Vasily N SukhorukovInstitute for Atherosclerosis Research, Russian Federation
Title: Atherosclerosis-associated mtDNA mutation g15059a can contribute to the disruption of mitophagy
Background: Dysfunctional autophagy (mitophagy) is associated with atherosclerosis progression. Dysfunctional mitophagy can promote apoptosis and lead to hyperactivation of inflammatory pathways in macrophages. Some mitochondrial mutations may provoke defective mitophagy in macrophages. One of this mutation, G15059A in CYB gene, was mainly found in atherosclerosis plaques and positively correlates with the age of patients with atherosclerosis and coronary artery disease.
Aim: The aim of this study was to assess the role of this mutation in dysfunctional mitophagy on macrophages.
Material and Methods: The control THP-1 cells and THP-1 cells with mitochondrial G15059A mutation (TCHSMAM1 cells) in CYB gene were used to assess mitophagy level. The mitoCAS9 vector and two sgRNAs to G15059A mutation was used to eliminate the mutation from TCHSMAM1 cells. Carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP) was used to stimulate mitophagy. Mitophagy was assessed by a confocal microscopy in the presence of MitoTracker Green and LysoTracker Deep Red.
Results: G15059A mutation was removed from TCHSMAM1 cells by CRISPR/Cas9 editing. As a result, the heteroplasmy level was decreased was reduced up to 3.7% from initial 68% in TCHSMAM1 cells. The mitophagy activity was increased in 1.3-fold (p<0.001) in THP-1 and in 1.5-fold (p<0.001) in CRISPR-treated TCHSMAM1 cells under induction of mitophagy by FCCP. In the intact TCHSMAM1 cells the mitophagy was dysfunctional under the same conditions.
Conclusion: Mutation G15059A can contribute to the disruption of mitophagy processes in cells, which can lead to the preservation of dysfunctional mitochondria in macrophages. This study was supported by Russian Science Foundation, Grant # 22-15-00064.
Vasily N Sukhorukov is researcher in Laboratory of Angiopathology, Institute of General Pathology and Pathophysiology, Moscow, Russia. I got my PhD in biology in the Institute of General Pathology and Pathophysiology. He is member of European Atherosclerotic Society. I got Young Investigator Award of Asian-Pacific Society of Atherosclerosis and Vascular Disease in 2018 and 2019 and guest editor in the International Journal of Molecular Science (MDPI) and Frontiers in Cardiovascular Medicine (Frontiers). His research is focused on mitochondrial mutations and their role in the metabolism of cholesterol and inflammatory response in atherogenesis. Moreover, I take part in a study of genes defined by bioinformatics approach as key master regulators which control dozens of metabolic pathways in macrophages exposed to modified lipoproteins.