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Striated muscle contraction is powered by actin-activated myosin ATPase. This process is regulated by Ca2+via the troponin complex. Slow- and fast-twitch fibers of vertebrate skeletal muscle express type I and type II myosin, respectively, and these myosin isoenzymes confer different ATPase activities, contractile velocities, and force. Skeletal muscle troponin has also diverged into fast and slow isoforms, but their functional significance is not fully understood. To investigate the expression of troponin isoforms in mammalian skeletal muscle and their functional relationship to that of the myosin isoforms, we concomitantly studied myosin, troponin T (TnT), and troponin I (TnI) isoform contents and isometric contractile properties in single fibers of rat skeletal muscle. We characterized a large number of Triton X-100-skinned single fibers from soleus, diaphragm, gastrocnemius, and extensor digitorum longus muscles and selected fibers with combinations of a single myosin isoform and a single class (slow or fast) of the TnT and TnI isoforms to investigate their role in determining contractility. Types IIa, IIx, and IIb myosin fibers produced higher isometric force than that of type I fibers. Despite the polyploidy of adult skeletal muscle fibers, the expression of fast or slow isoforms of TnT and TnI is tightly coupled. Fibers containing slow troponin had higher Ca2+sensitivity than that of the fast troponin fibers, whereas fibers containing fast troponin showed a higher cooperativity of Ca2+activation than that of the slow troponin fibers. These results demonstrate distinct but coordinated regulation of troponin and myosin isoform expression in skeletal muscle and their contribution to the contractile properties of muscle.

Summary The release of Ca 2+ from intracellular stores mediated by ryanodine receptors (RyR) Ca 2+ release channels is essential for striated muscle contraction and contributes to diverse neuronal functions including synaptic plasticity. Through Ca 2+ -induced Ca 2+ -release, RyR can amplify and propagate Ca 2+ signals initially generated by Ca 2+ entry into cardiac muscle cells or neurons. In contrast, RyR activation in skeletal muscle is under membrane potential control and does not require Ca 2+ entry. Nonphysiological or endogenous redox molecules can change RyR function via modification of a few RyR cysteine residues. This critical review will address the functional effects of RyR redox modification on Ca 2+ release in skeletal muscle and cardiac muscle as well as in the activation of signaling cascades and transcriptional regulators required for synaptic plasticity in neurons. Specifically, the effects of endogenous redox-active agents, which induce Snitrosylation or S-glutathionylation of particular channel cysteine residues, on the properties of muscle RyRs will be discussed. The effects of endogenous redox RyR modifications on cardiac preconditioning will be analyzed as well. In the hippocampus, sequential activation of ERKs and CREB is a requisite for Ca 2+ dependent gene expression associated with long lasting synaptic plasticity. Results showing that reactive oxygen/nitrogen species modify RyR channels from neurons and the RyR-mediated sequential activation of neuronal ERKs and CREB produced by hydrogen peroxide and other stimuli will be also discussed. IUBMB Life, 57: 315 – 322, 2005

Bindings of both myosin and Ca(2+) to the thin filament of vertebrate striated muscle are known to be strongly cooperative. Here the relation between these two sources of cooperativity and their consequences for physiological properties are assessed by comparing two models, with and without Monod-type myosin-binding cooperativity. In both models a thin filament regulatory unit (RU) is in either 'off' or 'on' state, and the equilibrium between them (K (on)) is [Ca(2+)]-dependent. The calculations predict the following: (1) In both models, myosin binding stabilizes the RU in the 'on' state, causing troponin to trap Ca(2+). This stabilization in turn increases the Ca(2+)-binding cooperativity, ensuring efficient regulation to occur in a narrow [Ca(2+)] range. (2) In the cooperative model, the RU is stabilized with a relatively low myosin affinity for actin (K approximately approximately 1), while the non-cooperative model requires a much higher affinity (K approximately approximately 10) to produce the same effect. (3) The cooperative model reproduces the known effects of [Ca(2+)] on the rate of force development and shortening velocity with a low K, but again the non-cooperative model requires a higher value. (4) Because of the finite value of K (on), the thin filaments can never be fully activated by increasing [Ca(2+)], indicating that contracting muscles are under strong influence of thin-filament cooperativity even at saturating [Ca(2+)]. Interpretation of data on muscle mechanics without considering these cooperative effects could therefore lead to a substantial (10-fold) overestimate of cross-bridge binding properties.

Troponin (Tn) plays the key roles in the regulation of striated muscle contraction. Tn consists of three subunits (TnT, TnC, and TnI). In combination with the stopped-flow method, fluorescence resonance energy transfer between probes attached to Cys-60 or Cys-250 of TnT and Cys-374 of actin was measured to determine the rates of switching movement of the troponin tail domain (Cys-60) and of the TnT-TnI coiled-coil C terminus (Cys-250) between three states (relaxed, closed, and open) of the thin filament. When the free Ca(2+) concentration was rapidly changed, these domains moved with rates of approximately 450 and approximately 85 s(-1) at pH 7.0 on Ca(2+) up and down, respectively. When myosin subfragment 1 (S1) was dissociated from thin filaments by rapid mixing with ATP, these domains moved with a single rate constant of approximately 400 s(-1) in the presence and absence of Ca(2+). The light scattering measurements showed that ATP-induced S1 dissociation occurred with a rate constant >800 s(-1). When S1 was rapidly mixed with the thin filament, these domains moved with almost the same or slightly faster rates than those of S1 binding measured by light scattering. In most but not all aspects, the rates of movement of the troponin tail domain and of the TnT-TnI coiled-coil C terminus were very similar to those of certain TnI sites (N terminus, Cys-133, and C terminus) previously characterized (Shitaka, Y., Kimura, C., Iio, T., and Miki, M. (2004) Biochemistry 43, 10739-10747), suggesting that a series of conformational changes in the Tn complex during switching on or off process occurs synchronously.

We prepared a new type of skeletal myosin subfragment 1 (S1-MLC1F) containing both, the essential and the regulatory light chains, intact, by exchanging the essential light chains of papain S1 with bacterially expressed longer isoform (MLC1F) of this light chain. We then compared the enzymatic and structural properties of chymotryptic S1, papain S1, and S1-MLC1F in the presence and in the absence of Ca(2+) ions bound to the regulatory light chain. In the presence of Ca(2+), subfragment 1 containing both intact light chains exhibited lower V(max) and lower K(m) for actin activation of S1 ATPase. When S1-MLC1F was cross-linked to actin via the N-terminus of the essential light chain, the yield was much higher when Ca(2+) ions saturated the regulatory light chain. Limited proteolysis of the essential light chain in S1-MLC1F was significantly inhibited in the presence of calcium as compared to chymotryptic S1. We conclude that the effect of binding of Ca(2+) to the regulatory light chain is transmitted to the N-terminal extension of the longer isoform of the essential light chain. The resulting structure of the N-terminus is less susceptible to proteolytic digestion, binds tighter to actin, and has an inhibitory effect on actin-activated myosin ATPase. This new conformation of the N-terminus may be responsible for calcium induced myosin-linked modulation of striated muscle contraction.

Striated (skeletal and cardiac) muscle is activated by the binding of Ca(2+) to troponin C and is regulated by the thin filament proteins, tropomyosin and troponin. Unlike in molluscan or smooth muscles, the myosin regulatory light chains (RLC) of striated muscles do not play a major regulatory role and their function is still not well understood. The N-terminal domain of RLC contains a 'Ca(2+)-Mg(2+)'-binding site and, analogous to that of smooth muscle myosin, also contains a phosphorylation site. During muscle contraction, the increase in Ca(2+) concentration activates the Ca(2+)/calmodulin-dependent myosin light chain kinase and leads to phosphorylation of the RLC. In agreement with other laboratories we have demonstrated that phosphorylation and Ca(2+) binding to the RLC play an important modulatory role in striated muscle contraction. Furthermore, the ventricular isoform of human cardiac RLC has been shown to be one of the sarcomeric proteins associated with familial hypertrophic cardiomyopathy (FHC), an autosomal dominant disease characterized by left ventricular hypertrophy, myofibrillar disarray and sudden cardiac death. Our recent studies have demonstrated that phosphorylation and Ca(2+) binding to human ventricular RLC are significantly altered by the FHC mutations and that their detrimental effects depend upon the specific position of the missense mutation, whether located in the proximity of the RLC 'Ca(2+)-Mg(2+)'-binding site or the phosphorylation site (Serine 15). We have also shown that there is a functional coupling between Ca(2+) and/or Mg(2+) binding to the RLC and phosphorylation and that the FHC mutations can affect this relationship. Further in vivo studies are necessary to investigate the mechanisms involved in the pathogenesis of RLC-linked FHC.

