Myosin assembly of smooth muscle: from ribbons and side polarity to a row polar helical model.

After decades of debate over the structure of smooth muscle myosin filaments, it is still unclear whether they are helical, as in all other muscle types, or square in shape. In both cases bipolar building units are proposed, but the deduced cross-bridge arrangements are fundamentally different. The opposite polarity of the adjusting longitudinal rows is proposed for the helical structure, while in the case of square filaments, or myosin ribbons, only their two faces are appositively polarized. Analysis of our unpublished archival data on light meromyosin (LMM) paracrystals and myosin rod assemblies as well as the filaments themselves indicated that the rods were assembled with a 6°-7° tilt angle from the rods' longitudinal axis, in contrast to the lack of tilt in LMM, both exhibiting a 14.3 nm myosin periodicity. Optical diffraction analysis of EM images of the rod assemblies and those of intact myosin confirmed their helical architecture characterized by 28 nm residue translations, 172 nm repeats and 516 nm pitch. A detailed helical model of these filaments was elucidated with bipolar tetramer building units made of two polar trimers. The filaments elongate at their two ends in a head-to-head manner, enabling targeted cross-bridge polarity of the adjacent rows, in the form of a unique Boerdijk-Coxeter type helix, similar to that of collagen or desmin fibers, with the covalent links replaced by a head-to-head clasp.

Self-assembly of smooth muscle myosin filaments: adaptation of filament length by telokin and Mg·ATP.

The contractile apparatus of smooth muscle is malleable to accommodate stress and strain exerted on the muscle cell and to maintain optimal contractility. Structural lability of smooth muscle myosin filaments is believed to play an important role in the cell's malleability. However, the mechanism and regulation of myosin filament formation is still poorly understood. In the present in vitro study, using a static light scattering method, length distributions were obtained from suspensions of short myosin filaments (SFs) formed by rapid dilution or long ones (LFs) formed by slow dialysis. The distributions indicated the presence of dynamic equilibriums between soluble myosin and the SFs; i.e.: trimers, hexamers and mini filaments, covering the range up to 0.75 µm. The LFs were more stable, exhibiting favorable sizes at about 1.25, 2.4 and 4.5 µm. More distinct distributions were obtained from filaments adsorbed to a glass surface, by evanescent wave scattering and local electric field enhancement. Addition of telokin (TL) to the suspensions of unphosphorylated SFs resulted in widening of the soluble range, while in the case of the LFs this shift was larger, and accompanied by reduced contribution of the soluble myosin species. Such changes were largely absent in the case of phosphorylated myosin. In contrast, the presence of Mg·ATP resulted in elongation of the filaments and clear separation of filaments from soluble myosin species. Thus, TL and Mg·ATP appeared to modify the distribution of myosin filament lengths, i.e., increasing the lengths in preparing for phosphorylation, or reducing it to aid dephosphorylation.

Helical model of smooth muscle myosin filament and the ribbons made of caldesmon: history revisited.

In early studies on smooth muscle, I described a crude myosin fraction (CMF) in which self-assembly of myosin filaments was observed. For the first time, the 14-nm periodicity stemming from regular arrangement of myosin heads on the filament surface was observed (Sobieszek in J Mol Biol 70:741-744, 1972). In this fraction, we also observed formation of long ribbon-shaped aggregates exhibiting a 5.6-nm periodicity, characteristic of tropomyosin (TM) paracrystals (Sobieszek and Small in Phil Trans R Soc Lond B 265:203-212, 1973). We therefore concluded that these ribbons were made of TM and they might be related to the myosin ribbons observed in electron micrographs (EM) of intact smooth muscle (Lowy and Small in Nature 227:46-51, 1970; Small and Squire in Mol Biol 67:117-149, 1972). Subsequently, Small (J Cell Sci 24:327-349, 1977) concluded that the ribbons observed in the EM sections were an artifact, but their observation in the CMF was not addressed. I have now revisited two aspects of the above studies. Firstly, based on my new multi-angle laser-scattering data and considering the length and stability of the building unit for the filament, a myosin trimer fit better to the previously proposed helical structure. Secondly, after two decades of systematic examinations of protein compositions in multiple smooth muscle extracts and isolated filaments, I concluded that the ribbons were made of caldesmon and not TM. Thirdly, actin-activated ATPase activity measurements indicated that modulation of this activity (by CaD and TM) was synergistic, cooperative and depended on myosin to actin ratio.

