Marion J. Siegman

Ca2+ can affect Vmax without changes in myosin light chain phosphorylation in smooth muscle.

The effects of elevated [Ca2+]o on crossbridge cycling rate, measured as maximum velocity of shortening (Vmax) and high energy phosphate usage (Δ∼P), and on the degree of phosphorylation of the 20,000-dalton light chain of myosin (MyLCP) during an isometric tetanus were determined in the rabbit taenia coli at 18°C. In an normal Krebs medium (1.9 mM Ca2+) the average rate of Δ∼P during force development is 4xhigher than during force maintenance. In 4.5 mM Ca2+-Krebs, the average rate of Δ∼P increases by 100% during force development and during force maintenance above that observed in normal Krebs medium, with no significant change in force output.Vmax increased in the high Ca2+ medium, in good agreement with the corresponding rates of Δ∼P, but without any significant change in the degree of MyLCP. Also, in both high and low calcium media,Vmax decreased with tetanus duration as did the Δ∼P; however, the degree of phosphorylation was not directly related to the average rate of energy usage during the two phases of the tetanus. Therefore, in intact smooth muscles Ca2+-dependent and time-dependent changes inVmax and average rate of Δ∼P can occur without corresponding changes in MyLCP. Modulation of crossbridge cycling rate may be accomplished by a Ca2+-dependent process in addition to MyLCP.

Phosphorylation of a high molecular weight (∼600 kDa) protein regulates catch in invertebrate smooth muscle

A unique property of smooth muscle is its ability to maintain force with a very low expenditure of energy. This characteristic is highly expressed in molluscan smooth muscles, such as the anterior byssus retractor muscle (ABRM) of Mytilus edulis, during a contractile state called ‘catch’. Catch occurs following the initial activation of the muscle, and is characterized by prolonged force maintenance in the face of a low [Ca2+]i, high instantaneous stiffness, a very slow cross-bridge cycling rate, and low ATP usage. In the intact muscle, rapid relaxation (release of catch) is initiated by serotonin, and mediated by an increase in cAMP and activation of protein kinase A. We sought to determine which proteins undergo a change in phosphorylation on a time-course that corresponds to the release of catch in permeabilized ABRM. Only one protein consistently satisfied this criterion. This protein, having a molecular weight of ∼600 kDa and a molar concentration about 30 times lower than the myosin heavy chain, showed an increase in phosphorylation during the release of catch. Under the mechanical conditions studied (rest, activation, catch, and release of catch), changes in phosphorylation of all other proteins, including myosin light chains, myosin heavy chain and paramyosin, are minimal compared with the cAMP-induced phosphorylation of the ∼600 kDa protein. Under these conditions, somewhat less than one mole of phosphate is incorporated per mole of ∼600 kDa protein. Inhibition of A kinase blocked both the cAMP-induced increase in phosphorylation of the protein and the release of catch. In addition, irreversible thiophosphorylation of the protein prevented the development of catch. In intact muscle, the degree of phosphorylation of the protein increases significantly when catch is released with serotonin. In muscles pre-treated with serotonin, a net dephosphorylation of the protein occurs when the muscle is subsequently put into catch. We conclude that the phosphorylation state of the ∼600 kDa protein regulates catch

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

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 Myosin Cross-Bridge Cycle and Its Control by Twitchin Phosphorylation in Catch Muscle

The anterior byssus retractor muscle of Mytilus edulis was used to characterize the myosin cross-bridge during catch, a state of tonic force maintenance with a very low rate of energy utilization. Addition of MgATP to permeabilized muscles in high force rigor at pCa > 8 results in a rapid loss of some force followed by a very slow rate of relaxation that is characteristic of catch. The fast component is slowed 3-4-fold in the presence of 1 mM MgADP, but the distribution between the fast and slow (catch) components is not dependent on [MgADP]. Phosphorylation of twitchin results in loss of the catch component. Fewer than 4% of the myosin heads have ADP bound in rigor, and the time course (0.2-10 s) of ADP formation following release of ATP from caged ATP is similar whether or not twitchin is phosphorylated. This suggests that MgATP binding to the cross-bridge and subsequent splitting are independent of twitchin phosphorylation, but detachment occurs only if twitchin is phosphorylated. A similar dependence of detachment on twitchin phosphorylation is seen with AMP-PNP and ATPgammaS. Single turnover experiments on bound ADP suggest an increase in the rate of release of ADP from the cross-bridge when catch is released by phosphorylation of twitchin. Low [Ca(2+)] and unphosphorylated twitchin appear to cause catch by 1) markedly slowing ADP release from attached cross-bridges and 2) preventing detachment following ATP binding to the rigor cross-bridge.

