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PubMed1988

Attenuation of ultrasound in homogenates of bovine skeletal muscle and other tissues.

The attenuation of ultrasound in homogenates of bovine skeletal muscle and suspensions of myofibrils was measured over the frequency range 1.5-7 MHz, and found to be proportional to protein concentration in both. In the homogenates it varied with frequency and temperature in a similar way to the attenuation in post rigor muscle tissue; myofibrils showed a higher frequency dependence. The attenuation in homogenates of bovine muscle, liver and kidney and in suspensions of myofibrils was measured over the pH range 3.5-13, and each showed a peak at about pH 11.5. This was thought to be due to a proton transfer process between NH3+ groups on the tissue proteins and OH- ions in the suspending fluid. A substantial peak at about pH 5 in the muscle and myofibril suspensions was not observed in homogenates of liver and kidney and was thought to be due to components of muscle that are absent from the other tissues. Myofibrils suspended in percoll solution of density 1.05 g cm-3, chosen to match approximately the density of the myofibrils, showed a slightly lower attenuation over the pH range 5-7, but a pH dependence similar to that of the myofibrils suspended in saline. The difference in the attenuations may be interpreted as the viscous component of the attenuation due to relative motion between the myofibril and its surrounding saline. The peak at pH 5 did not, however, appear to be due to the viscous loss mechanism peaking due to maximum shrinkage (and therefore maximum density) of the myofibril near this pH.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1979

Elevated levels of a calcium-activated muscle protease in rapidly atrophying muscles from vitamin E-deficient rabbits.

A Ca2+-activated proteolytic enzyme that partially degrades myofibrils was isolated from hind limb muscles of normal rabbits and rabbits undergoing rapid muscle atrophy as a result of vitamin E deficiency. Extractable Ca2+-activated protease activity was 3.6 times higher in muscle tissue from vitamin E-deficient rabbits than from muscle tissue of control rabbits. Ultrastructural studies of muscle from vitamin E-deficient rabbits showed that the Z disk was the first myofibrillar structure to show degradative changes in atrophying muscle. Myofibrils prepared from muscles from vitamin E-deficient rabbits showed partial or complete loss of Z-disk density. Sodium dodecyl sulfate polyacrylamide gel electrophoresis showed that the amount of troponin-T (37 000 daltons) and alpha-actinin (96 000 daltons) was reduced in myofibrils from atrophying muscle as compared to myofibrils prepared from control muscle. In vitro treatment of purified myofibrils with purified Ca2+-activated proteolytic enzyme produced alterations in myofibrillar ultrastructure that were identical to the initial alterations occurring in myofibrils from atrophying muscle (i.e. weakening and subsequent removal of Z disks). Additonally the electrophoretic banding pattern of Ca2+-activated proteolytic enzyme-treated myofibrils is very similar to that of myofibrils prepared from muscles atrophying as a result of nutritional vitamin E deficiency. The possible role of Ca2+-activated proteolytic enzyme in disassembly and degradation of the myofibril is discussed.

Animals↗
PubMed1955

Studies on the structure of muscle. III. Phase contrast and electron microscopy of dipteran flight muscle.

