J Lipid Atheroscler.
2024 May;13(2):166-183. 10.12997/jla.2024.13.2.166.
Mitochondrial Genome Editing: Exploring the Possible Relationship of the Atherosclerosis-Associated Mutation m.15059G>A With Defective Mitophagy
- Sukhorukov VN
1,2,3,4 - Khotina VA1,2
- Kalmykov VA1,2
- Zhuravlev AD1,2
- Sinyov VV1,2,3
- Popov DY4
- Vinokurov AY4
- Sobenin IA1,3
- Orekhov AN1,2,5
- Affiliations
-
- 1Laboratory of Angiopathology, Institute of General Pathology and Pathophysiology, Moscow, Russia
- 2Laboratory of Cellular and Molecular Pathology of Cardiovascular System, Petrovsky National Research Centre of Surgery, Moscow, Russia
- 3Laboratory of Medical Genetics, Institute of Experimental Cardiology, Russian Medical Research Center of Cardiology, Moscow, Russia
- 4Cell Physiology and Pathology Laboratory of R&D Center of Biomedical Photonics, Orel State University, Orel, Russia
- 5Institute for Atherosclerosis Research, Moscow, Russia
- KMID: 2556013
- DOI: http://doi.org/10.12997/jla.2024.13.2.166
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.
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