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PubMed1988

Regulation of binding of phosphofructokinase to myofibrils in the red and white muscle of the barred sand bass, Paralabrax nebulifer (Serranidae).

The binding of phosphofructokinase (PFK) to myofibrils from the white muscle of the fish Paralabrax nebulifer (Girard, 1854) is sensitive to factors known to be allosteric regulators of PFK activity. PFK in Triton-X-100-extracted muscle remains bound to myofibrils at pH 7.0 and is fully solubilized by increasing the pH to 8.0. The curve describing the pH-dependence of PFK binding to myofibrils is similar in its steepness to pH versus activity curves of PFK at low temperature. Nucleotides are also potent modulators, preventing the association of PFK with myofibrils at concentrations between 20 and 60 mumol l-1 of ATP, ADP, MgATP or GTP, listed in order of effectiveness. PFKs in the red and white muscle extracts differ in their pH-dependence of binding to myofibrils, and their kinetic and regulatory properties (response to citrate, pH and fructose-2,6-bisphosphate). Reversible binding of PFK to myofibrils may be important in the control of glycolysis, especially in the highly glycolytic white muscle fibres.

Animals↗
PubMed2005

Diminished myofibril organization in mutant axolotl hearts transfected with site-directed mutants of sarcomeric tropomyosins.

We used a model lacking endogenous sarcomeric tropomyosin, the cardiac mutant of the Mexican axolotl, to examine the effect of mutant tropomyosins on sarcomeric myofibril formation. Previous studies have introduced wild-type mouse alpha-tropomyosin into mutant hearts in organ culture with subsequent for-mation of organized myofibrils. This study examines the predominant embry-onic axolotl TPM-4 type tropomyosin (TPM4alpha), containing a conservative re- placement of glutamic acid for aspartic acid at the clinically important 175 site. In this study, ATmC-3 (TPM4alpha) promoted formation of organized myofibrils in hearts without endogenous tropomyosin. Site-directed mutagenesis of 175 glutamic acid with 175 glutamine or 175 lysine was toxic to the formation of organized myofibrils in mutant hearts in the absence of endogenous tropo-myosin. Cationic liposome co-transfection of both wild-type tropomyosin and Glu175Gln.TPM4alpha cDNA formed organized myofibrils in mutant hearts. A construct with GFP.Glu175Gln.TPM4alpha cDNA was used to confirm expression of the mutant fusion protein. Mutation at the 175 site in TPM4alpha type or TPM1alpha (striated muscle isoform of the TPM1 gene) was sufficient to alter the protein such that organized myofibrils would not form in ventricles of mutant hearts without endogenous tropomyosin.

Ambystoma↗
PubMed1978

Cardiac myofibril disorientation and Z band abnormalities in idiopathic cardiomyopathy. An electron microscope study.

Morphological alterations in idiopathic cardiomyopathy were examined and their clinicopathological significance was discussed. Six cases of congestive type and five of hypertrophic type chosen from 22 patients according to our strict criteria were examined by light and electron microscopy. The diameter values of cardiac muscle fibers were broadly distributed with multiple peaks in congestive cases and with 4 peaks in the hypertrophic cases in contrast to the 2 peaks in controls. The chief electron microscopic alterations consisted of myofibril disorientation and Z band abnormalities. The congestive cases were characterized by decreased and disdarrayed myofibrils (loose myofibril disorientation), wheras the hypertrophic cases by abundant myofibrils characteristically entangled with each other (tight myofibril disorientation). The Z band showed 3 types of abnormalities. Type 1 was characterized by widening, bifurcation and overriding of Z bands while the regular sarcomere was retained. The changes were more conspicuous in the hypertrophic cases than in the congestive cases. Type 2 was represented by clumping of Z band substance, and Type 3 by an interwoven form of electron dense fibers. Types 2 and 3 were seen in some of the hypertrophic and congestive cases. The tight myofibril disorientation and Type 1 of Z band abnormalities were considered important cytopathological clues for the diagnosis of the hypertrophic type of idiopathic cardiomyopathy.

Adult↗
PubMed1987

Myofibril formation in longterm-cultures of adult rat heart cells.

