Myoplasmic impedance of the barnacle muscle fiber.

Myoplasmic impedance was measured on a barnacle (Balanus nubilus) single muscle fiber that was placed in a cylindrical cavity to limit the volume and prevent the hydration of the myoplasm. At both ends of the cavity, the myoplasm was in direct contact with an electrolyte solution. When equilibrium with the external medium was reached, the myoplasmic impedance was measured at 10 degrees C with an impedance bridge at 1000 Hz. The results indicated that the myoplasmic impedance of the muscle fiber is mainly resistive. Treating the myoplasm as a suspension of small conductive particles, we deduced the specific conductivity of the contractile filaments kf and their volume fraction rho (kf = 2.78 X 10(-3) omega-1cm-1, and rho = 0.48). The experimental technique permits an estimate of the specific myoplasmic conductivity in vivo (6.27 X 10(-3) omega-1cm-1). Finally, a decrease in the pH of the external solution from 10.1 to 4.0 lowered the myoplasmic conductivity by 16%. This may be considered as indirect evidence that the conductivity of the contractile filaments is associated with the protein counter-ions, since Hinke et al. (1973. Ann. N.Y. Acad. Sci. 204, 274-296.) reported evidence that a lowering of pH decreases the number of counter-ions.     

Dedifferentiation and remodeling of motor endplates following experimental localized lesions of muscle fibers

Local anaesthetics, cardiotoxin and mechanical injuries may cause necrosis of muscle fibres while leaving the motor nerve fibres and their terminals intact. With local injuries to mouse muscles carried out by freezing or cutting we made a point of preserving both the nerve terminals and the muscle fibre portions on which these terminals were located. It was thus possible to follow the changes induced at endplates by these lesions. Within two or three days of the freezing or cutting, the muscle fibres underwent very different degrees of regression of the contractile material and T-system. The neuromuscular junctions also underwent changes, principally affecting their postsynaptic portion, in particular the folds of the subneural apparatus. After dedifferentiation of subsynaptic areas, we observed sprouting of the nerve terminal on muscle fibres which survived the amputation of one end and formed actively new myofibrils. This sprouting restored synaptic connections at the original sites, but with new structural features and correlative changes in the distribution of cholinergic receptors and cholinesterases. It is probable that after a phase of involution followed by a phase of recovery, the injured muscle fibres triggered off the nerve terminal sprouting which led to the remodelling of the endplates.     

The homozygous M712T mutation of UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase results in reduced enzyme activities but not in altered overall cellular sialylation in hereditary inclusion body myopathy

In vivo growth of bacterial flagellar filaments by self-assembly of flagellin is promoted by a capping structure composed of a pentameric assembly of hook associated protein 2 (HAP2). Isolated native filaments with intact HAP2 cap exhibited higher melting temperature (deltaTm = 4 degrees C) and significantly increased resistance against heat-induced depolymerization than non-capped ones. Reconstituted filaments were also stabilized by HAP2 binding, but the obtained filament-HAP2 complexes were less stable than native assemblies. Their fast depolymerization at elevated temperatures and sensitivity to proteolysis indicated that native-like filament-HAP2 complexes are rarely obtained by in vitro reconstitution. A procedure was developed to isolate perfectly capped native filaments to facilitate high-resolution structural analysis.     

