异常温度刺激影响心脏疾病的Bim相关机制及干细胞移植干预
|
摘要
背景：
近年来世界范围内极端气候事件频发,如严重冰雪灾害及反常高温暑热等。极端气候事件导致心血管疾病的病情加重,引起发病率和死亡率增高。针对寒冷与暑热刺激对心脏疾病产生的具体影响,及其在已有基础病变的心肌组织内所产生的具体作用,包括相关的细胞生物学、分子生物学机制,尚缺乏系统的研究报道。在寒冷及暑热环境下,患有心脏疾病个体的心脏功能恶化程度,心肌组织病变加重的具体病理学与解剖学参数,以及相关生化指标的变化,亦需要进行具体的量化分析。以便为明确寒冷及暑热刺激下心血管疾病的变化规律提供参考数据和资料。
心肌细胞损伤可体现在许多方面,包括细胞结构完整性破坏、线粒体能量代谢障碍、细胞内活性氧自由基(ROS)爆发、钙离子超载以及细胞凋亡改变等。冷／热刺激所造成的心肌细胞损伤,可能在上述各个方面有所体现。对冷／热刺激所造成心肌细胞损伤的方式进行观察分析,有助于在细胞水平展现冷／热刺激加重心肌病变的特点及规律。
心肌细胞凋亡是心肌损伤的重要病理进程之一。线粒体跨膜电位ΔΨm下降则是细胞凋亡级联反应过程中的起始事件。ΔΨm崩溃导致电子传递链中断,氧化磷酸化停止,最终改变细胞电化学氧化还原状态并引起细胞凋亡蛋白酶Caspase激活物释放,激活Caspase-9、Caspase-3导致细胞凋亡或死亡。关于冷／热刺激导致心肌细胞损伤是否与线粒体途径有关,需要研究证实。细胞内ROS爆发是心肌细胞损伤和凋亡的重要机制,而对于冷／热应激是否引起心肌细胞内ROS爆发,及其可能机制尚不明了。还原型烟酰胺腺嘌呤二核苷酸磷酸(NADPH)氧化酶复合物是体内活性氧ROS的重要来源之一,NADPH由多个不同的亚单位组成。其中最主要的是nox-1、p22-phox。冷／热应激在心肌细胞中是否引起nox-1、p22-phox的表达变化,及其所提示的ROS激活程度,有待实验加以探讨。
Bim是Bcl-2家族中只含BH3结构功能域(BH3-only)的重要促凋亡蛋白,广泛分布于机体各种组织,与细胞凋亡的调控功能有关。在不同的组织细胞,以及不同的凋亡诱导条件下,Bim蛋白表达的调节机制可能不同。目前,关于Bim蛋白在心肌细胞凋亡中所起到的作用尚报导不多。作为调节细胞凋亡的关键蛋白之一,Bim在冷／热刺激诱导的心肌细胞凋亡进程中,也极可能发挥着重要调控作用,并可能影响到心肌细胞结构完整性的维持、线粒体能量代谢、细胞内ROS的产生、钙离子超载等诸多方面。探究冷／热刺激下Bim介导心肌细胞损伤的机制,将对从凋亡角度深入认识冷／热刺激诱导心肌细胞损伤的具体机制,产生重要理论意义。
Bim可能与多种经典的凋亡相关信号通路有着密切关联。明晰冷／热刺激下Bim介导的心肌细胞损伤的信号传导途径及关联,可能在分子生物学水平提示新颖而有效的治疗干预靶点。Bim对心肌细胞凋亡的调控可能涉及众多信号分子途径,包括PI3K/Akt/GSK-3β信号途径、ERK信号途径等。这些信号通路其是否参与调节冷／热刺激诱导心肌损伤,及其与Bim表达的关系,目前尚未见报道。故此,Bim蛋白在冷／热刺激诱导心肌损伤中的作用机制以及调节机制,以及Bim与PI3K/Akt/GSK-3β信号途径、ERK信号途径中的相互作用,立为本课题的研究内容。
干细胞移植修复心肌损伤的效果,已经众多实验与临床研究证实。骨髓间充质干细胞(BM-MSCs)是目前常用于细胞移植治疗的组织工程种子细胞。BM-MSCs可能通过多种途径改善并恢复缺血梗死状态下的心肌功能。在极端气候条件下,对已有基础缺血病变的心肌组织预先行BM-MSCs移植干预,是否能够提升其对极端气候条件的耐受性,及其潜在的具体机制,尚未有研究涉及,也作为本研究探讨的内容之一。
方法：
在RXZ-300A型智能人工气候箱的基础上,建立模拟寒冷／暑热环境的模型装置。对兔心肌梗死动物模型进行寒冷／暑热干预。实验分组：Ⅰ、常温组(MI),Ⅱ、寒冷组(MI+Cold),Ⅲ、暑热组(MI+Heat)。标准化法测定心肌梗死面积变化情况；超声心动图测定心脏功能；生理记录仪测定血流动力学指标；梗死区心肌组织切片苏木精-伊红(HE)染色评价病理学改变；Real-time PCR法检测心肌组织内iNOS和eNOS mRNA表达水平；western blot检测梗死区心肌组织Bim蛋白的表达水平。同时,对大鼠心脏肥大模型进行寒冷／暑热干预。实验分组：Ⅰ、常温组(H)Ⅱ、寒冷组(H+Cold)Ⅲ、暑热组(H+Heat)。超声心动图测定寒冷／暑热干预后心脏功能与结构变化情况；多导生理记录仪测定血流动力学参数。
建立心肌细胞冷／热干预模型。对体外培养的新生乳鼠心肌细胞进行冷／热刺激干预,同时结合Bim siRNA干扰。实验分组：Ⅰ、正常对照组(Control),Ⅱ、冷刺激组(Cold),Ⅲ、热刺激组(Heat),Ⅳ、Bim siRNA+冷刺激组(BimsiRNA+Cold),Ⅴ、Bim siRNA+热刺激组(Bim siRNA+Heat),Ⅵ、非编码siRNA+冷刺激组(Non-coding siRNA+Cold),Ⅶ、非编码siRNA+热刺激组(Non-coding siRNA+Heat)。检测心肌细胞培养基中乳酸脱氢酶(LDH)活性；MTS法检测心肌细胞存活率；流式细胞仪检测心肌细胞内钙离子水平、ROS水平、线粒体膜电位水平；TUNEL法及AnnexinⅤPI荧光探针法检测心肌细胞凋亡情况；western blot检测心肌细胞中Bim蛋白的表达水平变化。
对体外培养的新生乳鼠心肌细胞进行冷／热干预,同时结合Bim siRNA干扰。实验分组：Ⅰ、正常对照组(Control),Ⅱ、冷刺激组(Cold),Ⅲ、热刺激组(Heat),Ⅳ、Bim siRNA+冷刺激组(Bim siRNA+Cold),Ⅴ、Bim siRNA+热刺激组(Bim siRNA+Heat),Ⅵ、非编码siRNA+冷刺激组(Non-coding siRNA+Cold),Ⅶ、非编码siRNA+热刺激组(Non-coding siRNA+Heat)。Western blot检测Caspase-9、nox-1、p22-phox蛋白表达水平,以及ERK与磷酸化ERK、PI3K与磷酸化PI3K、GSK-3β与磷酸化GSK-3β蛋白的表达水平。
对体外培养的新生乳鼠心肌细胞进行冷／热刺激,并结合ERK通路阻断剂PD98059、PI3K/Akt通路阻断剂LY294002干预。实验分组：Ⅰ、对照组(Control),Ⅱ、冷刺激组(Cold),Ⅲ、热刺激组(Heat),Ⅳ、冷刺激+LY294002组(Cold+LY294002),Ⅴ、热刺激+LY294002组(Heat+LY294002),Ⅵ、冷刺激+PD98059组(Cold+PD98059),Ⅶ、热刺激+PD98059组(Heat+PD98059)。Western blot检测Bim蛋白的表达水平。
骨髓穿刺法获得兔骨髓液,体外分离培养骨髓间充质干细胞(BM-MSCs)。建立兔心肌梗死模型,于梗死区域心肌组织内直接注射法进行自体干细胞移植。细胞移植后行寒冷／暑热干预。实验分组：Ⅰ、心梗+寒冷干预组(MIC),Ⅱ、心梗+暑热干预组(MIH),Ⅲ、心梗+细胞培养基注射(对照)+寒冷干预组(MIC+M),Ⅳ、心梗+细胞培养基注射(对照)+暑热干预组(MIH+M),Ⅴ、心梗+BM-MSCs移植+寒冷干预组(MIC+SCs),Ⅵ、心梗+BM-MSCs移植+暑热干预组(MIH+SCs)。标准化法测定心肌梗死面积；超声心动图检测心脏功能参数；western blot检测心肌组织内Bim蛋白表达水平变化。
结果：
心肌梗死动物模型接受寒冷／暑热干预后,寒冷组(MI+Cold)与暑热组(MI+Heat)的心肌梗死面积,较常温组(MI)均有扩大(p<0.01,MI:36.27±4.43%vs.MI+Cold:46.74±3.21%：MI+Heat:42.62±2.80%)；寒冷／暑热干预后,心肌梗死兔的左室舒张末径(LVEDD)与左室收缩末径(LVESD)较常温组增大；左室后壁厚度(LVWT)和收缩期室间隔厚度(IVST)较常温组减小；左室射血分数(LVEF)较常温组下降。寒冷／暑热组的左室收缩末压(LVESP)低于常温组；左室舒张末压(LVEDP)高于常温组；左室内压上升最大速率(+dp/dtmax)和左室内压下降最大速率(-dp/dtmax)皆较常温组明显降低。组织切片HE染色结果显示,寒冷组及暑热组的梗死部位心肌纤维组织增生,心肌细胞结构病理性改变均较常温组严重。寒冷／暑热干预后,寒冷刺激组的梗死心肌组织中iNOSmRNA表达水平约为无刺激心梗组的2倍；eNOS mRNA表达水平约为无刺激心梗组的60%。暑热刺激组的梗死心肌组织中iNOS mRNA表达水平约为无刺激心梗组的1.5倍；eNOS mRNA表达水平约为无刺激心梗组的40%。寒冷／暑热干预后,梗死区心肌组织中的Bim蛋白表达水平均增高(MI+Cold:0.59±0.07；MI+Heat:0.46±0.01;vs. MI:0.34±0.04, p<0.05)。
心脏肥大动物模型接受寒冷／暑热干预后,寒冷组和暑热组的左室后壁舒张末期厚度(LVPWTd)、室间隔舒张末期厚度(IVSTd)、左室舒张末期内径(LVDd),较常温组均有增加,提示心脏肥大加重。寒冷组和暑热组的左心室收缩压(LVSP),左心室压力变化最大速率(±dp/dtmax),较常温组均有增加,提示由心脏肥大引起的心脏收缩反常及左心室内高压进一步恶化。
冷／热刺激干预后,心肌细胞培养液中LDH活性明显增高(Cold:105.15±6.98 IU/L; Heat:96.49±5.94 IU/L vs. Control:41.09±4.59 IU/L, p<0.01)。针对Bim进行RNA干扰后再施加冷／热刺激干预,心肌细胞培养液中的LDH活性升高幅度较单纯冷／热刺激干预组有所下降(p<0.01,Bim siRNA+Cold:81.56±5.38 IU/L; Bim siRNA+Heat:62.79±3.90 IU/L)。冷／热刺激干预后,心肌细胞存活率显著下降(Cold:65.46±4.56%; Heat:68.56±3.92% vs. Control:90.13±5.01%, p<0.01)。针对Bim进行RNA干扰,冷刺激干预下心肌细胞存活率有所提升(p<0.01,BimsiRNA+Cold:80.65±3.05%),而对热刺激下心肌细胞存活率无明显影响(p>0.05，Bim siRNA+Heat:76.37±5.61%)。冷/热刺激干预后,心肌细胞内钙离子聚积加重(Cold:625±27 nmol/L; Heat:588±49 nmol/L vs. Control:361±52 nmol/L,p<0.01)。针对Bim进行RNA干扰下调,于冷/热干预下都能缓解心肌细胞内钙离子水平的增高(p<0.01, Bim siRNA+Cold:619±33 nmol/L; Bim siRNA+Heat: 591±61 nmol/L)。冷/热刺激干预后,心肌细胞内活性氧水平明显升高(Cold: 10.8±0.85;Heat:9.92±0.93 vs. Control:3.61±0.71, p<0.01)。接受Bim RNA干扰的实验组心肌细胞内活性氧水平较单纯冷/热刺激干预组明显下降(p<0.01,BimsiRNA+Cold:4.82±0.48; Bim siRNA+Heat:4.80±0.51)。
心肌细胞线粒体膜电位ΔΨm水平流式细胞仪检测结果显示：冷/热刺激干预后,心肌细胞线粒体膜电位ΔΨm水平明显降低(Cold51.32±5.57；Heat: 47.18±5.11 vs.Control:83.29±7.20, p<0.01)。RNA干扰下调Bim的实验组心肌细胞线粒体膜电位ΔΨm水平,较单纯冷/热刺激干预组的下降幅度明显减小(p<0.05, Bim siRNA+Cold:69.26±7.13; Bim siRNA+Heat:70.08±6.56)。心肌细胞线粒体膜电位荧光显微镜检测结果显示：冷/热刺激干预组心肌细胞的绿色荧光强于对照组,而红色荧光则减弱,表明线粒体跨膜电位受到破坏。Bim RNA干扰预处理后,心肌细胞绿色荧光强度减弱,红色荧光有所增强,提示通过RNA干扰下调Bim后,可减轻冷/热刺激所带来的心肌细胞线粒体ΔΨm下降。TUNEL检测和流式细胞仪AnnexinⅤPI检测结果均显示,冷/热刺激使心肌细胞凋亡率明显上升,而通过RNA干扰下调Bim后,可减轻冷/热刺激所造成的心肌细胞凋亡程度。
冷/热刺激干预后,心肌细胞内Bim蛋白表达水平明显升高(Cold:1.14±0.08; Heat:1.05±0.08 vs. Control:0.20±0.03, p<0.01)。RNA干扰下调Bim的实验组心肌细胞Bim蛋白表达水平较单纯冷/热刺激干预组显著下降(p<0.01,BimsiRNA+Cold:0.47±0.05;Bim siRNA+Heat:0.32±0.05)。
冷/热刺激干预后,心肌细胞Caspase-9蛋白表达水平显著上升(Cold: 1.05±0.09;Heat:1.38±0.13 vs.Control:0.11±0.01, p＜0.01)。针对Bim进行RNA干扰,则冷/热刺激干预下心肌细胞内升高的Caspase-9表达有所降低(p<0.01,BimsiRNA+Cold:0.22±0.01; Bim siRNA+Heat:0.32±0.01)。冷/热刺激干预后,心肌细胞nox-1蛋白表达水平有所上升(Cold:1.05±0.09; Heat:1.38±0.13 vs. Control:0.11±0.01,p<0.01)。针对Bim进行RNA干扰,热刺激干预下心肌细胞内升高的nox-1表达有所降低(p<0.01,Bim siRNA+Heat:0.32±0.01 vs. Heat:1.04±0.01)。冷／热刺激干预后,心肌细胞p22-phox蛋白表达增强(Cold:1.35±0.06; Heat: 1.58±0.03 vs. Control:0.39±0.01, p<0.01)。RNA干扰下调Bim后,可缓解冷／热刺激造成的心肌细胞内p22-phox高表达(p<0.01,Bim siRNA+Cold:0.87±0.03; Bim siRNA+Heat:1.05±0.06)。
冷／热干预刺激后,ERK5和p-ERK5蛋白表达均增高。通过Bim siRNA下调Bim表达后,冷刺激组心肌细胞内ERK5和p-ERK5蛋白表达均有所下降；热刺激组心肌细胞内ERK5无明显变化,但p-ERK5的表达下降。冷／热干预刺激后,心肌细胞内PI3K与p-PI3K蛋白表达略有增高趋势,但未观察到有统计学意义的改变(p>0.05)。通过Bim siRNA下调Bim后,冷／热刺激组心肌细胞内PI3K与p-PI3K蛋白表达亦无明显变化(p>0.05)。冷／热干预刺激后,未观察到心肌细胞内GSK-3β蛋白表达水平有统计学意义的改变(p>0.05)。通过BimsiRNA下调Bim后,冷／热刺激组心肌细胞内GSK-3β蛋白表达亦无明显变化。冷热干预刺激后,心肌细胞内p-GSK-3β蛋白水平均增高(p<0.01)。通过BimsiRNA下调Bim后,冷／热刺激组p-GSK-3β蛋白水平有所下降(p<0.01,Cold：1.15±0.06 vs. Bim siRNA+Cold:0.89±0.06; Heat:1.05±0.09 vs. Bim siRNA+Heat: 0.83±0.03).
LY294002干预对冷／热刺激下心肌细胞内Bim蛋白表达水平无明显影响。PD98059干预在冷刺激组使Bim蛋白表达有所增高(p<0.05,Cold：0.57±0.03 vs.Cold+PD98059:0.71±0.04)。PD98059干预在热刺激组使Bim蛋白表达显著增高(p<0.01, Heat:0.59±0.05 vs. Heat+PD98059:0.87±0.03)。
预先干细胞移植,再接受寒冷／暑热刺激后的心梗面积参数,与未接受干细胞移植实验组接受寒冷／暑热刺激后的心梗面积参数之间无统计学差异。预先干细胞移植组,在寒冷／暑热干预下,左室射血分数均有改善。预先接受细胞移植,可减轻寒冷／暑热刺激造成的心肌细胞内凋亡蛋白Bim的表达增高(p<0.01,MIC+SCs:0.52±0.02 vs. MIC:0.83±0.02; MIH+SCs:0.76±0.01 vs. MIH:0.85±0.02).
