Predominant myosin superrelaxed state in canine myocardium with naturally occurring dilated cardiomyopathy.

Dilated cardiomyopathy (DCM) is a naturally occurring heart failure condition in humans and dogs, notably characterized by a reduced contractility and ejection fraction. As the identification of its underlying cellular and molecular mechanisms remain incomplete, the aim of the present study was to assess whether the molecular motor myosin and its known relaxed conformational states are altered in DCM. For that, we dissected and skinned thin cardiac strips from left ventricle obtained from six DCM Doberman Pinschers and six nonfailing (NF) controls. We then used a combination of Mant-ATP chase experiments and X-ray diffraction to assess both energetic and structural changes of myosin. Using the Mant-ATP chase protocol, we observed that in DCM dogs, the amount of myosin molecules in the ATP-conserving conformational state, also known as superrelaxed (SRX), is significantly increased when compared with NF dogs. This alteration can be rescued by applying EMD-57033, a small molecule activating myosin. Conversely, with X-ray diffraction, we found that in DCM dogs, there is a higher proportion of myosin heads in the vicinity of actin when compared with NF dogs (1,0 to 1,1 intensity ratio). Hence, we observed an uncoupling between energetic (Mant-ATP chase) and structural (X-ray diffraction) data. Taken together, these results may indicate that in the heart of Doberman Pinschers with DCM, myosin molecules are potentially stuck in a nonsequestered but ATP-conserving SRX state, that can be counterbalanced by EMD-57033 demonstrating the potential for a myosin-centered pharmacological treatment of DCM.NEW & NOTEWORTHY The key finding of the present study is that, in left ventricles of dogs with a naturally occurring dilated cardiomyopathy, relaxed myosin molecules favor a nonsequestered superrelaxed state potentially impairing sarcomeric contractility. This alteration is rescuable by applying a small molecule activating myosin known as EMD-57033.

Structure of the Flight Muscle Thick Filament from the Bumble Bee, Bombus ignitus, at 6 Å Resolution.

Four insect orders have flight muscles that are both asynchronous and indirect; they are asynchronous in that the wingbeat frequency is decoupled from the frequency of nervous stimulation and indirect in that the muscles attach to the thoracic exoskeleton instead of directly to the wing. Flight muscle thick filaments from two orders, Hemiptera and Diptera, have been imaged at a subnanometer resolution, both of which revealed a myosin tail arrangement referred to as "curved molecular crystalline layers". Here, we report a thick filament structure from the indirect flight muscles of a third insect order, Hymenoptera, the Asian bumble bee Bombus ignitus. The myosin tails are in general agreement with previous determinations from Lethocerus indicus and Drosophila melanogaster. The Skip 2 region has the same unusual structure as found in Lethocerus indicus thick filaments, an α-helix discontinuity is also seen at Skip 4, but the orientation of the Skip 1 region on the surface of the backbone is less angled with respect to the filament axis than in the other two species. The heads are disordered as in Drosophila, but we observe no non-myosin proteins on the backbone surface that might prohibit the ordering of myosin heads onto the thick filament backbone. There are strong structural similarities among the three species in their non-myosin proteins within the backbone that suggest how one previously unassigned density in Lethocerus might be assigned. Overall, the structure conforms to the previously observed pattern of high similarity in the myosin tail arrangement, but differences in the non-myosin proteins.

Rapid recovery of phytic acid from rice brans using chitosan nanofiber-based porous hydrogels.

Phytic acid (PA) is a new type of naturally occurring pharmaceutical for afflictions such as cancer, diabetes, and renal calculi. The efficient, low-cost extraction of PA from biowaste is much sought after. Herein, highly pure PA was obtained from rice bran by adsorption at low pH onto porous chitosan nanofiber hydrogels. Due to the large surface area of the chitosan nanofiber-based porous hydrogels, the adsorption equilibrated within 60 min. Adsorption of PA was influenced by the buffer pH, temperature, and the ratio of chitosan in the hydrogel. PA was recovered by soaking the hydrogel in alkaline solution. After concentrating the solution and washing the residue with ethanol, highly pure sodium phytate was obtained with 32.2%-38.7% yield, as confirmed by Fourier transform infrared and high-performance liquid chromatography. To our knowledge, this is the first report on the recovery of pure PA in high yield without using toxic solvents.