Conformational stability of the myosin rod

Chymotryptic cleavage patterns of myosin rods from pig stomach, chicken gizzard, and rabbit skeletal muscle indicate that short (approximately 45 nm) heavy meromyosin subfragment 2 (SF2) is a consistent product of all three rods, whereas long (approximately 60 nm) SF2 is derived only from skeletal muscle myosin. Differential scanning calorimetry was used to follow the thermally induced melting transition of the rods and certain of their subfragments. In 0.12 M KCl, sodium phosphate buffer, pH 6.2-7.6, the light meromyosin (LMM) and SF2 domains of each rod had essentially identical conformational stabilities. Temperature midpoints for the melting transitions were 54-56 degrees C for the two smooth muscle myosin rods and 50-53 degrees C for the skeletal muscle myosin rod. In 0.6 M K Cl buffer, melting transitions for the smooth muscle myosin rods were essentially unchanged, but skeletal muscle myosin rods showed multiphase melting, with major transitions at 43 degrees C and 52 degrees C. The first of these was tentatively attributed to LMM, and the second to SF2. In 0.12 M K Cl buffer, the LMM transition was stabilised so that it superimposed on that of SF2. No melting was observed in any of the rods at physiological temperature. These results indicate that, excluding a possible but only narrow hinge region, the entire myosin rod has essentially uniform conformational stability at physiological pH and ionic strength, and thus that the contractile and elastic properties of the cross-bridge exist in the heavy meromyosin subfragment 1 (SF1) domains of the molecule.     

Structural and functional reconstitution of thin filaments in the contractile apparatus of cardiac muscle.

The muscle contractile apparatus has a highly ordered liquid crystalline structure. The molecular mechanism underlying the formation of this apparatus remains, however, to be elucidated. Selective removal and reconstitution of the components are useful means of examining this mechanism. In addition, this approach is a powerful technique for examining the structure and function of a specific component of the contractile system. In this study we have achieved the structural and functional reconstitution of thin filaments in the cardiac contractile apparatus. First, all thin filaments other than short fragments at the Z line were removed by treatment with gelsolin. Under these conditions no active tension could be generated. By incorporating exogenous actin into these thin filament-free fibers, actin filaments were reconstituted, and active tension, which was insensitive to Ca2+, was restored. The active tension after the reconstitution of thin filaments reached 135 +/- 64% of the original level. The augmentation of tension was attributable to the elongation of reconstituted filaments. As another possibility for augmented tension generation, we suggest the presence of an inhibitory system that was not reconstituted. In any case, the thin filaments of the cardiac contractile apparatus are considered to be assembled so as not to develop the highest degree of tension. Incorporation of the tropomyosin-troponin complex fully restored Ca2+ sensitivity without affecting maximum tension. The present results indicate that a muscle contractile apparatus with a higher order structure and function can be constructed by the self-assembly of constituent proteins.     

Impact of freezing/thawing procedures on the post-thaw viability of cryopreserved human saphenous vein conduits

BACKGROUND: Cryopreserved human blood vessels are important tools in reconstructive surgery. However, patency of frozen/thawed conduits depends largely on the freezing/thawing procedures employed. METHODS: Changes in tone were recorded on rings from human saphenous vein (SV) and used to quantify the degree of cryoinjury after different periods of exposure at room temperature to the cryomedium (Krebs-Henseleit solution containing 1.8M dimethyl sulfoxide and 0.1M sucrose) and after different cooling speeds and thawing rates following storage at -196 degrees C. RESULTS: Without freezing, exposure of SV to the cryomedium for up to 240 min did not modify contractile responses to noradrenaline (NA). Pre-freezing exposure to the cryomedium for 10-120 min attenuated significantly post-thaw maximal contractile responses to NA, endothelin-1 (ET-1) and potassium chloride (KCl) by 30-44%. Exposure for 240 min attenuated post-thaw contractile responses to all tested agents markedly by 62-67%. Optimal post-thaw contractile activity was obtained with SV frozen at about -1.2 degrees C/min and thawed slowly at about 15 degrees C/min. In these SV maximal contractile responses to NA, ET-1 and KCl amounted to 66%, 70% and 60% of that produced by unfrozen controls. Following cryostorage of veins for up to 10 years the responsiveness of vascular smooth muscle to NA was well maintained. CONCLUSION: Cryopreservation allows long-term banking of viable human SV with only minor loss in contractility.     

Studies on the origin of resting tension of skeletal muscle

Glycerol extracted frog skeletal muscle fibres at 2.2 μm sarcomere length (in situ-length) in a solution free of Ca⁺⁺ and Mg⁺⁺ but containing ATP, show a decrease in both their resting tension and their elastic modulus, if the ionic strength of the bathing solution is increased. This finding is compared with the behaviour of intact skeletal muscle fibres in hypertonic solution. It is concluded that the resting tension of intact skeletal muscle fibres at in situ-length is caused by the longitudinal sarcoplasmic reticulum as well as by interactions between the contractile filaments.     

