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. 2023 Feb 17;28(1):82.
doi: 10.1186/s40001-023-01051-4.

Autophagy impairment in patients with obstructive sleep apnea modulates intermittent hypoxia-induced oxidative stress and cell apoptosis via hypermethylation of the ATG5 gene promoter region

Yung-Che Chen  1   2   3 , I-Chun Lin  4 , Mao-Chang Su  5   6   7 , Po-Yuan Hsu  5 , Chang-Chun Hsiao  5   8 , Te-Yao Hsu  9 , Chia-Wei Liou  10 , Yu-Mu Chen  5   6 , Chien-Hung Chin  5   6 , Ting-Ya Wang  5 , Jen-Chieh Chang  11 , Yong-Yong Lin  5 , Chiu-Ping Lee  5 , Meng-Chih Lin  12   13   14
Affiliations

Autophagy impairment in patients with obstructive sleep apnea modulates intermittent hypoxia-induced oxidative stress and cell apoptosis via hypermethylation of the ATG5 gene promoter region

Yung-Che Chen et al. Eur J Med Res. .

Abstract

Background: Autophagy is a catabolic process that recycles damaged organelles and acts as a pro-survival mechanism, but little is known about autophagy dysfunction and epigenetic regulation in patients with obstructive sleep apnea (OSA).

Methods: Protein/gene expressions and DNA methylation levels of the autophagy-related genes (ATG) were examined in blood leukocytes from 64 patients with treatment-naïve OSA and 24 subjects with primary snoring (PS).

Results: LC3B protein expression of blood monocytes, and ATG5 protein expression of blood neutrophils were decreased in OSA patients versus PS subjects, while p62 protein expression of cytotoxic T cell was increased, particularly in those with nocturia. ATG5, ULK1, and BECN1 gene expressions of peripheral blood mononuclear cells were decreased in OSA patients versus PS subjects. LC3B gene promoter regions were hypermethylated in OSA patients, particularly in those with excessive daytime sleepiness, while ATG5 gene promoter regions were hypermethylated in those with morning headache or memory impairment. LC3B protein expression of blood monocytes and DNA methylation levels of the LC3B gene promoter region were negatively and positively correlated with apnea hyponea index, respectively. In vitro intermittent hypoxia with re-oxygenation exposure to human THP-1/HUVEC cell lines resulted in LC3B/ATG5/ULK1/BECN1 down-regulations and p62 up-regulation along with increased apoptosis and oxidative stress, while rapamycin and umbilical cord-mesenchymal stem cell treatment reversed these abnormalities through de-methylation of the ATG5 gene promoter.

Conclusions: Impaired autophagy activity in OSA patients was regulated by aberrant DNA methylation, correlated with clinical phenotypes, and contributed to increased cell apoptosis and oxidative stress. Autophagy enhancers may be novel therapeutics for OSA-related neurocognitive dysfunction.