Synchrotron Radiation X-ray Diffraction Techniques Applied to Insect Flight Muscle.

X-ray fiber diffraction is a powerful tool used for investigating the molecular structure of muscle and its dynamics during contraction. This technique has been successfully applied not only to skeletal and cardiac muscles of vertebrates but also to insect flight muscle. Generally, insect flight muscle has a highly ordered structure and is often capable of high-frequency oscillations. The X-ray diffraction studies on muscle have been accelerated by the advent of 3rd-generation synchrotron radiation facilities, which can generate brilliant and highly oriented X-ray beams. This review focuses on some of the novel experiments done on insect flight muscle by using synchrotron radiation X-rays. These include diffraction recordings from single myofibrils within a flight muscle fiber by using X-ray microbeams and high-speed diffraction recordings from the flight muscle during the wing-beat of live insects. These experiments have provided information about the molecular structure and dynamic function of flight muscle in unprecedented detail. Future directions of X-ray diffraction studies on muscle are also discussed.

Regulatory phosphorylation of poly-γ-glutamic acid with phosphate salts in the culture of Bacillus subtilis (natto).

Poly-γ-glutamic acid (PGA) was easily phosphorylated by direct addition of phosphorylating agents into the culture medium of Bacillus subtilis (natto). Tetrapolyphosphate salt was the most incorporated into PGA molecules of all used reagents. Phosphorylation occurred at the α-carboxyl side chains of PGA molecule. The amounts of bound phosphate to PGA were dependent on the amounts of added phosphorylating agent. In low molecular weight regions of less than 100 kDa, a cross-linked peak was observed in the phosphorylated PGAs, whereas their peaks at approximately 1000 kDa shifted to a higher molecular weight due to the bound phosphate. The PGA derivatives had a wide range in viscosity up to 15/1000 to 15 times when compared to the native PGA, depending on the degree of phosphorylation (DP) in the PGA derivatives. The PGA with low DP had a high viscosity due to the unfolding conformation whereas highly phosphorylated PGA had aggregation with low viscosity. Heat treatment at 80 °C after the addition of phosphate salt elicited a novel collagen-like helix structure. These observations show that phosphorylation is an effective way to diversify the physicochemical properties of PGA.

Myopathy-inducing mutation H40Y in ACTA1 hampers actin filament structure and function.

In humans, more than 200 missense mutations have been identified in the ACTA1 gene. The exact molecular mechanisms by which, these particular mutations become toxic and lead to muscle weakness and myopathies remain obscure. To address this, here, we performed a molecular dynamics simulation, and we used a broad range of biophysical assays to determine how the lethal and myopathy-related H40Y amino acid substitution in actin affects the structure, stability, and function of this protein. Interestingly, our results showed that H40Y severely disrupts the DNase I-binding-loop structure and actin filaments. In addition, we observed that normal and mutant actin monomers are likely to form distinctive homopolymers, with mutant filaments being very stiff, and not supporting proper myosin binding. These phenomena underlie the toxicity of H40Y and may be considered as important triggering factors for the contractile dysfunction, muscle weakness and disease phenotype seen in patients.

A Beetle Flight Muscle Displays Leg Muscle Microstructure.

