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PubMedDate not supplied

In situ study of myofibrils, mitochondria and bound creatine kinases in experimental cardiomyopathies.

Human cardiomyopathy has been extensively studied in the last decade, and knowledge of the functional and structural alterations of the heart has grown. However, understanding of the pathogenesis has come mostly from experimental studies. A number of work have been designed to elucidate if alterations of the contractile apparatus of cardiac cells contribute to the impairment of heart mechanics in cardiomyopathies. As well, an important question is to be solved: whether energy supply of the contraction-relaxation cycle is sufficient in the myopathic heart. Use of cardiac fibers skinned by different techniques allows to evaluate functional ability of myofibrils, mitochondria and bound creatine kinase which plays an important role in cardiomyocyte energy metabolism. The data presented in this chapter show that experimental cardiomyopathies of various types have some common features. These are an increase in calcium sensitivity of myofibrils and a depression of functional activity of mitochondrial creatine kinase. Possible mechanisms and physiological significance of these changes are discussed.

Adenosine Diphosphate↗
PubMed2000

Probing the functional roles of titin ligands in cardiac myofibril assembly and maintenance.

Sarcomeres of cardiac muscle are comprised of numerous proteins organized in an elegantly precise order. The exact mechanism of how these proteins are assembled into myofibrils during heart development is not yet understood, although existing in vitro and in vivo model systems have provided great insight into this complex process. It has been proposed by several groups that the giant elastic protein titin acts as a "molecular template" to orchestrate sarcomeric organization during myofibrillogenesis. Titin's highly modular structure, composed of both repeating and unique domains that interact with a wide spectrum of contractile and regulatory ligands, supports this hypothesis. Recent functional studies have provided clues to the physiological significance of the interaction of titin with several titin-binding proteins in the context of live cardiac cells. Improved models of cardiac myofibril assembly, along with the application of powerful functional studies in live cells, as well as the characterization of additional titin ligands, is likely to reveal surprising new functions for the titin third filament system.

Animals↗
PubMed1998

Measurement of ATP turnover during shortening and lengthening of rabbit psoas myofibrils using a fluorescent ATP analog.

In order to study ATP turnover during shortening and lengthening of rabbit psoas myofibrils, we have used fluorescence microscopy in which the displacement of a fluorescent nucleotide analog, 2'(3')-O-[N-[2-[[Cy3] amido] ethyl] carbamoyl]-adenosine 5' triphosphate (Cy3-EDA-ATP) bound to cross-bridge on flash photolysis of caged ATP was measured [Chaen et al. (1997) Biophys. J. 73, 2033-2042]. In the previous paper, we reported that when a myofibril was imposed to shorten with a constant velocity by a piezo-electric actuator, the nucleotide displacement rate constant initially increased to 0.7 s-1 with increasing shortening velocity and then declined with a further increase in shortening velocity. The rate constant during lengthening measured in the present experiment was found to be not significantly affected. These results suggest that the cross-bridge kinetics show a asymmetrical dependence on the mechanical strain in the cross-bridges, namely, the rate constants are not significantly affected at higher strain during lengthening but depend on the lower strain during shortening.

Adenosine Triphosphate↗
PubMed1978

Immunofluorescent subcellular localization of some muscle proteins: a comparison between tissue sections and isolated myofibrils.

The localization of parvalbumin in fish white muscle and of the calcium binding protein, of arginine kinase and of glycogen phosphorylase in crayfish tail muscle have been investigated by immunofluorescence using isolated myofibrils and muscle sections as starting materials. It is shown that the four proteins appear to be localized on the thin filaments when myofibrils are used as starting material. This result contrasts with previous observations where it appeared that parvalbumin in fish muscle and arginine kinase in crayfish muscle were distributed uniformly within the cell. This discrepancy is discussed in relation to the high solubility of these proteins. In the light of the present knowledge about striated muscles from these two organisms, it seems that the roles of parvalbumin in fish and of the calcium binding protein in crayfish are probably different.

Animals↗
PubMed1984

Sarcomere and filament lengths in passive muscle fibres with wavy myofibrils.

