FOXO3a 通过上调有丝分裂自噬诱导心肌纤维化。

FOXO3a Induces Myocardial Fibrosis by Upregulating Mitophagy.

机构信息

Department of Cardiovascular Medicine, The First Hospital of Changsha, 410005 Changsha, Hunan, China.

Department of Hematology & Oncology, The First Hospital of Changsha, 410005 Changsha, Hunan, China.

出版信息

Front Biosci (Landmark Ed). 2024 Feb 5;29(2):56. doi: 10.31083/j.fbl2902056.

DOI:10.31083/j.fbl2902056

PMID:38420796

Abstract

BACKGROUND

Atrial fibrillation is one of the most common cardiac arrhythmias. Myocardial fibrosis is closely associated with atrial remodeling, which leads to heightened risk of atrial fibrillation. This study aimed to explore whether forkhead box protein O3 (FOXO3a) impacts myocardial fibrosis incidence by regulating mitophagy.

METHODS

Cell viability was assessed by cell counting kit-8 (CCK-8) assays. The expression of vimentin and cytochrome C was detected by immunofluorescence assays. Quantitative real-time polymerase chain reaction (PCR) was used to analyze the relative mRNA level of FOXO3a. Expression of FOXO3a, phosphorylated FOXO3a, Collagen I, Collagen III, alpha-smooth muscle actin (α-SMA), matrix metalloprotease 9 (MMP9), light chain 3 (LC3), phosphatase and tensin homolog (PTEN)-induced kinase 1 (PINK1), Parkin, and sequestosome-1 (p62) proteins were determined by western blotting. 5-ethynyl-2'-deoxyuridine (EDU) incorporation was employed to measure cell proliferation. Changes in mitochondrial membrane potential were determined by 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethyl-imidacarbocyanine iodide (JC-1) staining. A wound healing assay was used to examine cell migration, and the levels of reactive oxygen species were determined by flow cytometry.

RESULTS

The expression of FOXO3a was upregulated in cardiac fibroblasts treated with angiotensin II (AngII), while the expression of phosphorylated FOXO3a was downregulated under these conditions. FOXO3a knockdown significantly inhibited the proliferation, migration and collagen secretion of cardiac fibroblasts treated with AngII. The ratio of LC3 II/I as well as expression of PINK1 and Parkin was increased, and the expression of p62 was decreased, in cardiac fibroblasts treated with AngII. Moreover, these effects were limited by FOXO3a knockdown. Finally, the mitophagy inducer everolimus (RAD001) attenuated the suppressive effect of FOXO3a knockdown on cardiac fibroblast activation.

CONCLUSIONS

FOXO3a promotes the progress of myocardial fibrosis by triggering mitophagy in cardiac fibroblasts.

摘要

背景

心房颤动是最常见的心律失常之一。心肌纤维化与心房重构密切相关，这会增加心房颤动的风险。本研究旨在探讨叉头框蛋白 O3（FOXO3a）是否通过调节线粒体自噬来影响心肌纤维化的发生。

方法

通过细胞计数试剂盒-8（CCK-8）测定细胞活力。通过免疫荧光测定检测波形蛋白和细胞色素 C 的表达。采用定量实时聚合酶链反应（PCR）分析 FOXO3a 的相对 mRNA 水平。通过蛋白质印迹法测定 FOXO3a、磷酸化 FOXO3a、胶原 I、胶原 III、α-平滑肌肌动蛋白（α-SMA）、基质金属蛋白酶 9（MMP9）、微管相关蛋白轻链 3（LC3）、磷酸酶和张力蛋白同源物（PTEN）诱导激酶 1（PINK1）、Parkin 和自噬相关蛋白 1（p62）的表达。采用 5-乙炔基-2'-脱氧尿苷（EDU）掺入法测定细胞增殖。通过 5,5',6,6'-四氯-1,1',3,3'-四乙基碘化丙啶（JC-1）染色测定线粒体膜电位变化。采用划痕愈合实验检测细胞迁移，通过流式细胞术测定活性氧水平。

结果

血管紧张素 II（AngII）处理的心肌成纤维细胞中 FOXO3a 的表达上调，而在这些条件下，磷酸化 FOXO3a 的表达下调。AngII 处理的心肌成纤维细胞中 FOXO3a 敲低显著抑制其增殖、迁移和胶原分泌。AngII 处理的心肌成纤维细胞中 LC3 II/I 的比值以及 PINK1 和 Parkin 的表达增加，而 p62 的表达减少。此外，FOXO3a 敲低可限制这些作用。最后，线粒体自噬诱导剂依维莫司（RAD001）减弱了 FOXO3a 敲低对心肌成纤维细胞激活的抑制作用。