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

Minozzo FC, Rassier DE. Effects of blebbistatin and Ca2+ concentration on force produced during stretch of skeletal muscle fibers. Am J Physiol Cell Physiol 299: C1127-C1135, 2010. First published August 18, 2010; doi:10.1152/ajpcell.00073.2010.-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 mu M blebbistatin(+/-) or its inactive isomer (+/+). All stretches started at a sarcomere length (SL) of 2.5 mu m. 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 Ca2+. L-c was not Ca2+ 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.