【作者】 刘惠良；

【导师】 齐晓勇；

【作者基本信息】 河北医科大学 ， 内科学， 2014， 博士

【摘要】 慢性心力衰竭（Chronic heart failure，CHF）是心血管疾病的常见并发症，其发病率逐年上升，正日益威胁着人们的身体健康。心力衰竭基础研究的深入和循证医学的开展，使得对心力衰竭的发病原因、病理生理机制以及临床预防和治疗的研究取得了巨大进步，而成功的心力衰竭动物模型建立为此提供了客观保障和积极贡献。动物心衰模型制作常用方法包括：心肌缺血型、压力负荷型、容量负荷型以及心肌病变型，不同的动物模型可以模拟相应疾病的发病机制。目前常用于研究代偿性肥大到失代偿心力衰竭的病理生理改变的动物模型之一是压力超负荷心衰模型，主动脉缩窄模型是左心室压力负荷心衰模型中较为理想的一种。其病理生理机制为早期心肌细胞发生代偿性肥大以对抗增高的后负荷，维持正常的射血功能，晚期发展为心力衰竭，而基因表达的改变、激素的影响、凋亡、能量代谢障碍、氧化应激、心律失常、血管功能障碍、胶原的沉积等是多重促进因素。心脏收缩力调节(cardiac contractility modulation，CCM)是指在心肌动作电位绝对不应期施加的一种非兴奋性电刺激信号（non-ExcitatoryCurrents，NEC）即绝对不应期电刺激（absolute refractory period electricalstimulation，ARPES）。ARPES不引发动作电位，但是能够使心肌细胞的收缩力增强。CCM的治疗可以改善左心室收缩功能，提高左室射血分数，而不增加额外耗氧量，可用于心力衰竭的治疗。自由基对生物细胞膜损伤的最终产物丙二醛(Mafonaldehyde，MDA)具有很强的生物毒性，而体内重要的自由基清除剂是超氧化物歧化酶（Superoxide Dismutase，SOD），SOD能够有效地清除体内自由基，保护细胞免受损伤；谷胱甘肽过氧化物酶(Glutathione-peroxidase，GSH-Px)作为催化过氧化氢分解的酶，也可以起到保护细胞膜结构和功能的作用。已有研究表明CHF患者的抗氧化能力和清除氧自由基能力降低。血浆MDA水平均随CHF程度增加而上升，而SOD活性随之下降，能够反映CHF程度及其预后。然而，有关CCM对心衰患者氧化应激影响的研究尚未见报道，需进一步研究。CHF的发生和发展过程中，肾素-血管紧张素-醛固酮系统(renin-angiotensin-aldosterone system，RAAS)的激活发挥着重要的作用。心衰时出现心室重塑，其重要表现是心肌纤维化（myocardial fibrosis，MF），而血管紧张素Ⅱ(angiotonin II，AngⅡ)是诱导MF、心肌细胞肥大过程中的主要介质，在CHF患者中明显升高，且升高水平与CHF病死率明显相关。AngⅡ过度表达导致心肌代谢及功能异常，促进压力超负荷诱导的心脏功能障碍，可使血管和心肌重构，这些病理改变又进一步激活RAAS，形成恶性循环。心肌收缩蛋白主要包括三种，即肌球蛋白、肌动蛋白和原肌凝蛋白，肌球蛋白是心肌的主要结构蛋白，占心脏成份的60%。心脏仅有2种肌球蛋白重链（myosin heavy chain，MHC）表达，即α-MHC和β-MHC，形成α-α和β-β同二聚体及α-β异二聚体，它们分别形成同功酶V1、V2和V3。V1具有最高的Ca2+和actin-activated ATPase活性，V2次之，V3为最低。正常心室肌以α-MHC表达为主，在左心室压力负荷增加时，机械牵张或神经体液因子可以刺激胚胎型β-MHC基因再表达增加。β-MHC与肌动蛋白的亲和力低于α-MHC，β-MHC基因再表达增加使心肌收缩力减弱、收缩速度减慢，导致心肌收缩功能减退。CCM可以增强心肌收缩力，其作用是否通过改变肌球蛋白重链表达尚需进一步研究。细胞凋亡是一种主动的细胞死亡，其整个过程需要蛋白的合成以及相关基因的调控才能完成，其中Bcl-2家族和Caspase家族是最重要的凋亡调控蛋白。凋亡的调控由十分复杂的信号网络系统控制，目前已知有3条主要信号通路：①线粒体通路；②死亡受体通路；③内质网通路。半胱胺酸-天冬氨酸蛋白酶（cysteine aspartate protease，Caspases-3）是体内细胞凋亡的执行者，凋亡的信号转导通路最终都能激活Caspases-3而水解各种细胞成分使细胞凋亡。乙醛脱氢酶2(acetaldehyde dehydrogenase，ALDH2)在依赖于线粒体提供能量的心脏表达特别多，研究发现其在心功能衰竭心肌中表达显著下降，ALDH2有抑制心肌细胞凋亡的作用。心肌细胞凋亡程度与心肌细胞表达Bcl-2/Bax的比率有关，Bcl-2/Bax的比率升高可以抑制心肌细胞凋亡，而Bcl-2/Bax的比率下降则促进心肌细胞凋亡。肌浆网是细胞内重要钙贮存器，其在胞浆中调节钙（calcium，Ca2+）浓度发挥着重要作用。Ca2+是心肌收缩和舒张活动的中心环节，肌浆网Ca2+-ATP酶（sarcoplasmic-endoplasmic-reticulum calcium ATPase，SERCA）在Ca2+的摄入、贮存和释放中发挥着关键作用。SERCA2分为SERCA2a，SERCA2b和SERCA2c三种亚型，心脏中主要是SERCA2a。肌浆网Ca2+摄取的两个关键因素是SERCA和受磷蛋白(phospho lamban，PLN)， PLN通过磷酸化和去磷酸化与SERCA2a作用，调节其Ca2+摄取功能。在人和实验动物心力衰竭模型中发现SERCA2a蛋白的表达及活性与正常心肌相比有明显降低。在慢性心衰情况下，心肌细胞功能失调的特征变化之一就是SERCA2a的活性及其mRNA表达下降，而增加SERCA2a的表达后可促进下降的心肌收缩力得到恢复，心功能得到改善。亦有研究发现在心力衰竭心肌中SERCA2amRNA表达与SERCA2a蛋白水平并不呈正相关，心肌心力衰竭发展的不同时期，SERCA2a的活性也不一样，而CCM增强心肌收缩力是否与SERCA2a及PLN对Ca2+摄入、贮存和释放的调节有关亦需进一步研究。本实验通过建立升主动脉缩窄心力衰竭兔模型，观察CCM对心力衰竭心功能影响，并探讨其可能机制。第一部分心脏收缩力调节对慢性心力衰竭兔心功能影响目的：通过升主动脉环扎法建立兔慢性心力衰竭模型，观察CCM对心功能的影响。方法：6月龄健康新西兰兔32只随机分为3组：假手术组（SHAM，n=8）、心衰模型组（HF，n=12）、CCM治疗组（CCM，n=12）。HF组与CCM组开胸后于升主动脉根部远端1.0cm处行环扎缩窄术；CCM组造模术中于左心室前壁近心尖部预置小儿临时起搏电极；SHAM组仅开胸，不给予主动脉环扎。三组分别于术前、术后4周、8周、12周行超声心动图检查，SHAM组、HF组于术后12周取血标本检测ANP、NT-proBNP。CCM组12周时给予绝对不应期电刺激（ARPES），每日6小时，持续一周，ARPES后采血标本检测。结果：1一般情况：32只新西兰兔，其中SHAM组8只术中无死亡，12周后均存活；HF组12只兔术中因气胸死亡2只，术后1只兔死于心力衰竭，12周时存活9只；CCM组12只兔术中死亡1只，术后死亡2只，1只兔自啮断电极导线，无法行CCM刺激而去组，12周时有效模型8只。24只兔造模成功17只，成功率70.8%；术前3组新西兰兔比较组间体重（Weight）、呼吸频率（R）和心率（HR）无明显差别；12周时HF组、CCM组兔临床表现为精神不振，活动少，口唇轻微紫绀，呼吸、心率比对照组增加，体重比对照组减少。2超声心动图指标变化：常规测量心脏超声指标，以LVEF＜50%为达到心衰标准。术前三组超声各项指标无明显差别。术后4周时三组兔的超声指标（如LVEF）比较虽有差异，但无统计学意义；8周时HF与CCM组有部分兔达到心衰标准；12周时HF与CCM组兔超声检查均达到LVEF＜50%的心衰标准。HF组和CCM组的IVS、IVPW、LVESD和LVEDD比SHAM组明显升高，而LVEF、LVFS和E/A比值在HF与CCM组比SHAM组明显下降。CCM组ARPES电刺激后心功能较HF组改善，差异有统计学意义。3血清心力衰竭标志物水平的比较：12周时HF组、CCM组血清ANP、NT-pro-BNP比对照组显著升高。而CCM组与HF组比较则显著降低，差异有统计学意义。结论：升主动脉环扎缩窄法可有效建立慢性心衰兔动物模型，CCM可增强心肌收缩力改善心衰兔的心功能。第二部分心脏收缩力调节对慢性心力衰竭兔的氧化应激的影响目的：观察慢性心力衰竭兔体内氧化应激的变化，探讨CCM对慢性心力衰竭兔的氧化应激的影响及机制。方法：8只CCM组兔，每天给予ARPES电刺激6小时，连续刺激一周，ARPES结束时采血标本，应用比色法检测血浆SOD、MDA、GSH-Px水平。