Mitochondrial genome editing: a possible interplay of atherosclerosis-associated mutation m.15059G>A with defective mitophagy

BackgroundIt was assumed that the cause of chronic inflammation in atherosclerosis is a disturbance of the innate immunity response, caused, among other factors, by mitochondrial dysfunctions. It was also suggested that mitochondrial dysfunction may be caused by heteroplasmic mutations in mitochondrial DNA. The aim of this study was to evaluate the effect of the mitochondrial nonsense mutation m.15059G>A on cellular functions in atherosclerosis: lipoidosis, pro-inflammatory response, and mitophagy. MethodsThe 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 for the elimination of mtDNA copies carrying the m.15059G>A mutation in the MT-CYB gene. Using this approach, the Cas9-TC-HSMAM1 cells with an eliminated m.15059G>A mutation was obtained. The gene expression levels of genes encoding enzymes related to cholesterol metabolism were analyzed by quantitative RT-PCR. The evaluation of pro-inflammatory cytokine secretion was assessed using ELISA. Mitophagy in cells was detected using confocal microscopy. ResultsIn contrast to intact TC-HSMAM1 cybrids, in Cas9-TC-HSMAM1 cells, incubation with atherogenic LDL led to a decrease in the expression of the gene encoding fatty acid synthase (FASN). It was found that TC-HSMAM1 cybrids are characterized by defective mitophagy and are also unable to reduce the production of pro-inflammatory cytokines (to form immune tolerance) in response to repeated LPS stimulation. Elimination of mtDNA carrying the m.15059G>A mutation led to the restoration of immune tolerance and activation of mitophagy in the studied cells. ConclusionsThe m.15059G>A mutation was found to be associated with defective mitophagy, immune tolerance, and impaired metabolism of intracellular lipids due to upregulation of the FASN. Thus, this mutation may play an important role in atherogenesis due to its contribution to the chronification of inflammation, which aggravates the progression of atherosclerosis.