Macrocompartmentation of total creatine in cardiomyocytes revisited

Distribution of total creatine (free creatine + phosphocreatine) between tw o subcellular macrocompartments-mitochondrial matrix space and cytoplasm - in heart and skeletal muscle cells was reinvestigated by using a permeabili zed cell technique. Isolated cardiomyocytes were treated with saponin (50 m ug/ml for 30 min or 600 mug/ml for 1 min) to open the outer cellular membra ne and release the metabolites from cytoplasm (cytoplasmic fraction, CF). A ll mitochondrial population in permeabilized cells remained intact: the out er membrane was impermeable for exogenous cytochrome c, the acceptor contro l index of respiration exceeded 10, the mitochondrial creatine kinase react ion was fully coupled to the adenine nucleotide translocator. Metabolites w ere released from mitochondrial fraction (MF) by 2-5% Triton X100. Total ce llular pool of free creatine + phosphocreatine (69.6 +/- 2.1 nmoles per mg of protein) was found exclusively in CF and was practically absent in MF. W hen fibers were prepared from perfused rat hearts, cellular distribution of creatine was not dependent on functional state of the heart and only sligh tly modified by ischemia. It is concluded that there is no stable pool of c reatine or phosphocreatine in the mitochondrial matrix in the intact muscle cells, and the total creatine pool is localized in only one macrocompartme nt-cytoplasm.