SHAM、HF组兔12周时采血进行相应指标的检测结果：1CCM对慢性心力衰竭兔血浆SOD、GSH-Px水平的影响：HF组与SHAM组比较，血浆SOD、GSH-Px水平有明显下降，CCM组与SHAM组比较SOD、GSH-Px水平亦有下降，ARPES电刺激后CCM组SOD、GSH-Px水平比HF组有明显升高，差异有统计学意义。2CCM对慢性心力衰竭兔血浆MDA水平的影响：HF组与SHAM组比较，血浆MDA水平有明显升高，CCM组与SHAM组比较MDA水平亦有明显升高，CCM组MDA水平比HF组有明显下降，差异有统计学意义。3慢性心力衰竭兔的氧化应激与心衰程度关系分析：直线相关分析表明：NT-proBNP与SOD、GSH-Px呈负相关，相关系数r分别为（r=-0.809）、（r=-0.895）；NT-proBNP与MDA呈正相关（r=0.848）。ANP与NT-proBNP呈正相关性（r=0.914）。结论：心衰兔体内存在明显的过氧化损伤，CCM能够升高SOD及GSH-Px水平，降低MDA水平，这可能与CCM刺激能增加心肌收缩力，改善心脏的收缩和舒张功能有关；血浆MDA、SODG、SH-Px水平变化能够客观反映出CHF程度及其预后。第三部分心脏收缩力调节对慢性心力衰竭兔肌球蛋白重链表达的影响目的：观察慢性心力衰竭兔心室重构的发生发展，探讨CCM改善心力衰竭兔左心室重构的分子机制。方法：应用实时荧光定量反转录PCR、Western-blot方法对α-MHC、β-MHC mRNA及蛋白表达进行检测；应用酶联免疫吸附法测定血清中AngⅡ的含量；分离称重左心室心肌质量，计算左室心肌质量指数。结果：1HF组、CCM组血浆AngⅡ比SHAM组均有显著升高，差异有统计学意义；CCM组比HF组血浆AngⅡ虽有下降，但差异无统计学意义。2AngⅡ与ANP、NT-pro-BNP均呈正相关，相关系数分别为(r=0.946，r=0.875，P=0.000)。3CCM对α-MHC和β-MHC mRNA表达影响：α-MHC mRNA、β-MHC mRNA在三组中表达存在明显差异，α-MHC mRNA在HF组比SHAM组明显下降。CCM组与SHAM组比较α-MHC mRNA表达有下降，但两组差异无统计学意义；CCM在ARPES电刺激后α-MHC mRNA表达比HF组明显提高。β-MHC mRNA在HF组及CCM组均比SHAM组明显升高；CCM组在ARPES电刺激后β-MHC mRNA表达明显下降，与HF组比较差异有统计学意义。4CCM刺激对α-MHC and β-MHC蛋白表达影响：α-MHC蛋白表达水平在SHAM组与β-action参照比较处于较高水平表达，而β-MHC蛋白处于较低水平表达。HF组与CCM组的α-MHC蛋白表达水平明显下降而β-MHC蛋白表达明显升高。CCM组与HF组比较，ARPES电刺激后出现α-MHC蛋白表达水平的升高和β-MHC蛋白表达水平的下降。5左室质量指数LVMI在HF组与CCM组均比SHAM组明显增加，HF组与CCM组数值虽有差别，但差异无统计学意义。结论：心衰时LVMI及血浆AngⅡ增高，ARPES电刺激短期内对LVMI及AngⅡ的影响不显著；心肌α-MHC mRNA及蛋白表达减少，β-MHCmRNA及蛋白表达增多，心肌发生了明显重构。CCM治疗后α-MHCmRNA及蛋白表达水平增高，β-MHC mRNA及蛋白表达水平减少，显示CCM是通过改善心力衰竭兔心肌肌球蛋白重链蛋白表达，使心肌肌球蛋白重链逆重构而增强心脏收缩力的。第四部分心脏收缩调节对慢性心力衰竭兔心肌细胞钙收缩调节的影响目的：观察CCM对慢性心力衰竭兔心肌SERCA2mRNA、PLN mRNA及其蛋白水平的影响，探讨其在分子水平的作用机制。方法：应用RT-PCR、Western-blot方法检测心肌组织中的SERCA2a及PLN的mRNA及蛋白表达水平。结果：1CCM对SERCA2a及PLN mRNA表达影响：SERCA2a mRNA表达在HF组比SHAM组明显下降；CCM组与SHAM组比较有差异，但无统计学意义，CCM组在ARPES电刺激后SERCA2a mRNA基因表达较HF组明显提高。PLN mRNA表达在HF组、CCM组均比SHAM组明显升高，CCM组在ARPES电刺激后PLN mRNA表达明显下降，与HF组比较差异有统计学意义。SHAM组、HF组及CCM组的SERCA2a/PLN（S/P）比值的均值分别为0.86±0.07、0.057±0.001、0.38±0.07（P＜0.01）；CCM组经ARPES电刺激后，S/P比值在CCM组比HF组明显升高。2CCM对SERCA2a及PLN蛋白表达影响：以β-action为内参照，SHAM组SERCA2a蛋白表达水平与参照水平相似，处于较高水平表达，而PLN蛋白处于较低水平表达。HF组与CCM组中SERCA2a蛋白表达水平明显下降而PLN蛋白表达水平明显升高，ARPES电刺激后CCM组与HF组比较，SERCA2a和PLN蛋白表达水平出现反向变化，而PLN的变化更明显。3相关性分析显示NT-proBNP与SERCA2a呈负相关，相关系数r为（r=-0.856P=0.000）；NT-proBNP与PLN呈正相关（r=0.918P=0.000）；SERCA2a与PLN呈负相关性（r=-0.892P=0.000）。结论：兔心力衰竭时心肌SERCA2a mRNA及蛋白表达水平降低，PLNmRNA及蛋白表达水平升高；CCM治疗后SERCA2a mRNA及蛋白表达水平提高而PLN mRNA及蛋白表达水平下降，CCM通过影响钙调节蛋白mRNA及蛋白的表达而改善心脏收缩舒张功能。ARPES对PLNmRNA及蛋白表达影响更大，PLN可能是比SERCA2a更敏感的观察指标，S/P比值可能更能决定和反映心功能的改善。第五部分心脏收缩力调节对慢性心力衰竭兔心肌细胞凋亡的影响目的：通过制作兔升主动脉缩窄心力衰竭模型，观察在心力衰竭发展过程中心肌细胞的凋亡发生发展，探讨CCM改善心功能的抗细胞凋亡机制。方法：CCM组在电刺激术完成后、SHAM和HF组在12周时，处死动物模型直接留取左室心肌标本，应用RT-PCR、Western-blot方法检测心肌组织中的的Bcl-2、Bax、ALDH2和Caspases-3mRNA以及蛋白基因表达水平；流式细胞仪观察心肌细胞凋亡比率，TUNEL方法观察心肌细胞凋亡。结果：1CCM对Bcl-2及Bax mRNA表达影响：以GAPDH为内参照基因，Bcl-2、Bax mRNA表达在三组中存在明显差异，Bcl-2mRNA表达在HF组和CCM组均较SHAM组明显下降，而Bax mRNA表达明显升高，差异均有统计学意义。CCM组在ARPES刺激后Bcl-2mRNA表达比HF组明显提高，而Bax mRNA表达明显下降，差异均有统计学意义。三组中Bcl-2/Bax比率为分别为0.92±0.22、0.05±0.14、0.17±0.04，ARPES刺激后Bcl-2/Bax比率在CCM组比HF组明显升高。2以β-action为内参照，SHAM组Bcl-2蛋白处于较高水平表达，而Bax蛋白处于较低水平表达；在HF组和CCM组，Bcl-2蛋白表达水平明显下降而Bax蛋白表达明显升高。ARPES刺激后，CCM组与HF组比较，出现了Bcl-2蛋白表达水平的升高和Bax蛋白表达水平的下降。3ALDH2、Caspase-3mRNA表达在三组中差异有统计学意义。HF组与SHAM组比较，ALDH2表达明显下降而Caspase-3mRNA表达明显升高，差异有统计学意义；CCM组与SHAM组比较，ALDH2mRNA表达下降，但两组差异无统计学意义，而Caspase-3mRNA表达则有升高，差异有统计学意义。ARPES刺激后CCM组与HF组比较，ALDH2mRNA明显提高而Caspase-3mRNA明显下降，差异有统计学意义。4SHAM组与β-action内参照水平相似，ALDH2蛋白处于较高表达水平，Caspase-3处于较低水平表达；在心力衰竭模型HF组和CCM组中，ALDH2蛋白表达水平明显下降，Caspase-3蛋白表达水平明显升高；ARPES刺激后，CCM组与HF组比较可见ALDH2蛋白表达水平的升高和Caspase-3蛋白表达水平的明显下降。5流式细胞术观察心肌细胞凋亡变化：三组比较心肌细胞凋亡%存在明显差异，HF组与CCM组为心力衰竭模型，较SHAM组心肌细胞凋亡百分比明显增加，三组比较有统计学差异；CCM组经一周的ARPES刺激后凋亡细胞数比HF组明显下降，差异有统计学意义。6TUNEL法观察心肌细胞凋亡：可见SHAM组与正常参照相似，心衰模型组HF+CCM组比SHAM组凋亡小体增多，CCM组与HF组比较凋亡小体减少。结论：兔心衰时抗凋亡基因Bcl-2、ALDH2mRNA及蛋白表达减少，促凋亡基因Bax mRNA及蛋白表达增多，Caspase-3mRNA及蛋白表达增多，凋亡细胞百分比升高，表明慢性心力衰竭心肌存在明显凋亡；经CCM治疗后Bcl-2、ALDH2mRNA及蛋白表达增多，而Bax mRNA、Caspase-3mRNA及蛋白表达减少，Bcl-2/Bax比率升高，心肌凋亡细胞减少。表明CCM刺激在增强心肌收缩力、改善心功能同时可以逆转心肌细胞的凋亡的发生和发展，可能是其能增强心肌收缩力的途径之一。更多还原

