Vasily N Sukhorukov, Victoria A Khotina, Vladislav A Kalmykov, Alexander D Zhuravlev, Vasily V Sinyov, Daniil Y Popov, Andrey Y Vinokurov, Igor A Sobenin, Alexander N Orekhov
J Lipid Atheroscler . 2024 May;13(2):166-183. doi: 10.12997/jla.2024.13.2.166. Epub 2024 Apr 18.
Abstract
Objective: The aim of this study was to evaluate the effect of the m.15059G>A mitochondrial nonsense mutation on cellular functions related to atherosclerosis, such as lipidosis, pro-inflammatory response, and mitophagy. Heteroplasmic mutations have been proposed as a potential cause of mitochondrial dysfunction, potentially disrupting the innate immune response and contributing to the chronic inflammation associated with atherosclerosis.
Methods: The human monocytic cell line THP-1 and cytoplasmic hybrid cell line TC-HSMAM1 were used. An original approach based on the CRISPR/Cas9 system was developed and used to eliminate mitochondrial DNA (mtDNA) copies carrying the m.15059G>A mutation in the MT-CYB gene. The expression levels of genes encoding enzymes related to cholesterol metabolism were analyzed using quantitative polymerase chain reaction. Pro-inflammatory cytokine secretion was assessed using enzyme-linked immunosorbent assays. Mitophagy in cells was detected using confocal microscopy.
Results: In contrast to intact TC-HSMAM1 cybrids, Cas9-TC-HSMAM1 cells exhibited a decrease in fatty acid synthase (FASN) gene expression following incubation with atherogenic low-density lipoprotein. TC-HSMAM1 cybrids were found to have defective mitophagy and an inability to downregulate the production of pro-inflammatory cytokines (to establish immune tolerance) upon repeated lipopolysaccharide stimulation. Removal of mtDNA harboring the m.15059G>A mutation resulted in the re-establishment of immune tolerance and the activation of mitophagy in the cells under investigation.
Conclusion: The m.15059G>A mutation was found to be associated with defective mitophagy, immune tolerance, and impaired metabolism of intracellular lipids due to upregulation of FASN in monocytes and macrophages.
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