Keywords: Autophagy; DNA methylation; Intermittent hypoxia with re-oxygenation; Mesenchymal stem cell; Obstructive sleep apnea; Rapamycin.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1
Impaired autophagy flux in patients with obstructive sleep apnea (OSA) and those with specific phenotypes. A LC3B protein expression of blood monocyte was decreased in OSA patients versus subjects with primary snoring (PS). B ATG5 protein expression of blood neutrophil was decreased in OSA patients versus PS subjects. C LC3B protein expression of blood monocyte was negatively correlated with apnea hyponea index (AHI). D P62 protein expression of blood cytotoxic T cell was increased in OSA patients versus PS subjects, and E positively correlated with oxygen desaturation index (ODI). F LC3B protein expression of blood helper T cell was decreased in sleep disordered breathing (SDB) patients with depression versus those without depression. G ATG5 protein expression of blood monocyte was decreased in SDB patients with subjective daytime fatigue versus those without fatigue. H Percentage of blood ATG5 ( +) helper T cell was negatively correlated with arousal index of total sleep time. I P62 protein expression of blood cytotoxic T cell was increased in SDB patients with nocturia versus those with nocturia
Fig. 2
Fig. 2
Decreased gene expressions of the autophagy-related genes (ATG) in OSA patients and those with excessive daytime sleepiness. A Percentage of blood p62( +) neutrophil was reduced after > 6-month continuous positive airway pressure treatment at home in five OSA patients. B ATG5 gene expression of peripheral blood mononuclear cells (PBMCs) was decreased in OSA patients versus PS subjects, and C negatively correlated with snoring index of total sleep time. D BECN1 gene expression of PBMCs was decreased in OSA patients versus PS subjects, and E negatively correlated with percent time of SpO2 less than 90% during rapid eye movement sleep. F ULK1 gene expression of PBMCs was decreased in OSA patients versus PS subjects, and G positively correlated with minimum SpO2 of total sleep time. H ULK1 gene expression was further decreased in sleep disordered breathing (SDB) patients with excessive daytime sleepiness (EDS) versus those without EDS, and I negatively correlated with Epworth Sleepiness Scale
Fig. 3
Fig. 3
Aberrant DNA methylation patterns of the ATG genes in OSA patients and those with specific phenotypes. A Mean DNA methylation levels over − 172, − 157, − 153, − 141, − 134, and − 118 CpG sites of the LC3B gene were increased in OSA patients versus PS subjects. B Mean DNA methylation levels over − 53, − 46, − 44, − 42, and − 37 CpG sites of the LC3B gene were increased in OSA patients versus PS subjects. C Mean DNA methylation levels over − 172, − 157, − 153, − 141, − 134, and − 118 CpG sites of the LC3B gene were positively correlated with apnea hyponea index. D DNA methylation levels over -153 CpG site of the LC3B gene were further increased in SDB patients with EDS versus those without EDS, and E positively correlated with Epworth Sleepiness Scale. F Mean DNA methylation levels over − 32, − 25, − 18, − 11 and − 1 CpG sites of the ATG5 gene were increased in SDB patients with morning headache versus those without headache. G Mean DNA methylation levels over − 108, − 106, − 96, − 86, − 80, − 65, and − 61 CpG sites of the ATG5 gene were increased in SDB patients with subjective memory impairment versus those with memory impairment. H Mean DNA methylation levels over − 164, − 145, − 142, and − 134 CpG sites of the p62 gene were decreased in SDB patients with nocturia versus those without nocturia. I Mean DNA methylation levels over − 105, − 101, − 99, and − 94 CpG sites of the p62 gene were decreased in SDB patients with cognitive dysfunction versus those without cognitive dysfunction
Fig. 4
Fig. 4
Autophagy enhancer and mesenchymal stem cell (MSC) treatment reversed intermittent hypoxia with re-oxygenation (IHR)-induced autophagy impairment, late apoptosis and oxidative stress. Either rapamycin (Rap) or human umbilical cord-derived MSC-condition medium (CM) reversed IHR-induced decreased A LC3B and B ATG5 protein expressions, and C increased p62 protein expression in THP-1 cells. D Rap and MSC-CM also reversed IHR-induced hypermethylation over − 32, − 25, − 18, − 11, and − 1 CpG sites of the ATG5 gene. Either Rap or MSC-CM reversed IHR-induced E late apoptosis and F over-production of reactive oxygen species (ROS) in HUVEC *p < 0.05, compared between IHR and normoxic (NOX) condition #p < 0.05, compared between IHR plus specific treatment and IHR alone condition
Fig. 5
Fig. 5
De-methylation and re-methylation agents reversed IHR-induced late apoptosis and autophagy impairment. Either de-methylation agent (5-Aza-2′-deoxycytidine; 5-AZA) or re-methylation agent (Folic Acid; FA) reversed IHR-induced A over-production of reactive oxygen species (ROS), as well as down-regulations of B LC3B and C ATG5 genes and D p62 up-regulation. E Only FA reversed IHR-induced late apoptosis. F Mean DNA methylation levels over − 108, − 106, − 96, − 86, − 80, − 65, and − 61 CpG sites of the ATG5 gene were increased with IHR stimuli versus normoxic (NOX) condition, but remained unchanged with either 5-AZA or FA treatment under IHR condition. *p < 0.05, compared between IHR and normoxic (NOX) condition #p < 0.05, compared between IHR plus specific treatment and IHR alone condition
Fig. 6
Fig. 6
Proposed model of epigenetics-regulated autophagy impairment in obstructive sleep apnea (OSA) and its clinical phenotypes. A schematic diaphragm depicts probably contributing effects of hypermethylated LC3B gene promoters and hypomethylated p62 gene promoters on the development of OSA and EDS/nocturia/depression/cognitive dysfunction phenotypes through inhibiting autophagy. In contrast, chronic intermittent hypoxia in OSA patients may lead to hypermethylated ATG5 gene promoters, which probably contribute to the development of morning headache, memory impairment, and fatigue through down regulating ATG5. Autophagy enhancer (rapamycin), mesenchymal stem cell, and re-methylation agent (folic acid) treatment may reduce late apoptosis and oxidative stress through augmenting autophagy. Red arrows indicate predisposition, and orange arrows indicate cause and effect relationships
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