结论：
寒冷／暑热干预可导致心肌梗死基础病变个体的梗死面积扩大,心脏收缩功能和舒张功能进一步恶化,并加重心肌组织坏死及凋亡；寒冷／暑热干预可通过诱导iNOS mRNA表达水平增高,eNOS mRNA表达水平降低,对心肌细胞产生损伤；寒冷／暑热干预可导致心脏肥大结构性病变和血流动力学紊乱程度加重；冷／热刺激通过引起心肌细胞内钙离子超载、ROS水平增高,以及线粒体膜电位降低,造成体外培养心肌细胞存活率下降、凋亡率上升；冷／热刺造成的损伤效应,与心肌细胞内促凋亡蛋白Bim的表达水平相关联。Bim可能介导了冷／热刺激对心肌细胞的损伤；Bim可能通过诱导Caspase-9表达增高,nox-1以及p22-phox蛋白合成增加,上调心肌细胞内ROS水平,介导冷／热刺激对心肌细胞的致凋亡效应；ERK5途径可反馈性抑制冷／热刺激造成的心肌细胞内促凋亡蛋白Bim的表达增高,发挥保护性作用。PI3K/Akt/GSK-3β信号通路可能调控冷／热刺激造成的心肌细胞内促凋亡蛋白Bim表达；BM-MSCs移植可减轻冷／热刺激造成的心肌梗死基础病变个体的心功能恶化,并可能通过下调促凋亡蛋白Bim的表达发挥保护性作用。
Background:
In recent years, extreme climate events occurred around the world frequently, such as severe snow or freezing weather disaster and the extraordinary summer heat. Extreme climate events can lead to aggravation of cardiovascular disease and higher morbidity and mortality. About the specific effects of cold and heat stress on heart disease, and the role of cold and heat stress in damage procedure in myocardial tissue, including relevant cellular biologic and molecular mechanisms, still lack of systematic research report yet. As to individuals with heart disease under cold and heat stress conditions, the deterioration of cardiac function, pathologic and anatomical parameters of myocardium damage, and related biochemical alterations, also require quantitative analysis, so as to provide data and information about the effects of cold and heat stress on cardiovascular disease.
Cardiomyocytes injury can be manifested in many ways, including the damage of cell structural integrity, mitochondrial energy metabolism disorder, intracellular reactive oxygen species (ROS) outbreak, calcium overload and apoptosis. Myocardial cell injury caused by cold/heat stimulation may be reflected in all aspects of the above. Analysis of myocardial cell damage caused by cold/heat stimulation in various ways of observation may help to demonstrate characteristics of aggravation of cardiac disease induced by cold/heat stimulation at the cellular level.
Cardiomyocyte apoptosis is one of important pathological processes of myocardial injury. Decrease of mitochondrial membrane potentialΔψm is the initiative event of apoptosis cascade procedure. Collapse ofΔψm leads to interruption of electron transport chain, arrest of oxidative phosphorylation, and eventually changes the electrochemical redox state of cells, and induces apoptosis protease-caspase activator release, then activation of Caspase-9, Caspase-3 lead to cell death or apoptosis. Study is needed to clarify that whether there is some relationship between cold/heat stimulation induced myocardial injury and the mitochondrial pathway. The outbreak of intracellular ROS is important mechanism in cardiomyocyte apoptosis. But in what way cold/heat stress to cause the outbreak of intracellular ROS is remained unclear. Reduced form of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase complex is major source of ROS in vivo. NADPH is composed by a number of different subunits. Most important ones among those subunits are nox-1 and p22-phox. Whether cold/heat stress causes expression alternations of nox-1 and p22-phox in cardiac cells, and the subsequence ROS activation, also needs experimental confirm.
Bim is member of Bcl-2 family that with only BH3 domain (BH3-only), and it's considered as important pro-apoptotic protein. Bim is widely distributed through the body in various organizations, and performs the function as apoptosis regulator. In different cells and different apoptosis-inducing conditions, the regulation mechanism of Bim protein expression may be different. Currently, role of Bim protein in cardiomyocytes apoptosis hasn't been reported frequently yet. As one of the key protein that regulating cell apoptosis, Bim may plays significant role in cold/heat stimulation-induced myocardial cell apoptosis, and likely to regulate maintenance of cellular structure integrity, mitochondrial energy metabolism, intracellular ROS generation, calcium overload, and many other aspects in myocardial cell. To explore the role of Bim in cold/hot stimulation induced myocardial cell injury may shed some new light on the understanding of this pathologic procedure from in-depth apoptotic attitude, and may generate important theoretical significance.
Bim may be closely associated with a variety of classic apoptosis-related signaling pathway. To investigate signal transduction pathway of Bim-mediated cold/ heat stimulation induced myocardial injury at level of molecular biology may suggest novel and effective therapeutic intervention target. Regulation to myocardial apoptosis performed by Bim may involve a number of signaling molecules, including PI3K/Akt/GSK-3β, ERK and other pathway. Whether these signaling pathways involved in the regulation of cold/heat stimulation induced myocardial injury and their relationship with the expression of Bim, has not been reported yet. Therefore, the role of Bim protein in the cold/heat stress induced myocardial injury and relative molecular mechanism, as well as interaction between PI3K/Akt/GSK-3βsignal pathway, ERK signaling pathway, will be established as topic of this current research.
Many studies have confirmed the beneficial effects of stem cells transplantation on repair of myocardial injury. Mesenchymal stem cells (BM-MSCs) were commonly used in cell transplantation therapy and tissue engineering. BM-MSCs can improve and restore the cardiac function under state of myocardial ischemia or infarction in many ways. In extreme weather conditions, whether pretreatment by BM-MSCs transplantation intervention can enhance the tolerance of individuals suffering from myocardial infarction (MI) to cold/heat stress, as well as what possible specific mechanisms may be involved, have not been reported yet and also as one of the aspects to be investigated in this study.
Methods:
Based on the RXZ-300A artificial mimic climate chamber, we established model unit of cold/heat environmental stress stimulation. Animal models of myocardial infarction were given the cold/heat stress. Experimental groups were designed as:Ⅰ. Normal temperature group (MI),Ⅱ. Cold group (MI+Cold),Ⅲ. Heat group (MI+Heat). Size of myocardial infarct area was determined by standard staining method; Cardiac function was assessed by echocardiography; Parameters of hemodynamic were recorded via physiological recorder; Pathological characteristics of myocardium was evaluated by hematoxylin-eosin (HE) staining of myocardial tissue section; iNOS and eNOS mRNA expression in myocardial tissue were detected via real-time PCR; Myocardial expression of Bim protein was detected by western blot. In addition, animal model of cardiac hypertrophy were given the cold/heat stress as well. Groups were designed as:Ⅰ. Normal temperature group (H),Ⅱ. Cold group (H+Cold),Ⅲ. Heat group (H+Heat). Echocardiography and detection by multi-channel physiological recorder were performed after the cold/heat stress intervention, to evaluated cardiac function and parameters of hemodynamic.
We established cellular cold/heat stimulation model in vitro. Neonatal rat myocardial cells were cultured to accept cold/heat stimulation, combined with Bim siRNA pretreatment. Experimental groups were designed as:Ⅰ. Control group (Control),Ⅱ. Cold stimulation group (Cold),Ⅲ. Heat stimulation (Heat),Ⅳ. Bim siRNA+Cold stimulation group (Bim siRNA+Cold),Ⅴ. Bim siRNA+Heat stimulation (Bim siRNA+Heat),Ⅵ. Non-coding siRNA+Cold stimulation (Non-coding siRNA+Cold),Ⅶ. Non-coding siRNA+Heat stimulation (Non-coding siRNA+Heat). Lactate dehydrogenase (LDH) activity in cell culture medium was detected; viability of cardiomyocytes was evaluated by MTS assay; levels of intracellular calcium, reactive oxygen species, mitochondrial membrane potential were detected via flow cytometry; TUNEL and AnnexinⅤPI fluorescence probe detection were performed to evaluate myocardial cell apoptosis; Bim protein expression in cardiomyocytes was analyzed by western blot detection.
Neonatal rat myocardial cells were cultured to accept cold/heat stimulation, combined with Bim siRNA pretreatment. Experimental groups were designed as:Ⅰ. Control group (Control),Ⅱ. Cold stimulation group (Cold),Ⅲ. Heat stimulation (Heat),Ⅳ.Bim siRNA+Cold stimulation group (Bim siRNA+Cold),Ⅴ. Bim siRNA+Heat stimulation (Bim siRNA+Heat),Ⅵ. Non-coding siRNA+Cold stimulation (Non-coding siRNA+Cold),Ⅶ. Non-coding siRNA+Heat stimulation (Non-coding siRNA+Heat). Protein expression of Caspase-9, nox-1, p22-phox, ERK and phosphorylated ERK, PI3K and phosphorylated PI3K, GSK-3βand phosphorylated GSK-3βwere detected by western blot.
Neonatal rat myocardial cells were cultured to accept cold/heat stimulation, combined with ERK pathway inhibitor-PD98059 or PI3K/Akt pathway inhibitor-LY294002 intervention. Experimental groups were designed as:Ⅰ. Control group (Control),Ⅱ. Cold stimulation group (Cold),Ⅲ. Heat stimulation group (Heat),Ⅳ. Cold stimulation+LY294002 group (Cold+LY294002),Ⅴ. Heat stimulation+LY294002 group (Heat+LY294002),Ⅵ. Cold stimulation+PD98059 group (Cold+PD98059),Ⅶ. Heat stimulation+PD98059 group (Heat+PD98059). Expression of Bim protein was detected by western blot.
Autologous bone marrow mesenchymal stem cells (BM-MSCs) were cultured from bone marrow of rabbit. Stem cell transplantation was performed via direct injection into infarcted area in heart of rabbit MI model. Then animals underwent cold/heat stress. Groups were designed as:Ⅰ. MI+Cold intervention group (MIC), Ⅱ. MI+Heat intervention group (MIH),Ⅲ. MI+cell culture medium injection (control)+Cold intervention group (MIC+M),Ⅳ. MI+cell culture medium injection (control)+Heat intervention group (MIH+M),Ⅴ. MI+BM-MSCs transplantation+ Cold intervention group (MIC+SCs),Ⅵ. MI+BM-MSCs transplantation+Heat intervention group (MIH+SCs). Size of myocardial infarct area was determined by standard staining method; cardiac function was assessed by echocardiography; expression of Bim protein was detected by western blot.
Results:
After animal model of myocardial infarction accepted cold/heat stress intervention, size of myocardial infarcted area in MI+Cold group and MI+Heat group were expanded compared with MI group (p<0.01, MI:36.27±4.43%vs. MI+Cold: 46.74±3.21% & MI+Heat:42.62±2.80%). After the cold/heat stress intervention, MI rabbit's left ventricular end diastolic diameter (LVEDD) and left ventricular end systolic diameter (LVESD) were increased compared with normal temperature group; left ventricular posterior wall thickness (LVWT) and systolic interventricular septal thickness (IVST) were decreased compared with normal temperature group; left ventricular ejection fraction (LVEF) was significantly decreased compared with normal temperature group. Left ventricular end systolic pressure (LVESP) in Cold/ Heat stress group was lower than normal temperature group; left ventricular end diastolic pressure (LVEDP) was higher than normal temperature group; maximal rate of left ventricular pressure rise (+dp/dtmax) and left maximal rate of ventricular pressure decline (-dp/dtmax) were significantly lower than normal temperature group. Tissue sections HE staining showed that the fibrosis of infarcted area and pathological changes of myocardial structure in Cold/Heat group were more serious than that in normal temperature group. After Cold/Heat stress intervention, the iNOS mRNA expression in infarcted myocardium in MI+Cold group was about 2 times of that in MI group; eNOS mRNA expression was about 60% of that in MI group. The iNOS mRNA expression in infarcted myocardium in MI+Cold group was about 1.5 times of that in MI group; eNOS mRNA expression was about 40% of that in MI group. After Cold/Heat stress intervention, Bim protein expression level in infarcted myocardium was increased (MI+Cold:0.59±0.07; MI+Heat:0.46±0.01; vs. MI: 0.34±0.04, p<0.05).
After animal model of cardiac hypertrophy accepted cold/heat stress intervention, end-diastolic left ventricular posterior wall thickness (LVPWTd), end-diastolic interventricular septal thickness (IVSTd), left ventricular end diastolic diameter (LVDd) in Cold/Heat group were higher than that in normal temperature group, suggested increased cardiac hypertrophy. Cold/Heat group's left ventricular systolic pressure (LVSP), maximum rate of left ventricular pressure (±dp/dtmax), were increased compared with normal temperature group, suggested further increase of the systolic pressure and deterioration of high-pressure in left ventricular caused by cardiac hypertrophy.
After Cold/Heat stimulation, LDH activity in myocardial cell culture medium increased significantly (Cold:105.15±6.98 IU/L; Heat:96.49±5.94 IU/L vs. Control:41.09±4.59 IU/L, p<0.01). In Bim RNA interference groups, LDH activity increasing in myocardial cell culture were alleviated than that in the pure Cold/Heat stimulation groups (p<0.01, Bim siRNA+Cold:81.56±5.38 IU/L; Bim siRNA+Heat: 62.79±3.90 IU/L). After Cold/Heat stimulation, cardiomyocytes viability was decreased significantly (Cold:65.46±4.56%; Heat:68.56±3.92% vs. Control: 90.13±5.01%, p<0.01). Bim RNA interference induced improved survival rate of cardiomyocytes under cold stimulation (p<0.01, Bim siRNA+Cold:80.65±3.05%), while with no effect on survival rate of cardiomyocytes in Heat condition (p> 0.05, Bim siRNA+Heat:76.37±5.61%). After Cold/Heat stimulation, accumulation of intracellular calcium in cardiomyocytes was increase (Cold:625±27 nmol/L; Heat: 588±49 nmol/L vs. Control:361±52 nmol/L, p<0.01). Bim RNA interference can alleviate intracellular calcium increasing under Cold/Heat condition (p<0.01, Bim siRNA+Cold:619±33 nmol/L; Bim siRNA+Heat:591±61 nmol/L). After Cold/ Heat stimulation, the level of intracellular reactive oxygen species was significantly increased (Cold:10.8±0.85; Heat:9.92±0.93 vs. Control:3.61±0.71, p<0.01). While accepted Bim RNA interference pretreatment, intracellular ROS was decreased significantly compared with pure Cold/Heat stimulation group (p<0.01, Bim siRNA+Cold:4.82±0.48; Bim siRNA+Heat:4.80±0.51).
Mitochondrial membrane potentialΔΨm level detection by flow cytometry resulted as:after Cold/Heat stimulation,ΔΨm was significantly lower (Cold: 51.32±5.57; Heat:47.18±5.11 vs. Control:83.29±7.20, p<0.01). Bim RNA interference can alleviateΔΨm decreasing when compared with pure Cold/Heat stimulation group (p<0.05, Bim siRNA+Cold:69.26±7.13; Bim siRNA+Heat: 70.08±6.56). Mitochondrial membrane potential detection via fluorescence microscopy resulted as:cardiomyocytes under Cold/Heat stimulation with higher green fluorescence intensity than control group, while the red fluorescence intensity was decreased, which indicated mitochondrial membrane potential damage. Bim RNA interference preconditioning weaken the intensity of green fluorescence, enhanced red fluorescence intensity in cardiomyocytes under Cold/Heat stimulation, suggested that reduced Bim by RNA interference can alleviate Cold/Heat stimulation induced decline in cardiac mitochondrialΔψm. Results of TUNEL detection and flow cytometry AnnexinⅤPI detection showed that the Cold/Heat stimulation significantly increased myocardial apoptosis rate. Bim RNA interference decreased cardiomyocytes apoptosis caused by Cold/Heat stimulation.
Cold/Heat stimulation significantly increased the expression of Bim protein in cardiomyocytes (Cold:1.14±0.08; Heat:1.05±0.08 vs. Control:0.20±0.03, p<0.01). Bim RNA interference significantly down-regulated high expression of Bim protein in cardiomyocytes induced by Cold/Heat stimulation, compared with pure Cold/ Heat stimulation group (p<0.01, Bim siRNA+Cold:0.47±0.05; Bim siRNA+Heat: 0.32±0.05).
After Cold/Heat stimulation, Caspase-9 protein level in cardiomyocytes increased significantly (Cold:1.05±0.09; Heat:1.38±0.13 vs. Control:0.11±0.01, p <0.01). Bim siRNA pretreatment alleviated intracellular Caspase-9 expression increasing in cardiomyocytes under Cold/Heat stimulation (p<0.01, Bim siRNA+Cold:0.22±0.01; Bim siRNA+Heat:0.32±0.01). After Cold/Heat stimulation, nox-1, p22-phox protein expression were increased (nox-1:Cold:1.05±0.09; Heat:1.38±0.13 vs. Control:0.11±0.01, p<0.01; p22-phox:Cold:1.35±0.06; Heat:1.58±0.03 vs. Control:0.39±0.01, p<0.01). Bim RNA interference attenuated heat stimulation caused high expression of nox-1(p<0.01, Bim siRNA+Heat:0.32±0.01 vs. Heat:1.04±0.01) and cold stimulation caused p22-phox high expression (p <0.01, Bim siRNA+Cold:0.87±0.03; Bim siRNA+Heat:1.05±0.06).
After Cold/Heat stimulation, ERK5 and p-ERK5 protein expression in cardiomyocytes were significantly increased. Bim siRNA alleviated intracellular high expression of ERK5 and p-ERK5 induced by cold stimulation and high expression of p-ERK5 induced by heat stimulation, but performed no significant change to heat stimulation induced intracellular ERK5 high expression. After Cold/Heat stimulation, PI3K and p-PI3K protein expression in cardiomyocytes tended to increase slightly, but no significant changes were observed (p> 0.05). Bim siRNA didn't make significant change to PI3K and p-PI3K protein expression in cardiomyocytes under the Cold/Heat stimulation (p> 0.05). Cold/Hot stimulation did not cause significantly change in GSK-3βprotein expression in cardiomyocytes (p> 0.05). Bim siRNA performed no significant effects on GSK-3βprotein expression in cardiomyocytes under Cold/Heat stimulation. Cold/Heat stimulation induced increasing of p-GSK-3βprotein in cardiomyocytes (p<0.01, vs. Control group). Bim siRNA alleviated increasing of p-GSK-3βprotein in cardiomyocytes under cold/heat stimulation (p<0.01, Cold:1.15±0.06 vs. Bim siRNA+Cold:0.89±0.06; Heat: 1.05±0.09 vs. Bim siRNA+Heat:0.83±0.03).
