[Effects of uncoupling protein 2 overexpression on myocardial mitochondrial dynamics in sepsis rats].
[Effects of uncoupling protein 2 overexpression on myocardial mitochondrial dynamics in sepsis rats].
复制标题
DOI：
10.3760/cma.j.issn.2095-4352.2019.10.018
复制
发表时间：
2019-10-01
影响因子：
--
通讯作者：
Fu, Xiaoyun
中科院分区：
文献类型：
--
作者：
Luo, Shiyu;Li, Guangsu;Fu, Xiaoyun
目的：探讨解偶联蛋白2（UCP 2）过表达对线粒体动力学的影响方法：将40只雄性SD大鼠随机分为4组（n = 10）：假手术组（Sham组）用生理盐水代替转染并模拟盲肠结扎穿孔（CLP）; CLP组用生理盐水代替转染，进行CLP诱导脓毒症;腺相关病毒（AAV）组用CLP转染后的心肌组织中加入空病毒; UCP 2过表达组（UCP 2组）在AAV-UCP 2（1 × 1015 vg/L，共60穆尔）心肌转染后3周进行CLP。CLP后24 h各组大鼠行超声心动图检查，然后立即处死大鼠，取心肌组织。电镜下观察心肌超微结构变化，Western Blot法检测心肌线粒体动力学相关调节蛋白Opa 1、动力蛋白相关蛋白Drp 1和裂变蛋白Fis 1的表达，化学发光法检测线粒体三磷酸腺苷（ATP）产生水平。（1）超声心动图结果显示，左室质量（LVM）和每搏输出量（SV）无显著性差异。与Sham组比较，CLP组和AAV组左室舒张期前壁厚度（LVAWd）、左室收缩期前壁厚度（LVAWs）、左室舒张期后壁厚度（LVPWd）、左室收缩期后壁厚度（LVPWs）、左室射血分数（LVEF）和左室短轴缩短率（LVFS）均显著增加，左室收缩期内径（LVEDs）、左室舒张期内径（LVEDd）、左室收缩末期容积（LVESV）、左室舒张末期容积（LVEDV）均明显减小。与CLP组和AAV组相比，UCP 2组LVAWs、LVEF、LVFS显著降低，LVEDs、LVEDV和LVESV显著增加[LVAWs（mm）：3.82±0.42 vs. 4.34±0.30，4.44±0.12; LVEF：0.921±0.038 vs. 0.979±0.019，0.991±0.010; LVFS：（65.33±6.56）% vs.（80.11±8.23）%，（85.31±6.11）%;左室舒张末内径（mm）：1.81±0.36 vs. 0.89±0.54，0.60±0.17;左室舒张末容积（穆尔）：137.09±50.05 vs. 89.72±53.04，85.42±40.99;左室收缩末容积（穆尔）：10.48±4.59 vs. 2.48±3.52，2.58±2.50，均P < 0.05]。(2)电镜显示Sham组心肌纤维结构清晰，排列整齐，椎间盘和线粒体结构完整，线粒体膜无损伤，嵴排列紧密。CLP组和AAV组肌纤维断裂，肌浆网扩张，线粒体肿胀严重，嵴结构紊乱。UCP 2组仅观察到心肌纤维水肿，肌纤维结构比Sham组和AV组更完整。线粒体轻度肿胀，嵴完整。(3)Western Blot检测显示，Opa 1和Fis 1在4组中的表达差异无统计学意义。CLP组和AAV组Drp 1的表达均显著高于Sham组。UCP 2组Drp 1的表达明显低于CLP组和AAV组（Drp 1/beta-actin：1.01±0.03 vs. 1.39±0.03，1.49±0.03，均P < 0.05）。(4)免疫荧光结果显示CLP组和AAV组ATP含量显著低于Sham组; UCP 2组ATP含量显著高于CLP组和AAV组（mumol/L：1.99±0.15 vs. 1.10±0.17，1.13±0.19，均P < 0.05）。UCP 2过表达可明显改善脓毒症大鼠心肌的全身收缩功能，保护心肌线粒体超微结构，抑制线粒体分裂，促进线粒体ATP合成。
OBJECTIVE: To investigate the effects of uncoupling protein 2 (UCP2) overexpression on mitochondrial dynamics (mitochondrial division and fusion) of sepsis myocardial injury in rats.METHODS: Forty male Sprague-Dawley (SD) rats were randomly divided into four groups (n = 10): sham operation group (Sham group) using normal saline instead of transfection and simulating cecal ligation and perforation (CLP); CLP group using normal saline instead of transfection, performing CLP to induce sepsis; adeno-associated virus (AAV) group using CLP after myocardial transfection with empty virus; UCP2 overexpression group (UCP2 group) CLP was performed 3 weeks after AAV-UCP2 (1*1015 vg/L, a total of 60 muL) myocardial transfection. The rats in each group were examined by echocardiography at 24 hours after the CLP, and then the rats were sacrificed immediately to harvest myocardial tissue. Myocardial ultrastructural changes were observed under the electron microscope, the expression of regulatory proteins related to myocardial mitochondrial dynamics [optic atrophy 1 (Opa1), dynamin-related protein 1 (Drp1) and fission 1 (Fis1)] were detected by Western Blot, and