Objective: To elucidate the role of bound creatine kinase in adenine nucleotide compartmentation in myofibrils, the effects of this enzyme's substrates and products on rigor tension were studied in using isolated skinned rat cardiomyocytes rather than fibres, to avoid restrictions due to concentration gradients within the multicellular preparations. Methods: A new experimental set-up was built to allow continuous and stable measurements of force developed by cells. Triton X-100-treated cardiomyocytes were glued between a glass holder and the needle of a galvanometer. A feedback system allowed the precise measurement of force by recording the coil current necessary to prevent movement of the needle. Results: At very low [Ca2+] (pCa 7), as MgATP level decreased, rigor tension appeared. In the absence of phosphocreatine (PCr), this tension started to rise at MgATP concentrations several times higher than in the presence of 12 mM PCr. In the absence of PCr, the pMgATP/tension curves of single cells usually had a complicated relationship which could not be analyzed by a simple Hill equation. In the absence of PCr, 250 μM MgADP strongly potentiated rigor tension development in the 1 mM-3 μM range of [MgATP]; at 100 μM MgATP, in the presence of MgADP, the tension was 4.6 times higher than in the absence of MgADP. Addition of 12 mM PCr immediately eliminated rigor. Finally, in the presence of 100 μM MgATP and 250 μM MgADP, a decrease in PCr resulted in rigor; the half-maximal contracture being recorded at 1 mM PCr. Conclusions: These results indicate a myofibrillar compartmentation of adenine nucleotides influenced by bound creatine kinase, since at equal MgATP concentrations in extramyofibrillar millieu the response of myofibrils strongly depends on the presence of PCr. Local accumulation of ADP in myofibrils due to a fall in cellular PCr and inability of myofibrillar creatine kinase to rephosphorylate ADP produced by myosin ATPase could be an important mechanism of diastolic tension rise in ischaemic conditions.
Veksler, V. I., Lechene, P., Matrougui, K., & Ventura-Clapier, R. (1997). Rigor tension in single skinned rat cardiac cell: Role of myofibrillar creatine kinase. Cardiovascular Research, 36(3), 354–362. https://doi.org/10.1016/S0008-6363(97)00178-8