The role of caldesmon and its phosphorylation by ERK on the binding force of unphosphorylated myosin to actin.

BACKGROUND: Studies conducted at the whole muscle level have shown that smooth muscle can maintain tension with low Adenosine triphosphate (ATP) consumption. Whereas it is generally accepted that this property (latch-state) is a consequence of the dephosphorylation of myosin during its attachment to actin, free dephosphorylated myosin can also bind to actin and contribute to force maintenance. We investigated the role of caldesmon (CaD) in regulating the binding force of unphosphorylated tonic smooth muscle myosin to actin. METHODS: To measure the effect of CaD on the binding of unphosphorylated myosin to actin (in the presence of ATP), we used a single beam laser trap assay to quantify the average unbinding force (Funb) in the absence or presence of caldesmon, extracellular signal-regulated kinase (ERK)-phosphorylated CaD, or CaD plus tropomyosin. RESULTS: Funb from unregulated actin (0.10±0.01pN) was significantly increased in the presence of CaD (0.17±0.02pN), tropomyosin (0.17±0.02pN) or both regulatory proteins (0.18±0.02pN). ERK phosphorylation of CaD significantly reduced the Funb (0.06±0.01pN). Inspection of the traces of the Funb as a function of time suggests that ERK phosphorylation of CaD decreases the binding force of myosin to actin or accelerates its detachment. CONCLUSIONS: CaD enhances the binding force of unphosphorylated myosin to actin potentially contributing to the latch-state. ERK phosphorylation of CaD decreases this binding force to very low levels. GENERAL SIGNIFICANCE: This study suggests a mechanism that likely contributes to the latch-state and that explains the muscle relaxation from the latch-state.

Unphosphorylated calponin enhances the binding force of unphosphorylated myosin to actin.

BACKGROUND: Smooth muscle has the distinctive ability to maintain force for long periods of time and at low energy costs. While it is generally agreed that this property, called the latch-state, is due to the dephosphorylation of myosin while attached to actin, dephosphorylated-detached myosin can also attach to actin and may contribute to force maintenance. Thus, we investigated the role of calponin in regulating and enhancing the binding force of unphosphorylated tonic muscle myosin to actin. METHODS: To measure the effect of calponin on the binding of unphosphorylated myosin to actin, we used the laser trap assay to quantify the average force of unbinding (Funb) in the absence and presence of calponin or phosphorylated calponin. RESULTS: Funb from F-actin alone (0.12±0.01pN; mean±SE) was significantly increased in the presence of calponin (0.20±0.02pN). This enhancement was lost when calponin was phosphorylated (0.12±0.01pN). To further verify that this enhancement of Funb was due to the cross-linking of actin to myosin by calponin, we repeated the measurements at high ionic strength. Indeed, the Funb obtained at a [KCl] of 25mM (0.21±0.02pN; mean±SE) was significantly decreased at a [KCl] of 150mM, (0.13±0.01pN). CONCLUSIONS: This study provides direct molecular level-evidence that calponin enhances the binding force of unphosphorylated myosin to actin by cross-linking them and that this is reversed upon calponin phosphorylation. Thus, calponin might play an important role in the latch-state. GENERAL SIGNIFICANCE: This study suggests a new mechanism that likely contributes to the latch-state, a fundamental and important property of smooth muscle that remains unresolved.

Molecular mechanical differences between isoforms of contractile actin in the presence of isoforms of smooth muscle tropomyosin.

