Shi X D, Cao L, Tan R, Zhou S Q, Li F, Liu F Z
Department of Cardiology, First Affiliated Hospital, Air Force Medical University, Xi'an 710032, China.
Department of Otolaryngology Head and Neck Surgery, Third People's Hospital of Yinchuan, Yinchuan 750001, China.
Zhonghua Lao Dong Wei Sheng Zhi Ye Bing Za Zhi. 2023 Jul 20;41(7):486-496. doi: 10.3760/cma.j.cn121094-20221025-00513.
To investigate the protective effect and its possible mechanism of A-kinase anchored protein 1 (AKAP1) on the myocardial injury induced by highland hypobaric hypoxia. From January 2021 to May 2022, male C57BL/6 SPF grade mice were divided into wild type control (WT) group and highland hypobaric hypoxia (HH) group with 6 mice in each group. HH group simulated 6000 m altitude with low pressure oxygen chamber for 4 weeks to build the model. Primary myocardial cells of SD rats were divided into normoxia control group and hypoxia experimental group (=3). Cell models were constructed in a three-gas hypoxia incubator with 1% oxygen concentration for 24 h. AKAP1 protein and mRNA expression in myocardial tissue and cells were detected by western blotting, immunohistochemistry and quantitative real-time polymerase chain reaction (qPCR). After myocardial point injection of the AKAP1 or the control adenovirus, the mice were divided into 3 groups (=6) : WT group, highland hypobaric hypoxia overexpression control group (HH+Ad-Ctrl group) and highland hypobaric hypoxia overexpression experimental group (HH+Ad-AKAP1 group). The cardiac function of mice was detected by noninvasive M-type ultrasonic cardiomotive, myocardial fibrosis was detected by Masson and Sirius Red staining, and cardiomyocyte hypertrophy was detected by wheat germ agglutinin. After the expression of AKAP1 in primary cardiomyocytes was downregulated by siRNA and upregulated by adenovirus, the cells were divided into three groups (=3) : normoxia control group, hypoxia interference control group (hypoxia+siCtrl group), hypoxia AKAP1 knockdown group (hypoxia+siAKAP1 group) ; normoxia control group, hypoxia overexpression control group (hypoxia+Ad-Ctrl group), hypoxia AKAP1 overexpression group (hypoxia+Ad-AKAP1 group). Apoptosis was detected by flow cytometry, AKAP1, apoptosis-related protein and mRNA expression levels were detected by western blotting and qPCR, mitochondrial membrane potential was detected by JC-1 staining, and mitochondrial reactive oxygen specie (ROS) level was detected by MitoSOX. The expression of AKAP1 in cardiac muscle of HH group was lower than that in the WT group, and the expression of AKAP1 in hypoxia experimental group was lower than that in normoxia control group (<0.01). Compared with WT group, the left ventricular ejection fraction and fraction shortening of left ventricle in HH+Ad-Ctrl group were decreased (<0.01), myocardial fibrosis and hypertrophy were aggravated (<0.01), and the expression of B-cell lymphoma-2 (BCL-2) was decreased, the expressions of BCL-2-associated X protein (BAX), Caspase 3 and Caspase 9 were increased (<0.01). After AKAP1 overexpression, compared with HH+Ad-Ctrl group, the left ventricular ejection fraction and left ventricular fraction shortening were increased in HH+Ad-AKAP1 group (<0.01), myocardial fibrosis and hypertrophy were reduced (<0.01), and the expression of BCL-2 was increased, the expressions of BAX, Caspase 3 and Caspase 9 were decreased (<0.01). Compared with normoxia control group, the expression of BCL-2 in hypoxia+siCtrl group was decreased, the expressions of BAX, Caspase 3, Caspase 9 were increased, the apoptosis level was increased (<0.01), the mitochondrial membrane potential was decreased and the production of ROS was increased (<0.01). After AKAP1 knockdown, compared with hypoxia+siCtrl group, the expression of BCL-2 in hypoxia+siAKAP1 group was decreased, the expressions of BAX, Caspase 3, Caspase 9 were increased, the apoptosis level was increased (<0.01), mitochondrial membrane potential was decreased, and the production of ROS was increased (<0.01). After AKAP1 overexpression, compared with hypoxia+Ad-Ctrl group, the expression of BCL-2 in hypoxia+Ad-AKAP1 group was increased, the expressions of BAX, Caspase 3 and Caspase 9 were decreased (<0.05), the apoptosis level was decreased (<0.01), and the mitochondrial membrane potential was enhanced, and the production of ROS was decreased (<0.01) . The downregulation of AKAP1 in cardiomyocytes under highland hypobaric hypoxia may lead to the decrease of mitochondrial membrane potential and the increase of ROS generation, leading to the apoptosis of cardiomyocytes, and thus aggravating the myocardial injury at highland hypobaric hypoxia.
