Effects of blebbistatin and Ca2+ concentration on force produced during stretch of skeletal muscle fibers.

When activated muscle fibers are stretched at low speeds [≤ 2 optimal length (L(o))/s], force increases in two phases, marked by a change in slope [critical force (P(c))] that happens at a critical sarcomere length extension (L(c)). Some studies attribute P(c) to the number of attached cross bridges before stretch, while others attribute it to cross bridges in a pre-power-stroke state. In this study, we reinvestigated the mechanisms of forces produced during stretch by altering either the number of cross bridges attached to actin or the cross-bridge state before stretch. Two sets of experiments were performed: 1) activated fibers were stretched by 3% L(o) at speeds of 1.0, 2.0, and 3.0 L(o)/s in different pCa(2+) (4.5, 5.0, 5.5, 6.0), or 2) activated fibers were stretched by 3% L(o) at 2 L(o)/s in pCa(2+) 4.5 containing either 5 μM blebbistatin(+/-) or its inactive isomer (+/+). All stretches started at a sarcomere length (SL) of 2.5 μm. When fibers were activated at a pCa(2+) of 4.5, P(c) was 2.47 ± 0.11 maximal force developed before stretch (P(o)) and decreased with lower concentrations of Ca(2+). L(c) was not Ca(2+) dependent; the pooled experiments provided a L(c) of 14.34 ± 0.34 nm/half-sarcomere (HS). P(c) and L(c) did not change with velocities of stretch. Fibers activated in blebbistatin(+/-) showed a higher P(c) (2.94 ± 0.17 P(o)) and L(c) (16.30 ± 0.38 nm/HS) than control fibers (P(c) 2.31 ± 0.08 P(o); L(c) 14.05 ± 0.63 nm/HS). The results suggest that forces produced during stretch are caused by both the number of cross bridges attached to actin and the cross bridges in a pre-power-stroke state. Such cross bridges are stretched by large amplitudes before detaching from actin and contribute significantly to the force developed during stretch.