Longitudinal compression of isolated skeletal muscle fibres of Rana pipiens caused waves to appear sharply at a critical striation spacing which was slightly less than the slack length measured at the same point. Both slack length and critical length varied between fibres and along the length of one fibre, being shortest near the tendons. The critical length varied from 1.93 to 2.11 microns. The troponin periodicity (Pdiff) was measured in embedded material by light diffraction of calibrated electron micrographs. Comparison between the troponin periodicities in a fibre made wavy at one end and stretched at the other showed that longitudinal compression did not cause shortening of the thin filaments. Comparison between Pdiff and the troponin periodicity of fresh muscle provided an estimate of the artefact mainly caused by shrinkage during specimen preparation. It varied from 3 to 11%. The gaps between the ends of the thin filaments in the M-line region were estimated from sarcomere length (corrected for shrinkage) and the assumed in vivo values for total thin-filament length or the length between the last troponin lines (1.975 microns and 1.925 microns respectively). The estimates were confirmed by a few direct measurements of thin-filament length and periodicity. Sarcomere length varied from fibre to fibre, from 1.91 to 2.12 microns, except at the inside of bends in wedge-shaped sarcomeres where it fell to 1.86 microns in some cases. This indicates that in one fibre the tips of the thin filaments overlapped at the level of the last troponin lines, while, at the other extreme, the tips of the thin filaments only just reached the bare zone of the thick filaments. The origin of the resistance to sliding and the force which restores an actively shortened fibre to its slack length are discussed. While there may be a well-defined barrier to sliding at the point where the troponins of opposite polarity meet, there must also be an additional length-dependent resistance to account for the appearance of waves at longer sarcomere lengths. The formation of waves is interpreted as a buckling phenomenon in which a longitudinal compressive force is applied to the myofibrils which have a finite stiffness bending and a finite elastic restraint against lateral displacement. The bending stiffness is largely and perhaps entirely accounted for by contributions from (1) the stiffness of the individual filaments and (2) the stiffness of myofibrils calculated from their Young's modulus.

Animals↗
PubMed1985

Immunocytochemical studies using a monoclonal antibody to bovine cardiac titin on intact and extracted myofibrils.

A monoclonal antibody specific to bovine cardiac titin has been identified. The antibody recognizes a common antigenic site in striated muscles of several species. In relaxed myofibrils, specific staining at the A-I junction resulted in a doublet of fluorescent bands within a sarcomere. The distance between the doublets in successive sarcomeres varied according to the degree of myofibrillar contraction. Staining on formamide-extracted myofibrils has confirmed that this epitope is located near the outer edges of isolated A bands. Selective extraction of myofibrillar proteins resulted in different staining patterns. Disrupting the structural integrity of the M-line or the A-band centre caused a significant amount of titin to translocate toward the Z-line region. In contrast, shortening of the A-band by removal of myosin from the ends of the thick filaments resulted in anti-titin staining moving closer to the M-line region. Several conclusions can be drawn from this study: (a) two aligned groups of titin molecules are placed symmetrically to the M-line in a sarcomere; (b) titin may attach directly or via intermediary protein(s) to sites near the M-line and Z-line such that the protein is under tension and (c) removal of proteins from either region results in titin staining in the opposite region. However, the edges of the A-band give some hindrance to collapse of the titin toward the M-line.

Animals↗
PubMed1991

Discrimination of Ca(2+)-ATPase activity of the sarcoplasmic reticulum from actomyosin-type ATPase activity of myofibrils in skinned mammalian skeletal muscle fibres: distinct effects of cyclopiazonic acid on the two ATPase activities.

We have developed a procedure to discriminate actomyosin-type ATPase activity from Ca(2+)-ATPase activity of sarcoplasmic reticulum (SR) in mechanically skinned fibres, determining simultaneously their Ca(2+)-induced tension and accompanying ATPase activity. When they were treated with an alkaline CyDTA-containing solution of low ionic strength which was reported to remove troponin C, the fibres showed a considerable amount of Ca(2+)-dependent ATPase activity, in spite of having little or no Ca(2+)-induced isometric tension. The residual ATPase activity is ascribed to the Ca(2+)-ATPase activity of SR, because it is completely abolished by 1% CHAPS treatment for 10 min. This conclusion is also supported by the finding that the Ca(2+)-dependence of the ATPase activity is very similar to that of Ca(2+)-ATPase of SR isolated from rabbit skeletal muscle, and that the estimated activity is consistent with the reported values of direct determinations. On the other hand, treatment with a detergent such as CHAPS or Triton X-100 removes SR activities (ATPase and Ca-uptake), leaving Ca(2+)-induced tension and actomyosin-type ATPase activity unchanged. This procedure indicated that the contribution of Ca(2+)-ATPase activity of SR may be minimal in total steady-state ATPase activity of mechanically skinned mammalian skeletal muscle fibres. Successive CyDTA and CHAPS treatments eliminated both Ca(2+)-induced tension and ATPase activity, which were recovered by the addition of troponin C. Using these procedures, we also examined the effect of cyclopiazonic acid (CPA) which was reported to be a specific inhibitor of Ca(2+)-ATPase of SR. Ca(2+)-ATPase activity of SR in skinned fibres was inhibited completely by 10 microM CPA and held to one-half by about 0.2 microM. This effect was only partially reversible. CPA at 10 microM or higher concentrations showed Ca(2+)-sensitizing action on myofibrils, which was readily reversible. CPA at 3 microM inhibited almost completely the Ca(2+)-ATPase activity of SR, while it had no effect on either actomyosin-type ATPase or isometric tension of myofibrils.

