Abstract Body (Do not enter title and authors here): Background: Augmented fatty acid oxidation (FAO) adversely affects cardiac function in diabetic patients. Mitochondrial protein lysine (K) acetylation is a critical mechanism modifying metabolic flux, yet specific acetylation targets enhancing FAO are unknown.
Purpose: This study aimed to identify proteins whose acetylation increases FAO activity in diabetic hearts using multi-omics analyses.
Methods: We used Otsuka-Long-Evans-Tokushima Fatty (OLETF) rats, a type 2 diabetes model aged 32-38 weeks, which display early-stage cardiomyopathy, and Long-Evans-Tokushima-Otsuka (LETO) rats as controls. Metabolomics, acetylomics, and proteomics were performed on samples from these hearts.
Results: OLETF had elevated blood glucose (194.0 vs 137.9 mg/dL, P = 0.0022) and Myh7 mRNA expression (7.11 vs 4.41 units, P = 0.0063) compared to LETO, indicating diabetes and cardiac dysfunction. Metabolomics revealed a decreased fumarate/succinate ratio in OLETF (0.51 vs 0.99 units, P = 0.0083), indicating suppressed activities of TCA cycle and complex II in the electron transport chain (ETC). FAO provides reducing equivalents through acetyl-CoA supply into TCA cycle and directly to ubiquinone via electron transfer flavoprotein (ETF), highlighting ETF's role in FAO upregulation in diabetic hearts. Acetylomics in mitochondrial fractions identified 55 acetylated K residues on 24 mitochondrial proteins increased by 1.5-fold (P < 0.05) in OLETF compared to LETO, with these proteins enriched in the FAO pathway. Specifically, K69-Ac, K75-Ac, and K126-Ac on ETFA, a subunit of ETF, were detected. To determine whether increased acetylation or protein expression contributed to the acetylomics signals, proteomics was performed on identical samples. Of 24 proteins, 22 were measurable, with several critical FAO-related proteins (e.g., ACADVL, ACADM, HADHA, HADHB, ACOT2) significantly more expressed in OLETF than in LETO, while ETFA expression remained similar (1.01 vs 1.00 units, P = 0.84). This suggests increased acetylation signals in FAO-related proteins are mainly due to upregulated protein expression, except for ETFA.
Conclusions: While increased expression is commonly seen in FAO genes in diabetic hearts, protein acetylation of ETFA appears to be a crucial compensatory mechanism to transfer reducing equivalent to ETC from the FAO under the reduced TCA cycle activity. These findings provide insights into the mechanisms underlying metabolic disturbance in the early stage of cardiomyopathy.