Troponin is essential in Ca(2+) regulation of skeletal and cardiac muscle contraction. It consists of three subunits (TnT, TnC and TnI) and, together with tropomyosin, is located on the actin filament. Here we present crystal structures of the core domains (relative molecular mass of 46,000 and 52,000) of human cardiac troponin in the Ca(2+)-saturated form. Analysis of the four-molecule structures reveals that the core domain is further divided into structurally distinct subdomains that are connected by flexible linkers, making the entire molecule highly flexible. The alpha-helical coiled-coil formed between TnT and TnI is integrated in a rigid and asymmetric structure (about 80 angstrom long), the IT arm, which bridges putative tropomyosin-anchoring regions. The structures of the troponin ternary complex imply that Ca(2+) binding to the regulatory site of TnC removes the carboxy-terminal portion of TnI from actin, thereby altering the mobility and/or flexibility of troponin and tropomyosin on the actin filament.

Vertebrate striated muscle contraction is regulated in a Ca(2+)-dependent fashion by tropomyosin (Tm) and troponin (Tn). This regulation involves shifts in the position of Tm and Tn on actin filaments and may include conformational changes in actin that are then communicated to myosin subfragment 1 (S1). To determine whether subdomain 2 of actin plays a role in this regulation, the DNase-I loop 38-52 of this subdomain was cleaved by subtilisin between residues Met(47) and Gly(48). Despite impaired unregulated function, the potentiation and regulation of cleaved actin movement in the in vitro motility assay was not significantly different from that of uncleaved actin. Stopped-flow measurements of ADP release from regulated and unregulated cleaved acto-S1 showed a marked increase in ADP release from acto-S1 in the presence of the regulatory complex. The enhancement of the actin affinity for S1 in the presence of regulatory proteins was greater for uncleaved than for cleaved F-actin. Finally, both cleaved and uncleaved actins protect myosin loop 1 from papain cleavage equally well. Our results suggest that the potentiation of actin function in the in vitro motility assay by regulatory proteins stems from changes in cross-bridge cycle kinetics. In addition, the unimpaired calcium-sensitive regulation of cleaved actin indicates that subdomain 2 conformation does not play an essential role in the regulation process.

Nebulin (600-900 kDa) and nebulette (107-109 kDa) are two homologous thin filament-associated proteins in skeletal and cardiac muscles, respectively. Both proteins are capped with a unique region at the amino terminus as well as a serine-rich linker domain and SH3 domains at the COOH terminus. Their significant size difference is attributed to the length of the central region wherein both proteins are primarily composed of approximately 35 amino acid repeats termed nebulin-like repeats or motifs. These motifs are marked by a conserved SXXXY sequence and high affinity binding to F-actin. To further characterize the effects that nebulin-like proteins may have on the striated muscle thin filament, we have cloned, expressed, and purified a five-motif chicken nebulette fragment and tested its interaction with the thin filament regulatory proteins. Both tropomyosin and troponin T individually bound the nebulette fragment, although the affinity of this interaction was significantly increased when tropomyosin-troponin T was tested as a binary complex. The addition of troponin I to the tropomyosin-troponin T complex decreased the binding to the nebulette fragment, indicating an involvement of the conserved T2 region of troponin T in this interaction. F-actin cosedimentation demonstrated that the nebulette fragment was able to significantly increase the affinity of the tropomyosin-troponin assembly for F-actin. The relationships provide a means for nebulin-like motifs to participate in the allosteric regulation of striated muscle contraction.

The troponin-tropomyosin-linked regulation of striated muscle contraction occurs through allosteric control by both Ca(2+) and myosin. The thin filament fluctuates between two extreme states: the inactive "off" state and the active "on" state. Intermediate states have been proposed from structural studies and transient kinetic measurements. However, in contrast to the well-characterised, on and off states, the mechanochemical properties of the intermediate states are much less well understood because of the instability of those states. In the present study, we have characterized a myosin-induced intermediate that is stabilized by cross-linking myosin motor domains (S1) to actin filaments (with a maximum of one S1 molecule for 50 actin monomers). A single S1 molecule is known to interact with two adjacent actin monomers. A detailed analysis revealed that thin filaments containing S1 molecules cross-linked to just one actin monomer (actin(1)-S1 complexes) are regulated with a 79% inhibition of the ATPase in the absence of Ca(2+). In contrast, filaments containing S1 molecules cross-linked at two positions, to two adjacent actin monomers (actin(2)-S1 complexes) totally lose their regulation in a highly cooperative manner. This loss of regulation was due both to an enhancement of the ATPase activity without calcium and an inhibition of the ATPase with calcium. Filaments containing actin(2)-S1 complexes, with significant ATPase activity in the absence of calcium (about 50%), did not move on a myosin-coated surface unless calcium was present. This partial uncoupling between the ATPase activity and in vitro motility in the absence of calcium demonstrates that the mechanical steps require actin-myosin contacts, which take place only in the on state and not in the off or intermediate states. These data provide new insights concerning the difference in cooperativity of Ca(2+) regulation that exists between the biochemical and mechanical cycles of the actin-myosin motor.

Troponin T (TnT) is an essential component of troponin (Tn) for the Ca 2 + -regulation of vertebrate striated muscle contraction. TnT consists of an extended NH 2 -terminal domain that interacts with tropomyosin (Tm) and a globular COOH-terminal domain that interacts with Tm, troponin I (TnI), and troponin C (TnC). We have generated two mutants of a rabbit skeletal β-TnT 25-kDa fragment (59-266) that have a unique cysteine at position 60 (N-terminal region) or 250 (C-terminal region). To understand the spatial rearrangement of TnT on the thin filament in response to Ca 2 + binding to TnC, we measured distances from Cys-60 and Cys-250 of TnT to Gln-41 and Cys-374 of F-actin on the reconstituted thin filament by using fluorescence resonance energy transfer (FRET). The distances from Cys-60 and Cys-250 of TnT to Gln-41 of F-actin were 39.5 and 30.0 A, respectively in the absence of Ca 2 + , and increased by 2.6 and 5.8 A, respectively upon binding of Ca 2 + to TnC. The rigor binding of myosin subfragment 1 (S1) further increased these distances by 4 and 5 A respectively, when the thin filaments were fully decorated with S1. This indicates that not only the C-terminal but also the N-terminal region of TnT showed the Ca 2 + - and S1-induced movement, and the C-terminal region moved more than N-terminal region. In the absence of Ca 2 + , the rigor S1 binding also increased the distances to the same extent as the presence of Ca 2 + when the thin filaments were fully decorated with S1. The addition of ATP completely reversed the changes in FRET induced by rigor S1 binding both in the presence and absence of Ca 2 + . However, plots of the extent of S1-incuced conformational change vs. molar ratio of S1 to actin showed hyperbolic curve in the presence of Ca 2 + but sigmoidal curve in the absence of Ca 2 + . FRET measurement of the distances from Cys-60 and Cys-250 of TnT to Cys-374 of actin showed almost the same results as the case of Gln-41 of actin. The present FRET measurements demonstrated that not only TnI but also TnT change their positions on the thin filament corresponding to three states of thin filaments (relaxed, Ca 2 + -induced or closed, and Sl-induced or open states).

Troponin T is a central component of the thin filament-associated troponin-tropomyosin system and plays an essential role in the Ca(2+) regulation of striated muscle contraction. The importance of the structure and function of troponin T is evident in the regulated isoform expression during development and the point mutations resulting in familial hypertrophic and dilated cardiomyopathies. We report here that turkeys with inherited dilated cardiomyopathy and heart failure express an unusual low molecular weight cardiac troponin T missing 11 amino acids due to the splice out of the normally conserved exon 8-encoded segment. The deletion of a 9-bp segment from intron 7 of the turkey cardiac troponin T gene may be responsible for the weakened splicing of the downstream exon 8 during mRNA processing. The exclusion of the exon 8-encoded segment results in conformational changes in cardiac troponin T, an altered binding affinity for troponin I and tropomyosin, and an increased calcium sensitivity of the actomyosin ATPase. Expression of the exon 8-deleted cardiac troponin T prior to the development of cardiomyopathy in turkeys indicates a novel RNA splicing disease and provides evidence for the role of troponin T structure-function variation in myocardial pathogenesis and heart failure.

Vertebrate striated (skeletal and cardiac) muscle contraction is regulated in a Ca2+-dependent manner by the troponin (Tn) complex through interactions with tropomyosin (Tm) and the actin filament. The Tn complex consists of three proteins: troponin I (TnI) which inhibits the actomyosin Mg2+-ATPase activity, troponin C (TnC) which binds Ca2+ ions and removes TnI inhibition, and troponin T (TnT) which makes primary interaction with Tm. These three subunits interact in a cooperative manner with each other and with Tm and actin. Binding of Ca2+ to TnC initiates structural changes in TnC which lead to structural changes in the other Tn subunits (TnI and TnT) as well as Tm. The movement of Tm on the actin filament facilitates the interaction between myosin and actin leading to muscle contraction. The cation, Ca2+, is the main regulatory and signaling molecule in striated muscle. Another cation, Mg2+, is also important in the regulation of striated muscle contraction mainly via its ability to inhibit the Ca2+ release channel, especially in skeletal muscle.

Regulation of contraction in vertebrate striated muscle is linked to the thin filament. The initial and critical step in activation is the binding of regulatory Ca2+ to specific sites in troponin. This binding triggers a series of protein-protein interactions which in turn induce structural changes in the proteins and lead to a cyclic interaction between actin and force-generating myosin crossbridge. These molecular interactions are dependent upon transduction of the initial Ca2+ signal along the thin filament. The transduction mechanism is viewed as a component of the overall contractile mechanism.