The proteins involved in smooth muscle's molecular contractile mechanism - the anti-parallel motion of actin and myosin filaments driven by myosin heads interacting with actin - are found as different isoforms. While their expression levels are altered in disease states, their relevance to the mechanical interaction of myosin with actin is not sufficiently understood. Here, we analyzed in vitro actin filament propulsion by smooth muscle myosin for [Formula: see text]-actin ([Formula: see text]A), [Formula: see text]-actin-tropomyosin-[Formula: see text] ([Formula: see text]A-Tm[Formula: see text]), [Formula: see text]-actin-tropomyosin-[Formula: see text] ([Formula: see text]A-Tm[Formula: see text]), [Formula: see text]-actin ([Formula: see text]A), [Formula: see text]-actin-tropomyosin-[Formula: see text] ([Formula: see text]A-Tm[Formula: see text]), and [Formula: see text]-actin-tropomoysin-[Formula: see text] ([Formula: see text]A-Tm[Formula: see text]). Actin sliding analysis with our specifically developed video analysis software followed by statistical assessment (Bootstrapped Principal Component Analysis) indicated that the in vitro motility of [Formula: see text]A, [Formula: see text]A, and [Formula: see text]A-Tm[Formula: see text] is not distinguishable. Compared to these three 'baseline conditions', statistically significant differences ([Formula: see text]) were: [Formula: see text]A-Tm[Formula: see text] - actin sliding velocity increased 1.12-fold, [Formula: see text]A-Tm[Formula: see text] - motile fraction decreased to 0.96-fold, stop time elevated 1.6-fold, [Formula: see text]A-Tm[Formula: see text] - run time elevated 1.7-fold. We constructed a mathematical model, simulated actin sliding data, and adjusted the kinetic parameters so as to mimic the experimentally observed differences: [Formula: see text]A-Tm[Formula: see text] - myosin binding to actin, the main, and the secondary myosin power stroke are accelerated, [Formula: see text]A-Tm[Formula: see text] - mechanical coupling between myosins is stronger, [Formula: see text]A-Tm[Formula: see text] - the secondary power stroke is decelerated and mechanical coupling between myosins is weaker. In summary, our results explain the different regulatory effects that specific combinations of actin and smooth muscle tropomyosin have on smooth muscle actin-myosin interaction kinetics.

Purification of Myosin from Bovine Tracheal Smooth Muscle, Filament Formation and Endogenous Association of Its Regulatory Complex.

Dynamic regulation of myosin filaments is a crucial factor in the ability of airway smooth muscle (ASM) to adapt to a wide length range. Increased stability or robustness of myosin filaments may play a role in the pathophysiology of asthmatic airways. Biochemical techniques for the purification of myosin and associated regulatory proteins could help elucidate potential alterations in myosin filament properties of asthmatic ASM. An effective myosin purification approach was originally developed for chicken gizzard smooth muscle myosin. More recently, we successfully adapted the procedure to bovine tracheal smooth muscle. This method yields purified myosin with or without the endogenous regulatory complex of myosin light chain kinase and myosin light chain phosphatase. The tight association of the regulatory complex with the assembled myosin filaments can be valuable in functional experiments. The purification protocol discussed here allows for enzymatic comparisons of myosin regulatory proteins. Furthermore, we detail the methodology for quantification and removal of the co-purified regulatory enzymes as a tool for exploring potentially altered phenotypes of the contractile apparatus in diseases such as asthma.

Catch muscle of bivalve molluscs contains myosin- and twitchin-associated protein kinase phosphorylating myorod.

We have shown previously that myorod, a molluscan thick filament protein of unknown function, is phosphorylated by vertebrate smooth myosin light chain kinase (MLCK) in N-terminal unique region. The aim of the present study was to clarify whether such phosphorylation may occur in molluscan muscles. We detected three kinases endogenous to molluscan catch muscle, namely, to the complex of surface thick filament proteins that consists of twitchin, myosin, and myorod. The first kinase was a protein kinase A because it was inhibited by a specific inhibitor; the second one was associated with twitchin and phosphorylated myorod at its N-terminal unique region independently of Ca(2+); and the third kinase was bound to myosin and phosphorylated myorod as well as myosin in the C-terminal part of both proteins. The myosin-associated kinase was inhibited by micromolar concentration of calcium ions. This enzyme could be separated from myosin by chromatography, whereas the kinase associated with twitchin could not be separated from twitchin. Since twitchin has a MLCK-like domain, it is possible that this domain was responsible for myorod phosphorylation. Phosphorylation of myorod within the twitchin-myosin-myorod complex increased the actin-activated Mg(2+)-ATPase activity of myosin. Taken together, these results indicate that phosphorylation of myorod by kinases associated with key proteins of catch contraction may contribute to the functional activity of myorod in molluscan smooth muscle.

