糖尿病心肌组织肌浆网钙转运ATP酶小泛素样修饰的研究

【作者】 姚晶；

【导师】 吴永健；

【作者基本信息】 北京协和医学院 ， 内科学， 2014， 博士

【摘要】 背景：肌浆网钙转运ATP酶(SERCA2a)是心肌舒张期钙离子转运出细胞质最重要的离子泵,SERCA2a活性与表达下降是糖尿病导致心肌舒张功能不全以及心肌损伤的重要机制。新近研究报道了小泛素样修饰可以提高SERCA2aATP酶活性、蛋白表达及稳定性。既往研究讨论了糖尿病损伤心肌组织内SERCA2a表达及活性的众多机制,然而,糖尿病是否可以作用于SERCA2a的小泛素样修饰进而影响SERCA2a的活性与表达尚未明确。研究方法：使用小动物超声和左室压力测定评价糖尿病大鼠和对照组大鼠心肌收缩和舒张功能；使用Western-blot和RT-PCR等手段比较糖尿病组和对照组大鼠心肌组织内SERCA2a的表达情况,使用免疫共沉淀以及小泛素样修饰试剂盒分析糖尿病大鼠和对照组大鼠心肌组织内SERCA2a小泛素样修饰程度；同时比较在不同葡萄糖和胰岛素浓度下心肌细胞内SERCA2a小泛素样修饰程度以及SERCA2a和其他小泛素样修饰相关蛋白的表达水平。结果：相比于对照组大鼠,2型糖尿病大鼠心肌收缩、舒张功能均下降,尤以舒张功能下降更为明显。糖尿病大鼠心肌组织内SERCA2a蛋白水平、基因表达和小泛素样修饰程度均下降；小泛素样修饰酶E2(Ubc9)的蛋白水平在糖尿病大鼠心肌中也下降,而SUMO1和小泛素样修饰酶E1(SAE1和SAE2组成的二聚体)与对照组大鼠相比没有显著差异。高葡萄糖单独作用下,心肌细胞内SERCA2a的小泛素样修饰程度升高并随着葡萄糖浓度的提高而提高,然而在加入100mlU/L的胰岛素之后,心肌细胞内SERCA2a的小泛素样修饰程度随着葡萄糖浓度的提高而降低。Ubc9的蛋白水平变化与SERCA2a小泛素样修饰程度变化保持一致,而SUM01、SAE1和SAE2的蛋白水平在不同葡萄糖和胰岛素浓度下无明显变化。结论：SERCA2a小泛素样修饰程度和Ubc9蛋白水平在糖尿病心肌组织和高糖高胰岛素培养的心肌细胞中下降,这些结果提示SERCA2a小泛素样修饰和Ubc9与糖尿病的心肌损伤密切相关。研究背景：糖尿病是心肌梗死后发生心力衰竭和死亡的独立危险因素。肌浆网钙转运ATP酶(SERCA2a)活性和表达水平下降是糖尿病导致心肌损伤和心肌舒缩功能不全的重要机制。新近研究报道了小泛素样修饰可以提高SERCA2a的ATP酶活性、蛋白表达及稳定性。本课题第一部分研究发现糖尿病降低心肌组织内SERCA2a小泛素样修饰程度,本部分研究试图明确SERCA2a小泛素样修饰是否同样参与到糖尿病加剧心肌梗死后心功能不全以及心衰进展中。研究方法：2型糖尿病大鼠和对照大鼠分别被随机分为心梗手术组和假手术组,分别评估各组大鼠心功能,SERCA2a小泛素样修饰程度以及相关蛋白的表达情况。通过对原代心肌细胞氧剥夺6小时或12小时,评估缺氧预适应和长时间缺氧对心肌细胞内SERCA2a小泛素样修饰程度以及相关蛋白的表达情况的作用。结果：糖尿病加剧了心肌梗死后的心脏收缩功能不全。相比于假手术组心肌梗死后1周大鼠非梗死区心肌组织内SERCA2a小泛素样修饰程度显著升高,而心肌梗死后4周大鼠心肌组织内SERCA2a小泛素样修饰程度则又回落到与假手术组相似的水平。对于糖尿病大鼠而言,非梗死区心肌组织内SERCA2a小泛素样修饰程度在心肌梗死后急性期和慢性期均无显著变化。相比于对照组心肌细胞,缺氧预适应后心肌细胞内SERCA2a小泛素样修饰程度显著升高,然而长时间缺氧后心肌细胞内SERCA2a小泛素样修饰程度又显著下降。在各项试验中,小泛素样修饰酶E2(Ubc9)的表达与SERCA2a小泛素样修饰程度变化保持一致,而SUMO1和小泛素样修饰酶E1却无显著变化。结论：非糖尿病大鼠心肌梗死后非梗死区Ubc9和SERCA2a小泛素样修饰代偿性升高,而糖尿病大鼠则无,缺氧预适应使得心肌细胞Ubc9和SERCA2a小泛素样修饰升高,而长时间缺氧则使其降低,以上结果显示Ubc9和SERCA2a小泛素样修饰在糖尿病加剧心肌梗死后心功能不全这一过程中起到了重要作用。更多还原

