Regulation of contraction in striated muscle

Ca(2+) 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 (A(7)TmTn) regulatory unit. Tm position on the actin filament is regulated by the occupancy of NH-terminal Ca(2+) binding sites on TnC, conformational changes resulting from Ca(2+) binding, and changes in the interactions among Tn, Tm, and actin and as well as by strong S1 binding to actin. Ca(2+) 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 Ca(2+)-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 A(7)TmTn regulatory units. The structural and biochemical findings support the physiological observations of steady-state and transient mechanical behavior. Physiological studies suggest the following. 1) Ca(2+) binding to Tn/Tm exposes sites on actin to which myosin can bind. 2) Ca(2+) regulates the strong binding of M.ADP.P(i) to 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 Ca(2+) 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 A(7)TmTn 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 ([Ca(2+)]) 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 Ca(2+) 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. (ABSTRACT TRUNCATED)     

The size and the speed of the working stroke of muscle myosin and its dependence on the force

Myosin II is the motor protein that produces force and shortening in muscle by ATP-driven cyclic interactions of its globular portion, the head, with the actin filament. During each interaction the myosin head undergoes a conformational change, the working stroke, which, depending on the mechanical conditions, can generate a force of several piconewtons or an axial displacement of the actin filament toward the centre of the sarcomere of several nanometres. However, the sizes of the elementary force and length steps and their dependence on the mechanical conditions are still under question. Due to the small fraction of the ATPase cycle time myosin II spends attached to actin, single molecule mechanics failed to produce definitive measurements of the individual events. In intact frog muscle fibres, however, myosin II's working stroke can be synchronised in the few milliseconds following a step reduction in either force or length superimposed on the isometric contraction. Here we show that with 150 μs force steps it is possible to separate the elastic response from the subsequent early rapid component of filament sliding due to the working stroke in the attached myosin heads. In this way we determine how the size and the speed of the working stroke depend on the clamped force. The relation between mechanical energy and force provides a molecular basis for muscle efficiency and an estimate of the isometric force exerted by a myosin head.     

Second harmonic imaging of intrinsic signals in muscle fibers in situ

We use second harmonic generation (SHG) imaging to study and quantify a strong intrinsic SHG signal in skeletal muscle fiber preparations and single isolated myofibrils. The intrinsic signal follows the striation pattern of the muscle cells and is positioned at the sarcomeric location of the myosin filaments. Interestingly, the signal is enhanced at the region where the myosin heads are located on the myosin filaments. As the intrinsic signal reflects the subcellular structure in an accurate way, SHG can be used for noninvasive high resolution structural imaging without exogenous labels in living muscle cells. This may be very important for detecting changes in myofibrillar organization occurring under pathophysiological conditions, e.g., in cardiac and skeletal myopathies. Due to the strong dependency of SHG on orientation and symmetries of the tissue, it may allow the study of dynamic interactions between the contractile proteins actin and myosin during force production and muscle shortening. Furthermore, SHG imaging can be combined with other nonlinear microscopical techniques, such as laser scanning multiphoton fluorescence microscopy, to simultaneously measure other dynamic cellular processes, representing a complementary method and extending the range of nonlinear microscopical methods.     

Functional heterogeneity of mammalian single muscle fibres: do myosin isoforms tell the whole story?

The large amount of data published in the last 10-15 years indicate that myosin isoforms are the major determinant of the large functional heterogeneity of the key contractile and biochemical properties of skeletal muscle fibres, including velocity of shortening, ATP consumption and power. Recent evidences are difficult to reconcile with such an idea and suggest that the properties of muscle fibres that are likely to depend on myosin, such as velocity of shortening, can change without a change in myosin isoform. That a given myosin isoform can modify its properties without shifting to another isoform is confirmed by some analyses of isolated myosin in vitro. The present review is mainly focused on findings that challenge the role of myosin isoforms in determining the functional heterogeneity of skeletal muscle. The work also reports on potential mechanisms behind such changes in myosin function independent of a shift in myosin isoform: the coexistence of different myosin heavy chain (MHC) isoforms in the same fibre, the existence of as yet undetected MHC isoforms, myosin light chain isoforms, post-translational modifications of myosin, the role of other myofibrillar proteins, geometry of the sarcomere and the myosin concentration in single fibres.     