Immunocytochemistry using antibodies against myofibrillar proteins such as heart-C-protein and myomesin but also the fluorescence of rhodamine conjugated phalloidin as marker for F-actin structures was employed to study the redifferentiation of cultured adult rat cardiomyocytes. It was demonstrated that freshly isolated rod shaped cells in culture round up before they attach to the substratum, while myofibrillar structures of the cells degenerate. As soon as they attached to the substratum and gradually flattened out, small cross-striated myofibrils reappeared in the central part of the cells. F-actin, in freshly isolated cells present only in the I-band of the myofibrils, extended throughout the cells into the processes in the form of filament cables. In later stages the myofibrillar distribution varied. Smaller myocytes were frequently filled with myofibrils whereas in larger cells with many processes the myofibrils were found either arranged in perinuclear regions or showed a mosaic distribution. Myofibrils appear to be directly aligned with bundles of actin stress fibers, thus further supporting the suggestion that the actin filaments might serve as a scaffold for myofibril formation.

Actins↗
PubMedDate not supplied

[Angle diagrams of light scattering in the suspension of isolated myofibrils in native, contracted and relaxed states].

An angle diagram of light scattering of myofibril suspensions was extremely asymmetric in the range of 0.05 to 10 degrees. The extent of asymmetry increased at relaxation and sharply decreased at contraction of myofibrils. Angle diagrams for myofibril light scattering calculated according to the approximate formulae of Mie theory, were close to those obtained in the experiment. It was shown that a high extent of asymmetry of calculated diagrams is determined by a low refractive index of myofibrils rather than by great dimensions of myofibrils. It is supposed that changes in the shape of scattering diagram and in other optical characteristics of myofibril and actomyosin suspensions associated with changes in their functional states depend mostly upon changes in the inner arrangement of particles rather than in their dimensions.

Animals↗
PubMed1985

Kinetic mechanism of 1-N6-etheno-2-aza-ATP and 1-N6-etheno-2-aza-ADP binding to bovine ventricular actomyosin-S1 and myofibrils.

The fluorescence emission of 1-N6-etheno-2-aza-ATP (epsilon-aza-ATP) at 410-460 nm is enhanced approximately 8-fold upon mixing substoichiometric concentrations of epsilon-aza-ATP with bovine cardiac actomyosin-S1 or myofibrils. The time course of nucleotide fluorescence measured in a front face stopped flow cell upon mixing epsilon-aza-ATP with bovine cardiac myofibrils ([Ca2+] less than 10(-7) M) is essentially the same as that with bovine cardiac actomyosin subfragment-1. In single turnover experiments, the fluorescence rapidly rises to a maximum value, then decreases with a rate constant of 0.04 s-1 at 0 degree C to a final value that is approximately twice the level of the unbound nucleotide. At concentrations of epsilon-aza-ATP greater than 40 microM the kinetics of epsilon-aza-ATP binding is clearly biphasic for both actomyosin-S1 and myofibrils. At 0 degree C, the rate of the more rapid phase is proportional to nucleotide concentration and has a second order rate constant of 1.7 X 10(5) M-1 s-1; the rate of the slower phase extrapolates to a maximum of 4-5 s-1 at high nucleotide concentration. The rate constants for dissociation of epsilon-aza-ADP from bovine cardiac actomyosin-S1 and myofibrils were measured from the decrease in epsilon-aza-ADP fluorescence enhancement observed upon displacement by ATP to be 20 and 18 s-1, respectively, at 0 degree C. These results indicate that most of the cross-bridges in cardiac myofibrils are bound to actin and that the geometric constraints imposed upon the interaction of actin and myosin by the three-dimensional structure of the myofibril do not modify the kinetics of epsilon-aza-ATP binding or epsilon-aza-ADP dissociation.

Adenosine Diphosphate↗
PubMed1994

Immunocytochemical localization of gelsolin in fibroblasts, myogenic cells, and isolated myofibrils.

Gelsolin was localized by immunofluorescence in fibroblasts and skeletal muscle cells using antibodies which eliminated the risk of detecting xenogenic plasma gelsolin. Gelsolin was consistently found to be closely associated with the elements of the microfilament system: In fibroblasts, a preferential labeling of the stress fibers was observed, whereas with myogenic cells and myofibrils isolated from skeletal muscle, a specific staining of the I-Z-I region in the sarcomeres was found. From double labeling of gelsolin and actin it became evident that the staining patterns for both proteins were practically coincident: The width and location of the fluorescent bands varied with the degree of contraction of the myofibrils. The region of cross-bridges in the A-zone, where thick and thin filaments overlap, remained unstained. The gelsolin staining of myofibrils was EGTA-resistant; it persisted after glycerol extraction and extensive washing. The presence of gelsolin in myofibrils after this treatment was also confirmed by immunoblotting. From these observations it was concluded that a significant part of the total gelsolin in skeletal muscle cells is tightly associated with the thin filaments, and is an integral part of the myofibrils even at low Ca(++)-concentrations. From the coincidence of actin and gelsolin staining in myofibrils it was concluded that gelsolin is localized along the whole length of the thin filaments in the sarcomere.(ABSTRACT TRUNCATED AT 250 WORDS)