探讨A激酶锚定蛋白1（AKAP1）对高原低压低氧所致心肌损伤的保护作用及其可能机制。2021年1月至2022年5月，将雄性C57BL/6 SPF级小鼠分为野生型对照组（WT）和高原低压低氧组（HH），每组6只。HH组采用低压氧舱模拟海拔6000 m环境4周建立模型。将SD大鼠原代心肌细胞分为常氧对照组和缺氧实验组（=3）。在三气缺氧培养箱中以1%氧浓度构建细胞模型24 h。采用蛋白质免疫印迹法、免疫组织化学法和实时定量聚合酶链反应（qPCR）检测心肌组织和细胞中AKAP1蛋白及mRNA表达。对小鼠进行心肌内注射AKAP1或对照腺病毒后，分为3组（=6）：WT组、高原低压低氧过表达对照组（HH+Ad-Ctrl组）和高原低压低氧过表达实验组（HH+Ad-AKAP1组）。采用无创M型超声心动图检测小鼠心功能，采用Masson染色和天狼星红染色检测心肌纤维化，采用麦胚凝集素检测心肌细胞肥大。通过小干扰RNA（siRNA）下调原代心肌细胞中AKAP1表达，腺病毒上调其表达后，分为3组（=3）：常氧对照组、缺氧干扰对照组（缺氧+siCtrl组）、缺氧AKAP1敲低组（缺氧+siAKAP1组）；常氧对照组、缺氧过表达对照组（缺氧+Ad-Ctrl组）、缺氧AKAP1过表达组（缺氧+Ad-AKAP1组）。采用流式细胞术检测细胞凋亡，采用蛋白质免疫印迹法和qPCR检测AKAP1、凋亡相关蛋白及mRNA表达水平，采用JC-1染色检测线粒体膜电位，采用MitoSOX检测线粒体活性氧（ROS）水平。HH组心肌中AKAP1表达低于WT组，缺氧实验组中AKAP1表达低于常氧对照组（<0.01）。与WT组比较，HH+Ad-Ctrl组左心室射血分数和左心室缩短分数降低（<0.01），心肌纤维化和肥大加重（<0.01），B细胞淋巴瘤-2（BCL-2）表达降低，BCL-2相关X蛋白（BAX）、半胱天冬酶3（Caspase 3）和半胱天冬酶9（Caspase 9）表达增加（<0.01）。AKAP1过表达后，与HH+Ad-Ctrl组比较，HH+Ad-AKAP1组左心室射血分数和左心室缩短分数增加（<0.01），心肌纤维化和肥大减轻（<0.01），BCL-2表达增加，BAX、Caspase 3和Caspase 9表达降低（<0.01）。与常氧对照组比较，缺氧+siCtrl组BCL-2表达降低，BAX、Caspase 3、Caspase 9表达增加，凋亡水平升高（<0.01），线粒体膜电位降低，ROS生成增加（<0.01）。AKAP1敲低后,与缺氧+siCtrl组比较，缺氧+siAKAP1组BCL-2表达降低，BAX、Caspase 3、Caspase 9表达增加，凋亡水平升高（<0.01），线粒体膜电位降低，ROS生成增加（<0.01）。AKAP1过表达后，与缺氧+Ad-Ctrl组比较，缺氧+Ad-AKAP1组BCL-2表达增加，BAX、Caspase 3和Caspase 9表达降低（<0.05），凋亡水平降低（<0.01），线粒体膜电位增强，ROS生成减少（<0.01）。高原低压低氧下心肌细胞中AKAP1下调可能导致线粒体膜电位降低和ROS生成增加，引起心肌细胞凋亡，从而加重高原低压低氧所致心肌损伤。