LY294002 intervention performed no effect on Bim expression in cardiomyocytes under Cold/Heat stimulation. PD98059 intervention furthered Bim expression increasing in cardiomyocytes under cold stimulation (p<0.05, Cold: 0.57±0.03 vs. Cold+PD98059:0.71±0.04) and caused more obvious higher expression of Bim protein under heat stimulation (p<0.01, Heat:0.59±0.05 vs. Heat+PD98059:0.87±0.03).
BM-MSCs transplantation pretreatment performed no effect on the size of infarcted area of MI in vivo. But the LVEF of MI individuals under Cold/Heat stress improved significantly by BM-MSCs transplantation pretreatment. BM-MSCs transplantation pretreatment also attenuated Cold/Heat stress induced high expression of pro-apoptotic protein Bim in infarcted myocardium (p<0.01, MIC+SCs: 0.52±0.02 vs. MIC:0.83±0.02; MIH+SCs:0.76±0.01 vs. MIH:0.85±0.02).
Conclusion:
Cold/Heat stress can lead to developed size of infarcted area of MI in vivo, aggravate cardiac systolic and diastolic function disorder, and further aggravate myocardial necrosis and apoptosis; Cold/Heat stress can induce increased expression of iNOS mRNA and decreased eNOS mRNA expression in infarcted myocardium to perform detrimental effect; Cold/Heat stress can lead to aggravation of pathologic structural change and hemodynamic disorder in cardiac hypertrophy situation; Cold/ Heat stimulation cause intracellular calcium overload, increased ROS level, and reduced mitochondrial membrane potential, resulting in decreased survival rate and increased apoptosis rate of cardiomyocytes in vitro; Intracellular pro-apoptotic protein Bim expression is associated with damage that Cold/Heat stimulation caused in cardiomyocytes. Bim may mediate the Cold/Heat stimulation induced myocardial cell injury; Bim can induce increased expression of Caspase-9, nox-1 and p22-phox protein expression, increase intracellular ROS level, to mediate apoptotic effect of Cold/Heat on cardiomyocytes; ERK5 signaling pathway suppresses Cold/Heat stimulation caused expression of Bim protein in feedback style, and plays a protective role against cardiomyocytes apoptosis. PI3K/GSK-3P signaling pathway may regulate the Cold/Heat stimulation caused expression of Bim protein; BM-MSCs transplantation pretreatment can alleviate deterioration of cardiac function disorder in MI individuals under Cold/Heat stress, and possibly play a protective role via down-regulation of pro-apoptotic protein Bim expression.
近年来世界范围内极端气候事件频发,如严重冰雪灾害及反常高温暑热等。极端气候事件导致心血管疾病的病情加重,引起发病率和死亡率增高。针对寒冷与暑热刺激对心脏疾病产生的具体影响,及其在已有基础病变的心肌组织内所产生的具体作用,包括相关的细胞生物学、分子生物学机制,尚缺乏系统的研究报道。在寒冷及暑热环境下,患有心脏疾病个体的心脏功能恶化程度,心肌组织病变加重的具体病理学与解剖学参数,以及相关生化指标的变化,亦需要进行具体的量化分析。以便为明确寒冷及暑热刺激下心血管疾病的变化规律提供参考数据和资料。
心肌细胞损伤可体现在许多方面,包括细胞结构完整性破坏、线粒体能量代谢障碍、细胞内活性氧自由基(ROS)爆发、钙离子超载以及细胞凋亡改变等。冷／热刺激所造成的心肌细胞损伤,可能在上述各个方面有所体现。对冷／热刺激所造成心肌细胞损伤的方式进行观察分析,有助于在细胞水平展现冷／热刺激加重心肌病变的特点及规律。
心肌细胞凋亡是心肌损伤的重要病理进程之一。线粒体跨膜电位ΔΨm下降则是细胞凋亡级联反应过程中的起始事件。ΔΨm崩溃导致电子传递链中断,氧化磷酸化停止,最终改变细胞电化学氧化还原状态并引起细胞凋亡蛋白酶Caspase激活物释放,激活Caspase-9、Caspase-3导致细胞凋亡或死亡。关于冷／热刺激导致心肌细胞损伤是否与线粒体途径有关,需要研究证实。细胞内ROS爆发是心肌细胞损伤和凋亡的重要机制,而对于冷／热应激是否引起心肌细胞内ROS爆发,及其可能机制尚不明了。还原型烟酰胺腺嘌呤二核苷酸磷酸(NADPH)氧化酶复合物是体内活性氧ROS的重要来源之一,NADPH由多个不同的亚单位组成。其中最主要的是nox-1、p22-phox。冷／热应激在心肌细胞中是否引起nox-1、p22-phox的表达变化,及其所提示的ROS激活程度,有待实验加以探讨。
Bim是Bcl-2家族中只含BH3结构功能域(BH3-only)的重要促凋亡蛋白,广泛分布于机体各种组织,与细胞凋亡的调控功能有关。在不同的组织细胞,以及不同的凋亡诱导条件下,Bim蛋白表达的调节机制可能不同。目前,关于Bim蛋白在心肌细胞凋亡中所起到的作用尚报导不多。作为调节细胞凋亡的关键蛋白之一,Bim在冷／热刺激诱导的心肌细胞凋亡进程中,也极可能发挥着重要调控作用,并可能影响到心肌细胞结构完整性的维持、线粒体能量代谢、细胞内ROS的产生、钙离子超载等诸多方面。探究冷／热刺激下Bim介导心肌细胞损伤的机制,将对从凋亡角度深入认识冷／热刺激诱导心肌细胞损伤的具体机制,产生重要理论意义。
Bim可能与多种经典的凋亡相关信号通路有着密切关联。明晰冷／热刺激下Bim介导的心肌细胞损伤的信号传导途径及关联,可能在分子生物学水平提示新颖而有效的治疗干预靶点。Bim对心肌细胞凋亡的调控可能涉及众多信号分子途径,包括PI3K/Akt/GSK-3β信号途径、ERK信号途径等。这些信号通路其是否参与调节冷／热刺激诱导心肌损伤,及其与Bim表达的关系,目前尚未见报道。故此,Bim蛋白在冷／热刺激诱导心肌损伤中的作用机制以及调节机制,以及Bim与PI3K/Akt/GSK-3β信号途径、ERK信号途径中的相互作用,立为本课题的研究内容。
干细胞移植修复心肌损伤的效果,已经众多实验与临床研究证实。骨髓间充质干细胞(BM-MSCs)是目前常用于细胞移植治疗的组织工程种子细胞。BM-MSCs可能通过多种途径改善并恢复缺血梗死状态下的心肌功能。在极端气候条件下,对已有基础缺血病变的心肌组织预先行BM-MSCs移植干预,是否能够提升其对极端气候条件的耐受性,及其潜在的具体机制,尚未有研究涉及,也作为本研究探讨的内容之一。
方法：
在RXZ-300A型智能人工气候箱的基础上,建立模拟寒冷／暑热环境的模型装置。对兔心肌梗死动物模型进行寒冷／暑热干预。实验分组：Ⅰ、常温组(MI),Ⅱ、寒冷组(MI+Cold),Ⅲ、暑热组(MI+Heat)。标准化法测定心肌梗死面积变化情况；超声心动图测定心脏功能；生理记录仪测定血流动力学指标；梗死区心肌组织切片苏木精-伊红(HE)染色评价病理学改变；Real-time PCR法检测心肌组织内iNOS和eNOS mRNA表达水平；western blot检测梗死区心肌组织Bim蛋白的表达水平。同时,对大鼠心脏肥大模型进行寒冷／暑热干预。实验分组：Ⅰ、常温组(H)Ⅱ、寒冷组(H+Cold)Ⅲ、暑热组(H+Heat)。超声心动图测定寒冷／暑热干预后心脏功能与结构变化情况；多导生理记录仪测定血流动力学参数。
建立心肌细胞冷／热干预模型。对体外培养的新生乳鼠心肌细胞进行冷／热刺激干预,同时结合Bim siRNA干扰。实验分组：Ⅰ、正常对照组(Control),Ⅱ、冷刺激组(Cold),Ⅲ、热刺激组(Heat),Ⅳ、Bim siRNA+冷刺激组(BimsiRNA+Cold),Ⅴ、Bim siRNA+热刺激组(Bim siRNA+Heat),Ⅵ、非编码siRNA+冷刺激组(Non-coding siRNA+Cold),Ⅶ、非编码siRNA+热刺激组(Non-coding siRNA+Heat)。检测心肌细胞培养基中乳酸脱氢酶(LDH)活性；MTS法检测心肌细胞存活率；流式细胞仪检测心肌细胞内钙离子水平、ROS水平、线粒体膜电位水平；TUNEL法及AnnexinⅤPI荧光探针法检测心肌细胞凋亡情况；western blot检测心肌细胞中Bim蛋白的表达水平变化。
对体外培养的新生乳鼠心肌细胞进行冷／热干预,同时结合Bim siRNA干扰。实验分组：Ⅰ、正常对照组(Control),Ⅱ、冷刺激组(Cold),Ⅲ、热刺激组(Heat),Ⅳ、Bim siRNA+冷刺激组(Bim siRNA+Cold),Ⅴ、Bim siRNA+热刺激组(Bim siRNA+Heat),Ⅵ、非编码siRNA+冷刺激组(Non-coding siRNA+Cold),Ⅶ、非编码siRNA+热刺激组(Non-coding siRNA+Heat)。Western blot检测Caspase-9、nox-1、p22-phox蛋白表达水平,以及ERK与磷酸化ERK、PI3K与磷酸化PI3K、GSK-3β与磷酸化GSK-3β蛋白的表达水平。
对体外培养的新生乳鼠心肌细胞进行冷／热刺激,并结合ERK通路阻断剂PD98059、PI3K/Akt通路阻断剂LY294002干预。实验分组：Ⅰ、对照组(Control),Ⅱ、冷刺激组(Cold),Ⅲ、热刺激组(Heat),Ⅳ、冷刺激+LY294002组(Cold+LY294002),Ⅴ、热刺激+LY294002组(Heat+LY294002),Ⅵ、冷刺激+PD98059组(Cold+PD98059),Ⅶ、热刺激+PD98059组(Heat+PD98059)。Western blot检测Bim蛋白的表达水平。
骨髓穿刺法获得兔骨髓液,体外分离培养骨髓间充质干细胞(BM-MSCs)。建立兔心肌梗死模型,于梗死区域心肌组织内直接注射法进行自体干细胞移植。细胞移植后行寒冷／暑热干预。实验分组：Ⅰ、心梗+寒冷干预组(MIC),Ⅱ、心梗+暑热干预组(MIH),Ⅲ、心梗+细胞培养基注射(对照)+寒冷干预组(MIC+M),Ⅳ、心梗+细胞培养基注射(对照)+暑热干预组(MIH+M),Ⅴ、心梗+BM-MSCs移植+寒冷干预组(MIC+SCs),Ⅵ、心梗+BM-MSCs移植+暑热干预组(MIH+SCs)。标准化法测定心肌梗死面积；超声心动图检测心脏功能参数；western blot检测心肌组织内Bim蛋白表达水平变化。
结果：
心肌梗死动物模型接受寒冷／暑热干预后,寒冷组(MI+Cold)与暑热组(MI+Heat)的心肌梗死面积,较常温组(MI)均有扩大(p<0.01,MI:36.27±4.43%vs.MI+Cold:46.74±3.21%：MI+Heat:42.62±2.80%)；寒冷／暑热干预后,心肌梗死兔的左室舒张末径(LVEDD)与左室收缩末径(LVESD)较常温组增大；左室后壁厚度(LVWT)和收缩期室间隔厚度(IVST)较常温组减小；左室射血分数(LVEF)较常温组下降。寒冷／暑热组的左室收缩末压(LVESP)低于常温组；左室舒张末压(LVEDP)高于常温组；左室内压上升最大速率(+dp/dtmax)和左室内压下降最大速率(-dp/dtmax)皆较常温组明显降低。组织切片HE染色结果显示,寒冷组及暑热组的梗死部位心肌纤维组织增生,心肌细胞结构病理性改变均较常温组严重。寒冷／暑热干预后,寒冷刺激组的梗死心肌组织中iNOSmRNA表达水平约为无刺激心梗组的2倍；eNOS mRNA表达水平约为无刺激心梗组的60%。暑热刺激组的梗死心肌组织中iNOS mRNA表达水平约为无刺激心梗组的1.5倍；eNOS mRNA表达水平约为无刺激心梗组的40%。寒冷／暑热干预后,梗死区心肌组织中的Bim蛋白表达水平均增高(MI+Cold:0.59±0.07；MI+Heat:0.46±0.01;vs. MI:0.34±0.04, p<0.05)。
心脏肥大动物模型接受寒冷／暑热干预后,寒冷组和暑热组的左室后壁舒张末期厚度(LVPWTd)、室间隔舒张末期厚度(IVSTd)、左室舒张末期内径(LVDd),较常温组均有增加,提示心脏肥大加重。寒冷组和暑热组的左心室收缩压(LVSP),左心室压力变化最大速率(±dp/dtmax),较常温组均有增加,提示由心脏肥大引起的心脏收缩反常及左心室内高压进一步恶化。
冷／热刺激干预后,心肌细胞培养液中LDH活性明显增高(Cold:105.15±6.98 IU/L; Heat:96.49±5.94 IU/L vs. Control:41.09±4.59 IU/L, p<0.01)。针对Bim进行RNA干扰后再施加冷／热刺激干预,心肌细胞培养液中的LDH活性升高幅度较单纯冷／热刺激干预组有所下降(p<0.01,Bim siRNA+Cold:81.56±5.38 IU/L; Bim siRNA+Heat:62.79±3.90 IU/L)。冷／热刺激干预后,心肌细胞存活率显著下降(Cold:65.46±4.56%; Heat:68.56±3.92% vs. Control:90.13±5.01%, p<0.01)。针对Bim进行RNA干扰,冷刺激干预下心肌细胞存活率有所提升(p<0.01,BimsiRNA+Cold:80.65±3.05%),而对热刺激下心肌细胞存活率无明显影响(p>0.05，Bim siRNA+Heat:76.37±5.61%)。冷/热刺激干预后,心肌细胞内钙离子聚积加重(Cold:625±27 nmol/L; Heat:588±49 nmol/L vs. Control:361±52 nmol/L,p<0.01)。针对Bim进行RNA干扰下调,于冷/热干预下都能缓解心肌细胞内钙离子水平的增高(p<0.01, Bim siRNA+Cold:619±33 nmol/L; Bim siRNA+Heat: 591±61 nmol/L)。冷/热刺激干预后,心肌细胞内活性氧水平明显升高(Cold: 10.8±0.85;Heat:9.92±0.93 vs. Control:3.61±0.71, p<0.01)。接受Bim RNA干扰的实验组心肌细胞内活性氧水平较单纯冷/热刺激干预组明显下降(p<0.01,BimsiRNA+Cold:4.82±0.48; Bim siRNA+Heat:4.80±0.51)。
心肌细胞线粒体膜电位ΔΨm水平流式细胞仪检测结果显示：冷/热刺激干预后,心肌细胞线粒体膜电位ΔΨm水平明显降低(Cold51.32±5.57；Heat: 47.18±5.11 vs.Control:83.29±7.20, p<0.01)。RNA干扰下调Bim的实验组心肌细胞线粒体膜电位ΔΨm水平,较单纯冷/热刺激干预组的下降幅度明显减小(p<0.05, Bim siRNA+Cold:69.26±7.13; Bim siRNA+Heat:70.08±6.56)。心肌细胞线粒体膜电位荧光显微镜检测结果显示：冷/热刺激干预组心肌细胞的绿色荧光强于对照组,而红色荧光则减弱,表明线粒体跨膜电位受到破坏。Bim RNA干扰预处理后,心肌细胞绿色荧光强度减弱,红色荧光有所增强,提示通过RNA干扰下调Bim后,可减轻冷/热刺激所带来的心肌细胞线粒体ΔΨm下降。TUNEL检测和流式细胞仪AnnexinⅤPI检测结果均显示,冷/热刺激使心肌细胞凋亡率明显上升,而通过RNA干扰下调Bim后,可减轻冷/热刺激所造成的心肌细胞凋亡程度。
冷/热刺激干预后,心肌细胞内Bim蛋白表达水平明显升高(Cold:1.14±0.08; Heat:1.05±0.08 vs. Control:0.20±0.03, p<0.01)。RNA干扰下调Bim的实验组心肌细胞Bim蛋白表达水平较单纯冷/热刺激干预组显著下降(p<0.01,BimsiRNA+Cold:0.47±0.05;Bim siRNA+Heat:0.32±0.05)。
冷/热刺激干预后,心肌细胞Caspase-9蛋白表达水平显著上升(Cold: 1.05±0.09;Heat:1.38±0.13 vs.Control:0.11±0.01, p＜0.01)。针对Bim进行RNA干扰,则冷/热刺激干预下心肌细胞内升高的Caspase-9表达有所降低(p<0.01,BimsiRNA+Cold:0.22±0.01; Bim siRNA+Heat:0.32±0.01)。冷/热刺激干预后,心肌细胞nox-1蛋白表达水平有所上升(Cold:1.05±0.09; Heat:1.38±0.13 vs. Control:0.11±0.01,p<0.01)。针对Bim进行RNA干扰,热刺激干预下心肌细胞内升高的nox-1表达有所降低(p<0.01,Bim siRNA+Heat:0.32±0.01 vs. Heat:1.04±0.01)。冷／热刺激干预后,心肌细胞p22-phox蛋白表达增强(Cold:1.35±0.06; Heat: 1.58±0.03 vs. Control:0.39±0.01, p<0.01)。RNA干扰下调Bim后,可缓解冷／热刺激造成的心肌细胞内p22-phox高表达(p<0.01,Bim siRNA+Cold:0.87±0.03; Bim siRNA+Heat:1.05±0.06)。
冷／热干预刺激后,ERK5和p-ERK5蛋白表达均增高。通过Bim siRNA下调Bim表达后,冷刺激组心肌细胞内ERK5和p-ERK5蛋白表达均有所下降；热刺激组心肌细胞内ERK5无明显变化,但p-ERK5的表达下降。冷／热干预刺激后,心肌细胞内PI3K与p-PI3K蛋白表达略有增高趋势,但未观察到有统计学意义的改变(p>0.05)。通过Bim siRNA下调Bim后,冷／热刺激组心肌细胞内PI3K与p-PI3K蛋白表达亦无明显变化(p>0.05)。冷／热干预刺激后,未观察到心肌细胞内GSK-3β蛋白表达水平有统计学意义的改变(p>0.05)。通过BimsiRNA下调Bim后,冷／热刺激组心肌细胞内GSK-3β蛋白表达亦无明显变化。冷热干预刺激后,心肌细胞内p-GSK-3β蛋白水平均增高(p<0.01)。通过BimsiRNA下调Bim后,冷／热刺激组p-GSK-3β蛋白水平有所下降(p<0.01,Cold：1.15±0.06 vs. Bim siRNA+Cold:0.89±0.06; Heat:1.05±0.09 vs. Bim siRNA+Heat: 0.83±0.03).