Preface. Introduction G. Stienen. Excitation-Contraction Coupling in Skeletal Muscle A. Gonzalez, E. Rios. Excitation-Contraction Coupling In Cardiac Muscle A.W. Trafford, D.A. Eisner. The Thick Filament of Vertebrate Striated Muscle R.J.C. Levine, R.W. Kensler. Structure and Regulation of Cardiac and Skeletal Muscle Thin Filaments L.S. Tobacman. Cation Signaling in Striated Muscle Contraction A.V. Gomes, et al. Calcium-Induced Molecular and Structural Signaling in Striated Muscle Contraction H.C. Cheung. Cooperativity in the Ca2+-regulation of Skeletal Muscle Contraction M.A. Geeves, S.S. Lehrer. Regulation of the Rate of Force Development in Heart and Skeletal Muscles R.L. Moss, et al. Modulation of Thin Filament Activity in Long and Short Term Regulation of Cardiac Function R.J. Solaro, et al. Thin Filament Regulation in Development P. Anderson. The Frank-Starling Relationship: Cellular and Molecular Mechanisms F. Fuchs. Determinants of Unloaded Shortening Velocity in Striated Muscle E. Homsher. Author index.

Fructose-bisphosphate aldolase A (ALDOA) is a key enzyme in glycolysis and is responsible for catalyzing the reversible conversion of fructose-1,6-bisphosphate to glyceraldehydes-3-phosphate and dihydroxyacetone phosphate. ALDOA contributes to various cellular functions such as muscle maintenance, regulation of cell shape and mobility, striated muscle contraction, actin filament organization and ATP biosynthetic process. Here, we reported that ALDOA is a highly expressed in lung squamous cell carcinoma (LSCC) and its expression level is correlated with LSCC metastasis, grades, differentiation status and poor prognosis. Depletion of ALDOA expression in the lung squamous carcinoma NCI-H520 cells reduces the capabilities of cell motility and tumorigenesis. These data suggest that ALDOA could be a potential marker for LSCC metastasis and a therapeutic target for drug development.

Remarkably we can tell our arms or legs to move and they do it. We can directly control or regulate the activity of our skeletal muscles. Striated muscle movement, produced by the interaction of filaments containing the proteins myosin and actin, is regulated by the proteins tropomyosin and troponin on the actin filaments. When an electrical signal passes down the motor nerve to a muscle it triggers a depolarisation of the muscle membrane (sarcolemma). In turn this triggers the sarcoplasmic reticulum to release calcium ions into the muscle interior where they bind to troponin, thus causing tropomyosin to shift from the face of the actin filament to which myosin heads need to bind to produce contraction. During relaxation calcium is pumped back into the sarcoplasmic reticulum, troponin loses its calcium and tropomyosin reverts to its off position. This is the steric blocking mechanism of muscle regulation. Key Concepts: Striated muscles contain repeating sarcomeres of overlapping arrays of long, thin actin and thicker myosin filaments. Myosin filaments carry projections, the myosin heads, which are enzymes that can bind to actin and can split and make use of the energy from ATP. During muscle contraction myosin heads bind to actin, change their configuration on actin along with releasing the products of ATP hydrolysis and cause relative sliding of the actin and myosin filaments. Vertebrate striated muscle contraction is controlled (regulated) by the action of the proteins troponin and tropomyosin on the actin filaments. Nervous stimulation causes a depolarisation of the muscle membrane (sarcolemma) which triggers the release of calcium ions from the sarcoplasmic reticulum. Calcium ions bind to troponin and thus cause or allow the tropomyosin strands on the actin filament to move so that the part of the actin surface where myosin heads need to bind is uncovered. Contraction (force generation) then occurs and only stops when the sarcoplasmic reticulum pumps calcium out of the muscle interior, troponin loses its bound calcium and tropomyosin shifts back to its off position. Keywords: myosin; actin; crossbridge cycle; tropomyosin; troponin; ryanodine receptor; steric blocking model

There is recent morphological evidence for an interaction of autonomic nerve fibers and extrinsic motor nerves of the rat esophagus. The aim of the present study was to investigate a possible functional role of this autonomic innervation of vagal motor fibers on rat esophageal smooth and striated muscle function in vitro. The entire esophagus with both Nn vagi, including the Nn recurrentes, was dissected and placed in an organ bath with oxygenated Krebs-Ringer buffer. Contractile activity was measured in longitudinal direction with a force transducer. Both Nn vagi were placed on a bipolar platinum electrode 2 cm apart from the esophagus. Vagal stimulation, applied for 1 s (40 V, 0.5 ms, 20 Hz) resulted in a biphasic contractile response, which was completely blocked by tetrodotoxin (10(-6) M). The first part consisted of a tetanic striated muscle contraction, which was abolished by tubocurarin (10(-5) M) but unaffected by atropine (10(-6) M) or hexamethonium (10(-4) M). In contrast, the second part was completely abolished by hexamethonium (10(-4) M) and atropine (10(-6) M), whereas tubocurarine (10(-5) M) showed no influence, suggesting a stimulation of preganglionic nerve fibers supplying esophageal smooth muscle (muscularis mucosae). In order to characterize possible autonomic transmitters of the ENS of the esophagus, the following experiments were carried out. The magnitude of the striated muscle response was unaffected by VIP (10(-7) M), 5-HT (10(-6) M) and galanin (10(-8) - 10(-7) M), whereas they caused an inhibition of the smooth muscle response (VIP: -53.8 +/- 4.2%; galanin 10(-8) M: - 18.5 +/- 2.2%; 10(-7) M: -40.4 +/- 2.9%; 5-HT: -78.2 +/- 2.1%). The inhibitory effects of VIP and galanin on smooth muscle were reversible by the antagonists VIP 10-28 and galanin 1-15. In the presence of the nitric oxide synthase (NOS) inhibitor L-NNA (10(-4) M), the smooth and striated muscle contraction were not significantly influenced. Exogenous application of the NO-donor DEA-NO (10(-4) M) reduced the smooth muscle contraction by -81.6 +/- 7.4%, but had no significant effect on the striated muscle contraction. Though immunohistochemical findings are highly suggestive of an nitrergic autonomic modulation of striated muscle contraction by enteric neurons, we could not demonstrate a NO-mediated action on striated muscle activity. Therefore, the physiological relevance of the immunohistochemical findings remain unclear.

Troponin T (TnT) is an essential element in the thin filament Ca2+-regulatory system controlling striated muscle contraction. Alternative RNA splicing generates developmental and muscle type-specific TnT isoforms differing in the hypervariable NH2-terminal region. Using avian fast skeletal muscle TnT containing a metal-binding segment, we have demonstrated a role of the NH2-terminal domain in modulating the conformation of TnT (Wang J and Jin JP. Biochemistry 37: 14519–14528, 1998). To further investigate the structure-function relationship of TnT, the present study constructed and characterized a recombinant protein in which the metal-binding peptide present in avian fast skeletal muscle TnT was fused to the NH2terminus of mouse slow skeletal muscle TnT. Metal ion or monoclonal antibody binding to the NH2-terminal extension induced conformational changes in other domains of the model TnT molecule. This was shown by the altered affinity to a monoclonal antibody against the COOH-terminal region and a polyclonal antiserum recognizing multiple epitopes. Protein binding assays showed that metal binding to the NH2-terminal extension had effects on the interaction of TnT with troponin I, troponin C, and most significantly, tropomyosin. The data indicate that the NH2-terminal Tx [4–7 repeats of a sequence motif His-(Glu/Ala)-Glu-Ala-His] extension confers a specific conformational modulation in the slow skeletal muscle TnT.

Striated muscle contraction is initiated when troponin C (TnC) binds Ca(2+), which activates actinomyosin ATPase. We investigated (i) the variation between cardiac TnC (cTnC) primary structure within teleost fish and (ii) the pattern of TnC expression in response to temperature acclimation. There were few differences between rainbow trout (Oncorhynchus mykiss), yellowfin tuna (Thunnus albacares), yellow perch (Perca flavescens), goldfish (Carassius auratus), white sucker (Catostomus commersoni), and icefish (Chaenocephalus aceratus) in cTnC amino acid sequence. No variation existed in the regulatory Ca(2+)-binding site (site 2). The site 3 and 4 substitutions were limited to residues not directly involved in Ca(2+) coordination. Fish cTnC primary structure was highly conserved between species (93%-98%) and collectively divergent from the highly conserved sequence seen in birds and mammals. Northern blots and polymerase chain reaction showed that thermal acclimation of trout (3 degrees, 18 degrees C) did not alter the TnC isoform pattern. While cardiac and white muscle had the expected isoforms-cTnC and fast troponin C (fTnC), respectively-red muscle unexpectedly expressed primarily ftnC. Cold acclimation did not alter myofibrillar ATPase Ca(2+) sensitivity, but maximal velocity increased by 60%. We found no evidence that TnC variants, arising between species or in response to thermal acclimation, play a major role in mitigating the effects of temperature on contractility of the adult fish heart.