Chemically skinned mammalian skeletal muscle. I. The structure of skinned rabbit psoas.

We studied the morphology of rabbit psoas muscle fixed at increasing intervals of time in a chemical skinning solution (Wood et al., 1975), or after skinning and storage for times up to 1 week. The storage solution, in which the chemically skinned muscled fibers were kept at -20 degrees C, had the same ionic composition as the skinning solution but was made with 50% (v/v) glycerol. Progressive structural changes occurred in fibers exposed to skinning solution. The structural changes were essentially complete after 24-48 hr in skinning solution and no further changes were detected in fibers stored for periods up to 1 week. Structural changes were: (i) holes or gaps in the plasma membrane; (ii) swelling of mitochondria and disorganization of their internal structure; (iii) slight swelling of the sarcoplasmic reticulum; (iv) disappearance of sarcoplasmic reticulum (SR) feet from triadic gaps. Other changes included loss of glycogen between fibrils and extraction of myoplasm, or the change of its staining properties. All architectural elements of the SR, except "feet", remained during skinning and storage, and the SR remained able to accumulate calcium. The morphology of the myofilaments during chemical skinning and during storage did not differ from control fibers. We conclude that chemical skinning alters the gross structure of the plasma membrane and mitochondria, but produces minimal changes in the sarcoplasmic reticulum and contractile proteins.     

Bridgelike interconnections between thick filaments in stretched skeletal muscle fibers observed by the freeze-fracture method

The ultrastructure of frog semitendinosus muscle was explored using the freeze-fracture, deep-etch, rotary-shadowing technique. Mechanically skinned fibers were stretched to decrease or eliminate the overlap of thick and thin filaments before rapid freezing with liquid propane. In relaxed, contracting, and rigor fibers, a significant number of bridgelike interconnections, distinct from those observed in the M-region, were observed between adjacent thick filaments in the non-overlap region. Their half-length and diameter corresponded approximately to the known dimensions of the cross-bridge (or myosin S-1). The interconnection may thus be formed by the binding of two apposed cross-bridges projecting from adjacent thick filaments. Fixation with 0.5% glutaraldehyde for 5-10 min before freezing effectively preserved these structures. The results indicate that the interconnections are genuine structures that appear commonly in stretched muscle fibers. They may play a role in stabilizing the thick filament lattice, and possibly in the contractile process.     

Thermal transitions in the low-density lipoprotein and lipids of the egg yolk of hens.

1. Differential sanning calorimetry and light-scattering have been used to investigate temperature-dependent transitions in low-density lipoprotein and in lipids from hens' egg yolk. Yolks of different fatty acid composition were obtained by varying the dietary lipid and by adding methyl sterculate to the hen's diet. 2. Lipoprotein solutions in 50 percent glycerol/water gave characteristic melting curves between -25 degrees C and 50 degrees C, and on cooling showed increases in light-scattering between 10 degrees C and -20 degrees C. The temperatures at which major changes occurred depended on the proportions of saturated and unsaturated fatty acids. 3. The thermal transitions in the intact lipoprotein in glycerol solution were reversible, but with marked hysteresis. Lipid extracted from the lipoprotein did not show temperature hystersis but the transition heats and melting curves similar to those of the intact lipoprotein. The results support the hypothesis of a "lipid-core" structure for low-density lipoproteins. 4. Scanning calorimetry of egg-yolk lecithins indicated a strong dependence of transition temperature on water content in the rane 3 percent-20 percent water. A rise in the mid-temperature of the liquid-crystalline to gel transition as the water content is lowered on freezing may be the primary event in the irreversible gelation of egg yolk and aggregation of lipoprotein.     

Troponin: structure, properties, and mechanism of functioning.