1. The flight muscles of blowflies are easily dispersed in appropriate media to form suspensions of myofibrils which are highly suitable for phase contrast observation of the band changes associated with ATP-induced contraction. 2. Fresh myofibrils show a simple band pattern in which the A substance is uniformly distributed throughout the sarcomere, while the pattern characteristic of glycerinated material is identical with that generally regarded as typical of relaxed vertebrate myofibrils (A, I, H, Z, and M bands present). 3. Unrestrained myofibrils of both fresh and glycerinated muscle shorten by not more than about 20 per cent on exposure to ATP. In both cases the A substance migrates during contraction and accumulates in dense bands in the Z region, while material also accumulates in the M region. It is proposed that these dense contraction bands be designated the C(z), and C(m) bands respectively. In restrained myofibrils, the I band does not disappear, but the C(z) and C(m) bands still appear in the presence of ATP. 4. The birefringence of the myofibrils decreases somewhat during contraction, but the shift of A substance does not result in an increase of birefringence in the C(z) and C(m) bands. It seems therefore that the A substance, if it is oriented parallel with the fibre axis in the relaxed myofibril, must exist in a coiled or folded configuration in the C hands of contracted myofibrils. 5. The fine structure of the flight muscle has been determined from electron microscopic examination of ultrathin sections. The myofibrils are of roughly hexagonal cross-section and consist of a regular single hexagonal array of compound myofilaments the cores of which extend continuously throughout all bands of the sarcomere in all states of contraction or relaxation so far investigated. 6. Each myofilament is joined laterally with its six nearest neighbours by thin filamentous bridges which repeat at regular intervals along the fibre axis and are present in the A, I, and Z, but not in the H or M bands. When stained with PTA, the myofilaments display a compound structure. In the A band, a lightly staining medullary region about 40 A in diameter is surrounded by a densely staining cortex, the over-all diameter of the myofilament being about 120 A. This thick cortex is absent in the I and H bands, but a thinner cortex is often visible. 7. It is suggested that the basic structure is a longitudinally continuous framework of F actin filaments, which are linked periodically by the lateral bridges (possibly tropomyosin). The A substance is free under certain conditions to migrate to the Z bands to form the C(z) bands. The material forming the C(m) bands possibly represents another component of the A substance. The results do not clearly indicate whether myosin is confined to the A bands or distributed throughout the sarcomere.

Actin Cytoskeleton↗
PubMed2004

Dynamics of obscurin localization during differentiation and remodeling of cardiac myocytes: obscurin as an integrator of myofibrillar structure.

Obscurin is a newly identified giant muscle protein whose functions remain to be elucidated. In this study we used high-resolution confocal microscopy to examine the dynamics of obscurin localization in cultures of rat cardiac myocytes during the assembly and disassembly of myofibrils. Double immunolabeling of neonatal and adult rat cells for obscurin and sarcomeric alpha-actinin, the major protein of Z-lines, demonstrated that, during myofibrillogenesis, obscurin is intensely incorporated into M-band areas of A-bands and, to a lesser extent, in Z-lines of newly formed sarcomeres. Presarcomeric structural precursors of myofibrils were intensely immunopositive for alpha-actinin and, unlike mature myofibrils, weakly immunopositive or immunonegative for obscurin. This indicates that most of the obscurin assembles in developing myofibrils after abundant incorporation of alpha-actinin and that massive integration of obscurin occurs at more advanced stages of sarcomere assembly. Immunoreactivity for obscurin in the middle of A-bands and in Z-lines of sarcomeres bridged the gaps between individual bundles of newly formed myofibrils, suggesting that this protein appears to be directly involved in their primary lateral connection and registered alignment into larger clusters. Close sarcomeric localization of obscurin and titin suggests that they may interact during myofibril assembly. Interestingly, the laterally aligned striated pattern of obscurin formed at a stage when desmin, traditionally considered as a molecular linker responsible for the lateral binding and stabilization of myofibrils at the Z-bands, was still diffusely localized. During the disassembly of the contractile system in adult myocytes, disappearance of the cross-striated pattern of obscurin preceded the disorganization of registered alignment and intense breakdown of myofibrils. The cross-striated pattern of desmin typical of terminally differentiated myocytes disappeared before or simultaneously with obscurin. During redifferentiation, as in neonatal myocytes, sarcomeric incorporation of obscurin closely followed that of alpha-actinin and occurred earlier than the striated arrangement of desmin intermediate filaments. The presence of obscurin in the Z-lines and its later assembly into the A/M-bands indicate that it may serve to stabilize and align sarcomeric structure when myosin filaments are incorporated. Our data suggest that obscurin, interacting with other muscle proteins and possibly with the sarcoplasmic reticulum, may have a role as a flexible structural integrator of myofibrils during assembly and adaptive remodeling of the contractile apparatus.

Actins↗
PubMed1981

Easily releasable myofilaments from skeletal and cardiac muscles maintained in vitro. Role in myofibrillar assembly and turnover.

