Integrating Multiomics to Reveal and Validate Key Proteins, Metabolites, and Pathways of ASB14 Affecting Mitochondrial Function in Cardiomyocytes AC16.

ASB14 may be involved in the heart failure progression through protein ubiquitination, but the involvement of downstream proteins and metabolites remains unknown. This study aimed to refine the regulatory mechanism of ASB14 on cardiomyocyte functions through in vitro experiments, proteomics, metabolomics, and integration analyses. In this study, pCDH-ASB14 was transfected into AC16 cells to construct an overexpression model of ASB14. CCK8, flow cytometry, and ATP levels were detected to explore the effects of ASB14 overexpression on cardiomyocytes. Tandem mass tags-isobaric tag for relative absolute quantitation-based proteomic and liquid chromatography-tandem mass spectrometry-based metabolomics studies were performed to screen differential proteins and metabolites and their enriched pathways. Integration analysis revealed common pathways, differential protein-metabolite relationship pairs, and protein-metabolite-pathway networks. Western blotting was used to verify expression levels of key proteins. In vitro experiments revealed that ASB14 overexpression promoted apoptosis and inhibited cell proliferation and mitochondrial function in AC16. Proteomics suggested 1306 differential proteins between AC16 cells transfected with pCDH-ASB14 and pCDH-NC, which were mainly enriched in linoleic acid metabolism. Meanwhile, metabolomics indicated 114 differential metabolites that were mainly enriched in purine metabolism. Integrative analysis suggested common pathways including diabetic cardiomyopathy and most significant correlations between 13 differential proteins and 16 differential metabolites. Validation of seven key proteins suggested that ASB14 overexpression significantly inhibited PIP4K2A, ferritin light chain, AMPK alpha-1, GLB1, ABCC9, and NME7, but promoted RRAGC protein expression levels. ASB14 overexpression regulates downstream proteins and inhibits mitochondrial function in cardiomyocytes through pathways of linoleic acid and purine metabolism.