自噬在自身抗体所致心力衰竭进程中对T小管重塑的影响

Effects of autophagy on transverse tubules of myocardiocytes during heart failure induced by autoantibodies

目的:通过大鼠自身抗体所致的心力衰竭模型的建立,研究细胞在β1-肾上腺素受体自身抗体(β1-adrenergic receptor autoantibody,β1-AA)所导致的心力衰竭中,自噬对T小管重塑的调控作用。方法:采用我校实验动物中心提供的健康Wistar大鼠24只为研究对象,将其随机分为4组,β1-AA组及β1-AA+Rapa组采用β1-AR-ECⅡ肽段对大鼠进行主动免疫,而其对照组(Control组)则采用溶剂对大鼠进行主动免疫,建立对照组模型,另设立阳性对照组(Rapa组)使用雷帕霉素对大鼠进行注射,与β1-AA+Rapa组进行对照。在免疫模型建立的前一天对每只大鼠进行尾静脉的血样采集,对血清进行分离,采用酶联免疫吸附法测量大鼠血样中的β1-AA的含量,此后定期测量β1-AA的含量。在建立模型后,分别于建立后的第8周和12周的第一天,每组分别选取3只大鼠,使用压力换能器及生物信号记录分析系统将输入的压力信号进行分析,测得大鼠的心功能参数。随后处死大鼠,对大鼠心脏进行活检,并取大鼠的血清和心室的心肌组织备用。采用共聚焦显微镜对大鼠左心室的横管重塑进行评估检测;采用Western blot法对大鼠心肌组织中的Beclin1蛋白的含量进行检测。结果:大鼠主动免疫模型建立后的第2周到第6周,β1-AA组及β1-AA+Rapa组大鼠体内的β1-AA水平快速升高,明显高于对照组大鼠,6周以后该两组大鼠的β1-AA水平比较稳定,但是仍显著高于对照组。β1-AA组大鼠心肌细胞内与自噬相关的Beclin1蛋白的表达水平从第8周起即明显低于其他3组。第8周时,β1-AA组及β1-AA+Rapa组心肌细胞横管均出现重塑改变,但不伴心功能明显改变;第12周时,β1-AA组大鼠心脏出现明显扩大,镜下可见心肌组织间隙有大量结缔组织增生,β1-AA组及β1-AA+Rapa组心肌细胞TT-power值均有进一步下降,但β1-AA组心肌细胞T小管重塑更为明显,心肌细胞横管亦显著破坏,其TT-power明显低于对照组,并伴心功能明显降低。结论:β1-AA可以抑制自噬活动,自噬活动减弱会导致心肌细胞横管系统的破坏,造成心肌细胞兴奋-收缩偶联障碍,从而导致心力衰竭的发生。在治疗因β1-AA所导致的心力衰竭中,上调自噬活性,可以延缓心力衰竭的发生,这为因β1-AA所导致的心力衰竭提供了新的治疗方法。

AIM： To study the role of autophagy that can regulate transverse tubules（ T-tubules）during the heart failure induced by β_1- adrenergic receptor autoantibodies（ β_1-AA） through the establishment of a rat model of heart failure induced by autoantibodies. METHODS： 24 healthy Wistar rats were provided by the Medical Lab as the research object of this study. All of them were divided into four groups randomly,each group had 6 rats. Two groups were the establishment of immunity model which were nominated as β_1-AA group and β_1-AA ＋Rapa group,these rats were actively immunized byβ1-AR-ECⅡ peptide,while the other two groups,established pseudo immune model,used solvent or Rapamycin to active immunize rats. Blood samples were collected from the caudal veins the rats one day before the active immunization models had been established. The serums were isolated by enzyme linked immunosorbent assay. After the model was established,the rats were killed at 8 weeks and 12 weeks after the establishment of the active immunization models. The rats of both groups were sacrificed via euthanasia. The autophagic related protein Beclin 1 of the rats were tested by Western blot. Meanwhile,the myocardiocytes of the rats were scanned by confocal microscope and optical microscope to evaluate the T-tubules and myocardiocytes of the hearts. The cardiac function was evaluated via blood pressure monitor. RESULTS： Two to six weeks after active immunization models had been established,β1-AA in the blood of the rats in the β_1-AA group and the β_1-AA ＋ Rapa group increased rapidly,significantly higher than the control group rats. After six weeks,the level of the β_1-AA in experimental rats was relatively stable,but it was still higher than the control group. Beclin1 protein in the experimental group of rats the level expressed lower than the other three groups. While T-tubules of the myocardiocytes in both the β_1-AA group and the β_1-AA ＋Rapa group had remodeled,the β_1-AA group ＇s remodeling was more significantly. The similar phenomenon was detected when we test these rats＇ cardiac function; the β_1-AA group ＇s heart cardiac function was declined dramatically 12 weeks after the establishment of the active immunization models.CONCLUSION： β_1-AA can suppress autophagy in myocardiocytes,and the remodeling of the T-tubules of the myocardiocytes will lead to heart failure.Stimulating autophagy of the myocardiocytes during the myocardiocytes remodeling will be a new method to treat heart failure.six weeks,the level of the β_1-AA in experimental rats was relatively stable,but it was still higher than the control group. Beclin1 protein in the experimental group of rats the level expressed lower than the other three groups. While T-tubules of the myocardiocytes in both the β_1-AA group and the β_1-AA ＋Rapa group had remodeled,the β_1-AA group ＇s remodeling was more significantly. The similar phenomenon was detected when we test these rats＇ cardiac function; the β_1-AA group ＇s heart cardiac function was declined dramatically 12 weeks after the establishment of the active immunization models.CONCLUSION： β_1-AA can suppress autophagy in myocardiocytes,and the remodeling of the T-tubules of the myocardiocytes will lead to heart failure.Stimulating autophagy of the myocardiocytes during the myocardiocytes remodeling will be a new method to treat heart failure.