Gentle treatment with an ATP-containing relaxing solution of isolated myofibrils from rat diaphragm, soleus, extensor digitorum longus, and left atria maintained in vitro releases a small amount of myofilaments constituting less than 5% of total myofibrillar protein. Successive extraction of myofibrils produced little further filament release. Releasable myofilaments lack alpha-actinin (Mr = 95,000), certain very high molecular weight proteins (greater than 200,000), and possibly M-line protein but contain other myofibrillar proteins. After pulse-labeling with [3H]leucine for 8 min, specific activity of the myosin heavy chain in the easily releasable myofilaments is 3-6 times higher than the specific activity of myosin heavy chain in the residual myofibrils, although 85-90% of total label is in the myofibrillar myosin. In the absence of protein synthesis, releasable filament specific activity decreases, with a half-time of 60-90 min, to that of the myofibrillar myosin. This labeling pattern appears inconsistent with a simple precursor-product relationship between releasable filaments and myofibrils suggesting that the filaments originate largely from myofibrils. Preincubation of muscles with several factors known to decrease proteolysis, i.e. passive stretch, leupeptin, colchicine, and cycloheximide, reduced the size of the releasable filament fraction. Treatment of muscles with the calcium ionophore A23187, which accelerates proteolysis, and pretreatment of myofibrils with either trypsin or calcium-dependent protease increased filament release. Therefore, the releasable filament fraction may contain intermediates in the breakdown of myofibrils. The labeling kinetics may indicate a mixing of myofilaments within myofibrils which functions in the movement of contractile protein to its possible site of degradation, i.e. the myofibrillar surface.

Animals↗
PubMed1990

Identification and localization of high molecular weight proteins in insect flight and leg muscle.

Thick and thin filaments in asynchronous flight muscle overlap nearly completely and thick filaments are attached to the Z-disc by connecting filaments. We have raised antibodies against a fraction of Lethocerus flight muscle myofibrils containing Z-discs and associated filaments and also against a low ionic strength extract of myofibrils. Monoclonal antibodies were obtained to proteins of 800 kd (p800), 700 kd (p700), 400 kd (p400) and alpha-actinin. The positions of the proteins in Lethocerus flight and leg myofibrils were determined by immunofluorescence and electron microscopy. p800 is in connecting filaments of flight myofibrils and in A-bands of leg myofibrils. p700 is in Z-discs of flight myofibrils and an immunologically related protein, p500, is in leg muscle Z-discs. p400 is in M-lines of both flight and leg myofibrils. Preliminary DNA sequencing shows that p800 is related to vertebrate titin and nematode twitchin. Molecules of p800 could extend from the Z-disc a short way along thick filaments, forming a mechanical link between the two structures. All three high molecular weight proteins probably stabilize the structure of the myofibril.

Amino Acid Sequence↗
PubMed1990

Differential response of cultured adult cardiac muscle cells to a tumor promotor: analysis of myofibrillar organization.

Cultured adult rat ventricular cardiac muscle cells were exposed to varying concentrations of 12-0-tetradecanoyl-phorbol-13 acetate (TPA) for two weeks. A considerable number of cardiac myocytes exposed to a medium with less than 200 ng/ml TPA were rich in myofibrils. The rest of the myocytes lacked organized myofibrils; the terminal parts of these myofibrils were transformed into cord-like structures largely consisting of dense Z-band materials. Some of these aberrant myofibrils contained short normal myofibrillar segments, with sarcomeres. A number of myocytes exposed to 200-250 ng/ml TPA contained myofibrils, which terminated in cord-like structures. The Z-band materials appeared as amorphous dense matrices and some sarcomeres were replaced completely or partially by leptomeres; the myocytes contained autophagosomes. The other myocytes did not contain myofibrils when exposed to the above higher concentrations of TPA. The patches of Z-band materials and structures containing Z-band materials attached to thin filaments on either side were scattered throughout the sarcoplasm of the cells, which were packed with myofilaments and 10 nm microfilaments. Some of these myocytes assumed a spindle shape and contained myofilaments, 10 nm microfilaments and leptomeres. Some of the myocytes, treated with TPA for 1-7 days and then allowed to recover in control medium for 7 days, contained various stages of myofibrillar organization, which did not differ significantly from those of the myofibril-containing cells exposed continuously to TPA as discussed above. The rest of the myocytes during the recovery period in control medium did not contain myofibrils. Rough endoplasmic reticulum and Golgi bodies in TPA-treated myocytes were found to be highly developed as compared to the controls. It is evident from these studies that the responsiveness of cardiac myocytes to TPA not only differs from that of skeletal muscle cells studied by others, but also varies within a population of cardiac myocytes.