LY294002干预对冷／热刺激下心肌细胞内Bim蛋白表达水平无明显影响。PD98059干预在冷刺激组使Bim蛋白表达有所增高(p<0.05,Cold：0.57±0.03 vs.Cold+PD98059:0.71±0.04)。PD98059干预在热刺激组使Bim蛋白表达显著增高(p<0.01, Heat:0.59±0.05 vs. Heat+PD98059:0.87±0.03)。
预先干细胞移植,再接受寒冷／暑热刺激后的心梗面积参数,与未接受干细胞移植实验组接受寒冷／暑热刺激后的心梗面积参数之间无统计学差异。预先干细胞移植组,在寒冷／暑热干预下,左室射血分数均有改善。预先接受细胞移植,可减轻寒冷／暑热刺激造成的心肌细胞内凋亡蛋白Bim的表达增高(p<0.01,MIC+SCs:0.52±0.02 vs. MIC:0.83±0.02; MIH+SCs:0.76±0.01 vs. MIH:0.85±0.02).
结论：
寒冷／暑热干预可导致心肌梗死基础病变个体的梗死面积扩大,心脏收缩功能和舒张功能进一步恶化,并加重心肌组织坏死及凋亡；寒冷／暑热干预可通过诱导iNOS mRNA表达水平增高,eNOS mRNA表达水平降低,对心肌细胞产生损伤；寒冷／暑热干预可导致心脏肥大结构性病变和血流动力学紊乱程度加重；冷／热刺激通过引起心肌细胞内钙离子超载、ROS水平增高,以及线粒体膜电位降低,造成体外培养心肌细胞存活率下降、凋亡率上升；冷／热刺造成的损伤效应,与心肌细胞内促凋亡蛋白Bim的表达水平相关联。Bim可能介导了冷／热刺激对心肌细胞的损伤；Bim可能通过诱导Caspase-9表达增高,nox-1以及p22-phox蛋白合成增加,上调心肌细胞内ROS水平,介导冷／热刺激对心肌细胞的致凋亡效应；ERK5途径可反馈性抑制冷／热刺激造成的心肌细胞内促凋亡蛋白Bim的表达增高,发挥保护性作用。PI3K/Akt/GSK-3β信号通路可能调控冷／热刺激造成的心肌细胞内促凋亡蛋白Bim表达；BM-MSCs移植可减轻冷／热刺激造成的心肌梗死基础病变个体的心功能恶化,并可能通过下调促凋亡蛋白Bim的表达发挥保护性作用。
Background:
In recent years, extreme climate events occurred around the world frequently, such as severe snow or freezing weather disaster and the extraordinary summer heat. Extreme climate events can lead to aggravation of cardiovascular disease and higher morbidity and mortality. About the specific effects of cold and heat stress on heart disease, and the role of cold and heat stress in damage procedure in myocardial tissue, including relevant cellular biologic and molecular mechanisms, still lack of systematic research report yet. As to individuals with heart disease under cold and heat stress conditions, the deterioration of cardiac function, pathologic and anatomical parameters of myocardium damage, and related biochemical alterations, also require quantitative analysis, so as to provide data and information about the effects of cold and heat stress on cardiovascular disease.
Cardiomyocytes injury can be manifested in many ways, including the damage of cell structural integrity, mitochondrial energy metabolism disorder, intracellular reactive oxygen species (ROS) outbreak, calcium overload and apoptosis. Myocardial cell injury caused by cold/heat stimulation may be reflected in all aspects of the above. Analysis of myocardial cell damage caused by cold/heat stimulation in various ways of observation may help to demonstrate characteristics of aggravation of cardiac disease induced by cold/heat stimulation at the cellular level.
Cardiomyocyte apoptosis is one of important pathological processes of myocardial injury. Decrease of mitochondrial membrane potentialΔψm is the initiative event of apoptosis cascade procedure. Collapse ofΔψm leads to interruption of electron transport chain, arrest of oxidative phosphorylation, and eventually changes the electrochemical redox state of cells, and induces apoptosis protease-caspase activator release, then activation of Caspase-9, Caspase-3 lead to cell death or apoptosis. Study is needed to clarify that whether there is some relationship between cold/heat stimulation induced myocardial injury and the mitochondrial pathway. The outbreak of intracellular ROS is important mechanism in cardiomyocyte apoptosis. But in what way cold/heat stress to cause the outbreak of intracellular ROS is remained unclear. Reduced form of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase complex is major source of ROS in vivo. NADPH is composed by a number of different subunits. Most important ones among those subunits are nox-1 and p22-phox. Whether cold/heat stress causes expression alternations of nox-1 and p22-phox in cardiac cells, and the subsequence ROS activation, also needs experimental confirm.
Bim is member of Bcl-2 family that with only BH3 domain (BH3-only), and it's considered as important pro-apoptotic protein. Bim is widely distributed through the body in various organizations, and performs the function as apoptosis regulator. In different cells and different apoptosis-inducing conditions, the regulation mechanism of Bim protein expression may be different. Currently, role of Bim protein in cardiomyocytes apoptosis hasn't been reported frequently yet. As one of the key protein that regulating cell apoptosis, Bim may plays significant role in cold/heat stimulation-induced myocardial cell apoptosis, and likely to regulate maintenance of cellular structure integrity, mitochondrial energy metabolism, intracellular ROS generation, calcium overload, and many other aspects in myocardial cell. To explore the role of Bim in cold/hot stimulation induced myocardial cell injury may shed some new light on the understanding of this pathologic procedure from in-depth apoptotic attitude, and may generate important theoretical significance.
Bim may be closely associated with a variety of classic apoptosis-related signaling pathway. To investigate signal transduction pathway of Bim-mediated cold/ heat stimulation induced myocardial injury at level of molecular biology may suggest novel and effective therapeutic intervention target. Regulation to myocardial apoptosis performed by Bim may involve a number of signaling molecules, including PI3K/Akt/GSK-3β, ERK and other pathway. Whether these signaling pathways involved in the regulation of cold/heat stimulation induced myocardial injury and their relationship with the expression of Bim, has not been reported yet. Therefore, the role of Bim protein in the cold/heat stress induced myocardial injury and relative molecular mechanism, as well as interaction between PI3K/Akt/GSK-3βsignal pathway, ERK signaling pathway, will be established as topic of this current research.
Many studies have confirmed the beneficial effects of stem cells transplantation on repair of myocardial injury. Mesenchymal stem cells (BM-MSCs) were commonly used in cell transplantation therapy and tissue engineering. BM-MSCs can improve and restore the cardiac function under state of myocardial ischemia or infarction in many ways. In extreme weather conditions, whether pretreatment by BM-MSCs transplantation intervention can enhance the tolerance of individuals suffering from myocardial infarction (MI) to cold/heat stress, as well as what possible specific mechanisms may be involved, have not been reported yet and also as one of the aspects to be investigated in this study.
Methods:
Based on the RXZ-300A artificial mimic climate chamber, we established model unit of cold/heat environmental stress stimulation. Animal models of myocardial infarction were given the cold/heat stress. Experimental groups were designed as:Ⅰ. Normal temperature group (MI),Ⅱ. Cold group (MI+Cold),Ⅲ. Heat group (MI+Heat). Size of myocardial infarct area was determined by standard staining method; Cardiac function was assessed by echocardiography; Parameters of hemodynamic were recorded via physiological recorder; Pathological characteristics of myocardium was evaluated by hematoxylin-eosin (HE) staining of myocardial tissue section; iNOS and eNOS mRNA expression in myocardial tissue were detected via real-time PCR; Myocardial expression of Bim protein was detected by western blot. In addition, animal model of cardiac hypertrophy were given the cold/heat stress as well. Groups were designed as:Ⅰ. Normal temperature group (H),Ⅱ. Cold group (H+Cold),Ⅲ. Heat group (H+Heat). Echocardiography and detection by multi-channel physiological recorder were performed after the cold/heat stress intervention, to evaluated cardiac function and parameters of hemodynamic.
We established cellular cold/heat stimulation model in vitro. Neonatal rat myocardial cells were cultured to accept cold/heat stimulation, combined with Bim siRNA pretreatment. Experimental groups were designed as:Ⅰ. Control group (Control),Ⅱ. Cold stimulation group (Cold),Ⅲ. Heat stimulation (Heat),Ⅳ. Bim siRNA+Cold stimulation group (Bim siRNA+Cold),Ⅴ. Bim siRNA+Heat stimulation (Bim siRNA+Heat),Ⅵ. Non-coding siRNA+Cold stimulation (Non-coding siRNA+Cold),Ⅶ. Non-coding siRNA+Heat stimulation (Non-coding siRNA+Heat). Lactate dehydrogenase (LDH) activity in cell culture medium was detected; viability of cardiomyocytes was evaluated by MTS assay; levels of intracellular calcium, reactive oxygen species, mitochondrial membrane potential were detected via flow cytometry; TUNEL and AnnexinⅤPI fluorescence probe detection were performed to evaluate myocardial cell apoptosis; Bim protein expression in cardiomyocytes was analyzed by western blot detection.
Neonatal rat myocardial cells were cultured to accept cold/heat stimulation, combined with Bim siRNA pretreatment. Experimental groups were designed as:Ⅰ. Control group (Control),Ⅱ. Cold stimulation group (Cold),Ⅲ. Heat stimulation (Heat),Ⅳ.Bim siRNA+Cold stimulation group (Bim siRNA+Cold),Ⅴ. Bim siRNA+Heat stimulation (Bim siRNA+Heat),Ⅵ. Non-coding siRNA+Cold stimulation (Non-coding siRNA+Cold),Ⅶ. Non-coding siRNA+Heat stimulation (Non-coding siRNA+Heat). Protein expression of Caspase-9, nox-1, p22-phox, ERK and phosphorylated ERK, PI3K and phosphorylated PI3K, GSK-3βand phosphorylated GSK-3βwere detected by western blot.
Neonatal rat myocardial cells were cultured to accept cold/heat stimulation, combined with ERK pathway inhibitor-PD98059 or PI3K/Akt pathway inhibitor-LY294002 intervention. Experimental groups were designed as:Ⅰ. Control group (Control),Ⅱ. Cold stimulation group (Cold),Ⅲ. Heat stimulation group (Heat),Ⅳ. Cold stimulation+LY294002 group (Cold+LY294002),Ⅴ. Heat stimulation+LY294002 group (Heat+LY294002),Ⅵ. Cold stimulation+PD98059 group (Cold+PD98059),Ⅶ. Heat stimulation+PD98059 group (Heat+PD98059). Expression of Bim protein was detected by western blot.
Autologous bone marrow mesenchymal stem cells (BM-MSCs) were cultured from bone marrow of rabbit. Stem cell transplantation was performed via direct injection into infarcted area in heart of rabbit MI model. Then animals underwent cold/heat stress. Groups were designed as:Ⅰ. MI+Cold intervention group (MIC), Ⅱ. MI+Heat intervention group (MIH),Ⅲ. MI+cell culture medium injection (control)+Cold intervention group (MIC+M),Ⅳ. MI+cell culture medium injection (control)+Heat intervention group (MIH+M),Ⅴ. MI+BM-MSCs transplantation+ Cold intervention group (MIC+SCs),Ⅵ. MI+BM-MSCs transplantation+Heat intervention group (MIH+SCs). Size of myocardial infarct area was determined by standard staining method; cardiac function was assessed by echocardiography; expression of Bim protein was detected by western blot.
Results:
After animal model of myocardial infarction accepted cold/heat stress intervention, size of myocardial infarcted area in MI+Cold group and MI+Heat group were expanded compared with MI group (p<0.01, MI:36.27±4.43%vs. MI+Cold: 46.74±3.21% & MI+Heat:42.62±2.80%). After the cold/heat stress intervention, MI rabbit's left ventricular end diastolic diameter (LVEDD) and left ventricular end systolic diameter (LVESD) were increased compared with normal temperature group; left ventricular posterior wall thickness (LVWT) and systolic interventricular septal thickness (IVST) were decreased compared with normal temperature group; left ventricular ejection fraction (LVEF) was significantly decreased compared with normal temperature group. Left ventricular end systolic pressure (LVESP) in Cold/ Heat stress group was lower than normal temperature group; left ventricular end diastolic pressure (LVEDP) was higher than normal temperature group; maximal rate of left ventricular pressure rise (+dp/dtmax) and left maximal rate of ventricular pressure decline (-dp/dtmax) were significantly lower than normal temperature group. Tissue sections HE staining showed that the fibrosis of infarcted area and pathological changes of myocardial structure in Cold/Heat group were more serious than that in normal temperature group. After Cold/Heat stress intervention, the iNOS mRNA expression in infarcted myocardium in MI+Cold group was about 2 times of that in MI group; eNOS mRNA expression was about 60% of that in MI group. The iNOS mRNA expression in infarcted myocardium in MI+Cold group was about 1.5 times of that in MI group; eNOS mRNA expression was about 40% of that in MI group. After Cold/Heat stress intervention, Bim protein expression level in infarcted myocardium was increased (MI+Cold:0.59±0.07; MI+Heat:0.46±0.01; vs. MI: 0.34±0.04, p<0.05).
After animal model of cardiac hypertrophy accepted cold/heat stress intervention, end-diastolic left ventricular posterior wall thickness (LVPWTd), end-diastolic interventricular septal thickness (IVSTd), left ventricular end diastolic diameter (LVDd) in Cold/Heat group were higher than that in normal temperature group, suggested increased cardiac hypertrophy. Cold/Heat group's left ventricular systolic pressure (LVSP), maximum rate of left ventricular pressure (±dp/dtmax), were increased compared with normal temperature group, suggested further increase of the systolic pressure and deterioration of high-pressure in left ventricular caused by cardiac hypertrophy.
After Cold/Heat stimulation, LDH activity in myocardial cell culture medium increased significantly (Cold:105.15±6.98 IU/L; Heat:96.49±5.94 IU/L vs. Control:41.09±4.59 IU/L, p<0.01). In Bim RNA interference groups, LDH activity increasing in myocardial cell culture were alleviated than that in the pure Cold/Heat stimulation groups (p<0.01, Bim siRNA+Cold:81.56±5.38 IU/L; Bim siRNA+Heat: 62.79±3.90 IU/L). After Cold/Heat stimulation, cardiomyocytes viability was decreased significantly (Cold:65.46±4.56%; Heat:68.56±3.92% vs. Control: 90.13±5.01%, p<0.01). Bim RNA interference induced improved survival rate of cardiomyocytes under cold stimulation (p<0.01, Bim siRNA+Cold:80.65±3.05%), while with no effect on survival rate of cardiomyocytes in Heat condition (p> 0.05, Bim siRNA+Heat:76.37±5.61%). After Cold/Heat stimulation, accumulation of intracellular calcium in cardiomyocytes was increase (Cold:625±27 nmol/L; Heat: 588±49 nmol/L vs. Control:361±52 nmol/L, p<0.01). Bim RNA interference can alleviate intracellular calcium increasing under Cold/Heat condition (p<0.01, Bim siRNA+Cold:619±33 nmol/L; Bim siRNA+Heat:591±61 nmol/L). After Cold/ Heat stimulation, the level of intracellular reactive oxygen species was significantly increased (Cold:10.8±0.85; Heat:9.92±0.93 vs. Control:3.61±0.71, p<0.01). While accepted Bim RNA interference pretreatment, intracellular ROS was decreased significantly compared with pure Cold/Heat stimulation group (p<0.01, Bim siRNA+Cold:4.82±0.48; Bim siRNA+Heat:4.80±0.51).
Mitochondrial membrane potentialΔΨm level detection by flow cytometry resulted as:after Cold/Heat stimulation,ΔΨm was significantly lower (Cold: 51.32±5.57; Heat:47.18±5.11 vs. Control:83.29±7.20, p<0.01). Bim RNA interference can alleviateΔΨm decreasing when compared with pure Cold/Heat stimulation group (p<0.05, Bim siRNA+Cold:69.26±7.13; Bim siRNA+Heat: 70.08±6.56). Mitochondrial membrane potential detection via fluorescence microscopy resulted as:cardiomyocytes under Cold/Heat stimulation with higher green fluorescence intensity than control group, while the red fluorescence intensity was decreased, which indicated mitochondrial membrane potential damage. Bim RNA interference preconditioning weaken the intensity of green fluorescence, enhanced red fluorescence intensity in cardiomyocytes under Cold/Heat stimulation, suggested that reduced Bim by RNA interference can alleviate Cold/Heat stimulation induced decline in cardiac mitochondrialΔψm. Results of TUNEL detection and flow cytometry AnnexinⅤPI detection showed that the Cold/Heat stimulation significantly increased myocardial apoptosis rate. Bim RNA interference decreased cardiomyocytes apoptosis caused by Cold/Heat stimulation.
Cold/Heat stimulation significantly increased the expression of Bim protein in cardiomyocytes (Cold:1.14±0.08; Heat:1.05±0.08 vs. Control:0.20±0.03, p<0.01). Bim RNA interference significantly down-regulated high expression of Bim protein in cardiomyocytes induced by Cold/Heat stimulation, compared with pure Cold/ Heat stimulation group (p<0.01, Bim siRNA+Cold:0.47±0.05; Bim siRNA+Heat: 0.32±0.05).