In contrast to major flight muscles in the Mecynorrhina torquata beetle, the third axillary (3Ax) muscle is a minor flight muscle that uniquely displays a powerful mechanical function despite its considerably small volume, ∼1/50 that of a major flight muscle. The 3Ax muscle contracts relatively slowly, and in flight strongly pulls the beating wing to attenuate the stroke amplitude. This attenuation leads to left-right turning in flight or wing folding to cease flying. What enables this small muscle to be so powerful? To explore this question, we examined the microstructure of the 3Ax muscle using synchrotron x-ray diffraction, optical microscopy, and immunoblotting analysis. We found that the 3Ax muscle has long (∼5 µm) myofilaments and that the ratio of thick (myosin) filaments to thin (actin) filaments is 1:5 or 1:6. These characteristics are not observed in the major flight muscles, which have shorter myofilaments (∼3.5 µm) with a smaller ratio (1:3), and instead are more typical of a leg muscle. Furthermore, the flight-muscle-specific troponin isoform, TnH, is not expressed in the 3Ax muscle. Since such a microstructure is suitable for generating large tension, the 3Ax muscle is appropriately designed to pull the wing strongly despite its small volume.

X-Ray Fiber Diffraction Recordings from Oriented Demembranated Chlamydomonas Flagellar Axonemes.

The high homology of its axonemal components with humans and a large repertoire of axonemal mutants make Chlamydomonas a useful model system for experiments on the structure and function of eukaryotic cilia and flagella. Using this organism, we explored the spatial arrangement of axonemal components under physiological conditions by small-angle x-ray fiber diffraction. Axonemes were oriented in physiological solution by continuous shear flow and exposed to intense and stable x rays generated in the synchrotron radiation facility SPring-8, BL45XU. We compared diffraction patterns from axonemes isolated from wild-type and mutant strains lacking the whole outer arm (oda1), radial spoke (pf14), central apparatus (pf18), or the α-chain of the outer arm dynein (oda11). Diffraction of the axonemes showed a series of well-defined meridional/layer-line and equatorial reflections. Diffraction patterns from mutant axonemes exhibited a systematic loss/attenuation of meridional/layer-line reflections, making it possible to determine the origin of various reflections. The 1/24 and 1/12 nm(-1) meridional reflections of oda1 and oda11 were much weaker than those of the wild-type, suggesting that the outer dynein arms are the main contributor to these reflections. The weaker 1/32 and 1/13.7 nm(-1) meridional reflections from pf14 compared with the wild-type suggest that these reflections come mainly from the radial spokes. The limited contribution of the central pair apparatus to the diffraction patterns was confirmed by the similarity between the patterns of the wild-type and pf18. The equatorial reflections were complex, but a comparison with electron micrograph-based models allowed the density of each axonemal component to be estimated. Addition of ATP to rigor-state axonemes also resulted in subtle changes in equatorial intensity profiles, which could report nucleotide-dependent structural changes of the dynein arms. The first detailed description of axonemal reflections presented here serves as a landmark for further x-ray diffraction studies to monitor the action of constituent proteins in functional axonemes.

X-ray recordings reveal how a human disease-linked skeletal muscle α-actin mutation leads to contractile dysfunction.

In humans, mutant skeletal muscle α-actin proteins are associated with contractile dysfunction, skeletal muscle weakness and a wide range of primarily skeletal muscle diseases. Despite this knowledge, the exact molecular mechanisms triggering the contractile dysfunction remain unknown. Here, we aimed to unravel these. Hence, we used a transgenic mouse model expressing a well-described D286G mutant skeletal muscle α-actin protein and recapitulating the human condition of contractile deregulation and severe skeletal muscle weakness. We then recorded and analyzed the small-angle X-ray diffraction patterns of isolated membrane-permeabilized myofibers. Results showed that upon addition of Ca(2+), the intensity changes of the second (1/19 nm(-1)) and sixth (1/5.9 nm(-1)) actin layer lines and of the first myosin meridional reflection (1/14.3 nm(-1)) were disrupted when the thin-thick filament overlap was optimal (sarcomere length of 2.5-2.6 µm). However these reflections were normal when the thin and thick filaments were not interacting (sarcomere length>3.6 µm). These findings demonstrate, for the first time, that the replacement of just one amino acid in the skeletal muscle α-actin protein partly prevents actin conformational changes during activation, disrupting the strong binding of myosin molecules. This leads to a limited myosin-related tropomyosin movement over the thin filaments, further affecting the amount of cross-bridges, explaining the contractile dysfunction.