the level of mitochondrial adenosine triphosphate (ATP) production was detected by chemiluminescence.RESULTS: (1) The echocardiographic results showed that there was no significant difference in left ventricular mass (LVM) and stroke volume (SV). Compared with Sham group, left ventricular diastolic anterior wall thickness (LVAWd), left ventricular systolic anterior wall thickness (LVAWs), left ventricular diastolic posterior wall thickness (LVPWd), left ventricular systolic posterior wall thickness (LVPWs), left ventricular ejection fraction (LVEF) and left ventricular short axis shortening rate (LVFS) were significantly increased in CLP group and AAV group, while left ventricular systolic diameter (LVEDs), left ventricular diastolic diameter (LVEDd), left ventricular end-systolic volume (LVESV), and left ventricular end-diastolic volume (LVEDV) were significantly decreased. Compared with CLP group and AAV group, LVAWs, LVEF, LVFS were significantly decreased in UCP2 group, and LVEDs, LVEDV and LVESV were significantly increased [LVAWs (mm): 3.82±0.42 vs. 4.34±0.30, 4.44±0.12; LVEF: 0.921±0.038 vs. 0.979±0.019, 0.991±0.010; LVFS: (65.33±6.56)% vs. (80.11±8.23)%, (85.31±6.11)%; LVEDs (mm): 1.81±0.36 vs. 0.89±0.54, 0.60±0.17; LVEDV (muL): 137.09±50.05 vs. 89.72±53.04, 85.42±40.99; LVESV (muL): 10.48±4.59 vs. 2.48±3.52, 2.58±2.50, all P < 0.05]. (2) Electron microscope showed that the structure of myocardial fibers in the Sham group was clear and aligned with complete intervertebral disc and mitochondrial structure, no damage to mitochondrial membranes, and tight arrangement of cristae. In CLP group and AAV group, muscle fiber breakage, sarcoplasmic reticulum expansion, severe mitochondrial swelling and even cristage structure disorder were observed. In the UCP2 group, only myocardial fiber edema was observed, and the muscle fiber structure was more complete than that of Sham group and AAV group. The mitochondria were slightly swollen and the cristae were intact. (3) Western Blot showed that there was no significant difference in the expression of Opa1 and Fis1 in the four groups. The expression of Drp1 in CLP group and AAV group were significantly higher than that in Sham group. The expression of Drp1 in UCP2 group was significantly lower than that in CLP group and AAV group (Drp1/beta-actin: 1.01±0.03 vs. 1.39±0.03, 1.49±0.03, both P < 0.05). (4) The results of immunofluorescence showed that the ATP content of CLP group and AAV group were significantly lower than that of Sham group; the ATP content of UCP2 group was significantly higher than that of CLP group and AAV group (mumol/L: 1.99±0.15 vs. 1.10±0.17, 1.13±0.19, both P < 0.05).CONCLUSIONS: UCP2 overexpression can significantly improve the systemic systolic function of myocardium in sepsis rats, protect myocardial mitochondrial ultrastructure, inhibit mitochondrial division, and improve mitochondrial ATP synthesis.