【Abstract】 Chronic heart failure (CHF) is a common complication of cardiovasculardisease; its incidence is increasing year by year and threatening the health ofus. Recently, along with the related basic study of heart failure andevidence-based development, we have more information about pathogenesis,pathophysiology and clinical prevention and treatment of heart failure.Successful establishment of heart failure animal model can provide objectivesupport and positive contributions to basic and clinical research.The animal models of CHF mainly include myocardial ischemia model,pressure load model, capacity load and cardiomyopathy. Pressure overloadheart failure model can be used to study the compensatory hypertrophy todecompensated heart failure pathophysiology changes. At present, coarctationof the aorta model is the most optimum model in all left ventricular pressureoverload heart failure model. Pig, dog, rabbit, rat, guinea pigs and monkeys,baboons are often used to establish the animal model of heart failure and dog,pig and rabbit are most common used. The advantage of rabbit is relativelyclose to human in some cell electrophysiological characteristics. The earlystage of pressure overload is mainly through compensatory hypertrophy,which by hypertrophy of myocardial cell, to overcome the load increased,guarantee the normal ejection fraction. Lately, suffering from heart failure iscombined results of various factors including changes in gene expression,hormones, apoptosis, energy metabolism, oxidative stress, vasculardysfunction, arrhythmia, collagen deposition and so on.Cardiac contraction modulation is a non-excitatory current (NEC) electricstimulation applied during absolute refractory period (ARPES), which isabsolute refractory period electrical stimulation (ARPES). It does not inducemyocardial cell action potentials but can boost the myocardial contractility and improve the heart function. Animal experiments and clinical trials showedthat treatment of CCM can improve left ventricular systolic function; increaseleft ventricular ejection fraction, and no extra consumption of oxygen, whichcan be used for the treatment of heart failure.Superoxide dismutase (SOD), an important free radical scavenger, caneffectively remove free radical in the body and protect cells from damage.Malondialdehyde (MDA), the final product of free radical damage tobiological membranes, has strong toxicity to the biological organisms.Glutathione peroxidase (GSH-Px) is a significant catalytic decomposition ofhydrogen peroxide enzyme, can protect the structure and function of cellmembrane function. Studies have shown that: oxygen free radicals areinvolved in the pathophysiological process of CHF. MDA levels in plasmaincreased with the degree of CHF while SOD activity decreased, which canreflect the degree and prognosis of CHF. Nevertheless, the effect of CCM onoxidative stress in patients with heart failure has not been reported and needfurther study.Renin angiotensin-aldosterone system (RAAS) plays an important role inthe occurrence and development process of CHF. Myocardial fibrosis (MF) isan important manifestation of ventricular remodeling. Angiotensin Ⅱ(Ang II)is a main medium induced MF and myocardial hypertrophy, which increasedsignificantly in CHF patients and the elevated level in CHF, was significantlyassociated with mortality. Ang II overexpression resulted in myocardialmetabolism and abnormal function, promoted the pressure overload inducedcardiac dysfunction, caused the vascular and myocardial remodeling, thesepathological changes can further activate RAAS, which is a vicious spiral.Myocardial