pThe measurement of cardiac Casup2+/sup transients using spectroscopic Casup2+/sup indicators is significantly affected by the buffering properties of the indicators. The aim of the present study was to construct a model of cardiac Casup2+/sup buffering that satisfied the kinetic constraints imposed by the maximum attainable rates of cardiac contraction and relaxation on the Casup2+/sup dissociation rate constants and which would account for the observed effects of sup19/supF-NMR indicators on the cardiac Casup2+/sup transient in the Langendorff-perfused ferret heart. It is generally assumed that the Casup2+/sup dependency of myofibril activation in cardiac myocytes is mediated by a single Casup2+/sup-binding site on troponin C. A model based on 1:1 Casup2+/sup binding to the myofilaments, however, was unable to reproduce our experimental data, but a model in which we assumed ATP-dependent co-operative Casup2+/sup binding to the myofilaments was able to reproduce these data. This model was used to calculate the concentration and dissociation constant of the ATP-independent myofilament Casup2+/sup binding, giving 58 and 2.0 iμ/iM respectively. In addition to reproducing our experimental data on the concentration of free Casup2+/sup ions in the cytoplasm ([Casup2+/sup]subi/sub), the resulting Casup2+/sup and ATP affinities given by fitting of the model also provided good predictions of the Casup2+/sup dependence of the myofibrillar ATPase activity measured under iin vitro/i conditions. Solutions to the model also indicate that the Casup2+/sup mobilized during each beat remains unchanged in the presence of the additional buffering load from Casup2+/sup indicators. The new model was used to estimate the extent of perturbation of the Casup2+/sup transient caused by different concentrations of indicators. As little as 10 iμ/iM of a Casup2+/sup indicator with a dissociation constant of 200 nM will cause a 20% reduction in peak-systolic [Casup2+/sup]subi/sub and 30 iμ/iM will cause approx. 50% reduction in the peak-systolic [Casup2+/sup]subi/sub in a heart paced at 1.0 Hz./p
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