Impact of tyrosine phosphorylation site mutation in FUNDC1 protein on mitophagy in H9c2 cardiomyocytes.
- Author:
Zhaoyang ZHANG
1
;
Yanli YU
2
;
Jieyun WU
1
;
Wei TIAN
3
;
Jingman XU
4
Author Information
1. School of Public Health, North China University of Science and Technology, Tangshan 063210, China.
2. Tangshan Maternal and Child Health Hospital, Tangshan 063000, China.
3. Hebei Key Laboratory of Organ Fibrosis, Tangshan 063210; Laboratory Animal Center, North China University of Science and Technology, Tangshan 063210, China.
4. School of Public Health, North China University of Science and Technology, Tangshan 063210; Hebei Key Laboratory of Organ Fibrosis, Tangshan 063210, China. *Corresponding author, E-mail: xujm@ncst.edu.cn.
- Publication Type:Journal Article
- MeSH:
Animals;
Rats;
Mitophagy/genetics*;
Myocytes, Cardiac/cytology*;
Mitochondrial Proteins/metabolism*;
Mutation;
Phosphorylation;
Tyrosine/genetics*;
Cell Line;
Membrane Proteins/metabolism*;
Membrane Potential, Mitochondrial
- From:
Chinese Journal of Cellular and Molecular Immunology
2025;41(7):629-636
- CountryChina
- Language:Chinese
-
Abstract:
Objective To investigate the effect of FUNDC1 tyrosine phosphorylation site mutations on mitophagy in H9c2 myocardial cells by constructing tyrosine site mutant plasmids (Y11 and Y18) of the FUN14 domain-containing protein 1 (FUNDC1). Methods The mutant plasmids constructed by whole-gene synthesis were transfected into rat myocardial H9c2 cells and divided into five groups: empty plasmid group, FUNDC1 overexpression group, Y11 mutant group, Y18 mutant group, and Y11 combined with Y18 mutant group. The viability of H9c2 cells was assessed using the CCK-8 assay. Additionally, tetramethylrhodamine ethyl ester (TMRE) staining was utilized to detect mitochondrial membrane potential. The protein expression levels of FUNDC1, translocase of the outer mitochondrial membrane 20 (TOM20), and cytochrome c oxidase IV (COX IV) were detected by Western blot analysis. Confocal microscopy was used to evaluate transfection efficiency as well as the co-localization of mitochondria and lysosomes. Results The FUNDC1 overexpression, Y11, Y18, and Y11 combined with Y18 mutant plasmids were successfully constructed. After plasmid transfection, widespread GFP fluorescence expression was observed under confocal microscopy. Compared with the empty plasmid group, FUNDC1 protein expression levels were significantly increased in the FUNDC1 overexpression group, Y11 mutation group, Y18 mutation group, and Y11 combined with Y18 mutation group, while cell viability and mitochondrial membrane potential showed no significant changes. Compared to the empty plasmid group, cells transfected with Y18 and Y11 combined with Y18 mutant plasmids showed increased TOM20 and COX IV expression levels and decreased mitochondrial-lysosomal co-localization. Conclusion Transfection with FUNDC1 Y18 or Y11 combined with Y18 mutant plasmids inhibited mitophagy in H9c2 myocardial cells.