contractile protein composed by myosin, actin, tropomyosin.Cardiac myosin accounted for60%of the protein, which is the majorstructural protein of myocardium. Heart only expressed myosin heavy chain(MHC) α-MHC and β-MHC, formed alpha-alpha and beta-beta with the twomer and alpha-beta ISO two dimers, which formed respectively the isoenzymeV1, V2and V3. V1has the highest activity of Ca2+and actin-activated ATPase, followed by V2, V3is the lowest. Ventricular muscle reexpressed of embryonicβ-MHC gene instead of α-MHC, increase proportion in total MHC expression,when left ventricular pressure overload and stimulated by mechanical stretchor neurohumoral factors. β-MHC has lower affinity to actin than α-MHC,consequently myocardial contractility is weak, and therefore slow down thespeed of contraction caused myocardial contractile dysfunction. WhetherCCM enhanced myocardial contractility is by changing the expression ofmyosin heavy chain still need further study.Cell apoptosis is an active cell death. Synthesis of related proteins andregulation genes was involved in apoptosis. The Caspase family and Bcl-2family are the most important apoptosis regulatory protein. Apoptosis iscontrolled by complex signal network system. As we all known, there arethree major signaling pathways: the mitochondrial pathway; the death receptorpathway and the endoplasmic reticulum pathway. These signal transductionpathways can activate apoptosis performer, cysteine aspartic acid protease(Caspases-3), it can hydrolysis of various cell components and cell apoptosis.The expression of aldehyde dehydrogenase2(ALDH2) is aboundant in themitochondrion provide energy dependent cardiac and its expressionsignificantly decreases in heart failure. ALDH2can inhibit the apoptosis ofmyocardial cells. Studies have found that the degree of the apoptosis ofmyocardial cells was related to the ratio of myocardial cells expressingBcl-2/Bax, the ratio of Bcl-2/Bax increased can inhibit myocardial cellapoptosis, and the ratio of Bcl-2/Bax decreased to promote apoptosis ofmyocardial cells.Sarcoplasmic reticulum, an important intracellular calcium storage device,plays an important role in the regulation of calcium concentration in thecytoplasm. Ca2+is a key factor in myocardial systolic and diastolic activity.Sarcoplasmic reticulum Ca2+-ATP enzyme (SERCA) plays a key role in Ca2+uptake, storage and release. SERCA2is composed by SERCA2a, SERCA2band SERCA2c, SERCA2a seems to be the mainly enzyme in the heart. Ca2+uptake of sarcoplasmic reticulum is mainly affected by SERCA and phospholamban (PLN). PLN through phosphorylation and dephosphorylationregulates Ca2+uptake of SERCA2a. In human and experimental animal modelsof heart failure was found the expression and activity of SERCA2a proteindecreased obviously compared with the normal myocardium. In chronic heartfailure, the expression of the activity and mRNA of SERCA2a decreased is thefeatures of the changes of myocardial dysfunction and increased theexpression of SERCA2a can promote the recovery of myocardial contractilityand cardiac function improvement. There some reports have found that theexpression of SERCA2amRNA in myocardium is not positive correlation withthe SERCA2a protein level in heart failure, in different period thedevelopment of heart failure, the SERCA2a activity of myocardial is not same.Whether there is relationship between CCM enhance myocardial contractilityand regulation of SERCA2a and PLN on Ca2+uptake, storage and release alsoneed further study.The aim of the present study is to observe the effects and mechanism ofCCM on cardiac function in rabbits with heart failure induced by ascendingaorta ring tied up.Part1Effects of cardiac contractility modulation on cardiac function inrabbits with chronic