Our previous data showed that static stiffness increase following the stimulation both in twitch or tetanic contractions has a time course distinct from that of tension and roughly similar to that of internal calcium concentration. We hypothesized that static stiffness could be attributed to elastic properties of elements of the sarcomere structure whose stiffness increases in a calcium-dependent way. The experiments reported here were made to test the validity of this hypothesis. To investigate the effects of intracellular calcium we measured the static stiffness in single frog muscle fibres under different conditions in which isometric tension was inhibited either by reducing calcium release or by a direct inhibition on the actomyosin interaction.
Frogs (Rana esculenta) were killed by decapitation followed by double pithing. Single fibres, dissected from tibialis anterior muscle, were mounted between the lever arms of a force transducer and a moving coil stretcher by means of aluminium clips. Average sarcomere length in a selected segment (1Ð2 mm long) of the fibre was measured using a striation follower device. The temperature was maintained constant at 14°C. The experiments were made in Ringer and test solutions containing one of the following agents: 2,3-butanedione monoxime (BDM) at 1Ð5 mM concentration, dantrolene and methoxyverapamil (D600) at 5Ð10 mM concentration, deuterium oxide Ringer (98 % of water substituted with D2O) and hypertonic solution up to 1.6 normal tonicity. To measure the static stiffness the fibres were rapidly stretched (up to 40 nm per half-sarcomere and about 0.5 ms duration) and hold for a period longer than the twitch time course. The fast tension transient was followed by a period during which the tension remained constant at a level that greatly exceeded the isometric force. This level is the static tension and the ratio between the static tension and the accompanying sarcomere length is the static stiffness. The complete time course of static stiffness was determined by applying stretches with different delays with respect to the stimulation.
The results show (Table 1) that static stiffness is almost unaffected by BDM and hypertonic solution, agents that inhibit tension generation mainly by affecting actomyosin interaction reducing cross-bridge formation, even at BDM concentration or Ringer tonicity that reduced twitch tension by more than 90 %. In contrast, a similar degree of tension inhibition was accompanied by strong static stiffness reduction when deuterium oxide, dantrolene or D600, agents which mainly reduce the calcium release, were used to inhibit tension generation.
Our data show that the static stiffness increase in frog skeletal muscle is calcium dependent, in line with our hypothesis, suggesting that the elastic sarcomere structure responsible for static stiffness may be calcium sensitive.
All procedures accord with current National guidelines.