This review discusses the structure and properties of the isolated components of troponin, their interaction, and the mechanisms of regulation of contractile activity of skeletal and cardiac muscle. Data on the structure of troponin C in crystals and in solution are presented. The Ca2+-induced conformational changes of troponin C structure are described. The structure of troponin I is analyzed and its interaction with other components of actin filaments is discussed. Data on phosphorylation of troponin I by various protein kinases are presented. The role of troponin I phosphorylation in the regulation of contractile activity of the heart is analyzed. The structural properties of troponin T and its interaction with other components of thin filaments are described. Data on the phosphorylation of troponin T are presented and the effect of troponin T phosphorylation on contractile activity of different muscles is discussed. Modern models of the functioning of troponin are presented and analyzed.     

Identification of a cytoskeletal protein localized in the myoplasm of ascidian eggs: localization is modified during anural development

The myoplasm of ascidian eggs is a localized cytoskeletal domain that is segregated to presumptive larval tail muscle cells during embryonic development. We have identified a cytoskeletal protein recognized by a vertebrate neurofilament monoclonal antibody (NN18) which is concentrated in the myoplasm in eggs and embryos of a variety of ascidian species. The NN18 antigen is localized in the periphery of unfertilized eggs, segregates with the myoplasm after fertilization, and enters the larval tail muscle cells during embryonic development. Western blots of one-dimensional and two-dimensional gels showed that the major component recognized by NN18 antibody is a 58 x 10(3) Mr protein (p58), which exists in at least three different isoforms. The enrichment of p58 in the Triton X-100-insoluble fraction of eggs and its reticular staining pattern in eggs and embryos suggests that it is a cytoskeletal protein. In subsequent experiments, p58 was used as a marker to determine whether changes in the myoplasm occur in eggs of anural ascidian species, i.e. those exhibiting a life cycle lacking tadpole larvae with differentiated muscle cells. Although p58 was localized in the myoplasm in eggs of four urodele ascidian species that develop into swimming tadpole larvae, this protein was distributed uniformly in eggs of three anural ascidian species. The eggs of two of these anural species contained the actin lamina, another component of the myoplasm, whereas the third anural species lacked the actin lamina. There was no detectible localization of p58 after fertilization or segregation into muscle lineage cells during cleavage of anural eggs. NN18 antigen was uniformly distributed in pre-vitellogenic oocytes and then localized in the perinuclear zone during vitellogenesis of urodele and anural ascidians. Subsequently, NN18 antigen was concentrated in the peripheral cytoplasm of post-vitellogenic oocytes and mature eggs of urodele, but not anural, ascidians. It is concluded that the myoplasm of ascidian eggs contains an intermediate filament-like cytoskeletal network which is missing in anural species that have modified or eliminated the tadpole larva.     

Thin filaments of molluscan catch muscles contain a calponin-like protein

A novel 40 kDa protein was detected in native thin filaments from catch muscles of the mussel Crenomytilus grayanus. The MALDY-TOF analysis of the protein showed a 40% homology with the calponin-like protein from the muscle of Mytilus galloprovincialis (45 kDa), which has a 36% homology with smooth muscle calponin from chicken gizzard (34 kDa). The amount of the calponin-like protein in thin filaments depends on isolation conditions and varies from the complete absence to the presence in amounts comparable with that of tropomyosin. The most significant factor that determines the contact of the protein in thin filaments is the temperature of solution in which thin filaments are sedimented by ultracentrifugation during isolation. At 22 degrees C and optimal values of both pH and ionic strength of the extraction solution, total calponin-like protein coprecipitates with thin filaments. At 2 degrees C it remains in the supernatant. The 40 kDa calponin-like protein from the mussel Crenomytilus grayanus has similar properties with smooth muscle calponin (34 kDa). It is thermostable and inhibits the actin-activated Mg -ATPase activity of actomyosin. In addition, the 40 kDa calponin-like protein isolated without using thermal treatment contains endogenous kinases. It was found that the calponin-like protein can be phosphorylated by endogenous kinases in the Ca -independent manner. These results indicate that the calponin-like protein from the catch muscle of the mussel Crenomytilus grayanus is a new member of the calponin family. The role of proteins from this family both in muscle and ponmuscle cells is still obscure. We suggest that the calponin-like protein is involved in the Ca -independent regulation of smooth muscle contraction.     

