Metabolic Enzyme Acetylation is Elicited in Response to Stress Induced by Cardiac Specific Overexpression of Human ADCY8

Abstract

INTRODUCTIONCardiac‐specific overexpression of human ADCY8 in mice (TGAC8), induces constant stimulation of the cAMP/PKA pathway in TGAC8 mice that results in a sustained, robust elevation of heart rate and cardiac contraction 24 hours a day, 7 days a week. Although progress has been made in understanding adaptive cardiac remodeling that occurs as a result of acute bouts of exercise, little is known about the effects of chronic, cardiac specific stress. Our initial transcriptomic, proteomic and metabolomic profile of the TGAC8 left ventricle (LV) has afforded some insight into how the heart adapts to this chronic stress: changes in key metabolic enzymes suggest an increase in glycolytic activity and reduction in fatty‐acid substrate support of metabolism. This prompted us to further investigate how this chronic cAMP‐PKA stress alters the bioenergetics of the heart. Due to its role in metabolic regulation, we were particularly interested in the acetylation status of metabolic enzymes in the TGAC8 model.METHODSAcetylation was quantified in the left ventricles of three 90‐day old wild type (WT) and TGAC8 mice. Acetylated peptides were isolated and enriched for label free quantification, using LC‐MS/MS analysis, and subsequent bioinformatic analysis was completed using STRING, Gene Ontology (GO) term enrichment, and Ingenuity Pathway Analysis. Acetylation sites were considered to be significantly different between TGAC8 and WT if the fold change was > 1.5 in either direction and significantly differed at the p < 0.05 level.RESULTSWe identified 6212 acetylated sites across 2206 proteins. Of these, 450 acetylation sites across 225 proteins were significantly different in the TGAC8 compared to WT, with 329 sites across 180 proteins being more acetylated in the TGAC8. STRING analysis revealed that 110 of these 225 proteins localize to the mitochondria: 81 of these being involved in the oxidation‐reduction process; and 58 being involved in oxidoreductase activity. The most significantly enriched GO terms shared common themes of cellular metabolism, fatty acid utilization and aerobic respiration (Fig. ). Proteins with the highest number of sites with significantly more acetylation in the TGAC8 had known roles in fat and glucose metabolism, such as ATP synthase, glycogen phosphorylase, isocitrate dehydrogenase, and various enzymes involved in the terminal steps of beta oxidation (Table ).CONCLUSIONAcetylation changes to key metabolic enzymes suggests a mechanism for alteration in TGAC8 fuel preferences deduced from the initial transcriptomic, proteomic, and metabolomic analysis. Specifically, increased acetylation of enzymes involved in beta oxidation, oxidative phosphorylation, and glucose metabolism, in combination with our previous omics data, suggests that the TGAC8 mouse heart may exhibit a preference for glucose utilization and exhibit impaired fatty acid metabolism.Support or Funding InformationThis research was funded by the Intramural Research Program of the National Institutes of Health, National Institute on Aging.Most significantly enriched GO terms from each subontology.Figure 1 Proteins with the greatest quantity of acetylation sites significantly higher in TGAC8 heart identified by LC‐MS/MS. Gene Name Description # of sites w/higher TGAC8 acetylation Atp5a1 ATP synthase subunit alpha, mitochondrial 10 Pygm Glycogen phosphorylase 10 Alb Serum albumin 7 Acaa2 3‐ketoacyl‐CoA thiolase 6 Eno3 Beta‐enolase 6 Hba‐a2 Alpha globin 1 6 Idh2 Isocitrate dehydrogenase 6 Aco2 Aconitate hydratase 5 Mb Myoglobin 5 Trf Serotransferrin 5 Acadm Medium‐chain specific acyl‐CoA dehydrogenase 4 Aldoa Fructose‐bisphosphate aldolase 4 Ckm Creatine kinase M‐type 4 Eno1 Alpha‐enolase 4 Fh1 Fumarate hydratase 4 Pgk1 Phosphoglycerate kinase 1 4 Sod2 Superoxide dismutase 4 Uqcrc1 Cytochrome b‐c1 complex subunit 1 4 Agl Amylo‐1,6‐glucosidase, 4‐alpha‐glucanotransferase 3 Atp5o ATP synthase subunit O 3