Skeletal and cardiac α-actin isoforms differently modulate myosin cross-bridge formation and myofibre force production.

Multiple congenital myopathies, including nemaline myopathy, can arise due to mutations in the ACTA1 gene encoding skeletal muscle α-actin. The main characteristics of ACTA1 null mutations (absence of skeletal muscle α-actin) are generalized skeletal muscle weakness and premature death. A mouse model (ACTC(Co)/KO) mimicking these conditions has successfully been rescued by transgenic over-expression of cardiac α-actin in skeletal muscles using the ACTC gene. Nevertheless, myofibres from ACTC(Co)/KO animals generate less force than normal myofibres (-20 to 25%). To understand the underlying mechanisms, here we have undertaken a detailed functional study of myofibres from ACTC(Co)/KO rodents. Mechanical and X-ray diffraction pattern analyses of single membrane-permeabilized myofibres showed, upon maximal Ca(2+) activation and under rigor conditions, lower stiffness and disrupted actin-layer line reflections in ACTC(Co)/KO when compared with age-matched wild-types. These results demonstrate that in ACTC(Co)/KO myofibres, the presence of cardiac α-actin instead of skeletal muscle α-actin alters actin conformational changes upon activation. This later finely modulates the strain of individual actomyosin interactions and overall lowers myofibre force production. Taken together, the present findings provide novel primordial information about actin isoforms, their functional differences and have to be considered when designing gene therapies for ACTA1-based congenital myopathies.

Pointed-end capping by tropomodulin modulates actomyosin crossbridge formation in skeletal muscle fibers.

In skeletal muscle, thick and thin filaments are arranged in a myofibrillar lattice. Tropomodulin 1 (Tmod1) is a pointed-end capping and tropomyosin-binding protein that controls thin-filament assembly, stability, and lengths. It remains unknown whether Tmods have other functional roles, such as regulating muscle contractility. To investigate this, we recorded and analyzed the mechanical properties and X-ray diffraction patterns of single membrane-permeabilized skeletal muscle fibers from mice lacking Tmod1. Results show that absence of Tmod1 and its replacement by Tmod3 and Tmod4 may impair initial tropomyosin movement over actin subunits during thin-filament activation, thus reducing both the fraction of actomyosin crossbridges in the strongly bound state (-29%) and fiber force-generating capacity (-31%). Therefore, Tmods are novel regulators of actomyosin crossbridge formation and muscle contractility, and future investigations and models of skeletal muscle force production must incorporate Tmods.

Revised stereochemistry of ficifolidione and its biological activities against insects and cells.

Ficifolidione (1), a moderately active insecticidal compound from two species of Myrtaceae, and its derivatives were synthesized to evaluate their insecticidal activity. X-ray crystallographic analyses and specific rotation values of ficifolidione and its C-4 (2) demonstrated that the structure of ficifolidione differs from the reported absolute structure; that is, the C-4 configuration of ficifolidione should have an S configuration. The reported insecticidal activity of ficifolidione (1) and its C-4 epimer (2) against adult houseflies (Musca domestica), mosquito larvae (Culex pipiens), and cutworms (Spodoptera litura) was not observed. The cytotoxicities of ficifolidione and its derivatives (1-4) against four cell lines, Sf9, Colon26, HL60, and Vero, were also measured because ficifolidione has a phloroglucinol-derived moiety, a motif that is often present in the structure of cytotoxic chemicals. Compound 1 exhibited IC50 values of ca. 32, 9, 3, and 12 µM for Sf9, Colon26, HL60, and Vero cells, respectively, indicating that ficifolidione possesses selective cytotoxicity against the four cell lines. In HL60 cells treated with 1, DNA fragmentation and the activation of procaspase 3 were observed, suggesting that the cytotoxicity is induced by apoptosis.