Animals↗
PubMed1994

Correlation of ActoS1, myofibrillar, and muscle fiber ATPases.

Our objective was to determine a good in vitro model for muscle fiber ATPase, and we compared the kinetics of Ca(2+)-activated myofibrils and cross-linked actoS1 in a buffer of physiological ionic strength. The myofibrils were cross-linked chemically to mimic the isometric condition of fibers or were un-cross-linked (the isotonic condition), and temperature perturbation was used to probe their ATPase mechanisms. At 4 degrees C, we have already shown that the kinetics of cross-linked actoS1 and myofibrils (cross-linked or not) are similar: there were large P(i) bursts and kcat values of about 1 s-1, close to that obtained with fibers [Herrmann, C., Sleep, J., Chaussepied, P., Travers, F. & Barman, T. (1993) Biochemistry 32, 7255-7263]. So, at 4 degrees C cross-linked actoS1 and myofibrils are equally good as models for fiber ATPase. At 20 degrees C, this similarity vanishes: progress curves with the myofibrils (cross-linked or not) had large P(i) bursts, but with cross-linked actoS1, bursts could not be discerned. This shows that at 20 degrees C the predominant steady-state intermediates are ATP complexes with actoS1 but are products complexes with the myofibrils, as with fibers [Ferenczi, M.A. (1986) Biophys. J. 50, 471-477]. Further, the kcat values were different: 15.5 s-1 with cross-linked actoS1, 8.3 s-1 for myofibrils, and 3.5 s-1 for cross-linked myofibrils. With fibers, kcat = 3.3 s-1. These results show that cross-linked myofibrillar ATPase is a good model for muscle fibers contracting isometrically.(ABSTRACT TRUNCATED AT 250 WORDS)

Actomyosin↗
PubMed2002

Myofibrillogenesis in skeletal muscle cells.

How are myofibrils assembled in skeletal muscles? The current authors present evidence that myofibrils assemble through a three-step model: premyofibrils to nascent myofibrils to mature myofibrils. This three-step sequence was based initially on studies of living and fixed cultured cells from cardiac muscle. Data from avian primary muscle cells and from a transgenic skeletal mouse cell line indicate that a premyofibril model for myofibrillogenesis also holds for skeletal muscle cells. Premyofibrils are characterized by minisarcomeres bounded by Z-bodies composed of the muscle isoform of alpha-actinin. Actin filaments are connected to these Z-bodies and to the mini-A-bands composed of nonmuscle myosin II filaments. Nascent myofibrils are formed when premyofibrils align and are modified by the addition of titin and muscle myosin II filaments. Mature myofibrils result when nonmuscle myosin II is eliminated from the myofibrils and the alpha-actinin rich Z-bodies fuse as the distance between them increases from 0.5 microm in premyofibrils to 2 to 2.5 microm in the mature myofibrils.

Actins↗
PubMed1995

Is Z-disk degradation responsible for postmortem tenderization?

A number of studies have suggested that Z-disk degradation is a major factor contributing to postmortem tenderization. These conclusions seem to have been based largely on experimental findings showing that the calpain system has a major role in postmortem tenderization, and that when incubated with myofibrils or muscle strips, purified calpain removes Z-disks. Approximately 65 to 80% of all postmortem tenderization occurs during the first 3 or 4 d postmortem, however, and there is little or no ultrastructurally detectable Z-disk degradation during this period. Electron microscope studies described in this paper show that, during the first 3 or 4 d of postmortem storage at 4 degrees C, both costameres and N2 lines are degraded. Costameres link myofibrils to the sarcolemma, and N2 lines have been reported to be areas where titin and nebulin filaments, which form a cytoskeletal network linking thick and thin filaments, respectively, to the Z-disk, coalesce. Filamentous structures linking adjacent myofibrils laterally at the level of each Z-disk are also degraded during the first 3 or 4 d of postmortem storage at 4 degrees C, resulting in gaps between myofibrils in postmortem muscle. Degradation of these structures would have important effects on tenderness. The proteins constituting these structures, nebulin and titin (N2 lines); vinculin, desmin, and dystrophin (three of the six to eight proteins constituting costameres); and desmin (filaments linking adjacent myofibrils) are all excellent substrates for the calpains, and nebulin, titin, vinculin, and desmin are largely degraded within 3 d postmortem in semimembranosus muscle. Electron micrographs of myofibrils used in the myofibril fragmentation index assay show that these myofibrils, which have been assumed to be broken at their Z-disks, in fact have intact Z-disks and are broken in their I-bands.

