Department of Cardiology, Tianjin Nankai Hospital, Tianjin, PR China.
Department of Medical Ultrasonics, Hainan General Hospital (Hainan Affiliated Hospital of Hainan Medical University), Haikou, PR China.
Bioengineered. 2021 Dec;12(1):4385-4396. doi: 10.1080/21655979.2021.1954841.
Myocardial ischemia/hypoxia-reperfusion injury mediates the progression of multiple cardiovascular diseases. It has been reported that knockdown of adaptor protein containing a PH domain, PTB domain and leucine zipper motif 1 (APPL1) is a significant factor for the progression of myocardial injury. However, the role of APPL1 in myocardial ischemia remains unclear. Hence, the aim of the present study was to investigate the specific mechanism underlying the role of APPL1 in myocardial ischemia.In our study, the mRNA level of APPL1 was detected by quantitative real-time PCR (RT-qPCR). The expressions of APPL1, Apoptotic protease activating factor-1 (APAF-1), cleaved caspase9 and other inflammation- and apoptosis-related proteins were determined by western blotting. The secretion of inflammatory cytokines and lactate dehydrogenase (LDH) levels were measured by commercial assay kits. The H9C2 cell viability was analyzed by cell counting kit-8 (CCK-8) assay. The apoptosis rate of H9C2 cells was analyzed by TUNEL assay. The interaction between APPL1 and APAF-1/caspase9 was determined by Immunoprecipitation (IP).Our findings demonstrated that APPL1 was low expressed in myocardial ischemia tissues and cells. APPL1 knockdown suppressed the viability of myocardial ischemia cells and aggravated hypoxia/reperfusion-induced LDH hypersecretion, inflammation and apoptosis. In addition, the overexpression of APPL1 induced inactivation of APAF-1/Caspase9 signaling pathway. Significantly, APAF1 inhibitor reversed the effect of APPL1 knockdown on viability, LDH secretion, inflammation and apoptosis.We conclude that APPL1 inhibits myocardial ischemia/hypoxia-reperfusion injury via inactivation of APAF-1/Caspase9 signaling pathway. Hence, APPL1 may be a novel and effective target for the treatment of myocardial ischemia.
心肌缺血/再灌注损伤介导多种心血管疾病的进展。据报道，衔接蛋白含 PH 结构域、PTB 结构域和亮氨酸拉链基序 1（APPL1）的敲低是心肌损伤进展的重要因素。然而，APPL1 在心肌缺血中的作用尚不清楚。因此，本研究旨在探讨 APPL1 在心肌缺血中的作用机制。
在本研究中，通过实时定量 PCR（RT-qPCR）检测 APPL1 的 mRNA 水平。通过蛋白质印迹法测定 APPL1、凋亡蛋白酶激活因子 1（APAF-1）、裂解 caspase9 和其他炎症和凋亡相关蛋白的表达。通过商业测定试剂盒测定炎症细胞因子的分泌和乳酸脱氢酶（LDH）水平。通过细胞计数试剂盒-8（CCK-8）测定 H9C2 细胞活力。通过 TUNEL 测定分析 H9C2 细胞的凋亡率。通过免疫沉淀（IP）测定确定 APPL1 与 APAF-1/caspase9 的相互作用。
我们的研究结果表明，APPL1 在心肌缺血组织和细胞中低表达。APPL1 敲低抑制心肌缺血细胞的活力，并加重缺氧/再灌注诱导的 LDH 过度分泌、炎症和凋亡。此外，APPL1 的过表达诱导 APAF-1/Caspase9 信号通路失活。值得注意的是，APAF1 抑制剂逆转了 APPL1 敲低对活力、LDH 分泌、炎症和凋亡的影响。
综上所述，APPL1 通过失活 APAF-1/Caspase9 信号通路抑制心肌缺血/再灌注损伤。因此，APPL1 可能是治疗心肌缺血的一种新的有效靶点。