Flexibility of the coordination geometry at the N-site of Cu(II)2 human serum-transferrin induced by the different orientations of Arg124.

The ESR spectra of dicupric human serum-transferrin (serum-Tf) were measured from -20 to 37°C in the liquid state (56% glycerol at pH 7.6). Two coordination geometries (types B-1 and B-2) with different ESR parameters were present at the N-site. The contents of the coordination geometry of type B-1 at the N-site increased as the temperature increased. The equilibrium constant between the coordination geometries of types B-1 and B-2 was determined by ESR spectra. The enthalpy value from type B-2 to B-1 was +5.3 kcal/mol, as obtained from a van't Hoff plot. The two conformational energies of the cluster models of the copper-binding site at the N-site of dicupric human serum-Tf, where the Arg124 residue was oriented in two different directions (conformations I and II), were calculated by Density Functional Theory, and the enthalpy value from conformation II to I was +2.1 kcal/mol. The enthalpy value was similar to that (+5.3 kcal/mol) obtained by the coordination geometrical change from type B-2 to B-1 in Cu(II)2 serum-Tf. In conformations I and II, the residue of Arg124 at the N-site is located either far from or near the copper-binding site, respectively, and in both cases the coordination geometry of the cupric ions at the N-site has changed from a flattened tetrahedron to a trigonal bipyramid. This result implies that the ESR spectral change from type B-2 to B-1 is caused by the presence of two different orientations of Arg124 in the change from conformation II to I.

Contrast variation method applied to structural evaluation of catalysts by X-ray small-angle scattering.

In the process of developing carbon-supported metal catalysts, determining the catalyst particle-size distribution is an essential step, because this parameter is directly related to the catalytic activities. The particle-size distribution is most effectively determined by small-angle X-ray scattering (SAXS). When metal catalysts are supported by high-performance mesoporous carbon materials, however, their mesopores may lead to erroneous particle-size estimation if the sizes of the catalysts and mesopores are comparable. Here we propose a novel approach to particle-size determination by introducing contrast variation-SAXS (CV-SAXS). In CV-SAXS, a multi-component sample is immersed in an inert solvent with a density equal to that of one of the components, thereby rendering that particular component invisible to X-rays. We used a mixture of tetrabromoethane and dimethyl sulfoxide as a contrast-matching solvent for carbon. As a test sample, we prepared a mixture of a small amount of platinum (Pt) catalyst and a bulk of mesoporous carbon, and subjected it to SAXS measurement in the absence and presence of the solvent. In the absence of the solvent, the estimated Pt particle size was affected by the mesopores, but in the presence of the solvent, the Pt particle size was correctly estimated in spite of the low Pt content. The results demonstrate that the CV-SAXS technique is useful for correctly determining the particle-size distribution for low-Pt-content catalysts, for which demands are increasing to reduce the use of expensive Pt.

Remodelling of Skeletal Muscle Myosin Metabolic States in Hibernating Mammals.