Myosin kinase of molluscan smooth muscle. Regulation by binding of calcium to the substrate and inhibition of myorod and twitchin phosphorylation by myosin.

Major contractile proteins were purified from relaxed actomyosin extracted from molluscan catch muscle myofibrils using ammonium sulfate fractionation and divalent cation precipitation. A fraction of this actomyosin was precipitated and purified as a supramolecular complex composed of twitchin (TW), myosin (MY), and myorod (MR). Another TW-MR complex was obtained via the removal of myosin. These supramolecular complexes and filaments assembled from purified myosin contained an endogenous protein kinase that phosphorylated myosin and myorod. Significantly, the activity of this novel myosin-associated (MA) kinase was inhibited at calcium concentrations of >0.1 microM. After partial purification of the kinase, we established that the inhibition resulted from binding of calcium to the substrate (myosin) and not from the binding to the enzyme (kinase). No inhibition was observed when myorod was used as a substrate, although the latter is identical to the rod portion of myosin lacking the head domains. Phosphorylation sites of myorod were identified, three at the C-terminal tip and three at the N-terminal domain. In the presence of calcium, addition of myosin to the TW-MR complex resulted in inhibition of this phosphorylation, while in the absence of myosin, this inhibition was negligible. Added myosin also inhibited phosphorylation of twitchin by PKA-like kinase, the latter also present in the complex. The opposite was true with the TW-MY-MR complex; that is, phosphorylation of myosin was inhibited by twitchin and/or myorod. Thus, in parallel to the well-established direct activation by calcium, molluscan catch muscle myosin also regulated its own phosphorylation. Therefore, in addition to the established phosphorylation of twitchin by PKA-like kinase, phosphorylation of myosin and myorod by myosin-associated kinase appears to play an important role in the development of the catch state.

Effect of actin C-terminal modification on tropomyosin isoforms binding and thin filament regulation.

Tropomyosins, a family of actin-binding regulatory proteins, are present in muscle and non-muscle cells. Multiple tropomyosin (TM) isoforms differ in actin affinity and regulatory properties, but little is known about the molecular bases of these differences. The C-terminus of actin stabilizes contacts between actin subunits in the filament and interacts with myosin and regulatory proteins. The goal of this work was to reveal how structural changes in actin and differences between TM isoforms affect binding between these proteins and affect thin filament regulation. Actin proteolytically truncated by three C-terminal amino acids exhibited 1.2-1.5 fold reduced affinity for non-muscle and smooth muscle tropomyosin isoforms. The truncation increased the cooperativity of myosin S1-induced tropomyosin binding for short tropomyosins (TM5a and TM1b9a), but it was neutral for long isoforms (smTM and TM2). Actin modification affected regulation of actomyosin ATPase activity in the presence of all tropomyosins by shifting the filament into a more active state. We conclude that the integrity of the actin C-terminus is important for actin-tropomyosin interactions, however the increased affinity of tropomyosin binding in the S1-induced state of the filament appears not to be involved in the tropomyosin isoform-dependent mechanism of the actomyosin ATPase activation.

Phosphorylation of caldesmon by myosin light chain kinase increases its binding affinity for phosphorylated myosin filaments.

Phosphorylation of myosin by myosin light chain kinase (MLCK) is essential for smooth muscle contraction. In this study we show that caldesmon (CaD) is also phosphorylated in vitro by MLCK. The phosphorylation is calcium- and calmodulin (CaM)-dependent and requires a MLCK concentration close to that found in vivo. On average, approximately 2 mol P(i) per mol of CaD are incorporated at Thr-626 and Thr-693, with additional partial phosphorylation at Ser-658 and Ser-702. The phosphorylation rate for CaD is 20- to 50-fold slower than that for filamentous myosin; faster relative rates were obtained with CaD added to purified actomyosin or myosin preparations containing endogenous MLCK/CaM complex. Addition of CaM also augmented CaD phosphorylation. We further demonstrate that [³²P] labeled CaD binds much more readily to phosphorylated filamentous myosin than to unphosphorylated myosin. For actomyosin, CaD binding affinity doubles after myosin phosphorylation, without a significant change in binding stoichiometry (approx. one CaD per myosin molecule). Unphosphorylated CaD is ineffective in competing with the phosphorylated protein for the binding site(s) on myosin filaments. The ATPase activity of reconstituted actomyosin is inhibited by unphosphorylated CaD, and this inhibition was removed by CaD phosphorylation. Our results suggest that CaD phosphorylation plays a role in modifying actomyosin interaction in vivo, particularly during prolonged muscle activation.