Ca2+regulation of contraction in vertebrate striated muscle is exerted primarily through effects on the thin filament, which regulate strong cross-bridge binding to actin. Structural and biochemical studies suggest that the position of tropomyosin (Tm) and troponin (Tn) on the thin filament determines the interaction of myosin with the binding sites on actin. These binding sites can be characterized as blocked (unable to bind to cross bridges), closed (able to weakly bind cross bridges), or open (able to bind cross bridges so that they subsequently isomerize to become strongly bound and release ATP hydrolysis products). Flexibility of the Tm may allow variability in actin (A) affinity for myosin along the thin filament other than through a single 7 actin:1 tropomyosin:1 troponin (A7TmTn) regulatory unit. Tm position on the actin filament is regulated by the occupancy of NH-terminal Ca2+binding sites on TnC, conformational changes resulting from Ca2+binding, and changes in the interactions among Tn, Tm, and actin and as well as by strong S1 binding to actin. Ca2+binding to TnC enhances TnC-TnI interaction, weakens TnI attachment to its binding sites on 1–2 actins of the regulatory unit, increases Tm movement over the actin surface, and exposes myosin-binding sites on actin previously blocked by Tm. Adjacent Tm are coupled in their overlap regions where Tm movement is also controlled by interactions with TnT. TnT also interacts with TnC-TnI in a Ca2+-dependent manner. All these interactions may vary with the different protein isoforms. The movement of Tm over the actin surface increases the “open” probability of myosin binding sites on actins so that some are in the open configuration available for myosin binding and cross-bridge isomerization to strong binding, force-producing states. In skeletal muscle, strong binding of cycling cross bridges promotes additional Tm movement. This movement effectively stabilizes Tm in the open position and allows cooperative activation of additional actins in that and possibly neighboring A7TmTn regulatory units. The structural and biochemical findings support the physiological observations of steady-state and transient mechanical behavior. Physiological studies suggest the following. 1) Ca2+binding to Tn/Tm exposes sites on actin to which myosin can bind. 2) Ca2+regulates the strong binding of M·ADP·Pito actin, which precedes the production of force (and/or shortening) and release of hydrolysis products. 3) The initial rate of force development depends mostly on the extent of Ca2+activation of the thin filament and myosin kinetic properties but depends little on the initial force level. 4) A small number of strongly attached cross bridges within an A7TmTn regulatory unit can activate the actins in one unit and perhaps those in neighboring units. This results in additional myosin binding and isomerization to strongly bound states and force production. 5) The rates of the product release steps per se (as indicated by the unloaded shortening velocity) early in shortening are largely independent of the extent of thin filament activation ([Ca2+]) beyond a given baseline level. However, with a greater extent of shortening, the rates depend on the activation level. 6) The cooperativity between neighboring regulatory units contributes to the activation by strong cross bridges of steady-state force but does not affect the rate of force development. 7) Strongly attached, cycling cross bridges can delay relaxation in skeletal muscle in a cooperative manner. 8) Strongly attached and cycling cross bridges can enhance Ca2+binding to cardiac TnC, but influence skeletal TnC to a lesser extent. 9) Different Tn subunit isoforms can modulate the cross-bridge detachment rate as shown by studies with mutant regulatory proteins in myotubes and in in vitro motility assays. These results and conclusions suggest possible explanations for differences between skeletal and cardiac muscle regulation and delineate the paths future research may take toward a better understanding of striated muscle regulation.

Thin filament-mediated regulation of striated muscle contraction involves conformational switching among a few quaternary structures, with transitions induced by binding of Ca(2+) and myosin. We establish and exploit Saccharomyces cerevisiae actin as a model system to investigate this process. Ca(2+)-sensitive troponin-tropomyosin binding affinities for wild type yeast actin are seen to closely resemble those for muscle actin, and these hybrid thin filaments produce Ca(2+)-sensitive regulation of the myosin S-1 MgATPase rate. Yeast actin filament inner domain mutant K315A/E316A depresses Ca(2+) activation of the MgATPase rate, producing a 4-fold weakening of the apparent Ca(2+) affinity and a 50% decrease in the MgATPase rate at saturating Ca(2+) concentration. Observed destabilization of troponin-tropomyosin binding to actin in the presence of Ca(2+), a 1.4-fold effect, provides a partial explanation. Despite the decrease in apparent MgATPase Ca(2+) affinity, there was no detectable change in the true Ca(2+) affinity of the thin filament, measured using fluorophore-labeled troponin. Another inner domain mutant, E311A/R312A, decreased the MgATPase rate but did not change the apparent Ca(2+) affinity. These results suggest that charged residues on the surface of the actin inner domain are important in Ca(2+)- and myosin-induced thin filament activation.

Ryanodine receptors are a family of intracellular Ca2+ release channel proteins, which exist as tetrameric complexes of large ( approximately 5000 amino acid residue) polypeptide monomers. As well as controlling striated muscle contraction and neurotransmitter release, these channel proteins have been implicated in several pathological states. In order to characterise ryanodine receptors in various tissues, mouse monoclonal antibodies were developed against the type 1 isoform isolated from skeletal muscle. Several of these antibodies recognise ryanodine receptor in skeletal muscle, as well as high molecular weight (k-HMW) protein in kidney microsomes. Like the ryanodine receptor, the k-HMW protein binds 45Ca2+ and sediments as a large complex upon sucrose density-gradient centrifugation. In contrast, the k-HMW protein does not bind ryanodine and is glycosylated. Furthermore, monoclonal and polyclonal antibodies generated against purified k-HMW protein do not recognise skeletal muscle ryanodine receptor. Characterisation of a cDNA clone encoding part of the k-HMW protein revealed that it is likely to be the rabbit homologue of human megalin, an autoimmune antigen in membranous glomerulonephritis. Potential consequences of immunological similarities between ryanodine receptors and megalin are discussed in terms of autoimmune disease.

The Distribution Moment (DM) model has simulated experimental data on skeletal muscle, but it has not been used previously to study the mechanics of active contraction in cardiac muscle. In contrast to previous models of striated muscle contraction, all parameters have physical meaning and assumptions concerning biophysical events within the cell are consistent with available data. In order to simulate cardiac muscle deactivation using the DM model it was necessary to make the cross-bridge detachment rates large for large displacements from the neutral equilibrium position of a cross-bridge. To examine the effect of cooperativity on cardiac muscle contraction, we used the DM model's tight coupling scheme with binding of one or two calcium sites regulating contraction. As observed experimentally, our model predicted a reduction of isometric tension development following rapid shortening lengthening transients when contraction is regulated by either one or two calcium binding sites. The predicted deactivating effect increased if the transient was applied late in the twitch when contraction is regulated by two calcium binding sites, but not when it is regulated by one site. This is the first study in which deactivation has been simulated without making any provisions for length-dependent calcium trononin dissociation.

The field of striated muscle regulation has changed tremendously over the last forty years. Many of the problems solved by Dr. Ebashi and by those stimulated by him offer new challenges for future generations of scientists. Many questions remain to be solved, and it should give particular pleasure to Dr. Ebashi to see how the seeds sown by him and his colleagues have now grown into a beautiful tree that bears rich fruit at present and will continue to do so for a long time in the future. (Mol Cell Biochem 190: 5-8, 1999)

The troponin complex forms the calcium-sensitive molecular switch which regulates striated muscle contraction in response to alterations in intracellular calcium concentration. It is located on the thin filament of the sarcomere and is composed of three subunits: troponin C, the calcium binding subunit; troponin T, which is involved in the attachment of the complex to tropomyosin; and troponin I, the inhibitory subunit. Multiple isoforms of each of these subunits have been identified (see table 1) which are expressed with distinct tissue-specificity and developmental regulation [1,2]. In the case of troponin I, three isoforms have been identified in vertebrate striated muscle and in the adult these are expressed in cardiac muscle, slow skeletal muscle and fast skeletal muscle respectively. In the adult heart, cardiac troponin I is the only troponin I isoform detected in the bulk of the myocardium. However, during development the predominant isoform expressed is slow skeletal troponin I [2]. We have previously documented aspects of troponin expression in the human heart [3–6] and demonstrated a developmental switch in troponin I expression during human development. Analysis of mRNA and protein levels suggests that the increase in expression of the cardiac troponin I gene seen in late fetal stages in man is due to an increase in transcription. Here, we present data on the basic machinery required for the expression of the human cardiac troponin I gene. In other studies we have analyzed the organization of the troponin gene families and revealed that the six human genes encoding the different troponin I and T isoforms are organized as paralogous pairs located at three different chromosomal sites. Analysis of the pair comprized of the cardiac troponin I and slow skeletal troponin T genes reveals that they are organized head to tail and lie within 3 kb of each other. Close physical linkage raises questions concerning the evolution of these two troponin gene families, for their regulation and for the analysis of mutations suspected to result in cardiomyopathy.