Twitchin as a regulator of catch contraction in molluscan smooth muscle

Molluscan catch muscle can maintain tension for a long time with little energy consumption. This unique phenomenon is regulated by phosphorylation and dephosphorylation of twitchin, a member of the titin/connectin family. The catch state is induced by a decrease of intracellular Ca2+ after the active contraction and is terminated by the phosphorylation of twitchin by the cAMP-dependent protein kinase (PKA). Twitchin, from the well-known catch muscle, the anterior byssus retractor muscle (ABRM) of the mollusc Mytilus, incorporates three phosphates into two major sites D1 and D2, and some minor sites. Dephosphorylation is required for re-entering the catch state. Myosin, actin and twitchin are essential players in the mechanism responsible for catch during which force is maintained while myosin cross-bridge cycling is very slow. Dephosphorylation of twitchin allows it to bind to F-actin, whereas phosphorylation decreases the affinity of the two proteins. Twitchin has been also been shown to be a thick filament-binding protein. These findings raise the possibility that twitchin regulates the myosin cross-bridge cycle and force output by interacting with both actin and myosin resulting in a structure that connects thick and thin filaments in a phosphorylation-dependent manner.

Comparison of the effects of 2,3-butanedione monoxime on force production, myosin light chain phosphorylation and chemical energy usage in intact and permeabilized smooth and skeletal muscles.

SummaryThe primary goal of this study was to determine the utility of 2,3-butanedione monoxime as a tool for determining and separating the chemical energy usage associated with force production from that of force-independent, or ‘activation’ processes in smooth and skeletal muscles. We determined the effects of 2,3-butanedione monoxime on force production, myosin light chain phosphorylation and high energy phosphate usage in intact and permeabilized smooth (rabbit taenia coli) and skeletal (mouse extensor digitorum longus) muscles. In the intact taenia coli, 2,3-butanedione monoxime depressed the tonic phase of the tetanus, contractures evoked by high potassium (90 mM) and by carbachol (10-5 M) and the small force response evoked by these agonists after treatment with D-600 (10-5 M). In the electrically stimulated intact taenia coli 2,3-butanedione monoxime (0–20 mM) caused a proportional inhibition of tetanic force output, myosin light chain phosphorylation and high energy phosphate usage (ED50 ∼ 7 mM for all these parameters). At 20 mM 2,3-butanedione monoxime, force and energy usage fell to near zero and the degree of myosin light chain phosphorylation decreased to resting values, indicating a shut-down of both force-dependent and force-independent energy usage at high concentrations of 2,3-butanedione monoxime. In permeabilized taenia coli, 2,3-butanedione monoxime had little or no depressant effects on force production, ATPase activity or calcium sensitivity. 2,3-butanedione monoxime had a very modest inhibitory effect on the in vitro motility of unregulated actin filaments interacting with thiophosphorylated myosin. In solution, 2,3-butanedione monoxime inhibited myosin light chain kinase, but not the phosphatase (SMP-IV). These results suggest that the major effect of 2,3-butanedione monoxime is not on the contractile proteins themselves, but rather on calcium delivery during excitation, thereby reducing the degree of activation of myosin light chain kinase and subsequent activation of myosin by light chain phosphorylation. Thus, 2,3-butanedione monoxime is not useful for the determination of the energetics of activation processes in smooth muscle because of its inhibition of both force-dependent and force-independent processes. In contrast, in the intact mouse extensor digitorum longus, 2,3-butanedione monoxime inhibits tetanic force production (ED50 ∼ 2 mM) to a much greater extent than myosin light chain phosphorylation. When 2,3-butanedione monoxime was used to manipulate force production in muscles at L0, it was found that ∼60% of the total energy usage was force-independent and the remainder was force-dependent. In the permeabilized extensor digitorum longus treated with 12 mM 2,3-butanedione monoxime, there was a decrease in calcium-activated force production and a decrease in calcium sensitivity. The effects of 2,3-butanedione monoxime were considerably greater in the intact than in the permeabilized mouse extensor digitorum longus. At 2,3-butanedione monoxime concentrations that block force production in the intact muscle, the effects on in vitro motility were small, yet far greater than those on smooth muscle myosin. These results suggest that 2,3-butanedione monoxime has a direct effect on the contractile proteins, but what cannot be ignored is the decrease in myosin light chain phosphorylation in the skeletal muscle, which, like the decreased force output, may result from a reduction in calcium release from the sarcoplasmic reticulum. For these reasons, the use of 2,3-butanedione monoxime to probe the components of energy usage during the contraction of skeletal muscle requires considerable caution and a full definition of its actions.