Animals↗
PubMed1992

Molecular analysis of protein sorting during biogenesis of muscle cytoarchitecture.

Isolated, rod-shaped adult rat cardiomyocytes (ARC) were kept in long-term cell cultures and the changes of the cardiomyocyte structure were investigated by confocal microscopy. The cells round up and make contact with the substrate by very flat, foot-like structures. After prolonged culture the amorphous cells regenerate a cardiomyocyte-like cytoarchitecture and myofibrils reemerge. In the perinuclear region myofibrils form continuously while in other cells discontinuous myofibrillogenesis was observed, where short sarcomeric segments occur all over the cytoplasmic space. During the regeneration of myofibrils certain proteins like a smooth muscle actin sort to non sarcomeric region, while myomesin or heart C-protein localize on myofibrils with high specificity. This culture system combined with method of epitope-tagging of contractile proteins are ideally suited to monitor the intracellular localization sites of exogenously introduced constructs to different cytoskeletal, since ARC exhibit at the same time stress fiber-like filaments (SFLF) and nascent myofibrils. The molecular properties of the different members of the myosin light chain isoprotein family were investigated by transfection experiments using epitope-tagged myosin light chain (MLC) cDNA. The sorting of the different types of MLC was shown to be isoprotein specific and with chimeric constructs it was shown that the isoprotein-specific incorporation into myofibrils was dependent on the presence of the middle domain of MLC-1f/3f. These MLC isoproteins can be arranged into a sequence of increasing affinity to myofibrils. A hierarchical order of myofibrillar assembly is postulated based on the association affinity. Similar experiments with constructs containing alpha-cardiac, alpha-smooth muscle and gamma-cytoplasmic actins have shown that expression of epitope-tagged actins in ARC result in different epitope staining patterns. While the alpha-cardiac actin showed a marked preference for sarcomeres, the alpha-smooth muscle isoproteins had an intermediate specificity and could either be preferentially incorporated into stress fiber-like filaments (SFLF) and in some cells to a lesser extent into myofibrils as well. Most striking results were obtained with gamma-cytoplasmic actin carrying a 5 or 11-mer epitope. This actin gave rise to large cells, induced the formation of filopodia filled with the transfected actin and depletion of the transfected actin from the perinuclear myofibrillar region.

Actins↗
PubMed1997

Premyofibrils in spreading adult cardiomyocytes in tissue culture: evidence for reexpression of the embryonic program for myofibrillogenesis in adult cells.

Do adult cardiomyocytes use the same pathways hypothesized for the formation of myofibrils in embryonic cardiomyocytes in tissue culture. [Rhee, et al., Cell Motil. Cytoskeleton 28:1-24, 1994]? Premyofibrils in embryonic cardiomyocytes are composed of short sarcomeric units of alpha-actinin (Z-bodies) and actin filaments held together by short nonmuscle myosin IIB filaments. Premyofibrils are believed to be transformed into nascent myofibrils by their capture of muscle-specific myosin II filaments aligned in aperiodic arrays. Nascent myofibrils are thought to transform into mature myofibrils by the loss of nonmuscle myosin IIB, the fusion of the Z-bodies into Z-bands, and the periodic alignment of muscle myosin II filaments into A-bands. Freshly isolated cat and rat adult cardiomyocytes placed in tissue culture lack premyofibrils and nascent myofibrils. Adult cardiomyocytes spreading in culture reinitiate the synthesis of nonmuscle myosin IIB. Moreover, patterns similar to the proposed embryonic myofibrillar program first detected in spreading chick embryonic hearts were also detected in these spreading adult mammalian cardiomyocytes. The isolated adult cardiomyocytes begin to spread after 1 day in culture by sending out lamellipodia. When these cells are injected with fluorescently labeled alpha-actinin, linear arrays of short spacings of beaded alpha-actinin bodies are detected in the spreading edges of the adult cardiomyocytes. These dense bodies (Z-bodies) stain positively for the same sarcomeric-specific isoform of alpha-actinin that is in the Z-bands of mature sarcomeres. These linear arrays of alpha-actinin-containing Z-bodies have other characteristics of premyofibrils and are detected only in the spreading regions of the cells. Thus, these premyofibrils at the edges of the spreading adult cardiomyocytes stain positively for nonmuscle myosin IIB but negatively for muscle-specific myosin II. Initially, no vinculin is associated with any parts of the premyofibrils in the spreading regions of the early spreading cardiomyocytes. However, later, vinculin is found to be associated with the ends of the premyofibrils. Fibers that stain solidly for muscle-specific myosin II (i.e., nascent myofibrils) are localized between the peripheral premyofibrils and the centrally positioned, mature myofibrils. It is suggested that the puzzling ability of cardiomyocytes in hypertrophic hearts to reinitiate the synthesis of fetal sarcomeric proteins may be related to the reinitiation of the embryonic premyofibril program for myofibrillogenesis.