Modulation of myosin filament activation by telokin in smooth muscle liberation of myosin kinase and phosphatase from supramolecular complexes.

The mechanism of telokin action on reversible phosphorylation of turkey gizzard myosin was investigated using a native-like filamentous myosin. This myosin contained endogenous calmodulin (CaM) and myosin light chain kinase (MLCK) at a molar ratio to myosin of about 1 to 40 or less depending on the initial extractions conditions. These levels were sufficient to fully phosphorylate myosin within 20-40 s or less after addition of [gamma-32P]ATP, but when the ATP was depleted, they became dephosphorylated indicating the presence of myosin light chain phosphatase (MLCP). Addition of telokin at the 1 to 1 or higher molar ratio to myosin caused a three- to five-fold inhibition of the initial phosphorylation rates (without reduction of the overall extent of phosphorylation) and produced a similar increase in the rate of dephosphorylation. The inhibition was also observed for myosin filaments free of MLCK and CaM together with constitutively active MLCKs produced by digestion, or by expression of a truncated mammalian kinase as well as for the wild-type enzyme. Thus, neither N- nor C-terminal of MLCK was necessary for interaction of myosin with telokin and the inhibition resulted from telokin-induced change of myosin head configuration within the filament that prevented their ordered, paracrystaline-like, aggregation. Sedimentation of the filamentous myosin in glycerol gradients showed that this change made the filaments less compact and facilitated release of the endogenous MLCK/CaM complex. For a mixture of the filaments with or without the complex, the configuration change resulted in an increase of the phosphorylation rate but not in its inhibition. The increase of the rate resulting from the liberation of the complex was also observed in mixtures of the filamentous myosin with added isolated regulatory light chain (ReLC) or soluble myosin head subfragment. This observation reinforces the above conclusions. The acceleration of the MLCP activity by telokin was shown to result from dissociation of its catalytic subunit from a MLCK/MLCP complex bound to the filamentous myosin. Analogous desensitizing effects of telokin were also demonstrated for the contraction and relaxation cycle of Triton-skinned fibers from guinea pig Teania coli. Taken together, our results indicate that telokin acted as an effective modulator or chaperone of the myosin filament and a scheme for its action in smooth muscle was proposed.

Catch muscle myorod modulates ATPase activity of Myosin in a phosphorylation-dependent way.

Myorod is expressed exclusively in molluscan catch muscle and localizes on the surface of thick filaments together with twitchin and myosin. Myorod is an alternatively spliced product of the myosin heavy-chain gene that contains the C-terminal rod part of myosin and a unique N-terminal domain. The unique domain is a target for phosphorylation by gizzard smooth myosin light chain kinase (smMLCK) and, perhaps, molluscan twitchin, which contains a MLCK-like domain. To elucidate the role of myorod and its phosphorylation in the catch muscle, the effect of chromatographically purified myorod on the actin-activated Mg2+-ATPase activity of myosin was studied. We found that phosphorylation at the N-terminus of myorod potentiated the actin-activated Mg2+-ATPase activity of mussel and rabbit myosins. This potentiation occurred only if myorod was phosphorylated and introduced into the ATPase assay as a co-filament with myosin. We suggest that myorod could be related to the catch state, a function specific to molluscan muscle.

Length adaptation of airway smooth muscle.

