Ca^(2+)/钙调蛋白依赖性蛋白激酶Ⅱ调节原肌球蛋白1对海洛因所致心肌细胞节律异常的影响  

Ca2+/calmodulin-dependent protein kinase Ⅱ modulates effect of tropomyosin1involved in role of heroin with causing abnormal cardiomyocyte rhythm in vitro

作　　者：季敏 苏丽萍[2] 刘丽[2] 管雅玲 肖锦玲 蒲红伟[3] JI Min;SU Liping;LIU Li;GUAN Yaling;XIAO Jinling;PU Hongwei(Pathology Teaching and Research Section of Basic Medical College,Xinjiang Medical University,Urumqi 830017,China;Department of Pathology,the First Affiliated Hospital of Xinjiang Medical University,Urumqi 830054,China;Subject Construction Department,the First Affiliated Hospital of Xinjiang Medical University,Urumqi 830054,China)

机构地区：[1]新疆医科大学基础医学院病理学教研室,乌鲁木齐830017 [2]新疆医科大学第一附属医院病理科,乌鲁木齐830054 [3]新疆医科大学第一附属医院学科建设科,乌鲁木齐830054

出　　处：《新疆医科大学学报》2022年第11期1231-1236,共6页Journal of Xinjiang Medical University

基　　金：国家自然科学基金地区科学基金(81860049,82160055)。

摘　　要：目的探讨Ca^(2+)/钙调蛋白依赖性蛋白激酶Ⅱ(Ca^(2+)/calmodulin-dependent protein kinaseII,CaMKII)调控原肌球蛋白1(Tropomyosin1,TPM1)在海洛因致心肌细胞节律异常中的作用。方法提取60只SPF级SD大鼠乳鼠心肌组织,培养原代心肌细胞,根据干预情况分为对照组(Control组)、海洛因处理组(HE组)、海洛因+KN-93处理组(HE+KN-93组)。Control组常规条件未作任何处理,HE组100μmol/L海洛因作用24 h,HE+KN-93组100μmol/L海洛因+1μmol/L抑制剂KN-93作用24 h。荧光倒置显微镜观察心肌细胞自发搏动频率的改变;Fluo-4 AM检测各组细胞内Ca^(2+)浓度的变化;串联质谱标记相对定量蛋白质组学筛查差异表达蛋白;Western blotting法检测TPM1、CaMKIIδ型及其T287位点[p-CaMKII(T287)]表达水平。结果心肌细胞自发搏动频率结果显示,Control组平均自发搏动频率(126.33±1.52)次/min高于HE组(88.00±2.64)次/min,HE+KN-93组自发搏动频率(110.33±1.52)次/min高于HE组(88.00±2.64)次/min,差异均有统计学意义(P<0.05)。Fluo-4 AM检测结果显示,HE组心肌细胞内Ca^(2+)浓度与Control组比较显著增加(P<0.05);HE+KN-93组细胞内Ca^(2+)浓度与HE组比较显著下降(P<0.05)。串联质谱标记相对定量蛋白质组学结果显示,HE组与Control组共筛选出784个差异表达蛋白质,其中上调差异表达蛋白质有442个,下调差异表达蛋白质有342个;HE组与HE+KN-93组共筛选出346个差异表达蛋白质,其中上调差异表达蛋白质有169个,下调差异表达蛋白质有177个。Western blotting结果显示,HE组p-CaMKII(T287)、TPM1蛋白相对表达量与Control组比较,均显著增加(P<0.05);HE+KN-93组p-CaMKII(T287)、TPM1蛋白相对表达量与HE组比较均降低(P<0.05);3组CaMKIIδ蛋白相对表达量均无统计学意义(P>0.05)。结论海洛因可造成心肌细胞节律异常改变,其机制可能与CaMKII激活后上调TPM1蛋白有关,这有望为临床上因海洛因导致的心律失常患者提供新的诊疗方向�Objective To investigate the role of Ca^(2+)/calmodulin-dependent protein kinase Ⅱ(CaMKⅡ)inregulation of proto-myosin 1(TPM1)in heroin-induced rhythm abnormalities in cardiac myocytes.Methods 60 SPF-grade SD rat mammary myocardial tissues were extracted and primary SD rat mammary myocardial cells were cultured and divided into the control group(control group),heroin-treated group(HE group),and heroin+KN-93-treated group(HE+KN-93 group)according to the intervention.The control group was left untreated under conventional conditions.The HE group was treated with 100μmol/L diacetylmorphine for 24 h,and the HE+KN-93 group was treated with 100μmol/L diacetylmorphine+1μmol/L KN-93 for 24 h.Fluorescence inverted microscopy was used to observe the altered systolic frequency rhythm in cardiomyocytes.Fluo-4 AM was used to detect changes in intracellular Ca^(2+) concentration in each group;tandem mass spectrometry labelling was used to screen indifferentially expressed proteins,and Western blotting was used to detect the expression levels of TPM1,CaMKⅡδ and its T287 site phosphorylation[p-CaMKⅡ(T287)].Results Spontaneous beat frequency results showed that the spontaneous beating frequency(126.33±1.52)times/min was higher than that of the HE group(88.00±2.64)times/min,and the spontaneous beating frequency(110.33±1.52)times/min was higher than that of the HE group(88.00±2.64)times/min was statistically significant(P<0.05).Fluo-4 AM fluorescence staining showed a significant increase(P<0.05)in intracellular Ca^(2+) concentration in the HE group compared with the control group,and a significant decrease(P<0.05)in intracellular Ca^(2+) concentration in the HE+KN-93 group compared with the HE group.Tandem mass spectrometry labeling relative quantitative proteomics results showed that a total of 784 differentially expressed proteins were screened in the HE and control groups,including 442 up-regulated differentially expressed proteins and 342 down-regulated differentially expressed proteins;and a total of 346 differ

关 键 词：海洛因 心律失常 CaMKⅡ TPM1 

分 类 号：R749.64[医药卫生—神经病学与精神病学]