Ca2+ uptake by cardiac sarcoplasmic reticulum ATPase in situ strongly depends on bound creatine kinase

The role of creatine kinase (CK) bound to sarcoplasmic reticulum (SR), in the energy supply of SR ATPase in situ, was studied in saponin-permeabilised rat ventricular fibres by loading SR at pCa 6. 5 for different times and under different energy supply conditions. Release of Ca2+ was induced by 5 mM caffeine and the peak of relative tension (T/Tmax) and the area under isometric tension curves, ST, were measured. Taking advantage of close localisation of myofibrils and SR, free [Ca2+] in the fibres during the release was estimated using steady state [Ca2+]/tension relationship. Peak [Ca2+] and integral of free Ca2+ transients (S[Ca2+]f) were then calculated. At all times, loading with 0.25 mM adenosine diphosphate, Mg2+ salt (MgADP) and 12 mM phosphocreatine (PCr) [when adenosine triphosphate (ATP) was generated via bound CK] was as efficient as loading with both 3.16 mM MgATP and 12 mM PCr (control conditions). However, when loading was supported by MgATP alone (3.16 mM), T/Tmax was only 40% and S[Ca2+]f 31% of control (P < 0.001). Under these conditions, addition of a soluble ATP-regenerating system (pyruvate kinase and phosphoenolpyruvate), did not increase loading substantially. Both ST and S[Ca2+]f were more sensitive to the loading conditions than T/Tmax and peak [Ca2+]. The data suggest that Ca2+ uptake by the SR in situ depends on local ATP/ADP ratio which is effectively controlled by bound CK.