The chemomechanical coupling mechanism in striated muscle contraction was examined by changing the nucleotide substrate from ATP to CTP. Maximum shortening velocity [extrapolation to zero force from force-velocity relation ( Vmax) and slope of slack test plots ( V0)], maximum isometric force (Po), power, and the curvature of the force-velocity curve [ a/Po(dimensionless parameter inversely related to the curvature)] were determined during maximum Ca2+-activated isotonic contractions of fibers from fast rabbit psoas and slow rat soleus muscles by using 0.2 mM MgATP, 4 mM MgATP, 4 mM MgCTP, or 10 mM MgCTP as the nucleotide substrate. In addition to a decrease in the maximum Ca2+-activated force in both fiber types, a change from 4 mM ATP to 10 mM CTP resulted in a decrease in Vmaxin psoas fibers from 3.26 to 1.87 muscle length/s. In soleus fibers, Vmaxwas reduced from 1.94 to 0.90 muscle length/s by this change in nucleotide. Surprisingly, peak power was unaffected in either fiber type by the change in nucleotide as the result of a three- to fourfold decrease in the curvature of the force-velocity relationship. The results are interpreted in terms of the Huxley model of muscle contraction as an increase in f1and g1coupled to a decrease in g2(where f1is the rate of cross-bridge attachment and g1and g2are rates of detachment) when CTP replaces ATP. This adequately accounts for the observed changes in Po, a/Po, and Vmax. However, the two-state Huxley model does not explicitly reveal the cross-bridge transitions that determine curvature of the force-velocity relationship. We hypothesize that a nucleotide-sensitive transition among strong-binding cross-bridge states following Pirelease, but before the release of the nucleotide diphosphate, underlies the alterations in a/Poreported here.

The contraction of vertebrate striated muscle contraction, and hence its work output, is controlled by Ca2+, which binds to troponin (Tn) associated with tropomyosin (TM) and actin in the thin filaments. Tn consists of three subunits: TnC, the Ca2+-receptor; TnI, an inhibitor of actomyosin activity; and TnT, anchoring Tn to T.M. Of the four Ca2+-binding sites, I and II in the N-terminal domain are Ca-specific sites, while sites III and IV, the high affinity Ca-Mg sites, are in the C-domain. The former are recognized as the functionally important triggering sites. TnC, whose structure has been solved by X-ray crystallography and recently by high-resolution NMR, contains two homologous globular domains connected by an unusual single α-helix. The C-terminal domain exhibits an open hydrophobic area regardless of whether Ca2+ or Mg2+ is bound to sites III and IV. In contrast, the N-terminal domain is a closed structure that opens a hydrophobic patch upon Ca2+-binding to its two “triggering” sites producing a TnI binding area. Crosslinking and fragment binding studies indicate that, in the main, the two polypeptide chains run in opposite directions in the complex of TnC with Tn. A model of TnC-TnI interactions based on low angle X-ray and neutron scattering is discussed in light of biochemical and other physico-chemical studies. The opening of the structure in the N-terminal domain of TnC may be regarded as a molecular switch. It activates a molecular switch in TnI, reflected in the movement of portions of its C-terminal half, including Cys l33, away from actin and closer to TnC, as well as other structural changes in Tnl. Finally the role of TnT in switching and transmitting the Ca2+-signal is discussed.

The structural basis of thin filament-linked regulation of muscle contraction is not yet understood. Here we have used electron microscopy and three-dimensional image reconstruction to observe the effects of Ca2+ and myosin head binding on thin filament structure, especially on the position of tropomyosin. Thin filaments isolated in EGTA were treated with Ca2+ or myosin heads (S-1) and negatively stained. Tropomyosin strands were directly visualized in electron micrographs, and distinct EGTA, Ca2+ and S-1-dependent positions were apparent in reconstructions. By fitting reconstructions to the atomic model of F-actin, clusters of amino acids on actin lying beneath tropomyosin were defined under each set of conditions. In the presence of Ca2+, tropomyosin moved 25 degrees away from its low Ca2+ position, exposing most, but not all, of the previously blocked myosin-binding sites. Saturation of filaments with myosin heads produced a further 10 degrees shift in tropomyosin position, thereby exposing the entire myosin-binding site. Our results thus suggest that full switching-on of thin filaments by reversal of steric-blocking requires both Ca2+ and the binding of myosin heads, acting in sequence. By using filaments which were partially decorated with heads, tropomyosin movement was shown to be cooperative, and the size of the actin-tropomyosin cooperative unit was estimated directly. Our results provide direct structural support for previous models of thin filament activation based on kinetics of actin-myosin interaction.

As pointed out by Dr Edman in his thoughtful review (Edman, 2012), residual force enhancement in striated muscles has been observed consistently over the past half-century, its properties have been well described, and its lack of explanation within the framework of the cross-bridge theory has been acknowledged. However, and this is the topic of Dr Edman's review, the primary mechanism producing force enhancement remains a matter of scientific debate. On the one hand, there is the classic explanation of the development of non-uniformities that develop upon muscle stretch; on the other hand, there is the idea of the ‘engagement’ of a passive structural element upon activation that produces the enhanced force following active muscle stretching. In his review, Dr Edman favours the idea that residual force enhancement is primarily a consequence of (half-) sarcomere length non-uniformities (e.g. [residual force enhancement]…‘is ultimately based on non-uniform sarcomere behaviour with the result that populations of sarcomeres, or half-sarcomeres, actually acquire a greater amount of filament overlap than expected from the overall sarcomere length recorded after stretch’; Edman, 2012). Here, we would like to make the argument that ‘the role of a passive structural element’, the second idea mentioned above, should not be dismissed prematurely. First, Dr Edman mentions the advantages of single fibre preparations over myofibril preparations, and all the points he makes are perfectly valid. However, the great advantage of myofibrils over single fibre preparations is that single myofibrils consist of serially arranged sarcomeres. Therefore, the forces measured at the ends of a myofibril reflect directly the force in each sarcomere. Furthermore, the instantaneous lengths of each and all sarcomeres of a myofibril can easily be quantified. In contrast, in a single fibre, sarcomeres are connected partly in parallel and partly in series forming a highly redundant network of force transmission and individual sarcomere lengths cannot be measured, thus making any inferences to sarcomere dynamics highly suspect. Regarding the involvement of a passive structural element in force enhancement, Dr Edman makes several statements that we do not agree with. For example, he states that: ‘It seems quite clear, however, that the phenomenon “force enhancement after stretch” is entirely limited to the active period of the fibre. It disappears completely as the fibre relaxes after the stimulation period.’ (Edman, 2012). This statement ignores the well-described passive force enhancement observed following active stretches of muscles (e.g. Herzog & Leonard, 2002), fibres (e.g. Lee et al. 2007) and myofibrils (e.g. Joumaa et al. 2007, 2008) which persists long after muscle stimulation has ceased. Dr Edman also mentions that: ‘Another strong indication that strain of elastic elements does not by itself determine the measured force is given by the fact that the force recorded during stretch is quite independent of the amplitude of the stretch ramp.’ (Edman, 2012). Again, we disagree; there is ample evidence that the forces during stretch increase with increasing stretch magnitudes, and that the forces at the end-length following stretches of different magnitudes are well correlated with the residual force enhancement (e.g. Bullimore et al. 2007). The sarcomere length non-uniformity theory, favoured in Dr Edman's review, also provides testable predictions. Arguably the most important of these is that a muscle cannot produce enhanced force that exceeds the maximal isometric force at optimal length (Edman et al. 1982). However, there are numerous studies showing peak force enhancements well in excess of 10% above the plateau forces (Lee & Herzog, 2008), and most importantly, force enhancement in single sarcomeres has been shown to exceed 35% on average, with peak values exceeding 50%, above the plateau of the force–length relationship (Leonard et al. 2010). These values cannot be attributed to random fluctuations or artifacts. In summary, we agree with Dr Edman that the mechanisms underlying residual force enhancement remain unknown. However, inferring single sarcomere mechanics from fibre experiments appears impossible. Myofibrils offer an elegant preparation to measure the mechanics of isolated sarcomeres directly and accurately, and have revealed enhanced forces exceeding 50% of the maximal isometric forces in single sarcomeres (Leonard et al. 2010). These results cannot be explained with increased myofilament overlap due to (half-) sarcomere length non-uniformities, but are consistent with the idea of the ‘engagement’ of a passive structural element upon activation. In agreement with Dr Edman, we speculate that titin might play a crucial role in this ‘passive’ force regulation (Herzog et al. 2012).

