Table5_A-Kinase Anchor Protein 1 deficiency causes mitochondrial dysfunction in mouse model of hyperoxia induced acute lung injury.xlsx

Background: Critically ill patients on supplemental oxygen therapy eventually develop acute lung injury (ALI). Reactive oxygen species (ROS) produced during ALI perturbs the mitochondrial dynamics resulting in cellular damage. Genetic deletion of the mitochondrial A-kinase anchoring protein 1 (Akap1) in mice resulted in mitochondrial damage, Endoplasmic reticulum (ER) stress, increased expression of mitophagy proteins and pro-inflammatory cytokines, exacerbating hyperoxia-induced Acute Lung Injury (HALI).
Objective: Despite a strong causal link between mitochondrial dysfunction and HALI, the mechanisms governing the disease progression at the transcriptome level is unknown.
Methods: In this study, RNA sequencing (RNA-seq) analysis was carried out using the lungs of Akap1 knockout (Akap1−/−) mice exposed to normoxia or 48 h of hyperoxia followed by quantitative real time PCR and Ingenuity pathway analysis (IPA). Western blot analysis assessed mitochondrial dysfunction, OXPHOS complex (I-V), apoptosis and antioxidant proteins. Mitochondrial enzymatic assays was used to measure the aconitase, fumarase, citrate synthase activities in isolated mitochondria from Akap1−/− vs. Wt mice exposed to hyperoxia.
Results: Transcriptome analysis of Akap1−/− exposed to hyperoxia reveals increases in transcripts encoding electron transport chain (ETC) and tricarboxylic acid cycle (TCA) proteins. Ingenuity pathway analysis (IPA) shows enrichment of mitochondrial dysfunction and oxidative phosphorylation in Akap1−/− mice. Loss of AKAP1, coupled with oxidant injury, significantly decreases the activities of TCA enzymes. Mechanistically, a significant loss of dynamin-related protein 1 (Drp1) phosphorylation at the protein kinase A (PKA) site Serine 637 (Ser637), decreases in Akt phosphorylation at Serine 437 (Ser47) and increase in the expression of pro-apoptotic protein Bax indicate mitochondrial dysfunction. Heme oxygenase-1 (HO-1) levels significantly increased in CD68 positive alveolar macrophages in Akap1−/− lungs, suggesting a strong antioxidant response to hyperoxia.
Conclusion: Overall these results suggest that AKAP1 overexpression and modulation of Drp1 phosphorylation at Ser637 is an important therapeutic strategy for acute lung injury.

背景：接受辅助氧疗的重症患者最终会发展为急性肺损伤（acute lung injury, ALI）。急性肺损伤过程中产生的活性氧（reactive oxygen species, ROS）会扰乱线粒体动力学，进而造成细胞损伤。在小鼠中敲除线粒体A激酶锚定蛋白1（mitochondrial A-kinase anchoring protein 1, Akap1）会引发线粒体损伤、内质网（endoplasmic reticulum, ER）应激、线粒体自噬相关蛋白与促炎细胞因子表达上调，加剧高氧诱导急性肺损伤（hyperoxia-induced Acute Lung Injury, HALI）。
目的：尽管线粒体功能障碍与HALI之间存在明确的因果关联，但目前仍不清楚该疾病进展在转录组层面的调控机制。
方法：本研究采用暴露于常氧或48小时高氧环境的Akap1敲除（Akap1−/−）小鼠肺组织进行RNA测序（RNA-seq）分析，随后开展定量实时PCR与Ingenuity通路分析（Ingenuity pathway analysis, IPA）。通过蛋白质免疫印迹（Western blot）分析评估线粒体功能障碍、氧化磷酸化复合物（I-V）、凋亡相关蛋白与抗氧化蛋白的表达情况。采用线粒体酶活性测定法，检测高氧暴露后Akap1−/−小鼠与野生型（Wt）小鼠分离线粒体中的乌头酸酶、延胡索酸酶、柠檬酸合酶活性。
结果：对高氧暴露后的Akap1−/−小鼠进行转录组分析发现，编码电子传递链（electron transport chain, ETC）与三羧酸循环（tricarboxylic acid cycle, TCA）相关蛋白的转录本表达上调。Ingenuity通路分析（IPA）显示，Akap1−/−小鼠中富集到线粒体功能障碍与氧化磷酸化相关通路。AKAP1缺失联合氧化应激损伤，会显著降低三羧酸循环酶的活性。从机制上来看，蛋白激酶A（protein kinase A, PKA）位点丝氨酸637（Ser637）处的动力相关蛋白1（dynamin-related protein 1, Drp1）磷酸化水平显著下降，丝氨酸437（Ser47）处的Akt磷酸化水平降低，且促凋亡蛋白Bax的表达上调，这些均提示线粒体功能障碍。在Akap1−/−小鼠肺组织的CD68阳性肺泡巨噬细胞中，血红素氧合酶1（heme oxygenase-1, HO-1）水平显著升高，表明机体对高氧环境产生了强烈的抗氧化应答。
结论：综合上述结果可知，AKAP1过表达以及调控动力相关蛋白1在丝氨酸637位点的磷酸化，可作为急性肺损伤的重要治疗策略。