Regulation of Catch Muscle by Twitchin Phosphorylation: Effects on Force, ATPase, and Shortening

Abstract

Recent experiments on permeabilized anterior byssus retractor muscle (ABRM) of Mytilus edulis have shown that phosphorylation of twitchin releases catch force at pCa > 8 and decreases force at suprabasal but submaximum [Ca2+]. Twitchin phosphorylation decreases force with no detectable change in ATPase activity, and thus increases the energy cost of force maintenance at subsaturating [Ca2+]. Similarly, twitchin phosphorylation causes no change in unloaded shortening velocity (Vo) at any [Ca2+], but when compared at equal submaximum forces, there is a higher Vo when twitchin is phosphorylated. During calcium activation, the force-maintaining structure controlled by twitchin phosphorylation adjusts to a 30% Lo release to maintain force at the shorter length. The data suggest that during both catch and calcium-mediated submaximum contractions, twitchin phosphorylation removes a structure that maintains force with a very low ATPase, but which can slowly cycle during submaximum calcium activation. A quantitative cross-bridge model of catch is presented that is based on modifications of the Hai and Murphy (1988. Am. J. Physiol. 254:C99-C106) latch bridge model for regulation of mammalian smooth muscle.

The N-terminal Region of Twitchin Binds Thick and Thin Contractile Filaments

“…On the other hand, Shelud'ko et al (21) described significant twitchin-actin interactions that were greatly decreased when twitchin was phosphorylated. Their results supported our previous suggestion (22) that twitchin might provide a mechanical link between thick and thin filaments. The idea that catch force is maintained by a mechanical link not involving the high force myosin cross-bridge is also supported by studies showing that agents that block force output by myosin cross-bridges have no effect when added to permeabilized muscles in catch (23,24) and actually increase catch force when added to activated muscles (24).…”

“…At calcium concentrations that produce submaximal force, the mechanical link responsible for catch force maintenance can adjust during muscle shortening and subsequently maintain catch force at a shorter length (22). We have proposed that this occurs by the transition of myosin to the high force state on actin, resulting in a displacement of twitchin as a link between thick and thin filaments (24).…”

Twitchin from Molluscan Catch Muscle

“…Mechanical Measurements-ABRM were dissected, and measurements of force production were performed under isometric conditions using an apparatus that was described previously (10,13,14). Intact ABRM were incubated in artificial seawater (10) alone or with additions of acetylcholine (50 M) or serotonin (10 M).…”

“…Intact ABRM were incubated in artificial seawater (10) alone or with additions of acetylcholine (50 M) or serotonin (10 M). Muscles were permeabilized by incubation in 1% Triton X-100 (14), and the detailed composition of the rigor (no ATP), relaxing (1 mM MgATP; pCa Ͼ 8), and activating (1 mM MgATP; pCa 5) solutions are the same as those used in earlier studies (10,13,14). Muscles were frozen in liquid nitrogen while on the mechanical apparatus, pulverized in frozen 0.5 N HClO 4 , and following thawing and centrifugation, the protein precipitate was solubilized in SDS sample buffer (16).…”

“…Based on a partial cDNA of the purified isolated protein, it was found to be a homologue of the mini-titin, twitchin (11), that is associated with the thick filament (12). The mechanism by which twitchin controls force output seems to reside in the ability of unphosphorylated twitchin to slow the detachment of calcium-free myosin from actin, primarily by slowing the rate of ADP release from attached cross-bridges (13,14). In the presence of phosphorylated twitchin, crossbridge detachment is fast at low intracellular [Ca 2ϩ ] (14).…”