Mechanical and structural properties underlying contraction of skeletal muscle fibers after partial 1-ethyl-3-[3-dimethylamino)propyl]carbodiimide cross-linking

Abstract

We show prolonged contraction of permeabilized muscle fibers of the frog during which structural order, as judged from low-angle x-ray diffraction, was preserved by means of partial cross-linking of the fibers using the zero-length cross-linker 1-ethyl-3-[3-dimethylamino)propyl]carbodiimide. Ten to twenty percent of the myosin cross-bridges were cross-linked, allowing the remaining 80-90% to cycle and generate force. These fibers displayed a well-preserved sarcomeric order and mechanical characteristics similar to those of intact muscle fibers. The intensity of the brightest meridional reflection at 14.5 nm, resulting from the projection of cross-bridges evenly spaced along the myofilament length, decreased by 60% as a relaxed fiber was deprived of ATP and entered the rigor state. Upon activation of a rigorized fiber by the addition of ATP, the intensity of this reflection returned to 97% of the relaxed value, suggesting that the overall orientation of cross-bridges in the active muscle was more perpendicular to the filament axis than in rigor. Following a small-amplitude length step applied to the active fibers, the reflection intensity decreased for both releases and stretches. In rigor, however, a small stretch increased the amplitude of the reflection by 35%. These findings show the close link between cross-bridge orientation and tension changes.

Myosin Regulatory Light Chain (RLC) Phosphorylation Change as a Modulator of Cardiac Muscle Contraction in Disease

“…Trabeculae were mounted in relaxing solution between hooks attached at one end to a force transducer (AE801, HJK Sensoren + Systeme, Freidburg, Germany) and at the other to a motor (32). Shellac dissolved in ethanol was used to fix the trabecular tissue in place.…”

The efficiency of contraction in rabbit skeletal muscle fibres, determined from the rate of release of inorganic phosphate

“…Calcium‐containing rigor solution was identical to calcium‐free rigor except that the free calcium concentration was 32 μ m achieved by addition of Ca‐EGTA and there was no apyrase and no P i ‐mop. Loading solution contained 60 mM TES, 10 mM EGTA, 1 mM free Mg 2+ , 40 mM glutathione, 32 μ m free Ca 2+ , 1.2 mM MDCC‐PBP, 5 mM NPE‐caged ATP (pre‐treated with 10 units ml −1 apyrase to remove ADP contamination: the final apyrase concentration in the loading solution was 0.0013 units ml −1 ), 10 mM creatine phosphate, 4 mg ml −1 creatine kinase obtained from chicken breast (338 units mg −1 at pH 7.1 and 25°C; Bershitsky et al 1996) and P i ‐mop.…”

Strong Binding of Myosin Heads Stretches and Twists the Actin Helix

“…6 and 7) indicates that the stretch causes a change in the shape of myosin heads bound to actin, probably by the elastic bending of the light chain domain of the heads to a position more perpendicular to the filament axis, also leading to an increase in the intensity of the M3 myosin meridional reflection (Fig. 5, Bershitsky et al, 1996;Dobbie et al, 1998;Reconditi et al, 2003). As the near meridional intensity of the 5.1-nm and 5.9-nm layer lines mainly comes from the light chain domains of myosin heads (Kraft et al, 2002), the change in the spacing of these layer lines induced by a stretch of rigor muscle depends not only on the compliance of actin filaments, but also on that of the myosin filaments.…”

“…Before mounting in the experimental trough, the bundles were additionally treated by soaking in relaxing solution with 0.5% Triton X-100 for 15-30 min. One end of a 3.1-3.6 mm long bundle was glued with shellac dissolved in ethanol (50/50% v/v) to the force transducer and another end to the motor, as described by Bershitsky and Tsaturyan (2002); the bundle was mounted horizontally and aligned and sarcomere length was adjusted to 2.4 mm by monitoring the position of the first-order diffraction lines from a He-Ne laser beam (Bershitsky et al, 1996). The bundles were put in rigor at 0°C in the presence of 10 mM 2,3-butanedione-monoxime (BDM) to maintain low rigor tension (,10 kN/m 2 ) and uniform sarcomere length (Bershitsky et al, 1996).…”

“…One end of a 3.1-3.6 mm long bundle was glued with shellac dissolved in ethanol (50/50% v/v) to the force transducer and another end to the motor, as described by Bershitsky and Tsaturyan (2002); the bundle was mounted horizontally and aligned and sarcomere length was adjusted to 2.4 mm by monitoring the position of the first-order diffraction lines from a He-Ne laser beam (Bershitsky et al, 1996). The bundles were put in rigor at 0°C in the presence of 10 mM 2,3-butanedione-monoxime (BDM) to maintain low rigor tension (,10 kN/m 2 ) and uniform sarcomere length (Bershitsky et al, 1996). Relaxing solution contained (in mM) 100 MOPS (3-[Nmorpholino]-propanesulfonic acid), 5 EGTA (ethylene glycol bis-(baminoethyl ether)N,N,N9,N9 tetraacetic acid), 5 MgATP, 2 Mg 21 , 80 potassium propionate, 5 DTT (dithiothreitol); rigor solution had the same composition, except no ATP was added and potassium propionate concentration was increased to 100 mM.…”