Activation of skinned muscle fibres from the Norway lobster Nephrops norvegicus L. by manganese ions

Effects of Mn2+ and Ca2+ on the mechanical properties of glycerinated myofibrillar bundles originating from slow S1 type muscle fibres of superficial flexor muscles of the lobster Nephrops norvegicus were investigated. Mn2+ (5–20μm) activated the preparations in a dose-dependent manner. The sensitivity of myofibrillar force generation for Mn2+ was around 30 times lower than that for Ca2+. The maximal tension produced under Mn2+ activation was about 75% of that under Ca2+ activation. At higher free Mn2+ concentrations (>2mm), the steady-state force decreased; it was completely abolished at 30mm free Mn2+. These high Mn2+ solutions were accompanied by changes in MgATP and MnATP concentrations, and in the ionic strength. Control experiments have shown that none of these parameters seemed fo account fully for the observed force depression in high Mn2+ solutions. It is likely that direct effects of Mn2+ such as a change of the myofilament surface charges are responsible. The maximal unloaded shortening velocity of the myofibrillar preparations was shown to be similar under maximal Mn2+ and Ca2+ activation. Conversely, the kinetics of stretch-induced delayed force increase were about two to three times faster under Mn2+ activation. These results suggest that certain steps of the cross-bridge cycle depend on the ion species bound to the regulatory proteins. This revised version was published online in July 2006 with corrections to the Cover Date.     

Radial mass transfer of cross-bridges in a tetanized ferret heart muscle

An X-ray diffraction experiment on ferret heart muscle was made to examine the relationship between tension and mass transfer from the thick to the thin filament associated with the interaction of cross-bridges with actin. A ferret papillary muscle was electrically stimulated for 8 s in the presence of 5 microM ryanodine to give a tetanus. At different extracellular Ca2+ concentrations (2-20 mM), a linear relationship was found between the tension and the mass transfer. It was concluded that a change in tension caused by altering the extracellular Ca2+ concentration is due to a change in the number of myosin heads bound to actin. This is in contrast to the results obtained on skinned preparations, which showed a markedly nonlinear relationship between the number of heads in the vicinity of the thin filaments and force. It was found that the tension per myosin head is similar in twitch and tetanus of cardiac muscle. Also, a similar fraction of myosin heads is recruited in tetanus of cardiac and skeletal muscles.     

Myofibrillar myopathy caused by a mutation in the motor domain of mouse MyHC IIb

Ariel is a mouse mutant that suffers from skeletal muscle myofibrillar degeneration due to the rapid accumulation of large intracellular protein aggregates. This fulminant disease is caused by an ENU-induced recessive mutation resulting in an L342Q change within the motor domain of the skeletal muscle myosin protein MYH4 (MyHC IIb). Although normal at birth, homozygous mice develop hindlimb paralysis from Day 13, consistent with the timing of the switch from developmental to adult myosin isoforms in mice. The mutated myosin (MYH4(L342Q)) is an aggregate-prone protein. Notwithstanding the speed of the process, biochemical analysis of purified aggregates showed the presence of proteins typically found in human myofibrillar myopathies, suggesting that the genesis of ariel aggregates follows a pathogenic pathway shared with other conformational protein diseases of skeletal muscle. In contrast, heterozygous mice are overtly and histologically indistinguishable from control mice. MYH4(L342Q) is present in muscles from heterozygous mice at only 7% of the levels of the wild-type protein, resulting in a small but significant increase in force production in isolated single fibres and indicating that elimination of the mutant protein in heterozygotes prevents the pathological changes observed in homozygotes. Recapitulation of the L342Q change in the functional equivalent of mouse MYH4 in human muscles, MYH1, results in a more aggregate-prone protein.     

