Effect of pH on the rate of myosin head detachment in molluscan catch muscle: are myosin heads involved in the catch state?

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 Ca2+-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.

No effect of twitchin phosphorylation on the rate of myosin head detachment in molluscan catch muscle: are myosin heads involved in the catch state?

Phosphorylation of twitchin is known to abolish the catch state of anterior byssus retractor muscle (ABRM) of the bivalve mollusc Mytilus edulis. To investigate the role of myosin head involvement in force maintenance during catch, the effect of twitchin phosphorylation on myosin head detachment was studied in saponin-skinned fibre bundles of ABRM. The detachment rate of myosin heads was deduced from two types of experiments: (1) force decay after stepwise stretch of maximally Ca2+-activated fibre bundles (pCa 4.5) and (2) force decay from high-force rigor, the former induced by a stepwise increase in ATP concentration elicited by photolysis of caged ATP (pCa<8). The rate of detachment was not affected by thiophosphorylation or phosphorylation of twitchin by 0.12 mM cAMP in the presence of the phosphatase inhibitor cyclosporine A (1 microM). Conversely, measurements of the rate of stretch-induced delayed force increase (stretch activation) and of the force increase following an ATP step in low-force rigor (pCa 4.5) suggest that the rate of myosin head attachment decreases after twitchin phosphorylation. We conclude that catch is not due to myosin heads remaining attached to actin filaments, but depends on myofilament interconnections that break down when twitchin is phosphorylated.

Effects of vanadate, phosphate and 2,3-butanedione monoxime (BDM) on skinned molluscan catch muscle.

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.