Animals↗
PubMed1999

Orientation change of cardiocytes induced by cyclic stretch stimulation: time dependency and involvement of protein kinases.

UNLABELLED: Mechanical stress has been implicated as one of the growth regulators in the heart. We investigated the effect of cyclic stretch stimulation on morphology and orientation of cultured cardiocytes. Embryonic rat (17 days postcoital) cardiomyocytes cultured on silicone dishes were cyclically stretched to 120% in length at a frequency of 30 cycles/min. After 12 h, in the initial stage of cultivation, cardiocytes and intracellular myofibrils oriented parallel to the stretch direction. When the stretch stimulus was prolonged to 24-48 h, myofibrils that oriented perpendicular to the stretch direction emerged. Furthermore, when the cells were stretched only in the later stage (after 24 h of cultivation), both cells and myofibrils tended to orient perpendicular to the stretch direction. Next we examined the effects of chemical compounds on these phase-related changes in myofibril orientation. None of the drugs tested (H-7, HA-1004, staurosporine, herbimycin A, genistein, GdCl3, and EGTA) blocked the parallel orientation of myofibrils induced by the initial-stage stretch. By contrast, H-7, staurosporine, herbimycin A, and genistein did inhibit almost completely the perpendicular orientation of the myofibrils induced by the late-stage stretch, but HA-1004, GdCl3, or EGTA did not. Immunoblotting study using anti-phsophotyrosine antibody indicated that tyrosine phosphorylation of a protein of about 125 kDa was enhanced in a time-dependent manner by the late-stage stretch, but not by the initial-stage stretch. IN CONCLUSION: the alignment change induced by cyclic stretch depends on the stage of cultivation: with stretch in the initial stage (within 12 h), cells and myofibrils orient parallel to the stretch; with stretch in the later stage (after 24 h), they orient perpendicular to the stretch. The effect of stretch in the later stage is likely mediated by protein kinase C and tyrosine kinase pathways.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1985

Location of C-protein, H-protein and X-protein in rabbit skeletal muscle fibre types.

The locations of C-protein, H-protein and X-protein in rabbit psoas, plantaris and soleus muscles have been investigated with fluorescently tagged specific antibodies. Two systems have been examined: isolated myofibrils allowed the locations of these proteins within the sarcomere to be determined, while cryosections allowed a comparison of the amounts of these proteins between different types of fibre in the three muscles. Using antibody-labelled cryosections, we find that the amounts of each of these proteins depends closely on the fibre type. In all the muscles studied, C-protein is present in the largest amounts in fast white and fast intermediate fibres and is absent from slow red fibres, while X-protein is absent from fast white fibres and is present in the largest amounts in fast and slow red fibres. In psoas muscle, H-protein is present in the largest amounts in fast white fibres and is absent in fast and slow red fibres. In plantaris muscle, however, H-protein is absent from fast white fibres but occurs in some slow red fibres. All psoas myofibrils label with anti-C and anti-H and a minority label with anti-X. In each case the pattern of labelling is a zone in each half of the A-band. Measured across the middle of the A-band, the zones for H-protein are much closer together than for C-protein; the centre-to-centre spacings are 0.35 micron for anti-H and 0.64 micron for anti-C. The fluorescent zones for X-protein are slightly but significantly closer (0.52 micron) than those for C-protein. All soleus myofibrils label with anti-X but the centre-to-centre spacing was greater (0.67 micron). With plantaris myofibrils, where labelling occurs with anti-C or anti-H, the spacings resemble those in psoas myofibrils, but with anti-X the spacing resembles that in soleus myofibrils. The spacing of the fluorescent zones in an A-band, whether produced by anti-C, anti-X or anti-H does not vary with sarcomere length. We conclude that X-protein and H-protein, like C-protein, are thick filament components. With both fibres and myofibrils, there is no simple relationship between the amount of X-protein and the amount of C-protein. Many fast intermediate fibres in psoas and plantaris muscle label as strongly with anti-C as do fast white fibres but also label as strongly with anti-X as do fast and slow red fibres.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1997