Mechanism of Catch Force: Tethering of Thick and Thin Filaments by Twitchin

Catch is a mechanical state occurring in some invertebrate smooth muscles characterized by high force maintenance and resistance to stretch during extremely slow relaxation. During catch, intracellular calcium is near basal concentration and myosin crossbridge cyctng rate is extremely slow. Catch force is relaxed by a protein kinase A-mediated phosphorylation of sites near the N- and C- temini of the minititin twitchin (~526 kDa). Some catch force maintenance car also occur together with cycling myosin crossbridges at submaximal calcium concentrations, but not when the muscle is maximally activated. Additionally, the link responsible for catch can adjust during shortening of submaximally activated muscles and maintain catch force at the new shorter length. Twitchin binds to both thick and thin filaments, and the thin filament binding shown by both the N- and Cterminal portions of twitchin is decreased by phosphorylation of the sites that regulate catch. The data suggest that the twitchin molecule itself is the catch force beanng tether between thick and thin filaments. We present a model for the regulation of catch in which the twitchin tether can be displaced from thin filaments by both (a) the phosphorylation of twitchin and (b) the attachment of high force myosin crossbridges.

Energetics and regulation of crossbridge states in mammalian smooth muscle

On the basis of measurements of the high energy phosphate usage associated with different mechanical states, as well as the degree of myosin light chain phosphorylation and mechanical properties, information has been gained concerning the existence and regulation of different crossbridge states in smooth muscle. Although incomplete, a general operational scheme is shown in figure 5. At very low intracellular calcium concentrations, actin and myosin are dissociated, as shown by a loss of resistance to stretch in resting muscles. At somewhat higher intracellular calcium concentrations in atonic, resting muscles, crossbridges can attach and be manifest mechanically as an increased resistance to stretch without ATP-driven crossbridge cycling and active force production. When the muscle is activated, intracellular calcium increases further, the light chains of myosin are phosphorylated through the calcium-calmodulin activation of myosin light chain kinase, actin-activated myosin ATPase activity increases and crossbridges cycle. Calcium also appears to modulate the ATPase activity and the rate of cycling of the phosphorylated crossbridge. The crossbridge cycling rate is highest during force development and slows with time as maximum isometric force is maintained reflecting a change in the rate at which phosphorylated crossbridges cycle. This may result from a decrease in the intracellular free calcium concentration with continued stimulation. During relaxation, the intracellular calcium concentration decreases, there is net dephosphorylation of the myosin light chains, the rate at which phosphorylated crossbridges cycle slows further with a gradual return to the attached, but non-cycling state or the detached state.

The N-terminal Region of Twitchin Binds Thick and Thin Contractile Filaments: REDUNDANT MECHANISMS OF CATCH FORCE MAINTENANCE*

Catch force maintenance in invertebrate smooth muscles is probably mediated by a force-bearing tether other than myosin cross-bridges between thick and thin filaments. The phosphorylation state of the mini-titin twitchin controls catch. The C-terminal phosphorylation site (D2) of twitchin with its flanking Ig domains forms a phosphorylation-sensitive complex with actin and myosin, suggesting that twitchin is the tether (Funabara, D., Osawa, R., Ueda, M., Kanoh, S., Hartshorne, D. J., and Watabe, S. (2009) J. Biol. Chem. 284, 18015–18020). Here we show that a region near the N terminus of twitchin also interacts with thick and thin filaments from Mytilus anterior byssus retractor muscles. Both a recombinant protein, including the D1 and DX phosphorylation sites with flanking 7th and 8th Ig domains, and a protein containing just the linker region bind to thin filaments with about a 1:1 mol ratio to actin and Kd values of 1 and 15 μm, respectively. Both proteins show a decrease in binding when phosphorylated. The unphosphorylated proteins increase force in partially activated permeabilized muscles, suggesting that they are sufficient to tether thick and thin filaments. There are two sites of thin filament interaction in this region because both a 52-residue peptide surrounding the DX site and a 47-residue peptide surrounding the D1 site show phosphorylation-dependent binding to thin filaments. The peptides relax catch force, confirming the regions central role in the mechanism of catch. The multiple sites of thin filament interaction in the N terminus of twitchin in addition to those in the C terminus provide an especially secure and redundant mechanical link between thick and thin filaments in catch.

Chemical energy usage during stimulation and stretch of mammalian smooth muscle.

High energy phosphate usage was measured in the rabbit taenia coli subjected to stimulation and stretch (0.12 L0/min) and was compared to that observed previously under isometric conditions. When the muscle was bathed in a medium containing 1.9 mM Ca2+, stretch during the period of initial force development substantially decreased the rate of chemical energy usage compared to that under isometric conditions. When crossbridge cycling rate under isometric conditions was increased by incubation of the muscle in a medium containing 4.5 mM Ca2+, there was a greater decrease in rate of high energy phosphate usage during stretch compared to isometric conditions. The low energy usage during stretch occurs even though average active force output was approximately 40% higher than that under isometric conditions. During the period of subsequent force maintenance when both energy usage and crossbridge cycling rate under isometric conditions were low, there was no significant effect of stretch on the average rate of energy usage at either Ca2+ level. These results are consistent with the hypothesis that during stimulation and stretch in smooth muscle, crossbridge attachment and force production can occur even though the actin-activated myosin ATPase activity normally associated with isometric force development is greatly suppressed.