Comparative force-velocity relation and analyses of myosin of dog atria and ventricles

The isolated muscle and purified myofibrillar proteins of canine atria and ventricles were compared relative to force-velocity relations and rate of adenosine 5'-triphosphatase (ATPase) activity as a function of calcium concentrations. The maximal stress development of isolated trabeculae of canine atria was similar to that of canine right ventricular papillary muscles when analyzed at saturating calcium concentrations (7.5 mM); however, stress was less in the atria when studied at normal calcium concentrations (2.5 mM). The maximal velocity of shortening of atrial trabeculae was about 2.3 times higher than that of ventricular muscle. Regulated actomyosin characterized from the myofibrillar proteins of the two tissues gave directionally similar calcium sensitivity. The maximum velocity of shortening for actin-activated atrial myosin of the dog was approximately 1.8 times higher when the latter was analyzed as a function of actin concentration. Both maximal tension of isolated muscle and regulated actomyosin ATPase activity were dependent on calcium concentration.     

Tuning the human heart molecular motors by myosin light chains

Cardiac contraction is triggered by the cyclic interaction of the "molecular motor" protein myosin with the actin filament, consuming ATP as the energy source to produce tension or shortening. The myosin heavy chain (MHC) contains the actin- and ATP-binding sites and represents the molecular motor of muscle contraction. This review describes the various subunits of human heart myosin in health and disease and discusses their functions. Two different MHC genes (alpha and beta) with distinct biochemical features are expressed in the human heart. Alpha-MHC confers a higher ATPase activity and higher shortening velocity to the heart than beta-MHC. Motor function is regulated by myosin light chain (MLC) isoforms. Expression of the atrial MLC-1 isoform in the hypertrophied human ventricle increases cross-bridge cycling and contractility. It is suggested that MLC-1 acts as a MHC/actin tether. Weakening of this tether increases myosin function. MLC-2 slows the rate of tension development of myosin. This relative inhibition is relieved upon phosphorylation of the MLC-2 perhaps caused by "swing-out" of cross-bridges from the myosin filament. Mutations in all ventricular myosin subunits have been found in patients with hypertrophic cardiomyopathy.     

The myogenic response in isolated rat cerebrovascular arteries: smooth muscle cell model

Previous models of the cerebrovascular smooth muscle cell have not addressed the interaction between the electrical, chemical, and mechanical components of cell function during the development of active tension. These models are primarily electrical, biochemical or mechanical in their orientation, and do not permit a full exploration of how the smooth muscle responds to electrical or mechanical forcing. To address this issue, we have developed a new model that consists of two major components: electrochemical and chemomechanical subsystem models of the smooth muscle cell. Included in the electrochemical model are models of the electrophysiological behavior of the cell membrane, fluid compartments, Ca2+ release and uptake by the sarcoplasmic reticulum (SR), and cytosolic Ca2+ buffering, particularly by calmodulin (CM). With this subsystem model, we can study the mechanics of the production of intracellular Ca2+ transient in response to membrane voltage clamp pulses. The chemomechanical model includes models of: (a) the chemical kinetics of myosin phosphorylation, and the formation of phosphorylated (cycling) myosin cross-bridges with actin, as well as attached (non-cycling) latch-type cross-bridges; and (b) a model of force generation and mechanical coupling to the contractile filaments and their attachments to protein structures and the skeletal framework of the cell. The two subsystem models are tested independently and compared with data. Likewise, the complete (combined) cell model responses to voltage pulse stimulation under isometric and isotonic conditions are calculated and compared with measured single cell length-force and force-velocity data obtained from literature. This integrated cell model provides biophysically based explanations of electrical, chemical, and mechanical phenomena in cerebrovascular smooth muscle, and has considerable utility as an adjunct to laboratory research and experimental design.     