Animals↗
PubMed1986

The effect of low ATP concentrations on relaxation in the myosin regulated myofibrils from scallop.

Troponin-tropomyosin-regulated myofibrils show a significant increase in ATPase activity and contract in the absence of calcium when the ATP concentration falls significantly below the saturation level. By contrast, the ATPase of the myosin-regulated myofibrils of scallop striated muscle was not activated in the absence of calcium when the ATP concentration was lowered to 10mM. Nevertheless, a very small fraction of crossbridges were active at 10mM ATP resulting in very slow myofibrillar shortening. In contrast to the behaviour of rabbit contractile proteins there was no correlation between myofibrillar shortening and ATP induced turbidity changes of actomyosin taken from scallop.

Actomyosin↗
PubMed1986

Actin-attached and detached crossbridges in myofibrils: segregation into two populations according to their sensitivity to proteolytic digestion of myosin heavy chain.

Tryptic digestion of myofibrils was used to assess the interaction of crossbridges with thin filaments in the presence of ATP analogues. The relative amounts of 200 kDa fragment produced by trypsin from myosin heavy chain when the crossbridge is attached to actin, and of 160 kDa fragment produced when the crossbridge is detached from actin, served as a measure of crossbridge-actin interaction. In rigor only the 200 kDa fragment was produced suggesting that a great majority of the crossbridges were strongly attached to actin; in the presence of MgPPi at 0 degrees C only the 160 kDa fragment was finally produced suggesting that eventually all crossbridges detached from actin. In the presence of MgPPi or MgAMPPNP at 25 degrees C both 200 and 160 kDa fragments were present for several minutes after myosin heavy chain had been completely digested, suggesting that two populations of crossbridges (attached and detached) co-existed at the same time within the myofibril. It is concluded that the addition of ATP analogues to muscle does not simply affect the chemical equilibrium of binding of myosin heads to actin but that it causes rapid dissociation of one crossbridge population without significant effect on binding to actin of the remaining crossbridge population.

Actins↗
PubMed1987

Action of a serine proteinase from fish skeletal muscle on myofibrils.

The action of a serine proteinase from fish skeletal muscle on myofibrils was studied. The enzyme was able to destroy the structural integrity of myofibrils, and to degrade both their major contractile and cytoskeletal constituent proteins. Proteolysis could be completely prevented by the addition of a trypsin inhibitor isolated from the same muscle.

Animals↗
PubMed1988

Increase in calcium sensitivity of cardiac myofibrils contributes to the cardiotonic action of sulmazole.

The aim of this study was to investigate whether increasing calcium sensitivity of myofibrils plays a role in the positive inotropic activity of the cardiotonic agent sulmazole. We studied the effects of the stereoisomers of sulmazole on cardiac contractility in vivo and in vitro, arterial blood pressure, cardiac (Na-K)ATPase activity, cAMP/cGMP-phosphodiesterase activity of cardiac and smooth muscle tissue and calcium sensitivity of skinned myocardial fibres. Both stereoisomers of sulmazole were equipotent vasodilators in vivo and this can be explained by their equipotent cAMP- and cGMP-phosphodiesterase inhibitory activities in smooth muscle tissue. However, (+)sulmazole was a much stronger positive inotropic agent than (-)sulmazole in vivo and in vitro. This difference in inotropic activity cannot be explained by cAMP- or cGMP-phosphodiesterase inhibition or (Na-K)ATPase inhibition in cardiac tissue. Only (+)sulmazole produced a dose-dependent increase in calcium sensitivity of skinned myocardial fibres. Therefore, the calcium sensitizing effect on myofibrils evoked by (+)sulmazole might be responsible for the difference in inotropic activity observed between the stereoisomers of sulmazole.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1991

The fub-1 mutation blocks initial myofibril formation in zebrafish muscle pioneer cells.