Identification and characterization of Myosin from rat testicular peritubular myoid cells

In the mammalian testis, peritubular myoid cells (PMCs) surround seminiferous tubules. These cells are contractile, express the cytoskeletal markers of true smooth muscle-alpha-isoactin and F-actin-and participate in the contraction of seminiferous tubules during the transport of spermatozoa and testicular fluid to the rete testis. Myosin from PMCs (PMC-myosin) was isolated from adult rat testis and purified by cycles of assembly-disassembly and sucrose gradient centrifugation. PMC-myosin was recognized by a monoclonal anti-smooth muscle myosin antibody, and the peptide sequence shared partial homology with rat smooth muscle myosin-II, MYH11 (also known as SMM-II). Most PMC-myosin (95%) was soluble in the PMC cytosol, and purified PMC-myosin did not assemble into filaments in the in vitro salt dialysis assay at 4 degrees C, but did at 20 degrees C. PMC-myosin filaments are stable to ionic strength to the same degree as gizzard MYH11 filaments, but PMC-myosin filaments were more unstable in the presence of ATP. When PMCs were induced to contract by endothelin 1, a fraction of the PMC-myosin was found to be involved in the contraction. From these results we infer that PMCs express an isoform of smooth muscle myosin-II that is characterized by solubility at physiological ionic strength, a requirement for high temperature to assemble into filaments in vitro, and instability at low ATP concentrations. PMC-myosin is part of the PMC contraction apparatus when PMCs are stimulated with endothelin 1.     

Skinning effects on skeletal muscle myowater probed by T2 relaxation of 1H-NMR

To find the cause of the skinning-induced fragility of frog skeletal muscle, the transverse relaxation process of 1H-NMR signals from skinned muscle was observed. A set of four characteristic exponentials well described the process. Aside from the extremely slow exponential component (time constant T2 > 0.4 s) representing surplus solution, the process was generally slower than that in living muscle. It had larger amplitudes of slow (T2 approximately 0.15 s) and intermediate (0.03 < T2 < 0.06 s) exponentials and had smaller amplitude and faster T2 in the rapid one (T2 < 0.03 s), suggesting that skinned muscle is more sol-like than intact myoplasm. To resolve their causes, we traced the exponentials following a stepwise treatment of living whole muscle to an isolated skinned fiber. Osmotic expansion of living muscle comparable to skinned muscle increased the intermediate exponential and decreased the rapid one without affecting T2. Subsequent chemical skinning markedly increased the slow exponential, decreased the rapid one, and slowed the intermediate one. The fiber isolation had no appreciable effect. Because l-carnosine at physiological concentration could not recover the skinning-induced difference, the difference would reflect the dilution and efflux of larger macromolecules, which stabilize myoplasm as a gel.     

Ultrastructural and physiological studies on the longitudinal body wall muscle of Dolabella auricularia. II. Localization of intracellular calcium and its translocation during mechanical activity

The localization of Ca-accumulating structures in the longitudinal body wall muscle (LBWM) of the opisthobranch mollusc Dolabella auricularia and their role in the contraction-relaxation cycle were studied by fixing the LBWM fibers at rest and during mechanical response to 400 mM K or to 10(-4)--10(-3) M acetylcholine in a 1% OsO4 solution containing 2% K pyroantimonate. In the resting fibers, electron-opaque pyroantimonate precipitate was mostly localized at the peripheral structures, i.e., along the inner surface of the plasma membrane, at the membrane of the surface tubules, and at the sarcoplasmic reticulum. In the fibers fixed during mechanical activity, the precipitate was diffusely distributed in the myoplasm in the form of numerous particles with corresponding decrease in the amount of the precipitate at the peripheral structures. Electron-probe X-ray microanalysis showed the presence of Ca in the precipitate, indicating that the precipitate may serve as a measure of Ca localization. These results are in accord with the view that, in the LBWM, the Ca stored in the peripheral structures is released into the myoplasm to activate the contractile mechanism.     