After Cold/Heat stimulation, Caspase-9 protein level in cardiomyocytes increased significantly (Cold:1.05±0.09; Heat:1.38±0.13 vs. Control:0.11±0.01, p <0.01). Bim siRNA pretreatment alleviated intracellular Caspase-9 expression increasing in cardiomyocytes under Cold/Heat stimulation (p<0.01, Bim siRNA+Cold:0.22±0.01; Bim siRNA+Heat:0.32±0.01). After Cold/Heat stimulation, nox-1, p22-phox protein expression were increased (nox-1:Cold:1.05±0.09; Heat:1.38±0.13 vs. Control:0.11±0.01, p<0.01; p22-phox:Cold:1.35±0.06; Heat:1.58±0.03 vs. Control:0.39±0.01, p<0.01). Bim RNA interference attenuated heat stimulation caused high expression of nox-1(p<0.01, Bim siRNA+Heat:0.32±0.01 vs. Heat:1.04±0.01) and cold stimulation caused p22-phox high expression (p <0.01, Bim siRNA+Cold:0.87±0.03; Bim siRNA+Heat:1.05±0.06).
After Cold/Heat stimulation, ERK5 and p-ERK5 protein expression in cardiomyocytes were significantly increased. Bim siRNA alleviated intracellular high expression of ERK5 and p-ERK5 induced by cold stimulation and high expression of p-ERK5 induced by heat stimulation, but performed no significant change to heat stimulation induced intracellular ERK5 high expression. After Cold/Heat stimulation, PI3K and p-PI3K protein expression in cardiomyocytes tended to increase slightly, but no significant changes were observed (p> 0.05). Bim siRNA didn't make significant change to PI3K and p-PI3K protein expression in cardiomyocytes under the Cold/Heat stimulation (p> 0.05). Cold/Hot stimulation did not cause significantly change in GSK-3βprotein expression in cardiomyocytes (p> 0.05). Bim siRNA performed no significant effects on GSK-3βprotein expression in cardiomyocytes under Cold/Heat stimulation. Cold/Heat stimulation induced increasing of p-GSK-3βprotein in cardiomyocytes (p<0.01, vs. Control group). Bim siRNA alleviated increasing of p-GSK-3βprotein in cardiomyocytes under cold/heat stimulation (p<0.01, Cold:1.15±0.06 vs. Bim siRNA+Cold:0.89±0.06; Heat: 1.05±0.09 vs. Bim siRNA+Heat:0.83±0.03).
LY294002 intervention performed no effect on Bim expression in cardiomyocytes under Cold/Heat stimulation. PD98059 intervention furthered Bim expression increasing in cardiomyocytes under cold stimulation (p<0.05, Cold: 0.57±0.03 vs. Cold+PD98059:0.71±0.04) and caused more obvious higher expression of Bim protein under heat stimulation (p<0.01, Heat:0.59±0.05 vs. Heat+PD98059:0.87±0.03).
BM-MSCs transplantation pretreatment performed no effect on the size of infarcted area of MI in vivo. But the LVEF of MI individuals under Cold/Heat stress improved significantly by BM-MSCs transplantation pretreatment. BM-MSCs transplantation pretreatment also attenuated Cold/Heat stress induced high expression of pro-apoptotic protein Bim in infarcted myocardium (p<0.01, MIC+SCs: 0.52±0.02 vs. MIC:0.83±0.02; MIH+SCs:0.76±0.01 vs. MIH:0.85±0.02).
Conclusion:
Cold/Heat stress can lead to developed size of infarcted area of MI in vivo, aggravate cardiac systolic and diastolic function disorder, and further aggravate myocardial necrosis and apoptosis; Cold/Heat stress can induce increased expression of iNOS mRNA and decreased eNOS mRNA expression in infarcted myocardium to perform detrimental effect; Cold/Heat stress can lead to aggravation of pathologic structural change and hemodynamic disorder in cardiac hypertrophy situation; Cold/ Heat stimulation cause intracellular calcium overload, increased ROS level, and reduced mitochondrial membrane potential, resulting in decreased survival rate and increased apoptosis rate of cardiomyocytes in vitro; Intracellular pro-apoptotic protein Bim expression is associated with damage that Cold/Heat stimulation caused in cardiomyocytes. Bim may mediate the Cold/Heat stimulation induced myocardial cell injury; Bim can induce increased expression of Caspase-9, nox-1 and p22-phox protein expression, increase intracellular ROS level, to mediate apoptotic effect of Cold/Heat on cardiomyocytes; ERK5 signaling pathway suppresses Cold/Heat stimulation caused expression of Bim protein in feedback style, and plays a protective role against cardiomyocytes apoptosis. PI3K/GSK-3P signaling pathway may regulate the Cold/Heat stimulation caused expression of Bim protein; BM-MSCs transplantation pretreatment can alleviate deterioration of cardiac function disorder in MI individuals under Cold/Heat stress, and possibly play a protective role via down-regulation of pro-apoptotic protein Bim expression.
引文
[1]Israel A, Zavala LE, Cierco M, et al. Effect of AT1 angiotensin Ⅱ receptor antagonists on the sympathetic response to a cold pressor test in healthy volunteers. Am J Ther,2007, Vol. 14(2):183-188.
[2]De Lorenzo F, Sharma V, Scully M, et al. Cold adaptation and the seasonal distribution of acute myocardial infarction. QJM,1999, Vol.92(12):747-751.
[3]Manfredini R, Portaluppi F, Salmi R, et al. Seasonal variation in the occurrence of nontraumatic rupture of thoracic aorta. An J Emerg Med,1999, Vol.17(7):672-674.
[4]Passero S, Reale F, Ciacci G, et al. Differing temporal patterns of onset in subgroups of patients with intracerebral hemorrhage. Stroke,2000, Vol.31(7):1538-1544.
[5]Aronow WS, Ahn C. Elderly nursing home patients with congestive heart failure after myocardial infarction living in New York city have a higher prevalence of mortality in cold weather and warm weather months. J Gerontol A Biol Sci Med Sci,2004, Vol.59(2): 146-147.
[6]吴自强,祝之明,祝善俊,等.降压药物干预高盐饮食复合冷应激诱导大鼠的心肌肥厚.第三军医大学学报,2003,Vol.25(14)：1240～1242.
[7]何丽华,张金良,张颖,等.高温对大鼠血压及血管活性物质的影响.北京大学学报(医学版),2005,Vol.37(4)：448～449.
[8]俞丽丽,李力,陈鸣.寒冷刺激诱发孕鼠妊娠高血压综合征动物模型研究.第三军医大学学报,2001,Vol.23(4)：419～421.
[9]Schildkopf P, Frey B, Mantel F, et al. Application of hyperthermia in addition to ionizing irradiation fosters necrotic cell death and HMGB1 release of colorectal tumor cells. Biochem Biophys Res Commun,2010, Vol.391(1):1014-1020.
[10]Cohen O, Kanana H, Zoizner R, et al. Altered Ca2+handling and myofilament desensitization underlie cardiomyocyte performance in normothermic and hyperthermic heat-acclimated rat hearts. J Appl Physiol,2007, Vol.103(1):266-275.
[11]Nakayama H, Chen X, Baines CP, et al. Ca2+and mitochondrial-dependent cardiomyocyte necrosis as a primary mediator of heart failure. J Clin Invest,2007, Vol.117(9):2431-2444.
[12]Yu T, Sheu SS, Robotham JL, et al. Mitochondrial fission mediates high glucose-induced cell death through elevated production of reactive oxygen species. Cardiovasc Res,2008, Vol. 79(2):341-351.
[13]Kim JK, Pedram A, Razandi M, et al. Estrogen prevents cardiomyocyte apoptosis through inhibition of reactive oxygen species and differential regulation of p38 kinase isoforms. J Biol Chem,2006, Vol.281(10):6760-6767.
[14]Tsutsui H, Kinugawa S, Matsushima S. Mitochondrial oxidative stress and dysfunction in myocardial remodelling. Cardiovasc Res,2009, Vol.81(3):449-56.
[15]Hool LC. Reactive oxygen species in cardiac signalling:from mitochondria to plasma membrane ion channels. Clin Exp Pharmacol Physiol,2006, Vol.33(1-2):146-151.
[16]Maitra U, Singh N, Gan L, et al.IRAK-1 contributes to lipopolysaccharide-induced reactive oxygen species generation in macrophages by inducing NOX-1 transcription and Racl activation and suppressing the expression of antioxidative enzymes. J Biol Chem,2009, Vol. 284(51):35403-35411.
[17]Min SC, Fang PX, Yan ZW, et al. Statins initiated after hypertrophy inhibit oxidative stress and prevent heart failure in rats with aortic stenosis. J Mol Cell Cardiol,2004, Vol.37(4): 889-896.
[18]Strasser A, Puthalakath H, Bouillet P, et al. The role of bim, a proapoptotic BH3-only member of the Bcl-2 family in cell-death control. Ann N Y Acad Sci,2000, Vol.917: 541-548.
[19]Pinon JD, Labi V, Egle A, et al. Bim and Bmf in tissue homeostasis and malignant disease. Oncogene,2008, Vol.27(Suppl 1):S41-52.
[20]Bouillet P, O'Reilly LA. CD95, BIM and T cell homeostasis. Nat Rev Immunol,2009, Vol. 9(7):514-519.
[21]Szegezdi E, Herbert KR, Kavanagh ET, et al. Nerve growth factor blocks thapsigargin-induced apoptosis at the level of the mitochondrion via regulation of Bim. J Cell Mol Med,2008, Vol.12(6A):2482-2496.
[22]Stang SL, Lopez-Campistrous A, Song X, A proapoptotic signaling pathway involving RasGRP, Erk, and Bim in B cells. Exp Hematol,2009, Vol.37(1):122-134.
[23]Rosas M, Birkenkamp KU, Lammers JW, et al. Cytokine mediated suppression of TF-1 apoptosis requires PI3K activation and inhibition of Bim expression. FEBS Lett,2005, Vol. 579(1):191-198.
[24]Chai WS, Zhu XM, Li SH, et al. Role of Bcl-2 family members in caspase-3/9-dependent apoptosis during Pseudomonas aeruginosa infection in U937 cells. Apoptosis,2008, Vol. 13(6):833-843.
[25]Zhang H, Song P, Tang Y, et al. Injection of bone marrow mesenchymal stem cells in the borderline area of infarcted myocardium:heart status and cell distribution. J Thorac Cardiovasc Surg,2007, Vol.134(5):1234-1240.
[26]Hermstad E, Adams B. Traumatic brain injury complicated by environmental hyperthermia. J Emerg Trauma Shock,2010, Vol.3(1):66-69.
[27]Gonzalez GE, Palleiro J, Monroy S, et al. Effects of the early administration of losartan on the functional and morphological aspects of postmyocardial infarction ventricular remodeling in rabbits. Cardiovasc Pathol,2005, Vol.14(2):88-95.
[28]张嘉宁,顾为望,许乙凯.结扎兔冠状动脉前降支与左室支的急性心肌梗塞比较.中国实验动物学报，1997, Vol.5(2):103-106。
[29]Vanagt WY, Cornelussen RN, Baynham TC, et al. Pacing-induced dyssynchrony during early reperfusion reduces infarct size. J Am Coll Cardiol,2007, Vol.49(17):1813-1819.
[30]Eng LF, Lee YL, Murphy GM, et al. A RT-PCR study of gene expression in a mechanical injury model. Prog Brain Res,1995, Vol.105:219-225
[31]Aptecar E, Dupouy P, Benvenuti C, et al. Sympathetic stimulation overrides flow-mediated endothelium-dependent epicardial coronary vasodilation in transplant patients. Circulation, 1996, Vol.94(10):2542-2550.
[32]Dzeka TN, Arnold JM. Prostaglandin modulation of venoconstriction to physiological stress in normals and heart failure patients. Am J Physiol Heart Circ Physiol,2003, Vol.284(3): H790-797.
[33]Seelig MS. Consequences of magnesium deficiency on the enhancement of stress reactions; preventive and therapeutic implications (a review). J Am Coll Nutr,1994, Vol.13(5): 429-446.
[34]Wang XL, Liu HR, Tao L, et al. Role of iNOS-derived reactive nitrogen species and resultant nitrative stress in leukocytes-induced cardiomyocyte apoptosis after myocardial ischemia/reperfusion. Apoptosis,2007, Vol.12(7):1209-1217.
[35]Sun Z. Cardiovascular responses to cold exposure. Front Biosci (Elite Ed),2010, Vol.2: 495-503.
[36]徐叔云,卞如濂,陈修.药理实验方法学[M].北京：人民卫生出版社,2002：1026～1028.
[37]王耀晟,何力鹏,周仪华,等.压力负荷型心脏肥大大鼠心电图分析.中华高血压杂志,2008,Vol.16(10)：933～936.
[38]Cook SA, Clerk A, Sugden PH. Are transgenic mice the'alkahest'to understanding myocardial hypertrophy and failure? J Mol Cell Cardiol,2009, Vol.46(2):118-129.
[39]Kenney MJ, Barney CC, Hirai T, et al. Sympathetic nerve responses to hyperthermia in the anesthetized rat. J Appl Physiol,1995, Vol.78(3):881-889.
[40]Scholz J, Roewer N, Troll U, et al. Malignant hyperthermia and inositol phosphate metabolism in the heart andskeletal musculature. Anasthesiol Intensivmed Notfallmed Schmerzther,1991, Vol.26(8):450-453.
[41]宋学立,钱令嘉,李风芝.冷应激对心肌细胞损伤的影响.解放军预防医学杂志,2001,Vol.19(2)：88～91.
[42]Chatterjee S, Premachandran S, Sharma D, et al. Therapeutic treatment with L-arginine rescues mice from heat stroke-induced death:physiological and molecular mechanisms. Shock,2005, Vol.24(4):341-347.
[43]Meneghini A, Ferreira C, de Abreu LC, et al. Memantine prevents cardiomyocytes nuclear size reduction in the left ventricle of rats exposed to cold stress. Clinics (Sao Paulo),2009, Vol.64(9):921-926.
[44]Patel MP, Masood A, Patel PS, et al. Targeting the Bcl-2. Curr Opin Oncol,2009,21(6): 516-523.
[45]Autret A, Martin SJ. Emerging role for members of the Bcl-2 family in mitochondrial morphogenesis. Mol Cell,2009, Vol.36(3):355-363.
[46]Gustafsson AB, Gottlieb RA. Bcl-2 family members and apoptosis, taken to heart. Am J Physiol Cell Physiol,2007, Vol.292(1):C45-51.
[47]Reeve JL, Duffy AM, O'Brien T, et al. Don't lose heart-therapeutic value of apoptosis prevention in the treatment of cardiovascular disease. J Cell Mol Med,2005, Vol.9(3): 609-622.
[48]Ogata Y, Takahashi M. Bcl-xL as an antiapoptotic molecule for cardiomyocytes. Drug News Perspect,2003, Vol.16(7):446-452.
[49]Billen LP, Shamas-Din A, Andrews DW. Bid:a Bax-like BH3 protein. Oncogene,2008, Vol. Suppl 1:S93-104.
[50]Giam M, Huang DC, Bouillet P. BH3-only proteins and their roles in programmed cell death. Oncogene,2008,27(Suppl 1):S128-136.
[51]Ewings KE, Wiggins CM, Cook SJ. Bim and the pro-survival Bcl-2 proteins:opposites attract, ERK repels. Cell Cycle,2007, Vol.6(18):2236-2240.
[52]Gillings AS, Balmanno K, Wiggins CM, et al. Apoptosis and autophagy:BIM as a mediator of tumour cell death in response to oncogene-targeted therapeutics. FEBS J,2009, Vol. 276(21):6050-6062.
[53]Czabotar PE, Colman PM, Huang DC. Bax activation by Bim? Cell Death Differ,2009, 16(9):1187-1191.
[54]Lim CL, Byrne C, Lee JK. Human thermoregulation and measurement of body temperature in exercise and clinical settings. Ann Acad Med Singapore,2008,37(4):347-353.
[55]Eyer F, Zilker T. Bench-to-bedside review:mechanisms and management of hyperthermia due to toxicity. Crit Care,2007, Vol.11(6):236.
[56]Gordon CJ. Thermophysiological responses to hyperthermic drugs:extrapolating from rodent to human. Prog Brain Res,2007, Vol.162:63-79.
[57]Deussen A. Hyperthermia and hypothermia. Effects on the cardiovascular system. Anaesthesist,2007, Vol.56(9):907-911.
[58]王耀晟,邹循锋,鲍晓明,等.去铁敏在大鼠心肌细胞原代培养中的保护性应用.中国循环杂志,2009,Vol.24(5)：387～390.
[59]Meyer G, Andre L, Tanguy S, et al. Simulated urban carbon monoxide air pollution exacerbates rat hearts ischemia-reperfusion injury. Am J Physiol Heart Circ Physiol,2010, Mar 5. [Epub ahead of print].
[60]Rabkin SW, Tsang MY. Interaction of the HMG-CoA reductase inhibitor lovastatinand nitric oxide in cardiomyocyte cell death. Pharmacology,2008, Vol.82(1):74-82.
[61]Lopez N, Varo N, Diez J, et al. Loss of myocardial LIF receptor inexperimental heart failure reduces cardiotrophin-1 cytoprotection. A role forneurohumoral agonists? Cardiovasc Res, 2007, Vol.75(3):536-545.
[62]Van Oort RJ, Respress JL, Li N, et al. Accelerated development of pressure overload-induced cardiac hypertrophy and dysfunction in an RyR2-R176Q knockin mouse model. Hypertension,2010, Feb 15. [Epub ahead of print].
[63]Mackiewicz U, Maczewski M, Klemenska E, et al. Brief postinfarction calcineurin blockade affects left ventricularremodeling and Ca(2+) handling in the rat. J Mol Cell Cardiol,2010, Jan 4. [Epub ahead of print].
[64]Lu F, Tian Z, Zhang W, et al. Calcium-sensing receptors induce apoptosis in rat cardiomyocytes via the endo (sarco) plasmic reticulum pathway during hypoxia/ reoxygenation. Basic Clin Pharmacol Toxicol,2009, Dec 22. [Epubahead of print].
[65]Redout EM, van der Toorn A, Zuidwijk MJ, et al. Antioxidant treatment attenuates pulmonary arterial hypertension-induced heart failure. Am J PhysiolHeart Circ Physiol,2010, Vol.298(3):H1038-1047.