Actin Cytoskeleton↗
PubMed1977

Connectin, an elastic protein of muscle. Its abundance in cardiac myofibrils.

Pure myofibrils were isolated from bovine heart by sucrose layer ultracentrifugation. Cardiac myofibrils thus prepared contained more protein as insoluble stroma than skeletal muscle. The insoluble stroma largely consisted of connectin, an elastic protein of muscle. The connectin content in cardiac myofibrils was about 18% of the total myofibrillar protein and was three times that in skeletal myofibrils. In view of the role of connectin as an elastic component of muscle, the abundance of connectin in cardiac myofibrils may be responsible for keeping myofibrils short at rest. This would account for the more effective tension generation in cardiac muscle on passive stretching due to blood inflow (Stirling's law).

Amino Acids↗
PubMed1996

Limits of titin extension in single cardiac myofibrils.

Passive force and dynamic stiffness were measured in relaxed, single myofibrils from rabbit ventricle over a wide range of sarcomere lengths, from approximately 2-5 microns. Myofibril stretch up to sarcomere lengths of approximately 3 microns resulted in a steady increase in both force and stiffness. The shape of the length-force and the length-stiffness curves remained fully reproducible for repeated extensions to a sarcomere length of approximately 2.7 microns. Above this length, myofibrillar viscoelastic properties were apparently changed irreversibly, likely due to structural alterations within the titin (connectin) filaments. Stretch beyond approximately 3 microns sarcomere length resulted in a markedly reduced slope of the passive force curve, while the stiffness curve became flat. Thus, cardiac sarcomeres apparently reach a strain limit near a length of 3 microns. Above the strain limit, both curve types frequently showed a series of inflections, which we assumed to result from the disruption of titin-thick filament bonds and consequent addition of previously bound A-band titin segments to the elastic I-band titin portion. Indeed, we confirmed in immunofluorescence microscopic studies, using a monoclonal antibody against titin near the A/I junction, that upon sarcomere stretch beyond the strain limit length, the previously stationary antibody epitopes suddenly moved into the I-band, indicating A-band titin release. Altogether, the passive force/stiffness-length relation of cardiac myofibrils was qualitatively similar to, but quantitatively different from, that reported for skeletal myofibrils. From these results, we inferred that cardiac myofibrils have an approximately two times greater relative I-band titin extensibility than skeletal myofibrils. This could hint at differences in the maximum passive force-bearing capacity of titin filaments in the two muscle types.

Animals↗
PubMed1988

Immunocytochemical analysis of the regeneration of myofibrils in long-term cultures of adult cardiomyocytes of the rat.

Dissociated adult rat ventricular cardiomyocytes obtained from hearts by retrograde perfusion with collagenase were investigated in long-term cultures. Myofibril regeneration, isoprotein transition of alpha- and beta-myosin heavy chain (MHC), and M-band localization of M-creatine kinase in the reconstituting heart cells were studied. Myofibril formation was demonstrated by the use of antibodies against either cardiac C-protein or myomesin as early differentiation markers. Four days after plating, small myofibrils could be identified in attached cells in a perinuclear fashion; later in culture the cells displayed various shapes and myofibril distribution. Frequently a patchy distribution of myofibrils within the extending peripheral processes could be observed. Colocalization of sarcomeres and phalloidin-stained F-actin filament bundles was demonstrated by double fluorescence staining and by the use of high intensifying video microscopy and computerized image processing. The immunofluorescence distribution of alpha- and beta-MHC isoproteins in newly isolated and cultured cardiomyocytes changed from 100% alpha-MHC and 70% beta-MHC in rod-shaped cells to about 100% beta-MHC and 70% alpha-MHC in spread out cultured cells. This shift was corroborated by a relative gradual decline in alpha-MHC at the expense of increasing amounts of beta-MHC with time in culture as assessed by sodium dodecyl sulfate gel electrophoresis of total cell homogenates. In addition, whereas rod-shaped newly isolated cardiomyocytes showed a clear M-band association of M-creatine kinase as found in adult heart tissue, adult cultivated spread out cells did not show a cross-striated pattern after incubation with antibody. Taken together, these observations suggest that adult cardiomyocytes not only undergo extensive morphological transitions in long-term cultures, but also generate new myofibrillar structures lacking M-creatine kinase and containing the beta-MHC, thus fitting the characteristics of fetal myofibrils. These results indicate a change from the adult terminally differentiated to a less differentiated state of the cardiac cells in culture.