Hibernation is a period of metabolic suppression utilized by many small and large mammal species to survive during winter periods. As the underlying cellular and molecular mechanisms remain incompletely understood, our study aimed to determine whether skeletal muscle myosin and its metabolic efficiency undergo alterations during hibernation to optimize energy utilization. We isolated muscle fibers from small hibernators, Ictidomys tridecemlineatus and Eliomys quercinus and larger hibernators, Ursus arctos and Ursus americanus. We then conducted loaded Mant-ATP chase experiments alongside X-ray diffraction to measure resting myosin dynamics and its ATP demand. In parallel, we performed multiple proteomics analyses. Our results showed a preservation of myosin structure in U. arctos and U. americanus during hibernation, whilst in I. tridecemlineatus and E. quercinus, changes in myosin metabolic states during torpor unexpectedly led to higher levels in energy expenditure of type II, fast-twitch muscle fibers at ambient lab temperatures (20°C). Upon repeating loaded Mant-ATP chase experiments at 8°C (near the body temperature of torpid animals), we found that myosin ATP consumption in type II muscle fibers was reduced by 77-107% during torpor compared to active periods. Additionally, we observed Myh2 hyper-phosphorylation during torpor in I. tridecemilineatus, which was predicted to stabilize the myosin molecule. This may act as a potential molecular mechanism mitigating myosin-associated increases in skeletal muscle energy expenditure during periods of torpor in response to cold exposure. Altogether, we demonstrate that resting myosin is altered in hibernating mammals, contributing to significant changes to the ATP consumption of skeletal muscle. Additionally, we observe that it is further altered in response to cold exposure and highlight myosin as a potentially contributor to skeletal muscle non-shivering thermogenesis.

Remodeling of skeletal muscle myosin metabolic states in hibernating mammals.

Hibernation is a period of metabolic suppression utilized by many small and large mammal species to survive during winter periods. As the underlying cellular and molecular mechanisms remain incompletely understood, our study aimed to determine whether skeletal muscle myosin and its metabolic efficiency undergo alterations during hibernation to optimize energy utilization. We isolated muscle fibers from small hibernators, Ictidomys tridecemlineatus and Eliomys quercinus and larger hibernators, Ursus arctos and Ursus americanus. We then conducted loaded Mant-ATP chase experiments alongside X-ray diffraction to measure resting myosin dynamics and its ATP demand. In parallel, we performed multiple proteomics analyses. Our results showed a preservation of myosin structure in U. arctos and U. americanus during hibernation, whilst in I. tridecemlineatus and E. quercinus, changes in myosin metabolic states during torpor unexpectedly led to higher levels in energy expenditure of type II, fast-twitch muscle fibers at ambient lab temperatures (20 °C). Upon repeating loaded Mant-ATP chase experiments at 8 °C (near the body temperature of torpid animals), we found that myosin ATP consumption in type II muscle fibers was reduced by 77-107% during torpor compared to active periods. Additionally, we observed Myh2 hyper-phosphorylation during torpor in I. tridecemilineatus, which was predicted to stabilize the myosin molecule. This may act as a potential molecular mechanism mitigating myosin-associated increases in skeletal muscle energy expenditure during periods of torpor in response to cold exposure. Altogether, we demonstrate that resting myosin is altered in hibernating mammals, contributing to significant changes to the ATP consumption of skeletal muscle. Additionally, we observe that it is further altered in response to cold exposure and highlight myosin as a potentially contributor to skeletal muscle non-shivering thermogenesis.

Many animals use hibernation as a tactic to survive harsh winters. During this dormant, inactive state, animals reduce or limit body processes, such as heart rate and body temperature, to minimise their energy use. To conserve energy during hibernation, animals can use different approaches. For example, garden dormice undergo periodic states of extremely low core temperatures (down to 4­8^oC); whereas Eurasian brown bears see milder temperature drops (down to 23­25^oC). An important organ that changes during hibernation is skeletal muscle. Skeletal muscle typically uses large amounts of energy, making up around 50% of body mass. To survive, hibernating animals must change how their skeletal muscle uses energy. Traditionally, active myosin ­ a protein found in muscles that helps muscles to contract ­ was thought to be responsible for most of the energy use by skeletal muscle. But, more recently, resting myosin has also been found to use energy when muscles are relaxed. Lewis et al. studied myosin and skeletal muscle energy use changes during hibernation and whether they could impact the metabolism of hibernating animals. Lewis et al. assessed myosin changes in muscle samples from squirrels, dormice and bears during hibernation and during activity. Experiments showed changes in resting myosin in squirrels and dormice (whose temperature drops to 4­8^oC during hibernation) but not in bears. Further analysis revealed that cooling samples from non-hibernating muscle to 4­8^oC increased energy use in resting myosin, thereby generating heat. However, no increase in energy use was found after cooling hibernating muscle samples to 4­8^oC. This suggest that resting myosin generates heat at cool temperatures ­ a mechanism that is switched off in hibernating animals to allow them to cool their body temperature. These findings reveal key insights into how animals conserve energy during hibernation. In addition, the results show that myosin regulates energy use in skeletal muscles, which indicates myosin may be a potential drug target in metabolic diseases, such as obesity.