The earliest muscle in zebrafish arises from iterated sets of two to six cells in each somite, the muscle pioneers (MP). MP develop synchronously in young trunk myotomes adjacent to the notochord, precisely where the horizontal myoseptum will form. They elongate without cell fusion and differentiate hours earlier than surrounding cells, thus providing a simple and accessible system for in vivo study of myogenesis and muscle patterning. Before the MP form definitive myofibrils they assemble long bundles of actin-containing filaments, similar to "stress-fiber-like structures" reported by others. In fub-1 mutants, in which myofibrils are disorganized in all skeletal muscle cells, the MP appear and elongate normally, but ordered actin filament bundles are not seen. This defect could underlie the later myofibrillar ones, consistent with the proposal that actin filament bundles are essential for proper formation of the muscle contractile apparatus.

Actins↗
PubMed1987

Alpha-actinin is absent from the terminal segments of myofibrils and from subsarcolemmal densities in frog skeletal muscle.

The presence and distribution of alpha-actinin, an actin-bundling protein, was investigated at sites where frog skeletal muscle forms junctions with tendon collagen fibers. These sites, called myotendinous junctions, are regions where myofibrils terminate and where the force of muscular contraction is transmitted from muscle cells to the substratum. An antibody manufactured to chicken smooth muscle alpha-actinin was used as a probe for alpha-actinin localization in this study. The cross-reactivity of this antibody with frog skeletal muscle alpha-actinin is demonstrated in immunoblots of one-dimensional (1D) electrophoretic separations of muscle proteins. Immunofluorescent localization of anti-alpha-actinin and electron microscopic immunolabelling confirms that the antibody binds to Z-discs with high affinity. However, in sections treated for electron microscopy with affinity-purified anti-alpha-actinin and a ferritin-conjugated, second antibody, there was no significant difference between experimental or control preparations in the number of ferritin grains overlying dense, subsarcolemmal material at junctional or non-junctional regions. Furthermore, Z-discs near myotendinous junctions displayed less binding of anti-alpha-actinin than Z-discs located several micrometers or more from the cells' termini. These findings indicate that thin filaments are not bundled by alpha-actinin near the sarcolemma. The results also provide evidence for molecular heterogeneity between Z-discs at the ends of muscle cells compared with other regions of the cell in that the terminal Z-discs of myofibrils contain very little or no alpha-actinin relative to non-terminal Z-discs.

Actinin↗
PubMed1984

ADP binds similarly to rigor muscle myofibrils and to actomyosin-subfragment one.

The binding of Mg2+ ADP to both rabbit skeletal and bovine cardiac myofibrils has been studied at two different temperatures. In each case a single class of binding sites was observed with a binding constant very close to that reported for the analogous actomyosin-subfragment one but much weaker than that seen with the analogous myosin subfragment one alone. These findings are discussed in terms of the constraints on the myosin cross-bridges imposed by the regular array of thick and thin filaments found in myofibrils.

Adenosine Diphosphate↗
PubMed1985

Degradation of alpha-actinin during Ca2+-sensitive proteolysis of myofibrils.

The noted loss of alpha-actinin from the Z-line of myofibrils during post-mortem autolysis, probably following the action of calcium-activated protease, has previously been attributed to its release without degradation. This report shows that in isolated myofibrils alpha-actinin is proteolysed in a Ca2+-sensitive manner presumably via the action of calcium-activated protease.

Actinin↗
PubMed1991

Extra actin filaments at the periphery of skeletal muscle myofibrils.

Myofibrils isolated from a variety of vertebrate muscle fibers have a set of peripheral filaments associated with the periphery of the Z line free to move away from the surface of the myofibril. Decoration with myosin subfragment 1 shows that these are actin filaments.

Actin Cytoskeleton↗
PubMed1984

A nonsense mutation within the act88F actin gene disrupts myofibril formation in Drosophila indirect flight muscles.

We have investigated the molecular basis of muscle abnormalities in the flightless Drosophila mutant lfm(3)7. This EMS-induced, semi-dominant allele was isolated by Mogami and Hotta (1981) and was shown to disrupt the organization of myofibrils in indirect flight muscles. Here we demonstrate that lfm(3)7 contains a nonsense mutation within codon 355 of the act88F actin gene. A single G greater than A transition converts a tryptophan (TGG) codon to an opal (TGA) terminator, thus deleting the carboxy-terminal 20 amino acids of an actin isoform that accumulates only in thoracic flight muscles. The truncated actin polypeptide is stable, and retains antigenicity to at least two anti-Drosophila actin monoclonal antibodies. We suggest that abnormalities in lfm(3)7 flight muscles result from incorporation of the mutant actin isoform into assembling myofibrils.

Actins↗