Mitochondrial Genome Editing: Exploring the Possible Relationship of the Atherosclerosis-Associated Mutation m.15059G>A With Defective Mitophagy
10.12997/jla.2024.13.2.166
- Author:
Vasily N. SUKHORUKOV
1
;
Victoria A. KHOTINA
;
Vladislav A. KALMYKOV
;
Alexander D. ZHURAVLEV
;
Vasily V. SINYOV
;
Daniil Y. POPOV
;
Andrey Y. VINOKUROV
;
Igor A. SOBENIN
;
Alexander N. OREKHOV
Author Information
1. Laboratory of Angiopathology, Institute of General Pathology and Pathophysiology, Moscow, Russia
- Publication Type:Original Article
- From:Journal of Lipid and Atherosclerosis
2024;13(2):166-183
- CountryRepublic of Korea
- Language:EN
-
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, proinflammatory 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 TCHSMAM1 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. Proinflammatory 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.