heart failureObjective: The aim of present study was to observe the effects of cardiaccontractility modulation on cardiac function in rabbits with chronic heartfailure induced by ligating ascending aortic.Methods:6months of32healthy New Zealand rabbits were randomlydivided into three groups: sham operation group (SHAM, n=8)；heart failuremodel group(HF, n=12)；CCM therapy group (CCM, n=12). In the HF groupand CCM group, we made cerclage constriction in the ascending aorta rootdistal1cm after thoracotomy. In the CCM group, we present a pediatrictemporary pacemaker electrode in the anterior left ventricular wall near apexduring the operation. In the SHAM group, we made a thoracotomy withoutaortic cerclage. Three groups respectively in the preoperative andpostoperative4weeks,8weeks and12weeks did electrocardiogram examinations. In SHAM group and HF group, the changes of ANP andNT-proBNP in serum were observed at12weeks after the operation. In theCCM group, we gave an absolute refractory period electrical stimulation at12weeks, lasting6hours everyday for7days, blood samples after ARPES.Results:1The general situation of32New Zealand rabbits: no death in SHAMgroup and after12weeks. In HF group,2rabbits died with pneumothorax and1rabbit died with heart failure after operation, at12weeks for9rabbits. InCCM group,1rabbits died during operation,2rabbits died after operation and1rabbit bitted off the electrode wire, no CCM stimulations was delivered andruled out,8effective models at12weeks. The successful rate of model was70.8％. Pre-operation weight (Weight), respiratory rate (R) and heart rate (HR)had no significant difference between the three groups of New Zealand rabbits.After12weeks HF group and CCM group rabbits showed spiritless, move less,cyanosis, respiratory. Compared with the control group, heart rate wasincreased, but body weight decreased.2Echocardiogram: Conventional measured the echocardiographicindexes, LVEF＜50％could meet the standard of heart failure. Preoperativethe indexes (for example LVEF) of the three groups had no significantdifference. The indexes(for example LVEF)between the three groups haddifference after4weeks of the operation, but the difference is not statisticallysignificant, some rabbits of the HF and CCM groups had meet the standard ofheart failure at8weeks and all rabbits of HF and CCM groups meet thestandard of heart failure at12weeks. Compared with SHAM group, IVS,IVPW, LVESD and LVEDD of HF and CCM group significantly increased,LVEF, LVFS and E/A were significantly decreased.Compared with HFgroup, the heart function had improved after ARPES electrostimulation inCCM group.3The level of serum marker in heart failure rabbits: Compared withcontrol group, serum ANP and NT-proBNP in HF and CCM groups significantly increased at12weeks. Compared with HF group, CCM groupsignificantly decreased.Conclutions: Ascending aortic root ligated allows effective establishmentof a rabbit model of chronic congestive heart failure. CCM signal treatmentcan improve cardiac function in rabbits with heart failure. The cardiaccontractility enhancement, which is the chang of LVESD, LVEF and E/A,implied the cardiac funtion improved.Part2Effects of cardiac contractility modulation in response to oxidativestress on chronic heart failure in rabbitsObjective: To observe the antioxidant capacity in chronic heart failure inrabbit and clearing oxygen free radical changes, study the effects of CCM onoxidative stress in rabbits with chronic heart failure and its mechanism.Methods:8rabbits in CCM group only give ARPES electricalstimulation6hours a day, to stimulate a week in a row. At the end of theARPES blood specimens, using colorimetry to detect the plasma SOD, MDA,GSH-Px levels. SHAM and HF groups at12weeks in