【Abstract】 Background:Sarcoplasmic Reticulum Calcium-Transporting ATPase2a(SERCA2a) is the main Ca2+pump of cardiomyocyte in diastolic period. In the condition of diabetic cardiomyopathy, the activity and expression of SERCA2a is decreased, leading to diastolic and systolic dysfunction of myocardium. It was recently reported that SUMOylation could enhance the activity and stability of SERCA2a. We assume that diabetes might bring down the intensity of SUMOylation of SERCA2a in myocardium.Methods:The cardiac functions of diabetic and control rats were measured by echocardiography and left ventricular pressure measurement. SUMOylation intensity was evaluated by co-immunoprecipitation and SUMOylation kit. The expressions of SUM01, SERCA2a, Ubc9and other enzymes of SUMOylation were measured by Western-blot and RT-PCR. In addition, we also test whether the SUMOylation intensity of SERCA2a can be regulated by different concentrations of glucose and insulin in vitro.Results:Diet-induced type2diabetic rats represented diastolic and systolic dysfunction, and the diastolic dysfunction is much more severe. The SUMOylation intensity of SERCA2a was attenuated simultaneously with the expression of Ubc9in diet-induced diabetic rats, while the expressions of SUM01, SAE1and SAE2were not changed. Interestingly, glucose alone increased Ubc9expression and the SUMOylation intensity of SERCA2a of cardiomyocytes in vitro in a concentration-dependent manner; however, with addition of100mIU/L insulin, glucose decreased Ubc9and SUMOylation intensity in a concentration-dependent manner on the contrary.Conclusions:SUMOylation intensity of SERCA2a decreased in diet-induced type2diabetic rats in vivo and also in cardiomyocytes with addition of high glucose and insulin in vitro. These observations provide evidence that Ubc9and SUMOylation is involved in diabetic cardiomyopathy. Background:Diabetes is an independent risk factor for heart failure and mortality after myocardial infarction(MI). In the condition of diabetes, the activity and expression of Sarcoplasmic Reticulum Calcium-transporting ATPase(SERCA2a) are decreased, leading to diastolic and systolic dysfunction of myocardium. It was recently reported that SUMOylation could elevate the activity and stability of SERCA2a. In the first part of this thesis, we found that diabetes attenuate SUMOylation intensity SERCA2a in myocardium.Methods:Diet-induced type2diabetic rats and controls were divided into suture ligation induced MI groups or sham groups. Primary cardiomyocytes were cultured in different concentrations of glucose and insulin, and underwent oxygen deprivation(OD) for6or12hours. Echocardiograph and left ventricular pressure were measured to analyze cardiac function. The intensity of SUMOylation of SERCA2a, expressions of SERCA2a, SUM01and enzymes of SUMOylation were evaluated.Results:Diabetes exacerbated diastolic and systolic dysfunction of myocardium after infarction. SUMOylation intensities of SERCA2a were enhanced respectively in1-week-post-MI non-diabetic rats and6-hour-OD cardiomyocytes but not in4-week-post-MI rats and12-hour-OD cardiomyocytes. This compensatory enhancement was almost completely blunted in1or4weeks post-MI diabetic rats. In all experiments, the expression of enzyme2of SUMOylation, namely Ubc9, was always in accordance with the SUMOylation intensity, while SUM01and enzyme1were not changed.Conclusions:SUMOylation intensity of SERCA2a was compensatorily enhanced in post-MI non-diabetic rats, but not in diabetic rats. These observations provide evidence that Ubc9and SUMOylation of SERCA2a are involved in diabetes-mediated exacerbation of left ventricular dysfunction after MI.更多还原