[66]Ceylan-Isik AF, Zhao P, Zhang B, et al. Cardiac overexpression of metallothionein rescues cardiac contractile dysfunction and endoplasmicreticulum stress but not autophagy in sepsis. J Mol Cell Cardiol,2010, Vol.48(2):367-378.
[67]Luo X, Cai H, Ni J, et al. c-Jun DNAzymes inhibit myocardial inflammation, ROS generation, infarct size, andimprove cardiac function after ischemia-reperfusion injury. Arterioscler ThrombVasc Biol,2009, Vol.29(11):1836-1842.
[68]Choudhury S, Bae S, Kumar SR, et al. Role of AIF in cardiac apoptosis in hypertrophic cardiomyocytes from Dahl salt-sensitive rats. Cardiovasc Res,2010, Vol.85(1):28-37.
[69]Bahi N, Zhang J, Llovera M, et al. Switch from caspase-dependent to caspase-independent death during heart development:essential role of endonuclease G in ischemia-induced DNA processing of differentiated cardiomyocytes. J Biol Chem,2006, Vol.281(32):22943-22952.
[70]Lemasters JJ, Theruvath TP, Zhong Z, et al. Mitochondrial calcium and the permeability transition in cell death. Biochim Biophys Acta,2009, Vol.1787(11):1395-1401.
[71]Burwell LS, Brookes PS. Mitochondria as a target for the cardioprotective effects of nitric oxide in ischemia-reperfusion injury. Antioxid Redox Signal,2008, Vol.10(3):579-599.
[72]Armstrong JS. Mitochondria:a target for cancer therapy. Br J Pharmacol,2006, Vol.147(3): 239-248.
[73]Labedzka K, Grzanka A, Izdebska M. Mitochondria and cell death. Postepy Hig Med Dosw (Online),2006, Vol.60:439-446.
[74]Tomicic MT, Christmann M, Fabian K, et al. Apaf-1 deficient mouse fibroblasts are resistant to MNNG and MMS-induced apoptotic death without attenuation of Bcl-2 decline. Toxicol Appl Pharmacol,2005, Vol.207(2 Suppl):117-122.
[75]Adams JM, Cory S. Apoptosomes:engines for caspase activation. Curr Opin Cell Biol,2002, Vol.14(6):715-720.
[76]Galluzzi L, Joza N, Tasdemir E, et al. No death without life:vital functions of apoptotic effectors. Cell Death Differ,2008, Vol.15(7):1113-1123.
[77]Plesnila N. Role of mitochondrial proteins for neuronal cell death after focal cerebral ischemia. Acta Neurochir Suppl,2004, Vol.89:15-19.
[78]Takemura Y, Goodson P, Bao HF, et al. Racl-mediated NADPH Oxidase release of 02-regulates epithelial sodium channel (ENaC) activity in the alveolar epithelium. Am J Physiol Lung Cell Mol Physiol,2010, [Epub ahead of print].
[79]Buggisch M, Ateghang B, Ruhe C, et al. Stimulation of ES-cell-derived cardiomyogenesis and neonatal cardiac cell proliferation by reactive oxygen species and NADPH oxidase. J Cell Sci,2007,120(Pt 5):885-894.
[80]Gill PS, Wilcox CS. NADPH oxidases in the kidney. Antioxid Redox Signal,2006, Vol. 8(9-10):1597-1607.
[81]Ley R, Ewings KE, Hadfield K, et al. Regulatory phosphorylation of Bim:sorting out the ERK from the JNK. Cell Death Differ,2005, Vol.12(8):1008-1014.
[82]Ewings KE, Hadfield-Moorhouse K, Wiggins CM, et al. ERK1/2-dependent phosphorylation of BimEL promotes its rapid dissociation from Mcl-1 and Bcl-xL. EMBO J,2007, Vol. 26(12):2856-2867.
[83]Hiibner A, Cavanagh-Kyros J, Rincon M, et al. Functional cooperation of the proapoptotic Bcl2 family proteins Bmf and Bim in vivo. Mol Cell Biol,2010, Vol.30(1):98-105.
[84]Ferreira IL, Nascimento MV, Ribeiro M, et al. Mitochondrial-dependent apoptosis in Huntington's disease human cybrids. Exp Neurol,2010, Vol.222(2):243-255.
[85]Tanaka S, Wakeyama H, Akiyama T, et al. Regulation of osteoclast apoptosis by bcl-2 family protein bim and caspase-3. Adv Exp Med Biol,2010, Vol.658:111-116.
[86]Ghosh J, Das J, Manna P, et al. Taurine prevents arsenic-induced cardiac oxidative stress and apoptotic damage:role of NF-kappa B, p38 and JNK MAPK pathway. Toxicol Appl Pharmacol,2009, Vol.240(1):73-87.
[87]Li XM, Ma YT, Yang YN, et al. Downregulation of survival signalling pathways and increased apoptosis in the transition of pressure overload-induced cardiac hypertrophy to heart failure. Clin Exp Pharmacol Physiol,2009, Vol.36(11):1054-1061.
[88]Liu D, He M, Yi B. Pim-3 protects against cardiomyocyte apoptosis in anoxia/reoxygenation injury via p38-mediated signal pathway. Int J Biochem Cell Biol,2009, Vol.41(11): 2315-2322.
[89]Tsang MY, Rabkin SW. p38 mitogen-activated protein kinase (MAPK) is activated by noradrenaline and serves a cardioprotective role, whereas adrenaline inducesp38 MAPK dephosphorylation. Clin Exp Pharmacol Physiol,2009, Vol.36(8):e12-19.
[90]Hii CS, Anson DS, Costabile M, et al. Characterization of the MEK5-ERK5 module in human neutrophils and its relationship to ERK1/ERK2 in the chemotactic response. J Biol Chem,2004, Vol.279(48):49825-49834.
[91]Liu CJ, Lo JF, Kuo CH, et al. Akt mediates 17 beta-estradiol and/or estrogen receptor-alpha inhibition of LPS-induced tumor necresis factor-alpha expression and myocardial cell apoptosis by suppressing the JNK1/2-NFkappaB pathway. J Cell Mol Med,2009, Vol. 13(9B):3655-3667.
[92]Song HP, Zhang L, Dang YM, et al. The PI3K/Akt pathway protects cardiomyocytes from Ischemic and hypoxic apoptosis via mitochondrial function. Clin Exp Pharmacol Physiol, 2010, Jan 17. [Epub ahead of print].
[93]Thimmaiah KN, Easton JB, Houghton PJ. Protection from rapamycin-induced apoptosis by insulin-like growth factor I is partially dependent on protein kinase C signaling. Cancer Res, 2010, Vol.70(5):2000-2009.
[94]Hu L, Sun Y, Hu J. Catalpol inhibits apoptosis in hydrogen peroxide-induced endothelium by activating the PI3K/Akt signaling pathway and modulating expression of Bcl-2 and Bax. Eur J Pharmacol,2010, Vol.628(1-3):155-163.
[95]Loriot Y, Calderaro J, Soria JC, et al. Anti-apoptotic mechanisms in small-cell lung carcinoma. Ann Pathol,2010, Vol.30(1):17-24.
[96]Lu Z, Cox-Hipkin MA, Windsor WT, et al.3-phosphoinositide-dependent protein kinase-1 regulates proliferation and survival of cancer cells with an activated mitogen-activated protein kinase pathway. Mol Cancer Res,2010, Mar 2. [Epub ahead of print].
[97]Hongisto V, Vainio JC, Thompson R, et al. The Wnt pool of glycogen synthase kinase 3-beta is critical for trophic-deprivation-induced neuronal death. Mol Cell Biol,2008, Vol.28(5): 1515-1527.
[98]Wollert KC, Meyer GP, Lotz J, et al. Intracoronary autologous bone marrow cell transfer after myocardial infarction:The BOOST randomized controlled clinical trial. Lancet,2004, Vol.364(9429):141-148.
[99]Strauer BE, Brehm M, Zeus T, et al. Regeneration of human infracted heart muscle by intracoronary autologous bone marrow cell transplantation in chronic coronary artery disease: The IACT Study. J Am Coll Cardiol,2005, Vol.46(9):1651-1658.
[100]Scha'chinger V, Erbs S, Elsasser A, et al. Improved clinical outcome after intracoronary administration of bone marrow-derived progenitor cells in acute myocardial infarction:Final 1-year results of the REPAIR-AMI trial. Eur Heart J,2006, Vol.27(17):2775-2783
[101]Mathiasen AB, Haack-Sorensen M, Kastrup J. Mesenchymal stromal cells for cardiovascular repair:current status and future challenges. Future Cardiol,2009, Vol.5(6):605-617.
[102]Abarbanell AM, Coffey AC, Fehrenbacher JW, et al. Proinflammatory cytokine effects on mesenchymal stem cell therapy for the ischemic heart. Ann Thorac Surg,2009, Vol.88(3): 1036-1043.
[103]Chachques JC. Cellular cardiac regenerative therapy in which patients? Expert Rev Cardiovasc Ther,2009, Vol.7(8):911-919.
[104]Joggerst SJ, Hatzopoulos AK. Stem cell therapy for cardiac repair:benefits and barriers. Expert Rev Mol Med,2009, Vol.11:e20.
[105]Trounson A. New perspectives in human stem cell therapeutic research. BMC Med,2009, Vol.11(7):29.
[106]Kao RL, Browder W, Li C. Cellular cardiomyoplasty:what have we learned? Asian Cardiovasc Thorac Ann,2009, Vol.17(1):89-101.
[107]王耀晟,程晓曙.骨髓间充质干细胞移植治疗心肌梗死的新技术.国际心血管病杂志,2007,Vol.34(1)：30～34.
[108]王耀晟,王伶,杨人强,等.表达hVEGF121/EGFP融合蛋白骨髓间充质干细胞的制备及缺氧干预.中国组织工程研究与临床康复,2008,Vol.12(21)：4001～4006. 参考文献
[109]王耀晟.表达hVEGF121/EGFP的间充质干细胞制备及缺氧对其表达影响.中国优秀硕士学位论文全文数据库,医药卫生科技辑,2008年3月,CNKI：CDMD：2.2008.011887.
[110]Engelhardt S, Hein L, Wiesmann F, et al. Progressive hypertrophy and heart failure in beta1-adrenergic receptor transgenic mice. Proc Natl Acad Sci USA,1999, Vol.96(12):7059～7064.
[111]王耀晟,程晓曙.细胞移植修复缺血性心肌损伤的哲学思考.南昌大学学报(人文社会科学版),2009,Vol.40(2)：65～68.
[112]Singh K, Communal C, Sawyer DB, et al. Adrenergic regulation of myocardial apoptosis. Cardiovasc Res,2000, Vol.45(3):713-719.
[113]Pfeffer MA, Braunwald E. Ventricular remodeling after myocardial infarction. Experimental observations and clinical implications. Circulation,1990, Vol.81(4):1161-1172.
[114]Li XY, Wang T, Jiang XJ, et al. Injectable hydrogel helps bone marrow-derived mononuclear cells restore infarcted myocardium. Cardiology,2010, Vol.115(3):194-199.
[115]Zhang Y, Wang H, Kovacs A, et al. Reduced expression of Cx43 attenuates ventricular remodeling after myocardial infarction via impaired TGF-beta signaling. Am J Physiol Heart Circ Physiol,2010, Vol.298(2):H477-487.
[116]Piao H, Youn TJ, Kwon JS, et al. Effects of bone marrow derived mesenchymal stem cells transplantation in acutely infarcting myocardium. Eur J Heart Fail,2005, Vol.7(5):730-738.
[117]Schuleri KH, Feigenbaum GS, Centola M, et al. Autologous mesenchymal stem cells produce reverse remodelling in chronic ischaemic cardiomyopathy. Eur Heart J,2009, Vol. 30(22):2722-2732.
[118]Hu X, Wang J, Chen J, et al. Optimal temporal delivery of bone marrow mesenchymal stem cells in rats with myocardial infarction. Eur J Cardiothorac Surg,2007, Vol.31(3):438-443.
[119]王耀晟,程晓曙,王伶,等.不同缺氧状态下骨髓间充质干细胞表达血管内皮生长因子的差异.中国组织工程研究与临床康复,2009,Vol.13(36)：7143～7146.
[120]Biondi-Zoccai GG, Abbate A, Vasaturo F, et al. Increased apoptosis in remote non-infarcted myocardium in muhivessel coronary disease. Int J Cardiol,2004, Vol.94(1):105-110.
[121]Chatterjee S, Stewart AS, Bish LT, et al. Viral gene transfer of the antiapoptotic factor Bcl-2 protects against chronic postischemic heart failure. Circulation,2002, Vol.106(12Suppl 1): 1212-217.
[122]Tsubokawa T, Yagi K, Nakanishi C, et al. Impact of anti-apoptotic and oxidative effects of bone marrow mesenchymal stem cells with transient overexpression of heme oxygenase-1 on myocardial ischemia. Am J Physiol Heart Circ Physiol,2010, Feb 12. [Epub ahead of print]
[123]张瑞成,董念国,侯剑峰,等.骨髓干细胞移植可影响心肌梗死后心肌细胞的凋亡.心脏杂志,2007, Vol.19(4):388-390,398.
[124]Kinnaird T, Stabile E, Burnett MS et al. Marrow-derived stromal cells express genes encoding a broad spectrum of arteriogenic cytokines and promote in-vitro and in-vivo. arteriogenesis through paracrine mechanisms, CirRes,2004, Vol.94:678-685.
[125]王耀晟,程晓曙,王伶,等.骨髓间充质干细胞在缺氧下VEGF的表达规律.第十九届 长城国际心脏病学会议,北京,2008,August：gw19～c0094.
[126]Yaosheng Wang, Xiaoshu Cheng, Lipeng He, et al. Expression of hVEGF121/EGFP in mesenchymal stem cells and angiogenic function of the fusion protein. World Congress of Cardiology Scientific Sessions 2010, Beijing,2010, June:28328.
[127]Berry C, Balachandran KP, L'Allier PL, et al. Importance of collateral circulation in coronary heart disease. Eur Heart J,2007, Vol.28(3):278-291.
[128]Sun Y, Weber KT. Infarct scar:a dynamic tissue. Cardiovasc Res,2000, Vol.46(2):250-256.
[129]王耀晟,程晓曙,王伶,等.骨髓间充质干细胞在缺氧条件下VEGF的表达规律.中国应用生理学杂志,2009,Vol.25(4)：475～477.
1130] Basso C, Rizzo S, Thiene G. The metamorphosis of myocardial infarction following coronary recanalization. Cardiovasc Pathol,2010, Vol.19(1):22-28.
[131]Gallagher GL, Jackson CJ, Hunyor SN. Myocardial extracellular matrix remodeling in ischemic heart failure. Front Biosci,2007, Vol.12:1410-1419.
[132]Bhindi R, Witting PK, McMahon AC, et al. Rat models of myocardial infarction. Pathogenetic insights and clinical relevance. Thromb Haemost,2006, Vol.96(5):602-610.
[133]Anversa P, Leri A, Kajstura J. Cardiac regeneration. J Am Coll Cardiol,2006, Vol.47(9): 1769-1776.
[134]Khan R, Sheppard R. Fibrosis in heart disease:understanding the role of transforming growth factor-beta in cardiomyopathy, valvular disease and arrhythmia. Immunology,2006, Vol.118(1):10-24.
[135]Liu L, Hu J, Sun B, et al. Mesenchymal stem cells inhibition of chronic ethanol-induced oxidative damage via upregulation of phosphatidylinositol-3-kinase/Akt and modulation of extracellular signal-regulated kinase 1/2 activation in PC12 cells and neurons. Neuroscience, 2010 Feb 11. [Epub ahead of print].
[136]Cho J, Rameshwar P, Sadoshima J. Distinct roles of glycogen synthase kinase (GSK)-3alpha and GSK-3beta in mediating cardiomyocyte differentiation in murine bone marrow-derived mesenchymal stem cells. J Biol Chem,2009,284(52):36647-36658.
[1]Israel A, Zavala LE, Cierco M, et al. Effect of AT1 angiotensin Ⅱ receptor antagonists on the sympathetic response to a cold pressor test in healthy volunteers. Am J Ther,2007, 14(2):183-188.
[2]Israel A, Zavala LE, Cierco M, et al. Effect of eprosartan on the sympathetic response to cold pressor test in healthy volunteers. Auton Neurosci,2006,126-127:179-184.
[3]Lindenblatt N, Menger MD, Klar E, et al. Sustained hypothermia accelerates microvascular thrombus formation in mice. Am J Physiol Heart Circ Physiol,2005,289(6):H2680-2687.
[4]Straub A, Azevedo R, Beierlein W, et al. Glycoprotein Ⅱb/Ⅲa inhibition reduces prothrombotic events under conditions of deep hypothermic circulatory arrest. Thromb Haemost,2005,94(1):115-22.
[5]Rothoerl RD, Brawanski A. The history and present status of deep hypothermia and circulatory arrest in cerebrovascular surgery. Neurosurg Focus,2006,20(6):E5.
[6]Downey JM, Cohen MV. Reducing infarct size in the setting of acute myocardial infarction. Prog Cardiovasc Dis,2006,48(5):363-371.
[7]Maeng M, Mortensson UM, Kristensen J, et al. Hypothermia during reperfusion does not reduce myocardial infarct size in pigs. Basic Res Cardiol 2006,101(1):61-68.
[8]Olivecrona GK, Gotberg M, Harnek J, et al. Mild hypothermia reduces cardiac post-ischemic reactive Hyperemia. BMC Cardiovasc Disord,2007,7:5.
[9]Mattu A, Brady WJ, Perron AD. Electrocardiographic manifestations of hypothermia. Am J Emerg Med,2002,20(4):314-326.
[10]Garcia-Dorado D, Rodriguez-Sinovas A, Ruiz-Meana M. Gap junction-mediated spread of cell injury and death during myocardial ischemia-reperfusion. Cardiovasc Res,2004, 61(3):386-401.
[11]Fedorov VV, Li L, Glukhov A, et al. Hibernator Citellus undulatus maintains safe cardiac conduction and is protected against tachyarrhythmias during extreme hypothermia:Possible role of Cx43 and Cx45 up-regulation. Heart Rhythm,2005,2(9):966-975.
[12]Aupperle H, Garbade J, Ullmann C, et al. Comparing the ultrastructural effects of two different cardiac preparation-and perfusion-techniques in a porcine model of extracorporal long-term preservation. Eur J Cardiothorac Surg,2007,31(2):214-221.