pursuance of rabbit testthe corresponding indicators.Results:1CCM on rabbit the levels of SOD, GSH-Px in serum: The influence ofHF group compared with the SHAM group, the plasma SOD, GSH-Px levelsdrop, statistically significant. CCM group compared with the SHAM group,the levels of SOD, GSH-Px had also decreased. After the ARPES electricalstimulation, SOD and GSH-Px of CCM group are obviously higher than theHF group.2CCM on the level of MDA in plasma in chronic heart failure: theinfluence of HF group compared with the SHAM group, the level of MDA inplasma significantly increases. CCM group compared with the SHAM group,the MDA level has also significantly increased. CCM group are significantlylower than the HF group, the MDA level was statistically difference.3rabbit chronic heart failure of oxidative stress and degree of heartfailure relationship analysis: linear correlation analysis showed that the NT- proBNP and negatively correlated with SOD, GSH-Px correlation coefficient(r=0.809), respectively (r=0.895); NT-proBNP and MDA were positivelycorrelated (r=0.848).ANP was positively correlation with the NT-proBNP (r=0.914).Conclusions: heart failure in rabbit body exist obvious peroxidationdamage, CCM can increase levels of SOD and GSH-Px, decrease MDA. Thismay be related to CCM stimulation can increase myocardial contraction andimprove cardiac systolic and diastolic function. Plasma MDA, SOD, GSH-Pxchanges can objectively reflect the degree of CHF and prognosis.Part3Effect of cardiac contractile modulation on the expression ofmyosin heavy chain in chronic heart failure rabbitObjective: To investigate the molecular mechanism of CCM improve theexpression of myosin heavy chain in heart failure induced by pressure loadincreaseMethods: The expression of protein and mRNA of α-MHC, β-MHC wasrespectively detected by real-time fluorescent quantitative reversetranscription PCR and Western-blot. The content of Ang II in serum was testedby ELISA. Separation of left ventricular mass weighed to calculate leftventricular mass index.Results:1The levels of AngⅡ in plasma of HF and CCM were significantlyhigher than SHAM group. The levels of Ang Ⅱin CCM group than HF aredecreased, but no statistical significance.2Ang Ⅱ and ANP, NT-proBNP were positively correlated, thecorrelation coefficient respectively (r=0.946, r=0.875, P=0.000).3Effect of CCM stimulation on the expression of α-MHC and β-MHCmRNA: The expression of α-MHC mRNA is obvious differences in the threegroups. HF group significantly decreased compared with the SHAM group.CCM group compared with the SHAM group is markedly reduced, but nostatistical difference was found between two groups. After ARPES electricalstimulation α-MHC mRNA in CCM is obviously higher than HF group. The expression of β-MHC mRNA is obvious difference in the three groups. HFgroup increased significantly compared with the SHAM group. CCM groupcompared with the SHAM group, the expression of β-MHC mRNA has alsoincreased significantly. CCM group after ARPES electrical stimulation β-MHC mRNA expression significantly decreased, compared with HF groupwas statistically significant.4CCM stimulation expression of α-MHC and β-MHC protein: theexpression of α-MHC protein in the SHAM group compared with beta actionreference expressed at a higher level, and expression of β-MHC protein atlower levels. HF and CCM group of expression of α-MHC protein decreasedobviously and expression of β-MHC increased significantly. CCM groupcompared with HF group, ARPES electrical stimulation after the expression ofα-MHC protein raised and β-MHC protein decreased.5Left ventricular mass indexes (LVMI) in HF and CCM group increasedcompared with SHAM group. There are differences between the HF and CCMgroups, but no statistical