Mitochondrial Genome Editing: Exploring the Possible Relationship of the Atherosclerosis-Associated Mutation m.15059G>A With Defective Mitophagy
Mitochondrial Genome Editing: Exploring the Possible Relationship of the Atherosclerosis-Associated Mutation m.15059G>A With Defective Mitophagy
Vasily N. Sukhorukov(Laboratory of Angiopathology, Institute of General Pathology and Pathophysiology); Victoria A. Khotina(Laboratory of Angiopathology, Institute of General Pathology and Pathophysiology); Vladislav A. Kalmykov(Laboratory of Angiopathology, Institute of General Pathology and Pathophysiology); Alexander D. Zhuravlev(Laboratory of Angiopathology, Institute of General Pathology and Pathophysiology); Vasily V. Sinyov(Laboratory of Angiopathology, Institute of General Pathology and Pathophysiology); Daniil Y. Popov(Cell Physiology and Pathology Laboratory of R&D Center of Biomedical Photonics); Andrey Y. Vinokurov(ell Physiology and Pathology Laboratory of R&D Center of Biomedical Photonics); Igor A. Sobenin(Laboratory of Angiopathology, Institute of General Pathology and Pathophysiology); Alexander N. Orekhov(Laboratory of Angiopathology, Institute of General Pathology and Pathophysiology)
13권 2호, 166~183쪽
초록
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.
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.
- 발행기관:
- 한국지질동맥경화학회
- 분류:
- 순환기내과