Muscle contraction results from a series of molecular events that involve transient interactions between myosin-containing thick and actin-containing thin filaments. The high affinity of myosin for actin suggests that without regulation, muscle would remain in a continuous state of contraction. Regulation of striated muscle contraction is primarily achieved by Ca2+-dependent modulation of myosin crossbridge cycling on actin by the thin filament (TF) troponin-tropomyosin complex. Alterations in various subunits of the complex trigger contractile dysregulation and myopathy. For example, point mutations located over a span of ten amino acids (130-39) of human cardiac troponin T (cTnT) are associated with distinct cardiomyopathic responses. The Drosophila up101 (E88K) mutation localizes to the end of this well-conserved region of TnT. Here we examined the consequences of the lesion on distinct muscle types from the fly. Cardiac performance and morphology were assessed using direct immersion DIC optics, high-speed video imaging and motion analysis. Relative to controls, up101 hearts displayed a phenotype reminiscent of human restrictive cardiomyopathy. End-diastolic and end-systolic dimensions and percent fractional shortening were significantly reduced. Furthermore systolic intervals were significantly prolonged. This suggests TF dysregulation initiates excessive periods of force production and diastolic dysfunction. To characterize the fundamental cause of cardiac remodeling, we isolated TFs from the indirect flight muscles (IFM). The constitutively expressed up101 mutation results in flightlessness due to severe IFM hypercontraction. Electron microscopy and three-dimensional reconstruction of IFM TFs revealed Ca+2-dependent tropomyosin movement, typical of control filaments, was not a general feature of the TnT mutants. The vast majority of Ca+2-free mutant TFs exhibited tropomyosin associated with successive actin monomers over actin subdomains 3 and 4, distal to known myosin binding sites. Thus, up101 TnT likely promotes TF dysinhibition and ubiquitous myopathic remodeling.

Troponin T (TnT) plays a major role in striated muscle contraction. We recently demonstrated that the fast skeletal muscle TnT3 is localized in the muscle nucleus, and either its full-length or COOH-terminus leads to muscle cell apoptosis. Here, we further explored the mechanism by which it enters the nucleus and promotes cytotoxicity and investigated its role in nuclear signaling pathway. Amino acid truncation and substitution showed that its COOH-terminus contains a dominant nuclear/nucleolar localization sequence (KLKRQK). Deleting this domain or substituting lysine and arginine residues (KLAAQK) resulted in a dramatic loss of TnT3 nuclear and nucleolar localization. In contrast, the GATAKGKVGGRWK domain-DsRed construct localized exclusively in the cytoplasm, indicating that a nuclear exporting sequence is possibly localized at the COOH-terminus. Additionally, we identified a classical DNA-binding Leucine Zipper Domain (LZD). Deletion of LZD or KLKRQK sequence significantly reduced cell apoptosis compared to full-length TnT3. shRNA knocking down of TnT3 in C2C12 myotubes down-regulated the expression of calcium channel α1s (CaVα1s) but not β1a subunit. Luciferase activity analysis further verified that knocking down TnT3 in C2C12 muscle cells inhibited while overexpression of TnT3-DsRed in mouse muscle in vivo increased the Cacna1 promoter activity. Additionally, chromatin immunoprecipitation using TnT3 antibody cocktails pulled down Cacna1 promoter fragments from C2C12 myotubes. In contrast, pre-rRNA processing in the nucleolus was not affected by TnT3 depletion. We conclude that TnT3 contains both a nuclear localization signal and a DNA binding domain, which may mediate its nuclear signaling in muscle cell apoptosis and gene expression regulation.

To better understand the regulation of striated muscle contraction, the effects of myosin subfragment 1 (S-1) on the actin binding of cardiac troponin and tropomyosin were investigated. Troponin's affinity for actin-tropomyosin was 4-fold stronger in the absence than in the presence of myosin S-1. CaCl2 had no effect on troponin binding to the thin filament in the presence of myosin S-1. The binding curve was weakly cooperative, implying interactions between adjacent troponin molecules. Myosin S-1 increased (40-200-fold) the affinity of tropomyosin for the thin filament, an effect opposite to the effect of myosin on troponin. This effect was highly cooperative and occurred in the presence of ADP or in the absence of nucleotide. Myosin altered the effect of ionic conditions on tropomyosin-actin binding, consistent with tropomyosin binding to a different site on F-actin in the presence of myosin. The results indicate that troponin-tropomyosin and strongly binding myosin cross-bridges do not compete for an F-actin binding site. Although repositioning of troponin-tropomyosin on the actin filament may be sterically required for tight myosin-actin binding, a myosin-induced conformational change in actin provides a better explanation for the complex effects of myosin on thin filament assembly.

Human fast skeletal troponin T (TnTf), the tropomyosin binding component of the multisubunit troponin complex, plays an important role in the Ca2+ regulation of striated muscle contraction. Specific primers designed from the 3' end of human TnTf cDNA were used to amplify an intronic region by polymerase chain reaction (PCR). This TnTf-specific PCR product was detected from two somatic cell hybrids containing human chromosomes 9 and 11, respectively, in NIGMS mapping panel 2. However, further studies with other somatic hybrid cell lines (Bios Laboratory) localized the TnTf gene (HGMW-approved symbol TNNT3) only to chromosome 11. This observation was further confirmed by fluorescence in situ hybridization with a 12-kb TnTf genomic probe generated by extended PCR, showing the sublocalization of the gene to band p15.5 on chromosome 11. This locus is of specific interest, as Beckwith-Wiedemann syndrome and various childhood and adult tumor-related abnormalities have been mapped to this region. The study also indicates the presence of an 11pter region in the NIGMS cell hybrid GM10611, which has previously been reported to contain only human chromosome 9.

▪ Abstract Myosin is a highly conserved, ubiquitous protein found in all eukaryotic cells, where it provides the motor function for diverse movements such as cytokinesis, phagocytosis, and muscle contraction. All myosins contain an amino-terminal motor/head domain and a carboxy-terminal tail domain. Due to the extensive number of different molecules identified to date, myosins have been divided into seven distinct classes based on the properties of the head domain. One such class, class II myosins, consists of the conventional two-headed myosins that form filaments and are composed of two myosin heavy chain (MYH) subunits and four myosin light chain subunits. The MYH subunit contains the ATPase activity providing energy that is the driving force for contractile processes mentioned above, and numerous MYH isoforms exist in vertebrates to carry out this function. The MYHs involved in striated muscle contraction in mammals are the focus of the current review. The genetics, molecular biology, and biochemical properties of mammalian MYHs are discussed below. MYH gene expression patterns in developing and adult striated muscles are described in detail, as are studies of regulation of MYH genes in the heart. The discovery that mutant MYH isoforms have a causal role in the human disease familial hypertrophic cardiomyopathy (FHC) has implemented structure/function investigations of MYHs. The regulation of MYH genes expressed in skeletal muscle and the potential functional implications that distinct MYH isoforms may have on muscle physiology are addressed.

Tropomyosins comprise a family of actin-binding proteins that are central to the control of calcium-regulated striated muscle contraction. To understand the functional role of tropomyosin isoform differences in cardiac muscle, we generated transgenic mice that overexpress striated muscle-specific beta-tropomyosin in the adult heart. Nine transgenic lines show a 150-fold increase in beta-tropomyosin mRNA expression in the heart, along with a 34-fold increase in the associated protein. This increase in beta-tropomyosin message and protein causes a concomitant decrease in the level of alpha-tropomyosin transcripts and their associated protein. There is a preferential formation of the alpha beta-heterodimer in the transgenic mouse myofibrils, and there are no detectable alterations in the expression of other contractile protein genes, including the endogenous beta-tropomyosin isoform. When expression from the beta-tropomyosin transgene is terminated, alpha-tropomyosin expression returns to normal levels. No structural changes were observed in these transgenic hearts nor in the associated sarcomeres. Interestingly, physiological analyses of these hearts using a work-performing model reveal a significant effect on diastolic function. As such, this study demonstrates that a coordinate regulatory mechanism exists between alpha- and beta-tropomyosin gene expression in the murine heart, which results in a functional correlation between alpha- and beta-tropomyosin isoform content and cardiac performance.

The relative potencies of several nicotinic cholinoceptor antagonists in producing tetanic fade and reduction of striated muscle contraction were investigated in the isolated guinea-pig oesophagus as well as the guinea-pig and rat phrenic nerve-diaphragm preparations. Contractile smooth muscle responses to vagal stimulation, which involves ganglionic activation, were also measured simultaneously with striated muscle responses in the oesophagus. The relative potency for inhibiting the response of oesophageal smooth muscle to vagal stimulation (20 Hz) was trimetaphan > mecamylamine > hexamethonium > tubocurarine > pancuronium. For oesophageal striated muscle, production of tetanic fade at 100 Hz and reduction in peak tetanic tension at 20 or 100 Hz showed a similar relative potency; pancuronium > tubocurarine > mecamylamine > trimetaphan > hexamethonium and similar results were obtained in the guinea-pig diaphragm for the antagonists investigated (pancuronium, tubocurarine and mecamylamine). In the rat phrenic nerve-diaphragm preparation, production of tetanic fade at 50 Hz and reduction in twitch or tetanic tension all showed the relative potency; tubocurarine > pancuronium > mecamylamine > trimetaphan > hexamethonium. These findings indicate differences in the nicotinic cholinoceptor subtypes involved in vagal ganglionic responses and those in tetanic fade.