difference.Conclusions: The LVMI and Ang Ⅱwas heighter in heart failure;ARPES electrical stimulation had no significant effect on LVMI and Ang Ⅱinsuch a short time. The expression of α-MHC mRNA and protein decreased inmyocardial of heart failure, the expression of β-MHC mRNA and proteinincreased. After treatment of CCM, the expression of α-MHC mRNA andprotein increased the expression of β-MHC mRNA and protein decreased.CCM enhanced cardiac contractility through the improvement ofreconstruction of myocardial myosin heavy chain in heart failure rabbit.Part4Cardiac contractile modulation effects on regulation of calciumcontraction in rabbit cardiomyocytes with chronic heart failureObjective：To observed the effects and mechanisms of CCM on themRNA and protein expression of PLN and SERCA2a in chronic heart failurerabbit myocardial.Methods: The mRNA and protein expression level of SERCA2a andPLN in myocardial tissue was detected by RT-PCR and Western-blot method. Results:1Effect of CCM stimulating on mRNA expression of SERCA2a andPLN: the mRNA expression of SERCA2a in HF group was obviously lowerthan that in SHAM group. There were no significant differences betweenCCM group and SHAM group. The expression of SERCA2a mRNA geneincreased significantly higher in CCM group after ARPES electricalstimulation than that in HF group. The mRNA expression of PLN in both HFgroup and CCM group were much higher than that in SHAM group. ThemRNA expression of PLN in CCM group after ARPES electrical stimulationdecreased significantly compared with HF group. SERCA2a/PLN (S/P) ratiosin SHAM group, HF group and the CCM group respectively were0.86±0.07,0.057±0.001,0.38±0.07, and there was statistics different in the threegroups (P <0.01). After ARPES electrical stimulation in CCM group, the ratioof S/P increased significantly in CCM group than that in HF group.2Effect of CCM stimulation on protein expression of SERCA2a andPLN: β-action as internal reference, SERCA2a protein expression at a highlevel that was similar to the reference level in SHAM group, while PLNprotein at the lower expression level that was similar to the reference level.SERCA2a protein expressions were significantly decreased in HF group andthe CCM group and expression of PLN protein was significantly increased.After ARPES electrical stimulation in CCM group compared with HF groupthat SERCA2a protein expression increased and the PLN protein expressiondecreased, while the change of PLN was more obviously.3Correlation analysis showed that NT-proBNP was negatively correlatedwith SERCA2a, and the correlation coefficient was obviously (r=-0.856P=0.000); NT-proBNP is positive correlation with PLN（r=0.918P=0.000）；SERCA2a was negatively correlated with PLN (r=0.918P=0.000).Conclusions: Protein and mRNA of SERCA2a were lower expression inheart failure myocardium of rabbits, while mRNA and protein of PLN werehigher expression. Protein and mRNA expression of SERCA2a increased afterCCM stimulated while PLN decreased. CCM improved cardiac systolic and diastolic function through influencing of calcium regulating protein mRNAand protein expression. ARPES had greater influence on mRNA and proteinexpression of PLN. PLN could be observation index that more sensitive thanSERCA2a, and the ratio of S/P may be more likely to determine and reflectthe improvement of heart function.Part5Cardiac contractility modulation effects on the apoptosis ofchronic heart failure in rabbit cardiomyocytesObjective: To observe the apoptosis in the development of heart failurein cardiomyocytes and investigate the mechanism. To observe theantiapoptotic mechanism of CCM improving cardiac function.Methods: After electrical stimulation in CCM group, SHAM group andHF group was killed animal model during12weeks