Calcium ion in skeletal muscle: its crucial role for muscle function, plasticity, and disease

Mammalian skeletal muscle shows an enormous variability in its functional features such as rate of force production, resistance to fatigue, and energy metabolism, with a wide spectrum from slow aerobic to fast anaerobic physiology. In addition, skeletal muscle exhibits high plasticity that is based on the potential of the muscle fibers to undergo changes of their cytoarchitecture and composition of specific muscle protein isoforms. Adaptive changes of the muscle fibers occur in response to a variety of stimuli such as, e.g., growth and differentition factors, hormones, nerve signals, or exercise. Additionally, the muscle fibers are arranged in compartments that often function as largely independent muscular subunits. All muscle fibers use Ca(2+) as their main regulatory and signaling molecule. Therefore, contractile properties of muscle fibers are dependent on the variable expression of proteins involved in Ca(2+) signaling and handling. Molecular diversity of the main proteins in the Ca(2+) signaling apparatus (the calcium cycle) largely determines the contraction and relaxation properties of a muscle fiber. The Ca(2+) signaling apparatus includes 1) the ryanodine receptor that is the sarcoplasmic reticulum Ca(2+) release channel, 2) the troponin protein complex that mediates the Ca(2+) effect to the myofibrillar structures leading to contraction, 3) the Ca(2+) pump responsible for Ca(2+) reuptake into the sarcoplasmic reticulum, and 4) calsequestrin, the Ca(2+) storage protein in the sarcoplasmic reticulum. In addition, a multitude of Ca(2+)-binding proteins is present in muscle tissue including parvalbumin, calmodulin, S100 proteins, annexins, sorcin, myosin light chains, beta-actinin, calcineurin, and calpain. These Ca(2+)-binding proteins may either exert an important role in Ca(2+)-triggered muscle contraction under certain conditions or modulate other muscle activities such as protein metabolism, differentiation, and growth. Recently, several Ca(2+) signaling and handling molecules have been shown to be altered in muscle diseases. Functional alterations of Ca(2+) handling seem to be responsible for the pathophysiological conditions seen in dystrophinopathies, Brody's disease, and malignant hyperthermia. These also underline the importance of the affected molecules for correct muscle performance.     

Stretch activation and isoforms of myosin heavy chain and troponin-T of rat skeletal muscle fibres

Recent studies on single mammalian skeletal muscle fibres revealed a correlation between the kinetics of stretch-induced delayed force increase (stretch activation) and the isoforms of the myosin heavy chain. This observation suggests a causal relation between stretch activation and myosin heavy chain. However, the assumption is weakened by the fact that isoforms of other myofibrillar proteins tend to be coexpressed with myosin heavy chain isoforms. The relation between the isoforms of the tropomyosin-binding troponin subunit and myosin heavy chain is unknown. For a variety of reasons, tropomyosin-binding troponin subunit is a possible candidate for being involved in stretch activation. Therefore, we measured stretch activation of single, maximally Ca2+-activated skinned rat skeletal muscle fibres and characterized them by their myosin heavy chain composition, as well as by the isoform species of tropomyosin-binding troponin subunit. Four myosin heavy chain isoforms (I, IIa, IId or IIx and IIb) and six tropomyosin-binding troponin subunit isoforms (TnT1s, TnT2s, TnT1f, TnT2f, TnT3f, TnT4f) were distinguis hed. The following preferential coexpression patterns of the myosin heavy chain and tropomyosin-binding troponin subunit isoforms were observed: MHCI-TnT1s, MHCIIa-TnT3f, MHCIId-TnT1f, and MHCIIb-TnT4f. Stretch activation kinetics was found to be correlated with the myosin heavy chain isoform complement also in fibres not displaying one of the preferential MHC-TnTf isoform coexpression patterns. This corroborates the assumption of a causal relation between myosin heavy chain and stretch activation This revised version was published online in July 2006 with corrections to the Cover Date.     

The role of sarcoplasmic reticulum in relaxation of mouse muscle; effects of 2,5-di(tert-butyl)-1,4-benzohydroquinone.