Interactions between troponin C (TnC) and troponin I (TnI) play an important role in the Ca(2+)-dependent regulation of vertebrate striated muscle contraction. Earlier studies have led to the proposal that the "inhibitory region" (residues 96-116) of TnI binds to an alpha-helical segment of TnC comprising residues 89-100 in the nonregulatory, C-terminal domain. Subsequently, on the basis of the results of zero-length cross-linking, we suggested that the inhibitory region of TnI also interacts with the N-terminal, regulatory domain of TnC [Leszyk, J., Grabarek, Z., Gergely, J., & Collins, J. H. (1990) Biochemistry 29, 299-304]. In the present study, we acetylated the epsilon-NH2 groups of the nine lysines of TnC in order to avoid complications which may arise from intramolecular cross-linking between NH2 and COOH groups of TnC. We then activated the COOH groups of acetylated TnC (AcTnC) with 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide and N-hydroxysuccinimide. The activated AcTnC was combined with TnI, and zero-length cross-links were formed between COOH groups in AcTnC and lysine epsilon-NH2 groups in TnI. The cross-linked heterodimer (AcCxI) was cleaved with CNBr and proteases, and the resulting cross-linked peptides were separated by HPLC and then sequenced. Our results show extensive cross-linking between AcTnC and TnI, involving both the N-terminal and C-terminal domains of TnC, as well as the N-terminal, C-terminal, and inhibitory regions of TnI.

Myosin regulatory light chain is phosphorylated by myosin light chain kinase at conserved serine and threonine residues in a number of species. Phosphorylation of myosin regulatory light chain regulates smooth muscle contraction, but appears to have a modulatory role in striated muscle contraction. We assessed the in vivo role of myosin regulatory light chain phosphorylation in the striated muscles of Drosophila melanogaster by substituting alanine at each or both conserved myosin light chain kinase-dependent phosphorylation sites, serine 66 and serine 67. We report here that myosin light chain kinase-dependent phosphorylation is not required for myofibrillogenesis or for the development of maximal isometric force in indirect flight muscles. However, mutants with substitutions at the major phosphorylation site (serine 66) or with the double substitutions had reduced power output in isolated flight muscle fibres and reduced flight ability, showing that myosin regulatory light chain phosphorylation is a key determinant of the stretch activation response in Drosophila.

The steric model of muscle regulation holds that tropomyosin strands running along thin filaments move away from myosin-binding sites on actin when muscle is activated. Exposing these sites would permit actomyosin interaction and contraction to proceed. This compelling and widely cited model is based on changes observed in X-ray diffraction patterns of skeletal muscle following activation. Although analysis of X-ray patterns can suggest models of filament structure, unambiguous interpretation is not possible. In contrast, three-dimensional reconstruction of thin-filament electron micrographs could, in principle, offer direct confirmation of the predicted tropomyosin movement, but so far tropomyosin in skeletal muscle has been resolved definitively only in the 'on' state but not in the 'off' state. Thin filaments from the arthropod Limulus have a similar composition to those from vertebrate skeletal muscle, and troponin-tropomyosin is distributed in both species with the same characteristic 38-nm periodicity. Limulus thin filaments activate skeletal muscle myosin ATPase at micromolar Ca2+ concentrations and confer a high calcium dependence on the enzyme. Arthropod and vertebrate troponin subunits form functional hybrids in vitro and the respective tropomyosins are functionally interchangeable, arguing for a common mechanism of thin-filament-linked regulation in the two phyla. Here we report that tropomyosin is readily resolved in native filaments of troponin-regulated Limulus muscle in both the 'on' and 'off' states, and demonstrate tropomyosin movement, providing support for the importance of steric effects in muscle activation.

When a swallowed liquid bolus is followed from mouth to stomach in man by contrast studies or manometry, it traverses its course without hesitation even though the bolus is propelled by striated muscle contraction in the first part of its journey and smooth muscle in the latter part. The striated muscle is innervated by excitatory cholinergic nicotinic cranial nerves whereas the smooth muscle of the esophagus is innervated by the enteric nervous system (ENS) through excitatory and inhibitory nerves. These differences can be demonstrated by observing the inhibitory effects of curare and atropine, the first blocking nicotinic receptors and the second muscarinic receptors. Early students of esophageal motility recognized that peristalsis could be initiated in two ways. The first is initiated by a swallow and is called primary peristalsis and the second called secondary peristalsis is initiated by distension of the esophagus. It was proposed that primary peristalsis was initiated by a single sensory input activated by the bolus entering the pharynx which in turn activated a motor program in the brain stem. Secondary peristalsis was believed to be stimulated by multiple afferent impulses arriving from the esophagus as the bolus passed down the esophagus. More recent studies using manometric techniques have suggested that the only difference between primary and secondary peristalsis is the afferent stimuli and the effector mechanism is the same. Subsequent studies of carefully timed, paired swallows, transection of vagus nerves and esophagus, and single nerve recordings suggest that the answer lies between the two extremes noted above. Primary peristalsis is initiated by afferent stimuli from the oropharyngeal junction that triggers a stereotyped motor output from swallowing center in the brain stem to both striated and smooth muscle segments of the esophagus. If motor nerves to the striated muscle segment are interrupted no peristalsis can be generated. Severing the central nervous system (CNS) input to the smooth muscle segment does not abolish the ability of local distension to elicit a peristaltic response. Thus the CNS provides direct stimulation to the striated muscles involved in swallowing and command signals to the ENS innervating the smooth muscle. The ENS generates the final peristaltic program for the smooth muscle segment. This program may be initiated by commands from the CNS and afferent stimuli from stretch receptors in the smooth muscle segment of the esophagus.

We analyzed the distribution and labeling patterns of troponin-T, a protein involved in the regulation of striated muscle contraction, in myocardiocytes obtained from chick embryos in Hamburger and Hamilton’s stage 25 and 39, and cultured for 8 days. Troponin-T expression was examined with indirect immunofluorescence, densitometry, fluorescence-activated cell sorting, electrophoresis and immunoblotting. The patterns of expression of troponin-T were compared with those of actin to determine possible correlations in different stages of chick embryo development and culture. Our findings show that in both stages of embryonic development, the cellular accumulation of troponin-T changed after 8 days of culture. Our results revealed a quantitative modification with time: after 4 days of culture there was a significant increase in this protein, followed by a slight additional increase after 8 days of culture. Flow cytometry findings confirmed these trends over time, showing a significant increase in positive cells after 4 days, followed by a smaller rise after 8 days of culture. In comparison with actin, this pattern was similar only in cells from Hamburger and Hamilton’s stage 25 embryos.

Activation of striated muscle contraction is a cooperative process initiated by calcium binding to troponin C (TnC). This is a solid-phase signal transduction process involving 3 states of thin filament: a blocked (B) state preventing myosin interaction, a closed (C) state allowing weak myosin interaction, and an fully activated open or M state. Here we evaluated the influence of ionic strength on Tn dissociation in relationship to myofibril thin filament state. The data we obtained showed that ionic strength had a significant influence on Tn dissociation with low ionic strength increasing Tn dissociation rate from non-overlap region (B or C state) and high ionic strength increasing Tn dissociation rate from overlap region (M state) of myofibril thin filament in both absence and presence of calcium. When ionic strength was decreased to ≤ 50 mM, Tn dissociation from non-overlap region became even faster than overlap region in the absence of calcium, suggesting that actin-tropomyosin in the non-overlap region actually changed from the inhibited B state to the C state at low ionic strength. Also, low ionic strength increased myofibril ATPase activity in the absence of calcium and the calcium-sensitivity of ATPase activity. Based on this and our current studies, we propose a refined kinetic scheme for Tn dissociation from myofibril thin filament, which suggests that the regulatory dissociation of TnI from actin-tropomyosin and association to TnC is not necessarily calcium coupled. Low ionic strength favors the B to C state equilibrium towards the C state (Head et al., Eur. J. Biochem, 227: 694) by favoring TnI dissociation from actin and possibly association with TnC in the absence of calcium. This well explains reported inconsistence between ATPase activity and S1 binding to actin under low ionic strength.

The Mexican axolotl, Ambystoma mexicanum, is an excellent animal model for studying heart development because it carries a naturally occurring recessive genetic mutation, designated gene c, for cardiac nonfunction. The double recessive mutants (c/c) fail to form organized myofibrils in the cardiac myoblasts resulting in hearts that fail to beat. Tropomyosin expression patterns have been studied in detail and show dramatically decreased expression in the hearts of homozygous mutant embryos. Because of the direct interaction between tropomyosin and troponin T (TnT), and the crucial functions of TnT in the regulation of striated muscle contraction, we have expanded our studies on this animal model to characterize the expression of the TnT gene in cardiac muscle throughout normal axolotl development as well as in mutant axolotls. In addition, we have succeeded in cloning the full-length cardiac troponin T (cTnT) cDNA from axolotl hearts. Confocal microscopy has shown a substantial, but reduced, expression of TnT protein in the mutant hearts when compared to normal during embryonic development.