[13]Bombig MT, Ferreira C, Mora O, et al. Pravastatin protection from cold stress in myocardium of rats. Jpn Heart J,2003,44(2):243-255.
[14]Steensrud T, Nordhaug D, Husnes KV, et al. Replacing potassium with nicorandil in cold St. Thomas'Hospital cardioplegia improves preservation of energetics and function in pig hearts. Ann Thorac Surg,2004,77(4):1391-1397.
[15]Jin SQ, Niu LJ, Deng CY, et al. Effect of temperature on the activation of myocardial KATP channel in guinea pig ventricular myocytes:a pilot study by whole cell patch clamp recording. Chin Med J (Engl),2006,119(20):1721-1726.
[16]Nakayama H, Chen X, Baines CP, et al. Ca2+-and mitochondrial-dependent cardiomyocyte necrosis as a primary mediator of heart failure. J Clin Invest,2007,117(9):2431-2444.
[17]Yatani A, Kim SJ, Kudej RK, et al. Insights into cardioprotection obtained from study of cellular Ca2+ handling in myocardium of true hibernating mammals. Am J Physiol Heart Circ Physiol,2004,286(6):H2219-2228.
[18]吴自强,祝善俊,祝之明,等.高盐饮食与冷应激复合诱导大鼠心肌肥厚的药物干预.临床心血管病杂志,2003,19(6)：369-372.
[19]Hoye AT, Davoren JE, Wipf P, et al. Targeting mitochondria. Acc Chem Res,2008, 41(1):87-97.
[20]宋学立,钱令嘉,李风芝.冷应激对心肌细胞损伤的影响.解放军预防医学杂志,2001,19(2)：88-91.
[21]Fairchild KD, Singh IS, Patel S, et al. Hypothermia prolongs activation of NF-kB and augments generation of inflammatory cytokines. Am J Physiol Cell Physiol,2004, 287(2):C422-31.
[22]Hamid AA, Mandai M, Fujita J, et al. Expression of cold-inducible RNA-binding protein in the normal endometrium, endometrial hyperplasia, and endometrial carcinoma. Int J Gynecol Pathol,2003,22(3):240-247.
[23]肖华,陈志坚,廖玉华,等.TNF-α早期表达在大鼠急性心肌梗死室性心律失常发生中的作用及机制研究.中国免疫学杂志,2008,24(1)：75-79.
[2]De Lorenzo F, Sharma V, Scully M, et al. Cold adaptation and the seasonal distribution of acute myocardial infarction. QJM,1999, Vol.92(12):747-751.
[3]Manfredini R, Portaluppi F, Salmi R, et al. Seasonal variation in the occurrence of nontraumatic rupture of thoracic aorta. An J Emerg Med,1999, Vol.17(7):672-674.
[4]Passero S, Reale F, Ciacci G, et al. Differing temporal patterns of onset in subgroups of patients with intracerebral hemorrhage. Stroke,2000, Vol.31(7):1538-1544.
[5]Aronow WS, Ahn C. Elderly nursing home patients with congestive heart failure after myocardial infarction living in New York city have a higher prevalence of mortality in cold weather and warm weather months. J Gerontol A Biol Sci Med Sci,2004, Vol.59(2): 146-147.
[6]吴自强,祝之明,祝善俊,等.降压药物干预高盐饮食复合冷应激诱导大鼠的心肌肥厚.第三军医大学学报,2003,Vol.25(14)：1240～1242.
[7]何丽华,张金良,张颖,等.高温对大鼠血压及血管活性物质的影响.北京大学学报(医学版),2005,Vol.37(4)：448～449.
[8]俞丽丽,李力,陈鸣.寒冷刺激诱发孕鼠妊娠高血压综合征动物模型研究.第三军医大学学报,2001,Vol.23(4)：419～421.
[9]Schildkopf P, Frey B, Mantel F, et al. Application of hyperthermia in addition to ionizing irradiation fosters necrotic cell death and HMGB1 release of colorectal tumor cells. Biochem Biophys Res Commun,2010, Vol.391(1):1014-1020.
[10]Cohen O, Kanana H, Zoizner R, et al. Altered Ca2+handling and myofilament desensitization underlie cardiomyocyte performance in normothermic and hyperthermic heat-acclimated rat hearts. J Appl Physiol,2007, Vol.103(1):266-275.
[11]Nakayama H, Chen X, Baines CP, et al. Ca2+and mitochondrial-dependent cardiomyocyte necrosis as a primary mediator of heart failure. J Clin Invest,2007, Vol.117(9):2431-2444.
[12]Yu T, Sheu SS, Robotham JL, et al. Mitochondrial fission mediates high glucose-induced cell death through elevated production of reactive oxygen species. Cardiovasc Res,2008, Vol. 79(2):341-351.
[13]Kim JK, Pedram A, Razandi M, et al. Estrogen prevents cardiomyocyte apoptosis through inhibition of reactive oxygen species and differential regulation of p38 kinase isoforms. J Biol Chem,2006, Vol.281(10):6760-6767.
[14]Tsutsui H, Kinugawa S, Matsushima S. Mitochondrial oxidative stress and dysfunction in myocardial remodelling. Cardiovasc Res,2009, Vol.81(3):449-56.
[15]Hool LC. Reactive oxygen species in cardiac signalling:from mitochondria to plasma membrane ion channels. Clin Exp Pharmacol Physiol,2006, Vol.33(1-2):146-151.
[16]Maitra U, Singh N, Gan L, et al.IRAK-1 contributes to lipopolysaccharide-induced reactive oxygen species generation in macrophages by inducing NOX-1 transcription and Racl activation and suppressing the expression of antioxidative enzymes. J Biol Chem,2009, Vol. 284(51):35403-35411.
[17]Min SC, Fang PX, Yan ZW, et al. Statins initiated after hypertrophy inhibit oxidative stress and prevent heart failure in rats with aortic stenosis. J Mol Cell Cardiol,2004, Vol.37(4): 889-896.
[18]Strasser A, Puthalakath H, Bouillet P, et al. The role of bim, a proapoptotic BH3-only member of the Bcl-2 family in cell-death control. Ann N Y Acad Sci,2000, Vol.917: 541-548.
[19]Pinon JD, Labi V, Egle A, et al. Bim and Bmf in tissue homeostasis and malignant disease. Oncogene,2008, Vol.27(Suppl 1):S41-52.
[20]Bouillet P, O'Reilly LA. CD95, BIM and T cell homeostasis. Nat Rev Immunol,2009, Vol. 9(7):514-519.
[21]Szegezdi E, Herbert KR, Kavanagh ET, et al. Nerve growth factor blocks thapsigargin-induced apoptosis at the level of the mitochondrion via regulation of Bim. J Cell Mol Med,2008, Vol.12(6A):2482-2496.
[22]Stang SL, Lopez-Campistrous A, Song X, A proapoptotic signaling pathway involving RasGRP, Erk, and Bim in B cells. Exp Hematol,2009, Vol.37(1):122-134.
[23]Rosas M, Birkenkamp KU, Lammers JW, et al. Cytokine mediated suppression of TF-1 apoptosis requires PI3K activation and inhibition of Bim expression. FEBS Lett,2005, Vol. 579(1):191-198.
[24]Chai WS, Zhu XM, Li SH, et al. Role of Bcl-2 family members in caspase-3/9-dependent apoptosis during Pseudomonas aeruginosa infection in U937 cells. Apoptosis,2008, Vol. 13(6):833-843.
[25]Zhang H, Song P, Tang Y, et al. Injection of bone marrow mesenchymal stem cells in the borderline area of infarcted myocardium:heart status and cell distribution. J Thorac Cardiovasc Surg,2007, Vol.134(5):1234-1240.
[26]Hermstad E, Adams B. Traumatic brain injury complicated by environmental hyperthermia. J Emerg Trauma Shock,2010, Vol.3(1):66-69.
[27]Gonzalez GE, Palleiro J, Monroy S, et al. Effects of the early administration of losartan on the functional and morphological aspects of postmyocardial infarction ventricular remodeling in rabbits. Cardiovasc Pathol,2005, Vol.14(2):88-95.
[28]张嘉宁,顾为望,许乙凯.结扎兔冠状动脉前降支与左室支的急性心肌梗塞比较.中国实验动物学报，1997, Vol.5(2):103-106。
[29]Vanagt WY, Cornelussen RN, Baynham TC, et al. Pacing-induced dyssynchrony during early reperfusion reduces infarct size. J Am Coll Cardiol,2007, Vol.49(17):1813-1819.
[30]Eng LF, Lee YL, Murphy GM, et al. A RT-PCR study of gene expression in a mechanical injury model. Prog Brain Res,1995, Vol.105:219-225
[31]Aptecar E, Dupouy P, Benvenuti C, et al. Sympathetic stimulation overrides flow-mediated endothelium-dependent epicardial coronary vasodilation in transplant patients. Circulation, 1996, Vol.94(10):2542-2550.
[32]Dzeka TN, Arnold JM. Prostaglandin modulation of venoconstriction to physiological stress in normals and heart failure patients. Am J Physiol Heart Circ Physiol,2003, Vol.284(3): H790-797.
[33]Seelig MS. Consequences of magnesium deficiency on the enhancement of stress reactions; preventive and therapeutic implications (a review). J Am Coll Nutr,1994, Vol.13(5): 429-446.
[34]Wang XL, Liu HR, Tao L, et al. Role of iNOS-derived reactive nitrogen species and resultant nitrative stress in leukocytes-induced cardiomyocyte apoptosis after myocardial ischemia/reperfusion. Apoptosis,2007, Vol.12(7):1209-1217.
[35]Sun Z. Cardiovascular responses to cold exposure. Front Biosci (Elite Ed),2010, Vol.2: 495-503.
[36]徐叔云,卞如濂,陈修.药理实验方法学[M].北京：人民卫生出版社,2002：1026～1028.
[37]王耀晟,何力鹏,周仪华,等.压力负荷型心脏肥大大鼠心电图分析.中华高血压杂志,2008,Vol.16(10)：933～936.
[38]Cook SA, Clerk A, Sugden PH. Are transgenic mice the'alkahest'to understanding myocardial hypertrophy and failure? J Mol Cell Cardiol,2009, Vol.46(2):118-129.
[39]Kenney MJ, Barney CC, Hirai T, et al. Sympathetic nerve responses to hyperthermia in the anesthetized rat. J Appl Physiol,1995, Vol.78(3):881-889.
[40]Scholz J, Roewer N, Troll U, et al. Malignant hyperthermia and inositol phosphate metabolism in the heart andskeletal musculature. Anasthesiol Intensivmed Notfallmed Schmerzther,1991, Vol.26(8):450-453.
[41]宋学立,钱令嘉,李风芝.冷应激对心肌细胞损伤的影响.解放军预防医学杂志,2001,Vol.19(2)：88～91.
[42]Chatterjee S, Premachandran S, Sharma D, et al. Therapeutic treatment with L-arginine rescues mice from heat stroke-induced death:physiological and molecular mechanisms. Shock,2005, Vol.24(4):341-347.
[43]Meneghini A, Ferreira C, de Abreu LC, et al. Memantine prevents cardiomyocytes nuclear size reduction in the left ventricle of rats exposed to cold stress. Clinics (Sao Paulo),2009, Vol.64(9):921-926.
[44]Patel MP, Masood A, Patel PS, et al. Targeting the Bcl-2. Curr Opin Oncol,2009,21(6): 516-523.
[45]Autret A, Martin SJ. Emerging role for members of the Bcl-2 family in mitochondrial morphogenesis. Mol Cell,2009, Vol.36(3):355-363.
[46]Gustafsson AB, Gottlieb RA. Bcl-2 family members and apoptosis, taken to heart. Am J Physiol Cell Physiol,2007, Vol.292(1):C45-51.
[47]Reeve JL, Duffy AM, O'Brien T, et al. Don't lose heart-therapeutic value of apoptosis prevention in the treatment of cardiovascular disease. J Cell Mol Med,2005, Vol.9(3): 609-622.
[48]Ogata Y, Takahashi M. Bcl-xL as an antiapoptotic molecule for cardiomyocytes. Drug News Perspect,2003, Vol.16(7):446-452.
[49]Billen LP, Shamas-Din A, Andrews DW. Bid:a Bax-like BH3 protein. Oncogene,2008, Vol. Suppl 1:S93-104.
[50]Giam M, Huang DC, Bouillet P. BH3-only proteins and their roles in programmed cell death. Oncogene,2008,27(Suppl 1):S128-136.
[51]Ewings KE, Wiggins CM, Cook SJ. Bim and the pro-survival Bcl-2 proteins:opposites attract, ERK repels. Cell Cycle,2007, Vol.6(18):2236-2240.
[52]Gillings AS, Balmanno K, Wiggins CM, et al. Apoptosis and autophagy:BIM as a mediator of tumour cell death in response to oncogene-targeted therapeutics. FEBS J,2009, Vol. 276(21):6050-6062.
[53]Czabotar PE, Colman PM, Huang DC. Bax activation by Bim? Cell Death Differ,2009, 16(9):1187-1191.
[54]Lim CL, Byrne C, Lee JK. Human thermoregulation and measurement of body temperature in exercise and clinical settings. Ann Acad Med Singapore,2008,37(4):347-353.
[55]Eyer F, Zilker T. Bench-to-bedside review:mechanisms and management of hyperthermia due to toxicity. Crit Care,2007, Vol.11(6):236.
[56]Gordon CJ. Thermophysiological responses to hyperthermic drugs:extrapolating from rodent to human. Prog Brain Res,2007, Vol.162:63-79.
[57]Deussen A. Hyperthermia and hypothermia. Effects on the cardiovascular system. Anaesthesist,2007, Vol.56(9):907-911.
[58]王耀晟,邹循锋,鲍晓明,等.去铁敏在大鼠心肌细胞原代培养中的保护性应用.中国循环杂志,2009,Vol.24(5)：387～390.
[59]Meyer G, Andre L, Tanguy S, et al. Simulated urban carbon monoxide air pollution exacerbates rat hearts ischemia-reperfusion injury. Am J Physiol Heart Circ Physiol,2010, Mar 5. [Epub ahead of print].
[60]Rabkin SW, Tsang MY. Interaction of the HMG-CoA reductase inhibitor lovastatinand nitric oxide in cardiomyocyte cell death. Pharmacology,2008, Vol.82(1):74-82.
[61]Lopez N, Varo N, Diez J, et al. Loss of myocardial LIF receptor inexperimental heart failure reduces cardiotrophin-1 cytoprotection. A role forneurohumoral agonists? Cardiovasc Res, 2007, Vol.75(3):536-545.
[62]Van Oort RJ, Respress JL, Li N, et al. Accelerated development of pressure overload-induced cardiac hypertrophy and dysfunction in an RyR2-R176Q knockin mouse model. Hypertension,2010, Feb 15. [Epub ahead of print].
[63]Mackiewicz U, Maczewski M, Klemenska E, et al. Brief postinfarction calcineurin blockade affects left ventricularremodeling and Ca(2+) handling in the rat. J Mol Cell Cardiol,2010, Jan 4. [Epub ahead of print].
[64]Lu F, Tian Z, Zhang W, et al. Calcium-sensing receptors induce apoptosis in rat cardiomyocytes via the endo (sarco) plasmic reticulum pathway during hypoxia/ reoxygenation. Basic Clin Pharmacol Toxicol,2009, Dec 22. [Epubahead of print].
[65]Redout EM, van der Toorn A, Zuidwijk MJ, et al. Antioxidant treatment attenuates pulmonary arterial hypertension-induced heart failure. Am J PhysiolHeart Circ Physiol,2010, Vol.298(3):H1038-1047.
[66]Ceylan-Isik AF, Zhao P, Zhang B, et al. Cardiac overexpression of metallothionein rescues cardiac contractile dysfunction and endoplasmicreticulum stress but not autophagy in sepsis. J Mol Cell Cardiol,2010, Vol.48(2):367-378.
[67]Luo X, Cai H, Ni J, et al. c-Jun DNAzymes inhibit myocardial inflammation, ROS generation, infarct size, andimprove cardiac function after ischemia-reperfusion injury. Arterioscler ThrombVasc Biol,2009, Vol.29(11):1836-1842.
[68]Choudhury S, Bae S, Kumar SR, et al. Role of AIF in cardiac apoptosis in hypertrophic cardiomyocytes from Dahl salt-sensitive rats. Cardiovasc Res,2010, Vol.85(1):28-37.
[69]Bahi N, Zhang J, Llovera M, et al. Switch from caspase-dependent to caspase-independent death during heart development:essential role of endonuclease G in ischemia-induced DNA processing of differentiated cardiomyocytes. J Biol Chem,2006, Vol.281(32):22943-22952.
[70]Lemasters JJ, Theruvath TP, Zhong Z, et al. Mitochondrial calcium and the permeability transition in cell death. Biochim Biophys Acta,2009, Vol.1787(11):1395-1401.
[71]Burwell LS, Brookes PS. Mitochondria as a target for the cardioprotective effects of nitric oxide in ischemia-reperfusion injury. Antioxid Redox Signal,2008, Vol.10(3):579-599.
[72]Armstrong JS. Mitochondria:a target for cancer therapy. Br J Pharmacol,2006, Vol.147(3): 239-248.
[73]Labedzka K, Grzanka A, Izdebska M. Mitochondria and cell death. Postepy Hig Med Dosw (Online),2006, Vol.60:439-446.
[74]Tomicic MT, Christmann M, Fabian K, et al. Apaf-1 deficient mouse fibroblasts are resistant to MNNG and MMS-induced apoptotic death without attenuation of Bcl-2 decline. Toxicol Appl Pharmacol,2005, Vol.207(2 Suppl):117-122.
[75]Adams JM, Cory S. Apoptosomes:engines for caspase activation. Curr Opin Cell Biol,2002, Vol.14(6):715-720.
[76]Galluzzi L, Joza N, Tasdemir E, et al. No death without life:vital functions of apoptotic effectors. Cell Death Differ,2008, Vol.15(7):1113-1123.
[77]Plesnila N. Role of mitochondrial proteins for neuronal cell death after focal cerebral ischemia. Acta Neurochir Suppl,2004, Vol.89:15-19.