Heterogeneity of smooth muscle-associated proteins in mammalian brain microvasculature

In the brain, the microvascular system is composed of endothelial cells surrounded by a layer of pericytes. The lack of smooth muscle cells in this tissue suggests that any contractile function must be performed by one or both of these cell types. The present study was undertaken in order to identify cells in terminal blood vessels that contain smooth muscle-like contractile machinery. Endothelial cells were reactive with antibodies against smooth muscle myosin but showed no other smooth muscle-related features. In contrast, pericytes of intact microvessels showed a pattern of protein expression similar to that of smooth muscle cells. Pericytes also behaved in tissue culture like cultured smooth muscle cells, with regard to the changes in expression of smooth muscle-related proteins. These data confirm the close relationship between smooth muscle cells and pericytes, and point to their contractile function in the brain microvessels.     

Blebbistatin specifically inhibits actin-myosin interaction in mouse cardiac muscle

Blebbistatin is a powerful inhibitor of actin-myosin interaction in isolated contractile proteins. To examine whether blebbistatin acts in a similar manner in the organized contractile system of striated muscle, the effects of blebbistatin on contraction of cardiac tissue from mouse were studied. The contraction of paced intact papillary muscle preparations and shortening of isolated cardiomyocytes were inhibited by blebbistatin with inhibitory constants in the micromolar range (1.3–2.8 µM). The inhibition constants are similar to those previously reported for isolated cardiac myosin subfragments showing that blebbistatin action is similar in filamentous myosin of the cardiac contractile apparatus and isolated proteins. The inhibition was not associated with alterations in action potential duration or decreased influx through L-type Ca²⁺ channels. Experiments on permeabilized cardiac muscle preparations showed that the inhibition was not due to alterations in Ca²⁺ sensitivity of the contractile filaments. The maximal shortening velocity was not affected by 1 µM blebbistatin. In conclusion, we show that blebbistatin is an inhibitor of the actin-myosin interaction in the organized contractile system of cardiac muscle and that its action is not due to effects on the Ca²⁺ influx and activation systems. heart; electrophysiology; permeabilized muscle     

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

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

Dissociation of Crk-associated substrate from the vimentin network is regulated by p21-activated kinase on ACh activation of airway smooth muscle

The intermediate filament protein vimentin has been shown to be required for smooth muscle contraction. The adapter protein p130 Crk-associated substrate (CAS) participates in the signaling processes that regulate force development in smooth muscle. However, the interaction of vimentin filaments with CAS has not been well elucidated. In the present study, ACh stimulation of tracheal smooth muscle strips increased the ratio of soluble to insoluble vimentin (an index of vimentin disassembly) in association with force development. ACh activation also induced vimentin phosphorylation at Ser(56) as assessed by immunoblot analysis. More importantly, CAS was found in the cytoskeletal vimentin fraction, and the amount of CAS in cytoskeletal vimentin was reduced in smooth muscle strips on contractile stimulation. CAS redistributed from the myoplasm to the periphery during ACh activation of smooth muscle cells. The ACh-elicited decrease in CAS distribution in cytoskeletal vimentin was attenuated by the downregulation of p21-activated kinase (PAK) 1 with antisense oligodeoxynucleotides. Vimentin phosphorylation at this residue, the ratio of soluble to insoluble vimentin, and active force in smooth muscle strips induced by ACh were also reduced in PAK-depleted tissues. These results suggest that PAK may regulate CAS release from the vimentin intermediate filaments by mediating vimentin phosphorylation at Ser(56) and the transition of cytoskeletal vimentin to soluble vimentin. The PAK-mediated dissociation of CAS from the vimentin network may participate in the cellular processes that affect active force development during ACh activation of tracheal smooth muscle tissues.