1. Intracellular calcium concentration ([Ca2+]i) and force were measured from isolated single mouse skeletal muscle fibres at rest and during tetani. The actions of 2,5-di(tert-butyl)-1,4-benzohydroquinone (TBQ), an inhibitor of the sarcoplasmic reticulum (SR) Ca2+ pump, were examined at a range of concentrations (100-1000 nM). 2. TBQ increased resting [Ca2+]i, increased tetanic [Ca2+]i and slowed the rate of decline of [Ca2+]i after a tetanus. TBQ produced a small increase in tetanic force and a large slowing of the rate of relaxation after a tetanus. All these effects were reversible. 3. TBQ had no important effects on the Ca2+ sensitivity or the maximum force produced by the myofibrillar proteins. 4. Analysis of the SR Ca2+ pump function confirmed that under control conditions and at very low levels of [Ca2+]i, the relationship between [Ca2+]i and SR pump rate was a 4th power function. TBQ caused a pronounced inhibition of the pump rate and reduced the power function to < 3. 5. Muscle fibres were fatigued by repeated tetani until tetanic [Ca2+]i and force were reduced and the rate of decline of [Ca2+]i after a tetanus was slowed. Under these conditions application of TBQ caused a further slowing of the rate of decline of [Ca2+]i but still increased tetanic [Ca2+]i and force. This result suggests that slowing of the SR pump rate is not the cause of the decline in tetanic [Ca2+]i and force at the late stage of fatigue. 6. A simple model of the interactions of Ca2+, TBQ and pump proteins is described, which predicts the 4th power function of the normal pump, inhibition by TBQ, and the reduced power function in the presence of TBQ. 7. A model of Ca2+ movements and force development in muscle is described, which closely matches the experimental results under control conditions. Inhibition of the SR pump by TBQ using the model of the pump described above simulates qualitatively all the observed effects of TBQ on [Ca2+]i and force. 8. In conclusion, TBQ is a potent, specific and reversible inhibitor of the SR Ca2+ pump in intact mouse skeletal muscle. Inhibition of the pump directly affects intracellular Ca2+ handling and force production.     

Distribution of isoforms of the myofibrillar proteins in myoid cells of thymus

Summary Myoid cells of calf and rat thymus have been identified by staining with a monoclonal antibody to the heavy chain of myosin that is not isoform specific. Heterogeneity in the protein composition of myoid cells has been demonstrated by staining with antibodies to the skeletal muscle isoforms of the myosin heavy chain, C-protein and components of the troponin complex. The immunochemical studies suggest that the myoid cells contain proteins closely resembling if not identical with those present in the myofibrils of skeletal muscle. The slow and fast skeletal muscle isoforms of the myofibrillar proteins are present in a large proportion of the myoid cells. A fraction of the myoid cells contains only the fast isoforms of the myofibrillar proteins but there is no sharp compartmentalization of the isoforms as occurs in type 1 and type 2 fibres of skeletal muscle. In general the pattern of gene expression is similar to that of developing skeletal muscle.     

Different effects of cardiac versus skeletal muscle regulatory proteins on in vitro measures of actin filament speed and force

Mammalian cardiac and skeletal muscle express unique isoforms of the thin filament regulatory proteins, troponin (Tn) and tropomyosin (Tm), and the significance of these different isoforms in thin filament regulation has not been clearly identified. Both in vitro and skinned cellular studies investigating the mechanism of thin filament regulation in striated muscle have often used heterogeneous mixtures of Tn, Tm and myosin isoforms, and variability in reported results might be explained by different combinations of these proteins. Here we used in vitro motility and force (microneedle) assays to investigate the influence of cardiac versus skeletal Tn and Tm isoforms on actin–heavy meromyosin (HMM) mechanics. When interacting with skeletal HMM, thin filaments reconstituted with cardiac Tn/Tm or skeletal Tn/Tm exhibited similar speed–calcium relationships and significantly increased maximum speed and force per filament length ( F / l ) at pCa 5 ( versus unregulated actin filaments). However, augmentation of F / l was greater with skeletal regulatory proteins. Reconstitution of thin filaments with the heterogeneous combination of skeletal Tn and cardiac Tm decreased sliding speeds at all [Ca²⁺] relative to thin filaments with skeletal Tn/Tm. Finally, for filaments reconstituted with any heterogeneous mix of Tn and Tm isoforms, force was not potentiated over that of unregulated actin filaments. Combined the results suggest (1) that cardiac regulatory proteins limit the allosteric enhancement of force, and (2) that Tn and Tm isoform homogeneity is important when studying Ca²⁺ regulation of crossbridge binding and kinetics as well as mechanistic differences between cardiac and skeletal muscle.     