Many types of smooth muscle, including airway smooth muscle (ASM), are capable of generating maximal force over a large length range due to length adaptation, which is a relatively rapid process in which smooth muscle regains contractility after experiencing a force decrease induced by length fluctuation. Although the underlying mechanism is unclear, it is believed that structural malleability of smooth muscle cells is essential for the adaptation to occur. The process is triggered by strain on the cell cytoskeleton that results in a series of yet undefined biochemical and biophysical events leading to restructuring of the cytoskeleton and contractile apparatus and consequently optimization of the overlap between the myosin and actin filaments. Although length adaptability is an intrinsic property of smooth muscle, maladaptation of ASM could result in excessive constriction of the airways and the inability of deep inspirations to dilate them. In this article, we describe the phenomenon of length adaptation in ASM and some possible underlying mechanisms that involve the myosin filament assembly and disassembly. We discuss a possible role of maladaptation of ASM in the pathogenesis of asthma. We believe that length adaptation in ASM is mediated by specific proteins and their posttranslational regulations involving covalent modifications, such as phosphorylation. The discovery of these molecules and the processes that regulate their activity will greatly enhance our understanding of the basic mechanisms of ASM contraction and will suggest molecular targets to alleviate asthma exacerbation related to excessive constriction of the airways.

Physical integrity of smooth muscle myosin filaments is enhanced by phosphorylation of the regulatory myosin light chain.

BACKGROUND AND AIMS: Smooth muscle myosin monomers self-assemble in solution to form filaments. Phosphorylation of the 20-kD regulatory myosin light chain (MLC20) enhances filament formation. It is not known whether the phosphorylated and non-phosphorylated filaments possess the same structural integrity. METHODS: We purified myosin from bovine trachealis to form filaments, in ATP-containing zero-calcium solution during a slow dialysis that gradually reduced the ionic strength. Sufficient myosin light chain kinase and phosphatase, as well as calmodulin, were retained after the myosin purification and this enabled phosphorylation of MLC20 within 20-40s after addition of calcium to the filament suspension. The phosphorylated and non-phosphorylated filaments were then partially disassembled by ultrasonification. The extent of filament disintegration was visualized and quantified by atomic force microscopy. RESULTS: MLC20 phosphorylation reduced the diameter of the filaments and rendered the filaments more resistant to ultrasonic agitation. Electron microscopy revealed a similar reduction in filament diameter in intact smooth muscle when the cells were activated. CONCLUSION: Modification of the structural and physical properties of myosin filaments by MLC20 phosphorylation may be a key regulation step in smooth muscle where formation and dissolution of the filaments are required in the cells' adaptation to different cell length.

Phosphorylation of myorod (catchin) by kinases tightly associated to molluscan and vertebrate smooth muscle myosins.

Myorod, also known as catchin, a newly discovered component of molluscan smooth muscle thick filaments, is an alternative product of the myosin heavy chain gene. It contains a C-terminal rod part that is identical to that part of myosin and a unique N-terminal domain that is very small relative to the myosin head domain. The role of myorod in contraction or relaxation of this muscle type is unknown. In the present study we demonstrated that myorod was phosphorylated not only by a kinase endogenous to molluscan myosin and twitchin but also to vertebrate smooth muscle myosin light chain kinase (MLCK). The rates and maximal levels of phosphorylation were up to threefold higher than those observed by protein kinase A with clear optima at the physiological salt concentrations. Using a mild digestion with chymotrypsin we isolated an 11 kDa phosphopeptide and showed that the phosphorylation site was located at the N-terminal domain of myorod at Thr 141 position. The sequence around this site exhibited a high degree of similarity to that expected for the substrate recognition site of MLCK. The phosphorylation rates strongly depended on the ionic conditions indicating that this site could be readily sterically blocked during myorod polymerization. Another component of the thick filaments involved in regulation of the catch state, twitchin, was phosphorylated by MLCK and exhibited endogenous myorod kinase and MLCK activities. A possible role of these phosphorylation reactions in the regulation of molluscan smooth muscles is discussed.

Vectorial activation of smooth muscle myosin filaments and its modulation by telokin.

