First, we activated the preparation at the slack SL (SL 1.90 μm) with a saturating Ca2+ concentration (pCa 4.5) to secure the ends of the preparation and to determine the quality of the contractile machinery. The values of maximal Ca2+-activated force (see below) were similar to those obtained in our previous studies conducted under the same experimental condition (Fukuda et al., 2003 (link), 2005 (link)). The preparation was stretched from the slack SL (i.e., 1.90 μm) to various SLs at a constant velocity of 0.1 muscle length/s and held for 30 min (15 min for gelsolin experiments; see Fig. 5 ), followed by a release to the slack SL (SL measured by later diffraction, Granzier and Irving, 1995 (link); Wu et al., 2000 (link); Fukuda et al., 2003 (link), 2005 (link)). After 1 h, the preparation was stretched again at the same velocity to determine the reproducibility of passive force. Only when the passive force development was reproducible (<3% reduction; used as a criterion), the preparation was incubated for 50 min at 22°C with purified PKA (catalytic subunit from bovine heart; Sigma-Aldrich) at a concentration of 1 U/μl, based on our previous study (Yamasaki et al., 2002 (link)). In some experiments, we used PKA-specific inhibitor (PKI; Sigma-Aldrich) at a concentration of 50 μM. Then, the same stretch-hold protocol was repeated and stress-relaxation data were obtained.
Finally, according to previous reports (Granzier and Irving, 1995 (link); Wu et al., 2000 (link); Fukuda et al., 2003 (link), 2005 (link)), the preparation was treated with KCl/KI, and titin-based passive force was obtained as total passive force minus collagen-based (KCl/KI insensitive) passive force. We found in collagen strips (prepared from rat ventricular trabeculae with KCl/KI treatment) that passive force is unaffected by PKA treatment (not depicted). Throughout the study, to minimize contraction-induced structural damage on the preparation and to ensure high reproducibility of passive force (Fukuda et al., 2003 (link), 2005 (link)), we measured passive and active forces at 12°C.
To extract thin filaments, some rat ventricular (RV) preparations were incubated overnight at ∼4°C in relaxing solution containing gelsolin fragment FX-45 (∼1 mg/ml) during continuous agitation. Maximal Ca2+-activated force was decreased to ∼5% of the control value with no significant difference in the value of steady-state passive force (see below). To ensure high reproducibility of passive force, we tested the effect of PKA on passive force (as described above), using a shorter (i.e., 15 min) hold period.
Finally, according to previous reports (Granzier and Irving, 1995 (link); Wu et al., 2000 (link); Fukuda et al., 2003 (link), 2005 (link)), the preparation was treated with KCl/KI, and titin-based passive force was obtained as total passive force minus collagen-based (KCl/KI insensitive) passive force. We found in collagen strips (prepared from rat ventricular trabeculae with KCl/KI treatment) that passive force is unaffected by PKA treatment (not depicted). Throughout the study, to minimize contraction-induced structural damage on the preparation and to ensure high reproducibility of passive force (Fukuda et al., 2003 (link), 2005 (link)), we measured passive and active forces at 12°C.
To extract thin filaments, some rat ventricular (RV) preparations were incubated overnight at ∼4°C in relaxing solution containing gelsolin fragment FX-45 (∼1 mg/ml) during continuous agitation. Maximal Ca2+-activated force was decreased to ∼5% of the control value with no significant difference in the value of steady-state passive force (see below). To ensure high reproducibility of passive force, we tested the effect of PKA on passive force (as described above), using a shorter (i.e., 15 min) hold period.
