/negative rates in the tests used as part of thescreening process.2 Genetic testing is help to screen HCM in athletes early and distinguish theathlete’s heart and HCM in athletes.3 The cTnI-R145W Genotype (+) mice showed an abnormal response toexercise. Compared with Genotype (-) mice swim group (8-month -old), thedeath rate of the Genotype (+) mice increased by 36%. Exercise canaggravate the diastolic function of Genotype (+) mice.4 The cause of increasing the rate of death in Genetype(+) mice (8-month-old)who died suddenly during exercise may largely due to the greater fibrosis,cardiomyocyte disarray, cardiomyocyte fragmentation and largehyperchromatic nuclei induced by exercise. Finally, these changes can alsolead to the Genetype(+) mice (8-month-old) death during exercise.5 The exercise can lead to the change of expression of PKCβs in Genetype(+)mice, However, the mechanisms of SCD is still unclear.更多还原