ALBERT

Notes: Abstract To determine if longitudinal bone growth affects the differentiation of fast- and slow-twitch muscles, the tibial bone was sectioned at 90 days gestation in foetal sheep so that the lower leg was permanently without structural support. At 140 days (term is ∼147 days) the contractile properties of whole muscles, activation profiles of single fibres and ultrastructure of fast- and slow-twitch muscles from the hindlimbs were studied. The contractile and activation profiles of the slow-twitch soleus muscles were significantly affected by tibial bone resection (TIBX). The soleus muscles from the TIBX hindlimbs showed: (1) a decrease in the time to peak of the twitch responses from 106.2 ± 10.7 ms (control, n = 4) to 65.1 ± 2.48 ms (TIBX, n = 5); (2) fatigue profiles more characteristic of those observed in the fast-twitch muscles; and (3) Ca2+- and Sr2+-activation profiles of skinned fibres similar to those from intact hindlimbs at earlier stages of gestation. In the FDL, TIBX did not significantly change whole muscle twitch contraction time, the fatigue profile or the Ca2+- and Sr2+-activation profiles of skinned fibres. Electron microscopy showed an increased deposition of glycogen in both soleus and FDL muscles. This study shows that the development of the slow-twitch phenotype is impeded in the absence of the physical support normally provided by the tibial bone. We suggest that longitudinal stretch is an important factor in allowing full expression of the slow-twitch phenotype.

Notes: Summary The properties of bi-directional sliding of F-actin prepared from rabbit skeletal muscle moving along clam thick filaments have been characterized in the presence of agents known to modify unloaded shortening velocity in muscle to determine if the sliding characteristics of actin are similar in the two directions of movement. Actin filaments moved at a fast velocity towards the central bare zone (11.1±0.2 μm s-1) and at a slower velocity away from the bare zone (3.9±0.3 μm s-1). Movement of filaments at the slow sliding velocity is thought to be sustained by a change in orientation of the myosin head. The Michaelis Menten constant (Km values) of ∼0.3 mm in the presence of MgATP concentrations of 0.01–2.0 mm at an ionic strength of 43.5 mm were reduced to ∼0.1 mm at low ionic strength (18.5 mm) although the Km values at the fast and slow sliding velocities at each ionic strength were similar. In the presence of constant concentrations of MgATP, increasing the MgADP concentrations from 0.5 to 2 mm, decreased the bi-directional sliding velocity of actin. The data were well fitted with an equation described by Michaelis Menten kinetics yielding mean absolute Km and Ki values of 0.41±0.01 and 0.44±0.05 mm for the fast velocity and 0.29±0.07 and 0.45±0.02 mm for the slow velocity of sliding, respectively. The Km and Ki values were not significantly different from each other at either the fast or slow sliding, velocities. The actin filament sliding velocity appeared to be controlled through the thick filament as actin was devoid of regulatory proteins and the presence of Ca2+ modified the MgATP dependent movement of actin. The pCa value for half maximal sliding velocity was 7.0 for both fast and slow velocities. The Km and Ki values and the Ca2+ sensitivity of the actin movement at the fast and slow sliding velocity are similar suggesting that no major biochemical changes have occurred in the myosin head as a result of a change in orientation.

Notes: Summary Single fibres of different sarcomere length at rest have been isolated from the claw muscle of the yabby (Cherax destructor), a decapod crustacean. Fibres of either long (SL 〉 6 μm) or short (SL 〈 4 μm) sarcomere length have been mechanically skinned and were maximally activated by Ca2+ and Sr2+ under various experimental conditions (ionic strength, in the presence of 2,3 butanedione monoxime (BDM)) to determine differences in their contractile properties. Isometric force was measured simultaneously with either myofibrillar MgATPase or fibre stiffness in both fibre types. The ultrastructure of individual long- and short-sarcomere fibres was also determined by electron microscopy. The long-sarcomere fibres developed greater tension (30.48±1.72 N cm−2) when maximally activated by Ca2+ compared with the short-sarcomere fibres (18.60±0.80 N cm−2). The difference in the maximum Ca2+-activated force can be explained by the difference in the amount of filament overlap between the two fibre types. The maximum Ca2+-activated myofibrillar MgATPase rate in the short-sarcomere fibres (1.60±0.27 mmol ATP l−1 s−1) was higher, but not significantly different from the ATPase rate in fibres with long-sarcomeres (1.09±0.14 mmol ATP l−1 s−1). As the concentration of myosin is estimated to be higher only by a factor of 1.22 in the short-sarcomere preparations there is no evidence to suggest that the myofibrillar MgATPase activity is different in the long- and short-sarcomere preparations. The maximum Ca2+-activated force (P 0) of both short- and long-sarcomere fibres was quite insensitive to BDM compared with vertebrate muscle. Force decreased to 60.2±5.3% and 76.1±2.7% in the short- and long-sarcomere fibres respectively in the presence of 100 mmol l−1 BDM. The difference in the force depression between the. long- and short-sarcomere fibres is statistically significant (p〈0.05). Fibre stiffness during maximum Ca2+-activation expressed as percentage maximum force per nm per half sarcomere was higher by a factor of 3.5 in short-sarcomere fibres than in long-sarcomere fibres suggesting that the compliance of the filaments in the long-sarcomere fibres is considerably higher than in the short-sarcomere fibres. Sr2+ could not activate the contractile apparatus to the same level as that seen by Ca2+ in either fibre type: the maximum Sr2+-activated force was (20±3%) and (63±3%) of the maximum Ca2+-activated force response in short- and long-sarcomere fibres, respectively. The ratio between fibre stiffness in the maximum Sr2+-activating solution and the Ca2+-activating solution was very similar to the ratio between the maximum Sr2+-activated force and Ca2+-activated force in either type of fibres, suggesting that the number of attached crossbridges is lower in the fibres when maximally activated by Sr2+ than when maximally activated by Ca2+. The short-sarcomere fibres were also more sensitive to changes in ionic strength than long-sarcomere fibres. In conclusion these results indicate that while several important specific characteristics of the short- and long-sarcomere length fibres (ATPase, maximum Ca2+-activated force and fibre stiffness) can be explained solely on differences in the ultrastructure (length and density per cross-sectional area of myosin filaments) there are also differences in the properties of the proteins involved in the force production and regulation evidenced by the differential effect of Sr2+, BDM and ionic strength on contractile activation in the two fibre types.

