微小RNA-499对心力衰竭大鼠心肌细胞凋亡的影响

MicroRNA-499 inhibits cardiomyocytes apoptosis in pacing-induced heart failure in rats

目的探究微小RNA-499(miR-499)在非缺血性心衰中对心肌细胞凋亡的影响。方法构建快速起搏致心衰大鼠,然后构建miR-499腺相关病毒(adeno-associated virus,AAV)载体并将其转染入大鼠体内,通过定量逆转录聚合酶链反应(qRT-PCR)使miR-499在大鼠体内的表达上调,用qRT-PCR检测大鼠体内miR-499的表达水平,用Western blot检测大鼠心衰模型中PDCD4和PACS2的含量,用免疫组化及TUNEL法检测心肌细胞凋亡程度。结果快速起搏致心衰大鼠模型建立成功,构建rno-miR-499腺相关病毒载体并将其转染入使大鼠模型体内miR-499表达上调,结果表明与假手术组相比,起搏组动物左心室射血分数(81.8%±2.4%vs 64.3%±2.2%,P <0.05)及左心室短轴缩短率(44.7%±2.4%vs 29.1%±0.9%,P <0.05)明显下降,而自主心率、心脏质量和左心室舒张末压明显上升。与单纯起搏组相比,miR-499过表达可使大鼠心衰模型自主心率下降[(441.6±22)/min vs (368.4±27)/min,P <0.05],左心室舒张末压降低[(34.8±11.4) mmHg vs (19.4±10.3) mmHg,P <0.05](1 mmHg=0.133 kPa)。心肌组织中程序性细胞凋亡因子4(programmed cell death 4,PDCD4)和PACS2蛋白的表达明显降低。快速起搏刺激可致大鼠心脏出现结构重构,心肌组织的凋亡程度增加,但miR-499的过表达则使其程度减小。结论 MiR-499可降低PDCD4和PACS2的表达,抑制快速起搏所致心衰大鼠心肌细胞的凋亡。

Objective To explore the protective effect of microRNA-499（miR-499） against myocardial apoptosis in non-ischemic heart failure. Methods Adeno-associated viral vector of rno-miR-499 and pacing-induced heart failure rat model was established, and then rats were infected by recombinant adeno-associated virus. The expression level of miR-499 was detected by qPT-PCR, protein concentrations of Pscd4 and PACS2 were determined using western blot assay. Apoptosis was detected via immunohistochemistry and TUNEL assay. Results Adeno-associated viral miR-499 was successfully transfected, and it led to specific overexpression of miR-499. Compared with the sham group, the LVEF（81.8%±2.4% vs 64.3%±2.2%） and LVFS（44.7%±2.4% vs 29.1%±0.9%） of the pacing＋NC group decreased significantly（P〈0.05, respectively）, while the autonomic heart rate and LVEDP increased significantly. Compared with the pacing＋NC group, the autonomic heart rate [（441.6±22）/min vs（368.4±27）/min] and LVEDP [（34.8±11.4） mmHg vs（19.4±10.3） mmHg] of the pacing group decreased significantly（all P〈0.05）. We found that enhancing the expression of miR-499 could significantly inhibit the expression of PDCD4 and PACS2 in pacing-induced heart failure rats. Furthermore, cell apoptosis increased by pacing, but attenuated by up-regulated miR-499. Conclusion MiR-499 may inhibit apoptosis in pacing-induced heart failure via PDCD4 and PACS2 pathway.