Ca2+-activation characteristics of single fibres from chemically skinned rat muscle incubated with glucose-6-phosphate

In this study we examined the effects of 3-24 h of incubation of chemically skinned rat fast-twitch muscle with the glycolytic metabolite glucose 6-phosphate (G6-P) on the contractile properties and myosin ATPase activity in single muscle fibres, and on the carbohydrate content of myosin heavy chains (MHCs). Exposure of the permeabilised muscle to 10 mM G6-P for 24 h at 22+/-1 degrees C in a rigor solution containing protease inhibitors and a reducing agent (dithiothreitol, DTT) significantly decreased maximum Ca(2+)-activated force output by 31%, lowered the Ca2+ threshold for contraction by 0.1 pCa units and produced shallower force-pCa curves compared with controls. Furthermore, under these conditions, G6-P-treated muscle displayed lower myofibrillar MgATPase activity and a markedly higher carbohydrate content of MHCs, as identified with an immunoblot protocol for glycoprotein detection. Shorter incubations under the same conditions or 24-h incubations with 5 mM G6-P generally resulted in smaller changes in the contractile activation parameters. These findings suggest that reducing sugars acting as metabolic intermediates in the glycolytic pathway can have important non-energy-related effects on the contractile activation characteristics of mammalian skeletal muscle. These effects are consistent with the glycation of muscle proteins, in particular that of the MHC.     

Intracellular effects of nitric oxide on force production and Ca2+ sensitivity of cardiac myofilaments

The gaseous free radical nitric oxide (NO*) has been implicated in a wide range of physiological functions and also has a role in the pathogenesis of cellular injury. It has been suggested that NO* and its congeners may exert effects on actin-myosin crossbridge cycling by modulating critical thiols on the myosin head. To understand the mode and site of actin of NO* in myofibrils, the effects of the NO* donor 3-(2-hydroxy-1-methyl-2-nitrosohydrazine)-N-methyl-1-propanamine (NOC-7) have been studied in Triton X-100-treated rabbit cardiac fibers, in which isometric force was measured at controlled degrees of activation. Experiments were undertaken after previous exposure of the preparations to NOC-7 (for 30 min). We found that NO* induced several alterations of myofibrillar function, i.e., decrease in Ca2+ sensitivity and Hill coefficient and potentiation of rigor contracture. We attributed the effect on rigor contracture to strong inhibition of myofibrillar creatine kinase (CK) activity, because it could be prevented by exogeneously added CK; such CK inactivation afforded by NO* may result in the myofibrillar ATP-to-ADP ratio. In further experiments, concentration of NO* released from NOC-7 was determined by the electron spin resonance spin-trapping technique; N-(dithiocarboxy)sarcosine-Fe2+ complex was used as the spin-trap. NO* at cumulative concentration of 0.69 microM was effective in producing both enhancement of rigor contracture and decrease of myofibrillar-bound CK activity; however, Ca2+-sensitivity (pCa50) was significantly decreased at >5.6 microM of NO*, suggesting a result from different mechanisms. Thus, the observed decrease in Ca2+ sensitivity seems to be associated with direct modification of the regulatory proteins by a relatively higher concentration of NO*, and possibly not via inhibition of myofibrillar CK activity. The data reported here indicate that CK may be a pathophysiologically main target for increased NO* formation at low molecular range in the disease state in cardiac muscle.     