Notes: Abstract The rate, magnitude and pharmacology of inorganic phosphate (Pi) transport into the sarcoplasmic reticulum were estimated in single, mechanically skinned skeletal muscle fibres of the rat. This was done, indirectly, by using a technique that measured the total Ca2+ content of the sarcoplasmic reticulum and by taking advantage of the 1:1 stoichiometry of Ca2+ and Pi transport into the sarcoplasmic reticulum lumen during Ca--Pi precipitation- induced Ca2+ loading. The apparent rate of Pi entry into the sarcoplasmic reticulum increased with increasing myoplasmic [Pi] in the 10 mm--50 mm range at a fixed, resting myoplasmic pCa of 7.15, as judged by the increase in the rate of Ca--Pi precipitation-induced sarcoplasmic reticulum Ca2+ uptake. At 20 mm myoplasmic [Pi] the rate of Pi entry was calculated to be at least 51 μm s−1 while the amount of Pi loaded appeared to saturate at around 3.5 mm (per fibre volume). These values are approximations due to the complex kinetics of formation of different species of Ca--Pi precipitate formed under physiological conditions. Phenylphosphonic acid (PhPA, 2.5 mm inhibited Pi transport by 37% at myoplasmic pCa 6.5 and also had a small, direct inhibitory effect on the sarcoplasmic reticulum Ca2+ pump (16%). In contrast, phosphonoformic acid (PFA, 1 mm) appeared to enhance both the degree of Pi entry and the activity of the sarcoplasmic reticulum Ca2+ pump, results that were attributed to transport of PFA into the sarcoplasmic reticulum lumen and its subsequent complexation with Ca2+. Thus, results from these studies indicate the presence of a Pi transporter in the sarcoplasmic reticulum membrane of mammalian skeletal muscle fibres that is (1) active at physiological concentrations of myoplasmic Pi and Ca2+ and (2) partially inhibited by PhPA. This Pi transporter represents a link between changes in myoplasmic [Pi] and subsequent changes in sarcoplasmic reticulum luminal [Pi]. It might therefore play a role in the delayed metabolic impairment of sarcoplasmic reticulum Ca2+ release seen during muscle fatigue, which should occur abruptly once the Ca--Pi solubility product is exceeded in the sarcoplasmic reticulum lumen

Notes: Summary Long-(SL〉6μm) and short-sarcomere (SL〈4μm) fibres were isolated from the claw muscle of the yabby (Cherax destructor) during limb regeneration and at different stages of the moult cycle. Long-sarcomere fibres were more susceptible to the changes resulting from the moult-induced atrophy compared with the short-sarcomere fibres. Signs of atrophy included fibre erosion, loss of myosin filaments, a reduction in the diameter of myosin filaments and changes associated with the Z line. The intracellular structure of the fibres, however, remained intact in both fibre types. Fibres taken immediately prior to ecdysis could not be fully activated with Ca2+ or Sr2+ without breaking. In contrast fibres taken within 4 h after ecdysis could develop and maintain full force when activated by Ca2+ or Sr2+. The results suggest that loss of myofibrillar proteins via the moult-induced atrophy and/or events associated with fibre elongation may occur in the period just prior to ecdysis and that these changes may be responsible for the fibres inability to function during the premoult stage. Results from this study showed that short-sarcomere fibres add sarcomeres by at least two different mechanisms (1) transverse sarcomere splitting and (2) Z line splitting. Long-sarcomere fibres appear to be elongated by mechanism (s) other than those used by short-sarcomere fibres which possibly involve large electron dense structures which are positioned between the myofibrils and within the A and I bands. Results from the regenerating chelae limb bud showed that sarcomeres form from separate units comprising myosin filaments and actin filaments anchored into Z lines respectively. These sub-sarcomeric units then join together to form sarcomeres. Myofibril formation is aided by electron dense regions which are closely associated with the membrane system. These fibres although short in length and still within the non-functional limb bud could be activated by Ca2+ and Sr2+ suggesting that full fibre function exists before the chelae become functional. Regenerating muscle fibres consisted predominately of fibres with short-sarcomeres.