then directly took leftventricular myocardial specimens, then mRNA and protein gene expression ofBcl-2, Bax, ALDH2and Caspases-3in myocardial tissue was detected byRT-PCR and Western-blot. Using flow cytometry to test the rate ofcardiomyocytes apoptosis. Cardiomyocytes apoptosis was observed byTUNEL methodResults:1GAPDH as internal reference, there was a marked difference about themRNA expression of Bcl-2、Bax in three groups. The mRNA expression ofBcl-2in CCM group decreased significantly comparied with SHAM group,while Bax increased significantly. The mRNA expression of Bcl-2increasedsignificantly after ARPES stimulation in CCM comparied with HF group,while mRNA expression of Bax decreased. Bcl-2/Bax ratios in three grouprespectively were0.92±0.22、0.05±0.14、0.17±0.04and there were statisticsdifferent. Bcl-2/Bax ratios after ARPES stimulation increased significantly inCCM group comparied with HF group.2With β-action as internal reference, Bcl-2protein at a higher expressionlevel in SHAM group, while Bax protein at the lower expression level. Theexpression of Bcl-2protein decreased significantly in HF group and CCMgroup, while the expression of Bax protein increased significantly. After ARPES stimulation, CCM group compared with HF group, there wasappeared that Bcl-2protein expression increased and Bax protein expressiondecreased.3The mRNA expression of ALDH2, Caspase-3in three groups wasremarkable difference. HF group compared with SHAM group, the expressionof ALDH2decreased significantly and mRNA expression of Caspase-3increased significantly. CCM group compared with SHAM group, the mRNAexpression of ALDH2decreased, but there was no significantly differencebetween two groups, while Caspase-3increased and there was statisticsdifference. After ARPES stimulation in CCM group compared with HF group,the mRNA of ALDH2increased significantly while Caspase-3decreasedsignificantly.4SHAM group was similar to β-action the reference levels and ALDH2protein at high expression level while Caspase-3was expressed at lower level.ALDH2protein expression decreased significantly in the HF group and CCMgroup while Caspase-3protein expression levels increased significantly. AfterARPES stimulation, CCM group compared with HF group showed thatALDH2protein expression increased and expression of Caspase-3proteindecreased significantly.5Flow cytometry was used to observe the changes of cardiomyocytesapoptosis: There were obvious differences between the three groups ofcardiomyocytes apoptosis. The percentage of cardiomyocytes apoptosis in HFgroup and CCM group increased obviously compared with SHAM group.There were significant differences among three groups. The number ofcardiomyocytes after ARPES stimulation durning one week of CCM groupdecreased significantly than that in HF group.6Cardiomyocyte apoptosis by TUNEL observed: SHAM group and thenormal reference seen similar, HF and SHAM group than the CCM groupincreased apoptotic bodies, but apoptotic bodies were lesser in the CCM groupthan HF group.Conclusions: The mRNA and protein expression of antiapoptosis gene Bcl-2, ALDH2in rabbits with heart failure reduced, while pro apoptotic geneBax and Caspase-3increased. The percentage of cardiomyocytes increased,which showed that obvious apoptosis in rabbits with chronic heart failure. ThemRNA and protein expression of Bcl-2, ALDH2increased after treatment withCCM, while Bax and Caspase-3decreased, and the Ratio of Bcl-2/Baxincreased, CCM group reduced myocardial apoptosis than HF group. Itsuggested that CCM stimulation not only enhanced myocardial contractilityand improved heart function but also reversal the occurrence and developmentof cardiomyocytes apoptosis in reverse. This might be able to enhancemyocardial contractility.更多还原