The thin filaments of smooth muscles

Summary Contraction in vertebrate smooth and striated muscles results from the interaction of the actin filaments with crossbridges arising from the myosin filaments. The functions of the actin based thin filaments are (1) interaction with myosin to produce force; (2) regulation of force generation in response to Ca²⁺ concentration; and (3) transmission of the force to the ends of the cell. The major protein components of smooth muscle thin filaments are actin, tropomyosin and caldesmon, present in molar ratios of 28:4:1 respectively. Other smooth muscle proteins which may be associated with the thin filaments in the cell are filamin, vinculin, -actinin, myosin light chain kinase and calmodulin. We have reviewed the structural and functional properties of these proteins and where possible we have suggested what their function and mechanism of action may be. We propose that actin and tropomyosin are involved in the force producing interaction with myosin, and that this interaction is controlled by a Ca²⁺-dependent mechanism involving caldesmon, tropomyosin and calmodulin. Vinculin, -actinin and filamin appear to be involved in the attachment of the thin filaments to the cell membrane and their spatial organization within the cell. We conclude that the filaments of smooth muscles share many common properties with those from skeletal muscle, but that they are also quite distinct in terms of both their caldesmon based regulatory mechanism and their mode of organization into a contractile apparatus.     

Smooth,cardiac and skeletal muscle myosin force and motion generation assessed by cross-bridge mechanical interactions in vitro

Summary Differences in the mechanical properties of mammalian smooth, skeletal, and cardiac muscle have led to the proposal that the myosin isozymes expressed by these tissues may differ in their molecular mechanics. To test this hypothesis, mixtures of fast skeletal, V1 cardiac, V3 cardiac and smooth muscle (phosphorylated and unphosphorylated) myosin were studied in an in vitro motility assay in which fluorescently-labelled actin filaments are observed moving over a myosin coated surface.Pure populations of each myosin produced actin filament velocities proportional to their actin-activated ATPase rates. Mixtures of two myosin species produced actin filament velocities between those of the faster and slower myosin alone. However, the shapes of the myosin mixture curves depended upon the types of myosins present. Analysis of myosin mixtures data suggest that: (1) the two myosins in the mixture interact mechanically and (2) the same force-velocity relationship describes a myosin's ability to operate over both positive and negative forces. These data also allow us to rank order the myosins by their average force per cross-bridge and ability to resist motion (phosphorylated smooth > skeletal = V3 cardiac > V1 cardiac). The results of our study may reflect the mechanical consequence of multiple myosin isozyme expression in a single muscle cell.     

Why choose myofibrils to study muscle myosin ATPase?

Our objective is to propose an overview of the usefulness of skeletal myofibril as an experimental system for studying mechanochemical coupling of skeletal muscles and myosin ATPase activity. The myofibril is a true functional mini-muscle that is able to contract in the presence of ATP. It also contains the machinery necessary for the calcium sensitivity of the contraction. In the absence of calcium, myofibrillar ATPase activity is basal, no shortening occurs and no active force is developed. In the presence of calcium, myofibrillar ATPase is activated and myofibrils either shorten with no external load (native myofibrils) or contract isometrically (cross-linked myofibrils). With this organised system, both chemical and mechanical studies can be carried out. For a decade, our laboratory has been using rabbit psoas myofibrils for exploring myosin ATPase activity. The first challenge was to successfully apply rapid kinetic approaches, such as rapid-flow-quench, to this organised system. Another challenge was to work with myofibrils in cryoenzymic conditions, i.e. in the presence of organic solvents and at sub-zero temperatures. In this overview, we highlight differences between the myosin ATPase in organised systems (myofibrils or fibres) and that of contractile proteins in solution (S1 or actoS1) that we observed using these approaches. We discuss the importance of these differences in terms of mechanochemical coupling. It is concluded that great care should be taken when extrapolating mechanochemical properties of the contractile proteins in solution to the whole muscle. This revised version was published online in August 2006 with corrections to the Cover Date.