This review summarizes recent work using caged compounds for the study of muscular contraction. We focus on what flash photolysis has taught us about the reaction mechanism and regulation of the actomyosin system of the intact fiber lattice, both during force generation and shortening. An excellent series of complementary reviews is available to the interested reader. The reviews of Lester & Nerbonne (71), Gurney & Lester (50), and McCray & Trentham (75) describe the theory, synthesis, photolysis, and use of caged compounds in detail. For those interested in the crossbridge mechanism, the reviews of Hibberd & Trentham (55) and Goldman (42) include many of the caged experiments, and other topics in muscle contraction are covered by the reviews of Cooke (18), Brenner (14), Homsher (58), and Thomas (94). The development of caged ATP by Kaplan et al (66) led to its first use in the study of muscle contraction in 1 982 (43). Progress from 1 982 to 1 986 was relatively slow for two reasons. First, the synthesis and purification of caged compounds was slow (several weeks) and the yield was low (ca. 25%). Second, the production of a rapid and uniform photolysis of the caged compounds required the use of an expensive (ca. $40,000) laser. In the past 3-4 years, however, two developments have occurred that should make these techniques available to most experimenters. First was the development of a

Inherited myopathies in skeletal and cardiac muscle can result from mutations in troponin or tropomyosin (Michele et al. 2002; Ahmad et al. 2005; Robinson et al. 2007) – components of the thin filament that confer Ca2+ regulation to striated muscle contraction (Gordon et al. 2000). Mechanisms connecting mutation to disease are elusive. The first physiological study on a novel tropomyosin mutation in distal arthrogryposis (DA) provides new and unexpected molecular insights into contractile dysfunction (Ochala et al. 2007). Troponin (Tn) is a complex of three distinct polypeptides (TnT, TnI and TnC), while tropomyosin (Tm) is a dimer of similar or identical polypeptides that form an α-helical coiled-coil. Tm molecules join end-to-end (head-to-tail), with two such strands extending the length of a thin filament (Perry, 2001). Ca2+ binding to Tn triggers contraction by dynamically displacing Tm on actin, exposing sites for myosin cross-bridge formation (Gordon et al. 2000). Tn and Tm mutations often alter Ca2+ sensitivity of contractility. In the heart, severity of familial hypertrophic cardiomyopathy is related to the degree to which Ca2+ sensitivity is increased by mutations (Gomes & Potter, 2004). Adding to this small but growing list of disease-related mutations in thin filament proteins, Tajsharghi et al. (2007) identified a point mutation in the β-Tm gene (TPM2) that correlated with DA type 2B; sequence analyses of genes for troponin subunits I (TNNI2) and T (TNNT3) were normal, and unaffected relatives did not have the TPM2 mutation. The two patients (mother and daughter) exhibited contractures of the distal joints at birth, and weakness as adults in proximal and distal muscles most prominent in the hands and feet (Tajsharghi et al. 2007) (additional details about DA type IIB are available in the Online Mendelian Inheritance in Man (OMIM) database entry 601680). Missense mutation R133W is near the middle of the Tm molecule, in the region coded for by exon 4 that is common to all alternatively spliced variants of β-Tm; splice variants of β-Tm transcript are found not only in skeletal muscle along with α-Tm (TPM1), but also in smooth muscle and fibroblasts (Perry, 2001). The R133W mutation removes a charge predicted to stabilize Tm's α-helical coiled-coil by its location at the g position of the seven-residue structural repeat (Tajsharghi et al. 2007). Based on this predicted destabilization of structure and the clinical observation of muscle hypercontraction at birth, β-Tm R133W would be expected to increase Ca2+ sensitivity of contractility and, perhaps, permit actomyosin interactions at nominally ‘relaxing’ Ca2+ levels; such results have been obtained from in vitro assays with four other DA mutations in Tn or β-Tm (Robinson et al. 2007). In this issue of The Journal of Physiology, Ochala et al. (2007) report that, surprisingly, there is no change in Ca2+ regulation of isometric force in slow type I muscle fibres – the fibre type with the highest proportion of β-Tm – biopsied from the two DA patients reported by Tajsharghi et al. (2007) compared with six unrelated control subjects. Both type I and type IIa fibres from the patients generally expressed less β-Tm (and more α-Tm), although this difference did not achieve significance. In type I fibres at saturating Ca2+, there were significant changes in force per cross-sectional area (decrease), velocity of unloaded shortening (VU; increase), and rate of tension redevelopment (kTR; decrease). These measurements point toward the R133W mutation in β-Tm increasing actomyosin cross-bridge detachment rate while decreasing attachment rate; motility assays with unregulated actin demonstrated that kinetic changes could not be explained by a modification of myosin function alone. Rather than altering Ca2+ sensitivity, this Tm mutation affects cross-bridge kinetics. Additional observations support the idea that Tn and Tm can modulate actomyosin function both in isometric and unloaded biomechanical assays (Gordon et al. 1997; Schoffstall et al. 2006; Moreno-Gonzalez et al. 2007). Muscle weakness experienced by the adult DA patients can be explained by a kinetically determined reduction in the proportion of strong binding cross-bridges. The elegant experiments of Ochala et al. (2007) raise new questions. Does the β-Tm R133W mutation in fact cause changes in Ca2+ sensitivity that are masked by other factors in the affected adults? What causes the progression from hypercontractility at birth to muscle weakness in adulthood; are normal developmental changes responsible, or are there adaptive changes within the muscle fibres? And what is the mechanism by which the β-Tm mutation affects actomyosin kinetics, particular the faster VU at saturating Ca2+? Perhaps destabilization of Tm structure alters its flexural rigidity. While it is tempting to speculate that such a change in Tm mechanics could alter its interactions with cross-bridges (Gordon et al. 2000), and possibly superactivation of actomyosin function (Gordon et al. 1997), these are certainly not the only possibilities. Now that we know the effects of β-Tm R133W in muscle fibres from patients, answers to these and other questions await in vitro experiments where the proportions of α- versus β-Tm, and homo- versus hetero-dimers, along with other factors, can be experimentally controlled.

Many studies have been conducted analyzing the manometric properties of patients with achalasia, but the striated portion of the esophagus has never been analyzed and is often overlooked. We retrospectively reviewed 120 manometric tracings (20 achalasia, 100 controls) performed between 1994 and 1997 and excluded tracings from patients with chronic cough and nutcracker esophagus. The data were assessed for age, sex, symptoms, duration of symptoms, lower esophageal sphincter pressure, gastroesophageal gradient, upper esophageal sphincter pressure, smooth muscle contraction amplitude and duration, striated muscle contraction amplitude and duration, length from upper esophageal sphincter to maximal striated muscle contraction, and esophageal length. The maximum striated muscle contraction amplitude was significantly decreased in achalasia patients with a median amplitude of 45 mm Hg (range 12-95) vs 76 mm Hg (range 30-210) in the control group (P = 0.002). Although the wave forms were similar, the maximum striated muscle contraction duration and the distance from the upper esophageal sphincter in achalasia patients was not significantly different from controls. The length of the esophagus was significantly longer in achalasia patients with a median value of 25 cm (range 21-30) vs 21 cm (range 17-26) in the control group (P < 0.001). Patients with achalasia have significantly lower maximum striated muscle contraction amplitudes and longer esophagi, but the duration of the contractions and the configuration of the wave forms are not different.

The esophagus is a region with three functional zones: (1) the upper esophageal sphincter; (2) the esophageal body; and (3) the lower esophageal sphincter. Control mechanisms within the central nervous system and peripherally serve to integrate these functional zones in a region where voluntary and involuntary control mechanisms and the activity of two different types of muscle are intimately coordinated. The distal 50 to 60 per cent of the esophagus in humans is entirely smooth muscle. Extrinsic control for esophageal motor function resides in a brainstem swallowing center with an afferent reception system, an efferent system of motor neurones, and a complex organizing or internuncial system of neurones. Sensory information from the esophagus is carried in the vagus nerves, but sensory pathways are also present in sympathetics entering the spinal cord. The vagus nerve receiving fibers both from the nucleus ambiguus and the dorsal motor nucleus innervates the striated and smooth muscle esophagus, respectively, including the sphincters. There is a myenteric nerve plexus in both the striated and smooth muscle segments. In the smooth muscle esophagus, there are two important effector neurones, an excitatory cholinergic neurone, and a nonadrenergic, noncholinergic (NANC) inhibitory neurone. The striated muscle contraction is directed and coordinated by sequential excitation through vagal fibers programmed by the central control mechanism. There are at least four potential control mechanisms for peristalsis in the smooth muscle esophagus: efferent motor fibers programmed by the swallowing center fire sequentially during peristalsis; the intramural neural mechanism can be excited to produce peristalsis near the onset of stimulation or with a delay after termination of stimulation; there is evidence for myogenic propagation of a peristaltic contraction. In humans, swallow-induced peristalsis is cholinergic and appears to result primarily from sequencing and activation of the intramural excitatory cholinergic neurones. Both central and peripheral levels of control are highly integrated to focus on the excitatory cholinergic neurones. It is likely that under normal circumstances, the central control mechanism exerts the dominant influence on these neurones for initiation and coordination of peristalsis in the smooth muscle esophagus. In humans, resting tone in the lower esophageal sphincter is predominantly cholinergic, but this tone is regulated by a balance between many excitatory and inhibitory influences. The relaxation on swallowing is caused by active inhibition of the muscle through NANC inhibitory neurones and cessation of tonic neural excitation to the

Most current textbooks of cell biology and histology use the steric blocking model to describe the protein mechanism by which vertebrate striated muscle contraction is regulated. Evidence accumulated in the past decade, however, reveals the regulation of muscle contraction to be far more complex than this model predicts.