Characteristics of troponin C binding to the myofibrillar thin filament: extraction of troponin C is not random along the length of the thin filament.

Troponin C (TnC) is the Ca(2+)-sensing subunit of troponin responsible for initiating the cascade of events resulting in contraction of striated muscle. This protein can be readily extracted from myofibrils with low-ionic-strength EDTA-containing buffers. The properties of TnC extraction have not been characterized at the structural level, nor have the interactions of TnC with the native myofibrillar thin filament been studied. To address these issues, fluorescein-labeled TnC, in conjunction with high-resolution digital fluorescence microscopy, was used to characterize TnC binding to myofibrils and to determine the randomness of TnC extraction. Fluorescein-5-maleimide TnC (F5M TnC) retained biological activity, as evidenced by reconstitution of Ca(2+)-dependent ATPase activity in extracted myofibrils and binding to TnI in a Ca(2+)-sensitive manner. The binding of F5M TnC to highly extracted myofibrils at low Ca2+ was restricted to the overlap region under rigor conditions, and the location of binding was not influenced by F5M TnC concentration. The addition of myosin subfragment 1 to occupy all actin sites resulted in F5M TnC being bound in both the overlap and nonoverlap regions. However, very little F5M TnC was bound to myofibrils under relaxing conditions. These results suggest that strong binding of myosin heads enhances TnC binding. At high Ca2+, the pattern of F5M TnC binding was concentration dependent: binding was restricted to the overlap region at low F5M TnC concentration, whereas the binding propagated into the nonoverlap region at higher levels. Analysis of fluorescence intensity showed the greatest binding of F5M TnC at high Ca2+ with S1, and these conditions were used to characterize partially TnC-extracted myofibrils. Comparison of partially extracted myofibrils showed that low levels of extraction were associated with greater F5M TnC being bound in the nonoverlap region than in the overlap region relative to higher levels of extraction. These results show that TnC extraction is not random along the length of the thin filament, but occurs more readily in the nonoverlap region. This observation, in conjunction with the influence of rigor heads on the pattern of F5M TnC binding, suggests that strong myosin binding to actin stabilizes TnC binding at low Ca2+.

Animals↗
PubMed1988

Microsecond rotational dynamics of phosphorescent-labeled muscle cross-bridges.

We have measured the microsecond rotational motions of myosin heads in muscle cross-bridges under physiological ionic conditions at 4 degrees C, by detecting the time-resolved phosphorescence of eosin-maleimide covalently attached to heads in skeletal muscle myofibrils. The anisotropy decay of heads in rigor (no ATP) is constant over the time range from 0.5 to 200 microsecond, indicating that they do not undergo rotational motion in this time range. In the presence of 5 mM MgATP, however, heads undergo complex rotational motion with correlation times of about 5 and 40 microsecond. The motion of heads in relaxed myofibrils is restricted out to 1 ms, as indicated by a nonzero value of the residual anisotropy. The anisotropy decay of eosin-labeled myosin, extracted from labeled myofibrils, also exhibits complex decay on the 200-microsecond time scale when assembled into synthetic thick filaments. The correlation times and amplitudes of heads in filaments (under the same ionic conditions as the myofibril experiments) are unaffected by MgATP and very similar to the values for heads in relaxed myofibrils. The larger residual anisotropy and longer correlation times seen in myofibrils are consistent with a restriction of rotational motion in the confines of the myofibril protein lattice. These are the first time-resolved measurements under physiological conditions of the rotational motions of cross-bridges in the microsecond time range.

