The Influence of Alternative Energy Transfer Systems on Respiration in Creatine-Deficient Mouse Cardiomyocytes

Both GAMT-/- and AGAT-/- mice lack a functional creatine kinase (CK) phosphotransfer system due to their deficiency of the enzymes guanidinoacetate methyltranferase (GAMT) and L-arginine:glycine amidinotransferase (AGAT) that synthesize creatine, respectively. As we have shown earlier, GAMT-deficiency is not associated with any changes in cardiomyocyte mitochondrial organization and both GAMT and AGAT-deficiencies do not alter intracellular compartmentation in relaxed cardiomyocytes.The aim of this study was to examine whether alternative energy transfer systems are up-regulated in GAMT-/- and AGAT-/- cardiomyocytes to compensate for the lack of CK phosphotransfer. The experiments were performed on freshly isolated permeabilized adult cardiomyocytes. ATP-stimulated respiration and the influence of activating adenylate kinase (AK) with AMP and hexokinase (HK) with glucose were measured and compared to the influence of activating CK with creatine. Their coupling was assessed using the competitive ADP-trapping PEP/PK assay. The respiration data were complemented with spectrophotometric recordings of total enzyme activities of CK, AK and HK using coupled enzyme assays.As expected on the basis of CK activity distribution between mitochondria and cytosol, the competitive PEP/PK consumed 70-80% of the ADP produced by CK before it reached the mitochondria in both GAMT-/- and AGAT-/- mice. In comparison, PEP/PK consumed 80-85% of the ADP produced by hexokinase and almost all ADP produced by AK in both mouse models. Our results confirm the coupling of CK to respiration in both GAMT-/- and AGAT-/- and their respective wildtype littermates. The coupling of HK is close to that of CK, consistent with some of HK binding to the mitochondria. In contrast, AK seems to be mainly cytosolic. Our results suggest no compensatory changes in the compartmentation of HK, AK or CK in GAMT-/- and AGAT-/- mice.