[78]Takemura Y, Goodson P, Bao HF, et al. Racl-mediated NADPH Oxidase release of 02-regulates epithelial sodium channel (ENaC) activity in the alveolar epithelium. Am J Physiol Lung Cell Mol Physiol,2010, [Epub ahead of print].
[79]Buggisch M, Ateghang B, Ruhe C, et al. Stimulation of ES-cell-derived cardiomyogenesis and neonatal cardiac cell proliferation by reactive oxygen species and NADPH oxidase. J Cell Sci,2007,120(Pt 5):885-894.
[80]Gill PS, Wilcox CS. NADPH oxidases in the kidney. Antioxid Redox Signal,2006, Vol. 8(9-10):1597-1607.
[81]Ley R, Ewings KE, Hadfield K, et al. Regulatory phosphorylation of Bim:sorting out the ERK from the JNK. Cell Death Differ,2005, Vol.12(8):1008-1014.
[82]Ewings KE, Hadfield-Moorhouse K, Wiggins CM, et al. ERK1/2-dependent phosphorylation of BimEL promotes its rapid dissociation from Mcl-1 and Bcl-xL. EMBO J,2007, Vol. 26(12):2856-2867.
[83]Hiibner A, Cavanagh-Kyros J, Rincon M, et al. Functional cooperation of the proapoptotic Bcl2 family proteins Bmf and Bim in vivo. Mol Cell Biol,2010, Vol.30(1):98-105.
[84]Ferreira IL, Nascimento MV, Ribeiro M, et al. Mitochondrial-dependent apoptosis in Huntington's disease human cybrids. Exp Neurol,2010, Vol.222(2):243-255.
[85]Tanaka S, Wakeyama H, Akiyama T, et al. Regulation of osteoclast apoptosis by bcl-2 family protein bim and caspase-3. Adv Exp Med Biol,2010, Vol.658:111-116.
[86]Ghosh J, Das J, Manna P, et al. Taurine prevents arsenic-induced cardiac oxidative stress and apoptotic damage:role of NF-kappa B, p38 and JNK MAPK pathway. Toxicol Appl Pharmacol,2009, Vol.240(1):73-87.
[87]Li XM, Ma YT, Yang YN, et al. Downregulation of survival signalling pathways and increased apoptosis in the transition of pressure overload-induced cardiac hypertrophy to heart failure. Clin Exp Pharmacol Physiol,2009, Vol.36(11):1054-1061.
[88]Liu D, He M, Yi B. Pim-3 protects against cardiomyocyte apoptosis in anoxia/reoxygenation injury via p38-mediated signal pathway. Int J Biochem Cell Biol,2009, Vol.41(11): 2315-2322.
[89]Tsang MY, Rabkin SW. p38 mitogen-activated protein kinase (MAPK) is activated by noradrenaline and serves a cardioprotective role, whereas adrenaline inducesp38 MAPK dephosphorylation. Clin Exp Pharmacol Physiol,2009, Vol.36(8):e12-19.
[90]Hii CS, Anson DS, Costabile M, et al. Characterization of the MEK5-ERK5 module in human neutrophils and its relationship to ERK1/ERK2 in the chemotactic response. J Biol Chem,2004, Vol.279(48):49825-49834.
[91]Liu CJ, Lo JF, Kuo CH, et al. Akt mediates 17 beta-estradiol and/or estrogen receptor-alpha inhibition of LPS-induced tumor necresis factor-alpha expression and myocardial cell apoptosis by suppressing the JNK1/2-NFkappaB pathway. J Cell Mol Med,2009, Vol. 13(9B):3655-3667.
[92]Song HP, Zhang L, Dang YM, et al. The PI3K/Akt pathway protects cardiomyocytes from Ischemic and hypoxic apoptosis via mitochondrial function. Clin Exp Pharmacol Physiol, 2010, Jan 17. [Epub ahead of print].
[93]Thimmaiah KN, Easton JB, Houghton PJ. Protection from rapamycin-induced apoptosis by insulin-like growth factor I is partially dependent on protein kinase C signaling. Cancer Res, 2010, Vol.70(5):2000-2009.
[94]Hu L, Sun Y, Hu J. Catalpol inhibits apoptosis in hydrogen peroxide-induced endothelium by activating the PI3K/Akt signaling pathway and modulating expression of Bcl-2 and Bax. Eur J Pharmacol,2010, Vol.628(1-3):155-163.
[95]Loriot Y, Calderaro J, Soria JC, et al. Anti-apoptotic mechanisms in small-cell lung carcinoma. Ann Pathol,2010, Vol.30(1):17-24.
[96]Lu Z, Cox-Hipkin MA, Windsor WT, et al.3-phosphoinositide-dependent protein kinase-1 regulates proliferation and survival of cancer cells with an activated mitogen-activated protein kinase pathway. Mol Cancer Res,2010, Mar 2. [Epub ahead of print].
[97]Hongisto V, Vainio JC, Thompson R, et al. The Wnt pool of glycogen synthase kinase 3-beta is critical for trophic-deprivation-induced neuronal death. Mol Cell Biol,2008, Vol.28(5): 1515-1527.
[98]Wollert KC, Meyer GP, Lotz J, et al. Intracoronary autologous bone marrow cell transfer after myocardial infarction:The BOOST randomized controlled clinical trial. Lancet,2004, Vol.364(9429):141-148.
[99]Strauer BE, Brehm M, Zeus T, et al. Regeneration of human infracted heart muscle by intracoronary autologous bone marrow cell transplantation in chronic coronary artery disease: The IACT Study. J Am Coll Cardiol,2005, Vol.46(9):1651-1658.
[100]Scha'chinger V, Erbs S, Elsasser A, et al. Improved clinical outcome after intracoronary administration of bone marrow-derived progenitor cells in acute myocardial infarction:Final 1-year results of the REPAIR-AMI trial. Eur Heart J,2006, Vol.27(17):2775-2783
[101]Mathiasen AB, Haack-Sorensen M, Kastrup J. Mesenchymal stromal cells for cardiovascular repair:current status and future challenges. Future Cardiol,2009, Vol.5(6):605-617.
[102]Abarbanell AM, Coffey AC, Fehrenbacher JW, et al. Proinflammatory cytokine effects on mesenchymal stem cell therapy for the ischemic heart. Ann Thorac Surg,2009, Vol.88(3): 1036-1043.
[103]Chachques JC. Cellular cardiac regenerative therapy in which patients? Expert Rev Cardiovasc Ther,2009, Vol.7(8):911-919.
[104]Joggerst SJ, Hatzopoulos AK. Stem cell therapy for cardiac repair:benefits and barriers. Expert Rev Mol Med,2009, Vol.11:e20.
[105]Trounson A. New perspectives in human stem cell therapeutic research. BMC Med,2009, Vol.11(7):29.
[106]Kao RL, Browder W, Li C. Cellular cardiomyoplasty:what have we learned? Asian Cardiovasc Thorac Ann,2009, Vol.17(1):89-101.
[107]王耀晟,程晓曙.骨髓间充质干细胞移植治疗心肌梗死的新技术.国际心血管病杂志,2007,Vol.34(1)：30～34.
[108]王耀晟,王伶,杨人强,等.表达hVEGF121/EGFP融合蛋白骨髓间充质干细胞的制备及缺氧干预.中国组织工程研究与临床康复,2008,Vol.12(21)：4001～4006. 参考文献
[109]王耀晟.表达hVEGF121/EGFP的间充质干细胞制备及缺氧对其表达影响.中国优秀硕士学位论文全文数据库,医药卫生科技辑,2008年3月,CNKI：CDMD：2.2008.011887.
[110]Engelhardt S, Hein L, Wiesmann F, et al. Progressive hypertrophy and heart failure in beta1-adrenergic receptor transgenic mice. Proc Natl Acad Sci USA,1999, Vol.96(12):7059～7064.
[111]王耀晟,程晓曙.细胞移植修复缺血性心肌损伤的哲学思考.南昌大学学报(人文社会科学版),2009,Vol.40(2)：65～68.
[112]Singh K, Communal C, Sawyer DB, et al. Adrenergic regulation of myocardial apoptosis. Cardiovasc Res,2000, Vol.45(3):713-719.
[113]Pfeffer MA, Braunwald E. Ventricular remodeling after myocardial infarction. Experimental observations and clinical implications. Circulation,1990, Vol.81(4):1161-1172.
[114]Li XY, Wang T, Jiang XJ, et al. Injectable hydrogel helps bone marrow-derived mononuclear cells restore infarcted myocardium. Cardiology,2010, Vol.115(3):194-199.
[115]Zhang Y, Wang H, Kovacs A, et al. Reduced expression of Cx43 attenuates ventricular remodeling after myocardial infarction via impaired TGF-beta signaling. Am J Physiol Heart Circ Physiol,2010, Vol.298(2):H477-487.
[116]Piao H, Youn TJ, Kwon JS, et al. Effects of bone marrow derived mesenchymal stem cells transplantation in acutely infarcting myocardium. Eur J Heart Fail,2005, Vol.7(5):730-738.
[117]Schuleri KH, Feigenbaum GS, Centola M, et al. Autologous mesenchymal stem cells produce reverse remodelling in chronic ischaemic cardiomyopathy. Eur Heart J,2009, Vol. 30(22):2722-2732.
[118]Hu X, Wang J, Chen J, et al. Optimal temporal delivery of bone marrow mesenchymal stem cells in rats with myocardial infarction. Eur J Cardiothorac Surg,2007, Vol.31(3):438-443.
[119]王耀晟,程晓曙,王伶,等.不同缺氧状态下骨髓间充质干细胞表达血管内皮生长因子的差异.中国组织工程研究与临床康复,2009,Vol.13(36)：7143～7146.
[120]Biondi-Zoccai GG, Abbate A, Vasaturo F, et al. Increased apoptosis in remote non-infarcted myocardium in muhivessel coronary disease. Int J Cardiol,2004, Vol.94(1):105-110.
[121]Chatterjee S, Stewart AS, Bish LT, et al. Viral gene transfer of the antiapoptotic factor Bcl-2 protects against chronic postischemic heart failure. Circulation,2002, Vol.106(12Suppl 1): 1212-217.
[122]Tsubokawa T, Yagi K, Nakanishi C, et al. Impact of anti-apoptotic and oxidative effects of bone marrow mesenchymal stem cells with transient overexpression of heme oxygenase-1 on myocardial ischemia. Am J Physiol Heart Circ Physiol,2010, Feb 12. [Epub ahead of print]
[123]张瑞成,董念国,侯剑峰,等.骨髓干细胞移植可影响心肌梗死后心肌细胞的凋亡.心脏杂志,2007, Vol.19(4):388-390,398.
[124]Kinnaird T, Stabile E, Burnett MS et al. Marrow-derived stromal cells express genes encoding a broad spectrum of arteriogenic cytokines and promote in-vitro and in-vivo. arteriogenesis through paracrine mechanisms, CirRes,2004, Vol.94:678-685.
[125]王耀晟,程晓曙,王伶,等.骨髓间充质干细胞在缺氧下VEGF的表达规律.第十九届 长城国际心脏病学会议,北京,2008,August：gw19～c0094.
[126]Yaosheng Wang, Xiaoshu Cheng, Lipeng He, et al. Expression of hVEGF121/EGFP in mesenchymal stem cells and angiogenic function of the fusion protein. World Congress of Cardiology Scientific Sessions 2010, Beijing,2010, June:28328.
[127]Berry C, Balachandran KP, L'Allier PL, et al. Importance of collateral circulation in coronary heart disease. Eur Heart J,2007, Vol.28(3):278-291.
[128]Sun Y, Weber KT. Infarct scar:a dynamic tissue. Cardiovasc Res,2000, Vol.46(2):250-256.
[129]王耀晟,程晓曙,王伶,等.骨髓间充质干细胞在缺氧条件下VEGF的表达规律.中国应用生理学杂志,2009,Vol.25(4)：475～477.
1130] Basso C, Rizzo S, Thiene G. The metamorphosis of myocardial infarction following coronary recanalization. Cardiovasc Pathol,2010, Vol.19(1):22-28.
[131]Gallagher GL, Jackson CJ, Hunyor SN. Myocardial extracellular matrix remodeling in ischemic heart failure. Front Biosci,2007, Vol.12:1410-1419.
[132]Bhindi R, Witting PK, McMahon AC, et al. Rat models of myocardial infarction. Pathogenetic insights and clinical relevance. Thromb Haemost,2006, Vol.96(5):602-610.
[133]Anversa P, Leri A, Kajstura J. Cardiac regeneration. J Am Coll Cardiol,2006, Vol.47(9): 1769-1776.
[134]Khan R, Sheppard R. Fibrosis in heart disease:understanding the role of transforming growth factor-beta in cardiomyopathy, valvular disease and arrhythmia. Immunology,2006, Vol.118(1):10-24.
[135]Liu L, Hu J, Sun B, et al. Mesenchymal stem cells inhibition of chronic ethanol-induced oxidative damage via upregulation of phosphatidylinositol-3-kinase/Akt and modulation of extracellular signal-regulated kinase 1/2 activation in PC12 cells and neurons. Neuroscience, 2010 Feb 11. [Epub ahead of print].
[136]Cho J, Rameshwar P, Sadoshima J. Distinct roles of glycogen synthase kinase (GSK)-3alpha and GSK-3beta in mediating cardiomyocyte differentiation in murine bone marrow-derived mesenchymal stem cells. J Biol Chem,2009,284(52):36647-36658.
[1]Israel A, Zavala LE, Cierco M, et al. Effect of AT1 angiotensin Ⅱ receptor antagonists on the sympathetic response to a cold pressor test in healthy volunteers. Am J Ther,2007, 14(2):183-188.
[2]Israel A, Zavala LE, Cierco M, et al. Effect of eprosartan on the sympathetic response to cold pressor test in healthy volunteers. Auton Neurosci,2006,126-127:179-184.
[3]Lindenblatt N, Menger MD, Klar E, et al. Sustained hypothermia accelerates microvascular thrombus formation in mice. Am J Physiol Heart Circ Physiol,2005,289(6):H2680-2687.
[4]Straub A, Azevedo R, Beierlein W, et al. Glycoprotein Ⅱb/Ⅲa inhibition reduces prothrombotic events under conditions of deep hypothermic circulatory arrest. Thromb Haemost,2005,94(1):115-22.
[5]Rothoerl RD, Brawanski A. The history and present status of deep hypothermia and circulatory arrest in cerebrovascular surgery. Neurosurg Focus,2006,20(6):E5.
[6]Downey JM, Cohen MV. Reducing infarct size in the setting of acute myocardial infarction. Prog Cardiovasc Dis,2006,48(5):363-371.
[7]Maeng M, Mortensson UM, Kristensen J, et al. Hypothermia during reperfusion does not reduce myocardial infarct size in pigs. Basic Res Cardiol 2006,101(1):61-68.
[8]Olivecrona GK, Gotberg M, Harnek J, et al. Mild hypothermia reduces cardiac post-ischemic reactive Hyperemia. BMC Cardiovasc Disord,2007,7:5.
[9]Mattu A, Brady WJ, Perron AD. Electrocardiographic manifestations of hypothermia. Am J Emerg Med,2002,20(4):314-326.
[10]Garcia-Dorado D, Rodriguez-Sinovas A, Ruiz-Meana M. Gap junction-mediated spread of cell injury and death during myocardial ischemia-reperfusion. Cardiovasc Res,2004, 61(3):386-401.
[11]Fedorov VV, Li L, Glukhov A, et al. Hibernator Citellus undulatus maintains safe cardiac conduction and is protected against tachyarrhythmias during extreme hypothermia:Possible role of Cx43 and Cx45 up-regulation. Heart Rhythm,2005,2(9):966-975.
[12]Aupperle H, Garbade J, Ullmann C, et al. Comparing the ultrastructural effects of two different cardiac preparation-and perfusion-techniques in a porcine model of extracorporal long-term preservation. Eur J Cardiothorac Surg,2007,31(2):214-221.
[13]Bombig MT, Ferreira C, Mora O, et al. Pravastatin protection from cold stress in myocardium of rats. Jpn Heart J,2003,44(2):243-255.
[14]Steensrud T, Nordhaug D, Husnes KV, et al. Replacing potassium with nicorandil in cold St. Thomas'Hospital cardioplegia improves preservation of energetics and function in pig hearts. Ann Thorac Surg,2004,77(4):1391-1397.
[15]Jin SQ, Niu LJ, Deng CY, et al. Effect of temperature on the activation of myocardial KATP channel in guinea pig ventricular myocytes:a pilot study by whole cell patch clamp recording. Chin Med J (Engl),2006,119(20):1721-1726.
[16]Nakayama H, Chen X, Baines CP, et al. Ca2+-and mitochondrial-dependent cardiomyocyte necrosis as a primary mediator of heart failure. J Clin Invest,2007,117(9):2431-2444.
[17]Yatani A, Kim SJ, Kudej RK, et al. Insights into cardioprotection obtained from study of cellular Ca2+ handling in myocardium of true hibernating mammals. Am J Physiol Heart Circ Physiol,2004,286(6):H2219-2228.
[18]吴自强,祝善俊,祝之明,等.高盐饮食与冷应激复合诱导大鼠心肌肥厚的药物干预.临床心血管病杂志,2003,19(6)：369-372.
[19]Hoye AT, Davoren JE, Wipf P, et al. Targeting mitochondria. Acc Chem Res,2008, 41(1):87-97.
[20]宋学立,钱令嘉,李风芝.冷应激对心肌细胞损伤的影响.解放军预防医学杂志,2001,19(2)：88-91.
[21]Fairchild KD, Singh IS, Patel S, et al. Hypothermia prolongs activation of NF-kB and augments generation of inflammatory cytokines. Am J Physiol Cell Physiol,2004, 287(2):C422-31.
[22]Hamid AA, Mandai M, Fujita J, et al. Expression of cold-inducible RNA-binding protein in the normal endometrium, endometrial hyperplasia, and endometrial carcinoma. Int J Gynecol Pathol,2003,22(3):240-247.
[23]肖华,陈志坚,廖玉华,等.TNF-α早期表达在大鼠急性心肌梗死室性心律失常发生中的作用及机制研究.中国免疫学杂志,2008,24(1)：75-79.