The effects of orthovanadate (V(i)), inorganic phosphate (P(i)) and 2,3-butanedione monoxime (BDM) on tension, force transients and the catch state (passive tension maintenance) were investigated in saponin-skinned fibre bundles of the anterior byssus retractor muscle (ABRM) of the bivalve mollusc Mytilus edulis at pH 6.7. During maximal Ca(2+) activation isometric force was depressed by V(i) (0.03-10 mM), P(i) (10 mM) and BDM (50 mM). Force transients following quick stretches (0.1-0.3% of fibre length) were accelerated substantially by 1 mM V(i), 10 mM P(i) or 50 mM BDM. These compounds also accelerated force responses in experiments in which ATP was released rapidly from caged ATP by flash photolysis at both pCa 4.7 (force rise) and at pCa>8 (force decline). The effects on the catch state were investigated in two types of experiments: (1) Ca(2+) removal after maximal Ca(2+) activation and (2) rapid ATP release during high-force rigor at pCa>8. In both cases rapid relaxation was followed by slow relaxation (slower than 2% of initial force per min). This later slow relaxation (catch) was insensitive to V(i) (1-10 mM), P(i) (10 mM) and BDM (50 mM) but was accelerated by 0.12 mM cAMP. Complete relaxation to almost zero force was attained by changing pH from 6.7 to 7.7 (pCa>8). We conclude that catch depends on cAMP- and pH-sensitive structures linking the myofilaments and not on the force-generating actomyosin cross-bridges that are sensitive to V(i), P(i) and BDM.

Galler. Kinetic properties of myosin heavy chain isoforms in mouse skeletal muscle: comparison with rat, rabbit, and human and correlation with amino acid sequence. Am J Physiol Cell Physiol 287: C1725-C1732, 2004. First published August 11, 2004 doi:10.1152/ ajpcell.00255.2004.-Stretch activation kinetics were investigated in skinned mouse skeletal muscle fibers of known myosin heavy chain (MHC) isoform content to assess kinetic properties of different myosin heads while generating force. The time to peak of stretchinduced delayed force increase (t3) was strongly correlated with MHC isoforms [t3 given in ms for fiber types containing specified isoforms; means Ϯ SD with n in parentheses: MHCI 680 Ϯ 108 (13) For rat, rabbit, and human skeletal muscles the same type of correlation was found previously. The kinetics decreases slightly with increasing body mass. Available amino acid sequences were aligned to quantify the structural variability of MHC isoforms of different animal species. The variation in t3 showed a correlation with the structural variability of specific actin-binding loops (so-called loop 2 and loop 3) of myosin heads (r ϭ 0.74). This suggests that alterations of amino acids in these loops contribute to the different kinetics of myosin heads of various MHC isoforms.isoform structure-function relationship; stretch activation; muscle mechanics SKELETAL MUSCLES ARE COMPOSED of different fiber types to fulfill various functional needs. This diversity relates to the existence of specific myofibrillar protein isoforms in different fiber types (for review, see Refs. 41 and 46). Muscle fiber types are generally categorized according to their specific myosin heavy chain (MHC) isoforms. The head portion of this protein is the essential component of the force-generating mechanism of muscle (for review, see Ref. 30). Three fast fiber types (type II) have been identified in limb muscles of adult rodents: types IIB, IIX (or IID), and IIA, containing the isoforms MHCIIb, MHCIIx(d), and MHCIIa, respectively. Conversely, only one slow fiber type (type I) has to date been characterized by its MHC isoform, called MHCI, but there is increasing evidence that the type I fibers do not represent a homogeneous population (2,11,15).MHC isoform content determines the contractile properties of muscle fibers (for review see Ref. 4). The relation between MHC content, stretch activation kinetics, and unloaded shortening velocity (V u ) has been clearly established for rat, rabbit, and human skeletal muscle fibers (4,7,9,17,35). Although the mouse is the most important mammalian model for genetic manipulations, the skeletal muscle fibers of this species have been investigated to a much lesser degree. The relationship between shortening velocity and load has been determined in different mouse muscle fiber types. Furthermore, V u was compared with the actin filament sliding propelled by the different myosin isoforms (28, 39). The kinetics of stretch activation has never been investigated in mouse skeletal muscle fibers, although a...