Flight muscle-specific Pro-Ala-rich extension of troponin is important for maintaining the insect-type myofilament lattice integrity.

Insect flight muscle (IFM) can oscillate at frequencies up to 1000Hz, owing to its capability of stretch activation (SA). It is a highly specialized form of cross striated muscles, and its peculiar features include the IFM-specific isoform of troponin-I (troponin-H or TnH) with an unusually long Pro-Ala-rich extension at the C-terminus. Although we have shown that this extension does not directly take part in SA, questions remain as to what its real role is and why it is expressed only in IFM. Here we explored the structural role of the extension, be comparing X-ray diffraction patterns and electron micrographs of bumblebee IFM fibers before and after enzymatic removal of the extension. The removal had a dramatic effect on diffraction patterns: In IFMs in general, the equatorial 2,0 reflection is much stronger than the 1,1 reflection, but after removal, their intensities became almost equal (stronger 1,1 is a feature of vertebrate skeletal muscle). Electron micrographs revealed that a substantial fraction of the thin filaments showed a tendency to move towards the vertebrate position (the trigonal position between three thick filaments), while the rest of the thin filaments remained in their original insect position (midway between two neighboring thick filaments). Therefore, one of the roles of the extension is suggested to keep the filament lattice in the correct configuration for IFM. This insect-type lattice structure is preserved among IFMs from varied insect orders but not in body muscles, suggesting that the maintenance of this lattice structure is important for flight functions.

The long C-terminal extension of insect flight muscle-specific troponin-I isoform is not required for stretch activation.

Stretch-induced enhancement of active force (stretch activation, SA) is observed in striated muscles in general, and most conspicuously in insect flight muscle (IFM). It remains unclear whether a common mechanism underlies the SA of all muscle types, or the SA of IFM relies on its highly specialized features. Recent studies suggest that IFM-specific isoforms of thin filament regulatory proteins (troponin and tropomyosin) are implicated in SA. Among others, IFM-specific troponin-I (troponin-H or TnH), with an unusually long Pro-Ala-rich extension at the C-terminus, has been speculated to transmit the mechanical signal of stretch to the troponin complex. To verify this hypothesis, it was removed by a specific endoproteinase in bumblebee IFM, expecting that it would eliminate SA while leaving intact the capacity for Ca(2+)-activated isometric force. Electrophoretic data showed that the extension was almost completely (97%) removed from IFM fibers after treatment. Unexpectedly, SA force was still conspicuous, and its rate of rise was not affected. Therefore, the results preclude the possibility that the extension is a main part of the mechanism of SA. This leaves open the possibility that SAs of IFM and vertebrate striated muscles, which lack the extension, operate under common basic mechanisms.

Myofilament lattice structure in presence of a skeletal myopathy-related tropomyosin mutation.

Human tropomyosin mutations deregulate skeletal muscle contraction at the cellular level. One key feature is the slowing of the kinetics of force development. The aim of the present study was to characterize the potential underlying molecular mechanisms by recording and analyzing the X-ray diffraction patterns of human membrane-permeabilized muscle cells expressing a particular ß-tropomyosin mutation (E41K). During resting conditions, the d1,0 lattice spacing, Δ1,0 and I1,1 to I1,0 ratio were not different from control values. These results suggest that, in presence of the E41K ß-tropomyosin mutation, the myofilament lattice geometry is well maintained and therefore may not have any detrimental influence on the contraction mechanisms and thus, on the rate of force generation.