Lu Feifei, Ding Le, Qiao Yanxiang
Taian Municipal Hospital, Cardiovascular Medicine, Taian, 271000, Shandong, China.
Healthy Management Department, Qingdao Eighth People's Hospital, Qingdao, 266000, Shandong, China.
Biochem Genet. 2024 Oct 24. doi: 10.1007/s10528-024-10926-y.
It was reported that serum apurinic/apyrimidinic endodeoxyribonuclease 1 (APEX1) level was higher in acute myocardial infarction (AMI) patients than in angina. This study aimed to investigate the role and mechanism of APEX1 in AMI progression. The mRNA and protein levels of APEX1 and zinc finger CCHC domain containing 9 (ZCCHC9) in blood specimens of AMI patients and normal controls were determined by RT-qPCR and Western blot assays, respectively. H9c2 cardiomyocytes were treated with angiotensin II (Ang II) to induce cardiomyocyte injury and then transfected with small interfering RNA against APEX1 (si-APEX1) or overexpression plasmids of ZCCHC9 (pcDNA-ZCCHC9). The cell viability, apoptosis, inflammatory cytokine levels, and fibrosis-associated protein expression in H9c2 cells were evaluated. ZCCHC9 promoter methylation were detected with methylation-specific PCR (MSP) assay. Then, rescue experiments were performed to explore whether APEX1 mediated cardiomyocyte functions by regulating ZCCHC9 expression. Furthermore, we explored whether the APEX1/ZCCHC9 axis regulated cardiomyocyte injury in AMI via the p38 MAPK signaling pathway. Additionally, an AMI rat model was established using the left anterior descending artery (LAD) ligation method and multipoint intramyocardial injection (5 points, 2 µL/point) of lentivirus (1 × 10 TU/mL) carrying scramble or si-APEX1 was conducted before modeling. The rats were euthanized four weeks after AMI modeling, and blood samples and myocardial tissues were harvested. The infarct area, cell apoptosis, inflammation, and fibrosis in myocardial tissues were detected. APEX1 was upregulated and ZCCHC9 was downregulated in blood samples of AMI patients compared with normal controls. APEX1 knockdown or ZCCHC9 overexpression attenuated Ang II-induced viability reduction, apoptosis, inflammation, and fibrosis in cardiomyocytes. APEX1 inhibited ZCCHC9 expression by promoting DNA methyltransferase 1 (DNMT1)-mediated ZCCHC9 promoter methylation. ZCCHC9 knockdown abolished the protective effects of APEX1 knockdown on Ang II-induced cardiomyocyte injury. APEX1 knockdown inhibited the p38 MAPK signal signaling, and anisomycin reversed the effect of APEX1 knockdown on cardiomyocyte functions. Additionally, APEX1 knockdown alleviated apoptosis, inflammation, and fibrosis in myocardial tissues of AMI rats. APEX1 knockdown attenuated Ang II-induced apoptosis, inflammation, and fibrosis in cardiomyocytes although promoting ZCCHC9 expression and inhibiting the p38 MAPK signaling pathway, thus relieving myocardial infarction, inflammation, and fibrosis in AMI rats.
据报道，急性心肌梗死（AMI）患者血清脱嘌呤/脱嘧啶核酸内切酶1（APEX1）水平高于心绞痛患者。本研究旨在探讨APEX1在AMI进展中的作用及机制。分别采用RT-qPCR和蛋白质免疫印迹法检测AMI患者和正常对照者血液标本中APEX1和含锌指CCHC结构域9（ZCCHC9）的mRNA和蛋白水平。用血管紧张素II（Ang II）处理H9c2心肌细胞以诱导心肌细胞损伤，然后转染针对APEX1的小干扰RNA（si-APEX1）或ZCCHC9的过表达质粒（pcDNA-ZCCHC9）。评估H9c2细胞的细胞活力、凋亡、炎性细胞因子水平和纤维化相关蛋白表达。用甲基化特异性PCR（MSP）法检测ZCCHC9启动子甲基化。然后进行拯救实验，以探讨APEX1是否通过调节ZCCHC9表达介导心肌细胞功能。此外，我们探讨了APEX1/ZCCHC9轴是否通过p38丝裂原活化蛋白激酶（MAPK）信号通路调节AMI中的心肌细胞损伤。另外，采用左冠状动脉前降支（LAD）结扎法建立AMI大鼠模型，并在建模前多点心肌内注射（5点，2 μL/点）携带乱序序列或si-APEX1的慢病毒（1×10 TU/mL）。AMI建模4周后对大鼠实施安乐死，采集血样和心肌组织。检测心肌组织的梗死面积、细胞凋亡、炎症和纤维化情况。与正常对照相比，AMI患者血液标本中APEX1上调而ZCCHC9下调。敲低APEX1或过表达ZCCHC9可减轻Ang II诱导的心肌细胞活力降低、凋亡、炎症和纤维化。APEX1通过促进DNA甲基转移酶1（DNMT1）介导的ZCCHC9启动子甲基化抑制ZCCHC9表达。敲低ZCCHC9可消除敲低APEX1对Ang II诱导的心肌细胞损伤的保护作用。敲低APEX1抑制p38 MAPK信号通路，茴香霉素可逆转敲低APEX1对心肌细胞功能的影响。此外，敲低APEX1可减轻AMI大鼠心肌组织的凋亡、炎症和纤维化。敲低APEX1可减轻Ang II诱导的心肌细胞凋亡、炎症和纤维化，尽管其促进了ZCCHC9表达并抑制了p38 MAPK信号通路，从而减轻了AMI大鼠的心肌梗死及炎症和纤维化。