Contractile properties of skinned muscle fibres from the masseter muscle and strips of heart atrium muscle from rabbits, both containing the alpha-cardiac myosin heavy chain isoform (alpha-cardiac MHC), were investigated and compared with those of other skeletal muscle fibre types. The stretch-induced delayed force increase (stretch activation) was investigated on maximally Ca(2+)-activated skinned preparations as an index of the kinetic properties of the myosin heads of various MHC isoforms. Skeletal muscle fibres containing exclusively alpha-cardiac MHC (type alpha) and muscle strips of heart atrium showed specific kinetics of stretch activation intermediate between those of types IIA and I fibres. In agreement with available data the unloaded shortening velocity V(u) of type alpha fibres was also intermediate between that of types IIA and I fibres. Compared with skeletal muscle type alpha fibres, muscle strips of heart atrium exhibited significantly (P<0.001) faster kinetics of both stretch activation and V(u). We conclude that type alpha fibres have characteristic kinetic properties that fill the gap between the fast type II and the slow type I fibres, and the following order of velocity can be established: IIB>IID(X)>IIA>alpha>I.

Cross-bridge kinetics underlying stretch-induced force transients was studied in fibres with different myosin light chain (MLC) isoforms from skeletal muscles of rabbit and rat. The force transients were induced by stepwise stretches (< 0.3% of fibre length) applied on maximally Ca 2+ -activated skinned fibres. Fast fibre types IIB, IID (or IIX) and IIA and the slow fibre type I containing the myosin heavy chain isoforms MHC-IIb, MHC-IId (or MHC-IIx), MHC-IIa and MHC-I, respectively, were investigated. The MLC isoform content varied within fibre types. Fast fibre types contained the fast regulatory MLC isoform MLC2f and different proportions of the fast alkali MLC isoforms MLC1f and MLC3f. Type I fibres contained the slow regulatory MLC isoform MLC2s and the slow alkali MLC isoform MLC1s. Slow MLC isoforms were also present in several type IIA fibres. The kinetics of force transients differed by a factor of about 30 between fibre types (order from fastest to slowest kinetics: IIB > IID > IIA I). The kinetics of the force transients was not dependent on the relative content of MLC1f and MLC3f. Type IIA fibres containing fast and slow MLC isoforms were about 1.2 times slower than type IIA fibres containing only fast MLC isoforms. We conclude that while the cross-bridge kinetics is mainly determined by the MHC isoforms present, it is affected by fast and slow MLC isoforms but not by the relative content of MLC1f and MLC3f. Thus, the physiological role of fast and slow MLC isoforms in type IIA fibres is a fine-tuning of the cross-bridge kinetics.

Moderate alkalisation is known to terminate the catch state of bivalve mollusc smooth muscles such as the anterior byssus retractor muscle (ABRM) of Mytilus edulis L. In the present study, we investigated the effect of moderate alkalisation (pH 7.2-7.7 vs control pH 6.7) on the myosin head detachment rate in saponin-skinned fibre bundles of ABRM in order to investigate the possible role of myosin heads in the force maintenance during catch. The detachment rate of myosin heads was deduced from two types of experiments. (1) In stretch experiments on maximally Ca 2+ -activated fibre bundles (pCa 4.5), the rate of force decay after stepwise stretch was assessed. (2) In ATP step experiments, the rate of force decay from high force rigor (pCa>8) was evaluated. The ATP step was induced by photolysis of caged ATP. We found that moderate alkalisation induces relaxation of skinned fibres in catch, thereby reducing both force and stiffness, whereas it does not accelerate the rate of myosin head detachment. This acceleration, however, would be expected if catch would be simply due to myosin heads remaining sustainably attached to actin filaments. Thus, the myosin heads may be less involved in catch than generally assumed. Catch may possibly depend on a different kind of myofilament interconnections, which are abolished by moderate alkalisation.