Smooth muscle myosin copurifies with myosin light chain kinase (MLCK) and calmodulin (CaM) as well as with variable amounts of myosin phosphatase. Therefore, myosin filaments formed in vitro also contain relatively high levels of these enzymes. Thus these filaments may be considered to be native-like because they are similar to those expected to exist in vivo. These endogenous enzymes are present at high concentrations relative to myosin, sufficient for rapid phosphorylation and dephosphorylation of the filaments at rates comparable to those observed for contraction and relaxation in intact muscle strips. The phosphorylation by MLCK/CaM complex appears to exhibit some directionality and is not governed by a random diffusional process. For the mixtures of myosin filaments with and without the endogenous MLCK/CaM complex, the complex preferentially phosphorylates its own parent filament at a higher rate than the neighboring filaments. This selective or vectorial-like activation is lost or absent when myosin filaments are dissolved at high ionic strength. Similar vectorial-like activation is exhibited by the reconstituted filament suspensions, but the soluble systems composed of isolated regulatory light chain or soluble myosin head subfragments exhibit normal diffusional kinetic behavior. At physiological concentrations, kinase related protein (telokin) effectively modulates the activation process by reducing the phosphorylation rate of the filaments without affecting the overall phosphorylation level. This results from telokin-induced liberation of the active MLCK/CaM complex from the filaments, so that the latter can also activate the neighboring filaments via a slower diffusional process. When this complex is bound at insufficient levels, this actually results in acceleration of the initial phosphorylation rates. In short, I suggest that in smooth muscle, telokin plays a chaperone role for myosin and its filaments.

(+)Insert smooth muscle myosin heavy chain (SM-B) isoform expression in human tissues.

Two smooth muscle myosin heavy chain isoforms differ in their amino terminus by the presence [(+)insert] or absence [(-)insert] of a seven-amino acid insert. Animal studies show that the (+)insert isoform is predominantly expressed in rapidly contracting phasic muscle and the (-)insert isoform is mostly found in slowly contracting tonic muscle. The expression of the (+)insert isoform has never been demonstrated in human smooth muscle. We hypothesized that the (+)insert isoform is present in humans and that its expression is commensurate with the organ's functional requirements. We report, for the first time, the sequence of the human (+)insert isoform and quantification of its expression by real-time PCR and Western blot analysis in a panel of human organs. The (+)insert isoform mRNA and protein expression levels are significantly greater in small intestine compared with all organs studied except for trachea and are significantly greater in trachea compared with uterus and aorta. To assess the functional significance of this differential myosin isoform expression between organs, we measured the rate of actin filament movement (nu(max)) when propelled by myosin purified from rat organs, because the rat and human inserts are identical and their remaining sequences show 93% identity. nu(max) exhibits a rank correlation from the most tonic to the most phasic organ. The selective expression of the (+)insert isoform observed among human organs suggests that it is an important determinant of tissue shortening velocity. A differential expression of the (+)insert isoform could also account for altered contractile properties observed in human pathology.

Slowing effects of Mg2+ on contractile kinetics of skinned preparations of rat hearts depending on myosin heavy chain isoform content.

The effects of changes in Mg2+ concentration on the kinetics of stretch activation were investigated on skinned rat heart preparations under maximal Ca2+ activation. Muscle strips of hyper- and hypothyroid rat hearts were investigated at 0.5 and 1 mM free Mg2+; the total ATP concentration was 8 mM which resulted in saturating MgATP2- concentrations above 5 mM. Preparations containing exclusively the cardiac alpha-myosin heavy chain (hyper- and hypothyroid atria, hyperthyroid ventricles) showed an acceleration of the kinetics of stretch activation by a factor of about 1.5 (P<0.01, paired t-test) when free Mg2+ was decreased from 1 to 0.5 mM. Conversely, preparations containing exclusively the beta-myosin heavy chain isoform showed only a small acceleration by a factor of 1.05 (P<0.05, paired t-test) under the same conditions. The fact that the Mg2+ sensitivity was dependent on the myosin heavy chain isoform excludes the possibility that Mg2+ exhibits only unspecific effects on contractile proteins. Several hypotheses for explaining the observed Mg2+ effects are discussed. The conditions used in our experiments might be close to the physiological situation and, thus, changes of Mg2+ concentration must be considered as possible factors modulating the contractile kinetics especially of atrial muscle tissue.