Normal cardiac function requires ATP to be synthesized at a sufficient rate to drive the molecular processes underlying electromechanical coupling. Myocardial metabolic dysfunction observed during heart failure leads to reductions in the chemical potential at which ATP is synthesized. We have observed reductions in oxidative capacity, and total adenine nucleotide (TAN) levels in a rat aortic constriction (TAC) induced heart failure model that parallel changes seen in large mammal models and humans with heart failure. Computer simulations of cardiac mechano-energetics also predict a marked increase in inorganic phosphate levels in diseased hearts compared to healthy control hearts. Furthermore, simulations predict that restoring the impaired energetic dysfunction of TAC rats improves systolic function. Metabolomic and proteomic analysis reveal increases in purine degradation by-products and AMP deaminase 3 (AMPD3) levels in failing versus control hearts. AMPD activity has been hypothesized to play a role in skeletal muscle in type 2 diabetes, however, there is still a need to decern the role, if any, of AMPD3 in the heart and in failure. A cardiac ventricle specific AMPD3 conditional knock out (cKO) rat model was developed to test the hypothesis that decreasing AMPD3 activity in the failing heart will lead to a preservation of TAN and improvement in cardiac mechanical function. Cardiac hypertrophy and decomposition were induced by placing a clip (TAC) in three-week old female and male rats. In the cKO model, AMPD3 exon 6 is flanked by loxP sites, and are either heterozygous for cre recombinase driven by the cardiac myosin light chain promoter (cre+/-) or lack the cre enzyme (cre-/-). Tamoxifen was administered to all rats both pre-surgery and post-surgery to induce AMPD3 knock down. Echocardiography was performed at 2- and 18-weeks post-surgery. At 18 weeks post-surgery, hearts were harvested for mitochondrial capacity, metabolite measurements, AMPD activity, and protein and mRNA measurements. Knock down efficiency in cre +/- animals compared to cre -/- was assessed based on enzyme activity, mRNA expression, and protein abundance. There is a 45% decrease in total AMPD activity in the AMPD3 cKO rats, where measured AMPD activity reflects the sum activity of all present AMPD isoforms. Additionally, there is 52% decrease in protein abundance and a 72% reduction in AMPD3 mRNA expression. Preliminary observations reveal significant differences in oxidative capacity and in cardiac function between TAC and sham rats, consistent with the hypothesis that knock down of AMDP3, an enzyme that is upregulated during TAC induced heart failure in rodents, results in preservation of myocardial nucleotide pools and improve cardiac mechanical function. Research is supported by the NIH grants HL144657 and F31-HL154605 (RL). We are grateful for support from the post-baccalaureate research program (2R25GM086262-09), the Systems and Integrative Biology (T32-GM008322) training grant and the Cardiovascular Research and Entrepreneurship training grant (T32-HL125242) and the University of Michigan Rackham Merit Fellowship. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.