Changes in the Ca2+-activation characteristics of demembranated rat singlemuscle fibres after prolonged incubation at room temperature in the presence and absence of DTT and protease inhibitors

Prolonged incubation (24 h) of chemically skinned rat muscle preparations i n rigor solutions at room temperature and in the absence of reducing agents and protease inhibitors modified the Ca2+-activation characteristics of th e contractile machinery. In the absence of reducing agents and protease inh ibitors, the contraction threshold for incubated fibres was shifted to lowe r Ca2+ concentrations and the steepness of the steady-state force-pCa (-log (10)[Ca2+]) curve was decreased compared to that of control muscle fibres. Mean myosin ATPase activity under these conditions was significantly lowere d by a factor of 2.7. Fibres incubated in the presence of 10 mM dithiothrei tol (DTT) and protease inhibitors (100 muM pepstatin A/200 muM leupeptin) p roduced a maximum Ca2+-activated force per cross-sectional area that compar ed favourably with that of freshly dissected muscle fibres and there were n o changes in the other contractile activation characteristics. Intermediate responses were obtained when fibres were incubated in the presence of eith er DTT or protease inhibitors. MgATPase activities of incubated preparation s increased significantly following the addition of protease inhibitors and /or DTT ts the incubation medium. Taken together, these results suggest tha t in the presence of DTT and protease inhibitors, most contractile properti es are maintained at levels seen in fresh mechanically skinned fibres. The extended viability of this preparation and its closely related properties w ith fresh muscle fibres make it a useful model for experiments requiring lo nger term incubations with biological agents.