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Guanidinoacetate-N-methyltransferase (GAMT) is a key enzyme in creatine biosynthesis, such that GAMT knockout mice lack phosphocreatine (PCr) as a substrate for energy transfer via the creatine kinase (CK) reaction. Despite undetectable levels of PCr and creatine, GAMT knockout mice exhibit only minor changes in baseline function and impaired contractile reserve, suggesting remarkable plasticity in myocardial energy metabolism. However, the precise nature of compensatory metabolic mechanisms remains unknown. The aim of this study was to examine the potential roles of F1F0 ATP synthase and the complementary phosphotransfer enzyme adenylate kinase in GAMT knockout hearts. Mitochondria were isolated from the 27-week GAMT knockout and age-matched wild-type hearts. To assay the F1F0 ATP synthase capacity, maximal F1 ATPase hydrolytic activity was measured spectrophotometrically in mitochondrial homogenate by coupling ATP hydrolysis to NADH oxidation. Total enzyme activities of CK, adenylate kinase and glycolytic enzymes (glyceraldehyde 3-phosphate dehydrogenase, phosphoglycerate kinase, pyruvate kinase) were measured in ventricular tissue extracts using coupled enzyme assays. GAMT knockout hearts were characterised by a marked increase in F1F0 ATP synthase activity (oligomycin sensitive activity 2.16Â±0.5 vs 4.2Â±1.1Â μmol ATP/minute per mg; n=6, p<0.05), decreased CK (6.8Â±0.6 vs 5.0Â±0.4 U/mg, p<0.01; n=13), and unaltered adenylate kinase activity (2.5Â±0.6 vs 2.6Â±0.6 U/mg; n=11), while glycolytic enzyme activities where consistently elevated in knockout hearts. Therefore, long-term adaptation to chronic perturbation of the CK/PCr system in GAMT knockout hearts does not include a compensatory increase in phosphotransfer via adenylate kinase. Rather, this study suggests increased ATP synthesis as a potential compensatory mechanism to maintain cardiac function close to normal.