Animals↗
PubMed1981

An immunological approach to myosin light-chain function in thick filament linked regulation. 2. Effects of anti-scallop myosin light-chain antibodies. Possible regulatory role for the essential light chain.

Specific antibodies directed against the regulatory light chains (R-LC) or essential light chains (SH-LC) of scallop myosin abolished calcium regulation in myofibrils, myosin, and heavy meromyosin by elevating the actin-activated Mg2+-ATPase activity in the absence of calcium. Calcium dependence was completely eliminated at molar ratios of 2.5-3 antibodies bound per myosin. Monovalent anti-R-LC Fab and anti-SH-LC Fab fragments also desensitized myofibrils fully. High Ca2+-ATPase activity remained unaffected by the antibodies. Anti-SH-LC IgG reduced to about one-half the actin-activated Mg2+-ATPase in the presence of calcium and the potassium-activated ethylenediaminetetraacetic acid (EDTA)-ATPase activities. Anti-SH-LC Fab, however, desensitized without inhibiting the actin-activated Mg2+-ATPase. The desensitizing effect of both antibodies was abolished by prior absorption with the homologous myosin light chain. Calcium binding and R-LC and anti-SH-LC IgG's and by anti-SH-LC Fab. The anti-R-LC Fab fragment induced a significant (70%) dissociation of R-LC from myofibrils and myosins with concomitant losses in calcium binding. In contrast, anti-R-LC IgG prevented the dissociation of R-LC from myosin by EDTA. Binding of anti-R-LC IgG to myofibrils was proportional to thier R-LC content. Increased amounts of anti-SH-LC IgG were bound by myofibrils devoid of R-LC. Bound anti-SH-LC antibody significantly inhibited the reuptake of R-LC by EDTA-treated myofibrils as well as the full binding of anti-R-LC antibody. Certain rabbits produced a population of anti-SH-LC antibodies which were specific for this light chain and bound extensively to myosin but failed to desensitize it (nondesensitizing anti-SH-LC antibody). The desensitizing and nondesensitizing anti-SH-LC populations bound to different regions of the SH-LC on the myosin, and the binding of the two types of antibody to the SH-LC was nearly additive. The nondesensitizing SH-antibody inhibited the reuptake of R-LC less, and its binding to myofibrils was not influenced by the absence of R-LC. These studies indicate a direct or indirect involvement of the SH-LC's in myosin-linked regulation, raise the possibility of an interaction between the R-LC and SH-LC, and confirm the regulatory function of the scallop R-LC. A model for a relative location of the two types of light chains and the involvement of the subfragment-2 region of myosin linked regulation is discussed.

Adenosine Triphosphatases↗
PubMed1997

Myofibrillogenesis visualized in living embryonic cardiomyocytes.

Myofibril formation was visualized in cultured live cardiomyocytes that were transfected with plasmids expressing green fluorescent protein (GFP) linked to the Z-band protein, alpha-actinin. The expression of this fluorescent protein provided an in vivo label for structures containing alpha-actinin. The GFP-alpha-actinin fusion protein was incorporated into Z-bands, intercalated discs, and attachment plaques, as well as into the punctate aggregates, or Z-bodies, that are thought to be the precursors of Z-bands. Observations of live cells over several days in culture permitted us to test aspects of several theories of myofibril assembly that had been proposed previously based on the study of fixed cells. Fine fibrils, called premyofibrils, that formed de novo at the spreading edges of cardiomyocytes, contained punctate concentrations of alpha-actinin, termed Z-bodies. The punctate Z-bodies grew and aligned with Z-bodies in adjacent fibrils. With increasing time, adjacent fibrils and Z-bodies appeared to fuse and form mature myofibrils and Z-bands in cytoplasmic regions where the linear arrays of Z-bodies had been. These new myofibrils became aligned with existing myofibrils at their Z-bands to form myofibrils that spanned the length of the spread cell. These results are consistent with a model that postulates that the fibrils that form de novo near the cell membrane are premyofibrils-i.e., the precursors of mature myofibrils.

Actinin↗