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

Abstract

When activated muscle fibers are stretched at low speeds [≤2 optimal length ( Lo)/s], force increases in two phases, marked by a change in slope [critical force (Pc)] that happens at a critical sarcomere length extension ( Lc). Some studies attribute Pc 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% Lo at speeds of 1.0, 2.0, and 3.0 Lo/s in different pCa2+ (4.5, 5.0, 5.5, 6.0), or 2) activated fibers were stretched by 3% Lo at 2 Lo/s in pCa2+ 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 pCa2+ of 4.5, Pc was 2.47 ± 0.11 maximal force developed before stretch (Po) and decreased with lower concentrations of Ca2+. Lc was not Ca2+ dependent; the pooled experiments provided a Lc of 14.34 ± 0.34 nm/half-sarcomere (HS). Pc and Lc did not change with velocities of stretch. Fibers activated in blebbistatin(+/−) showed a higher Pc (2.94 ± 0.17 Po) and Lc (16.30 ± 0.38 nm/HS) than control fibers (Pc 2.31 ± 0.08 Po; Lc 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.