Animals↗
PubMed2003

Mutations in the motor domain modulate myosin activity and myofibril organization.

We have investigated the functional impact on cardiac myofibril organization and myosin motor activity of point mutations associated with familial hypertrophic cardiomyopathies (FHC). Embryonic chicken cardiomyocytes were transfected with vectors encoding green fluorescent protein (GFP) fused to a striated muscle myosin heavy chain (GFP-myosin). Within 24 hours of transfection, the GFP-myosin is found co-assembled with the endogenous myosin in striated myofibrils. The wild-type GFP-myosin had no effect on the organization of the contractile cytoskeleton of the cardiomyocytes. However, expression of myosin with the R403Q FHC mutation resulted in a small but significant decrease in myofibril organization, and the R453C and G584R mutations caused a more dramatic increase in myofibril disarray. The embryonic cardiomyocytes beat spontaneously in culture and this was not affected by expression of the wild-type or mutant GFP-myosin. For the biochemical analysis of myosin motor activity, replication defective adenovirus was used to express the wild-type and mutant GFP-myosin in C2C12 myotubes. The R403Q mutation enhanced actin filament velocity but had no effect on the myosin duty ratio. The R453C and G584R mutations impaired actin filament movement and both increased the duty ratio. The effects of these mutations on myosin motor activity correlate with changes in myofibril organization of live cardiomyocytes. Thus, mutations associated with hypertrophic cardiomyopathies that alter myosin motor activity can also impair myofibril organization.

Adenoviridae↗
PubMed1979

Differential response of myofibrils and 10-nm filaments to a cocarcinogen.

Multinucleated myotubes containing large numbers of striated myofibrils and large numbers of longitudinally-oriented 10-nm filaments were treated with the cocarcinogen phorbol-12-myristate-13-acetate (PMA) for 24, 48 or 72 hours. The inhibitory effects of PMA on the accumulation of myofibrils was evident within 24 hours, and by 72 hours virtually all striated myofibrils had disappeared. In contrast, the density of the 10-nm filaments was greatly enhanced in these myofibril-depleted myotubes. These effects were not due to a generalized cytotoxicity, for PMA stimulated the replication of the presumptive myoblasts and fibroblasts present in these cultures. 24 hours after removing the PMA, these myotubes assembled a new set of striated myofibrils and the density of 10-nm filaments diminished proportionately.

Animals↗
PubMed2006

Targeted disruption of N-RAP gene function by RNA interference: a role for N-RAP in myofibril organization.

N-RAP is a muscle-specific protein concentrated in myofibril precursors during sarcomere assembly and at intercalated disks in adult heart. We used RNA interference to achieve a targeted decrease in N-RAP transcript and protein levels in primary cultures of embryonic mouse cardiomyocytes. N-RAP transcript levels were decreased by approximately 70% within 2 days following transfection with N-RAP specific siRNA. N-RAP protein levels steadily decreased over several days, reaching approximately 50% of control levels within 6 days. N-RAP protein knockdown was associated with decreased myofibril assembly, as assessed by alpha-actinin organization into mature striations. Transcripts encoding N-RAP binding proteins associated with assembling or mature myofibrils, such as alpha-actinin, Krp1, and muscle LIM protein, were expressed at normal levels during N-RAP protein knockdown, and alpha-actinin and Krp-1 protein levels were also unchanged. Transcripts encoding muscle myosin heavy chain and nonmuscle myosin heavy chain IIB were also expressed at relatively normal levels. However, decreased N-RAP protein levels were associated with dramatic changes in the encoded myosin proteins, with muscle myosin heavy chain levels increasing and nonmuscle myosin heavy chain IIB decreasing. N-RAP transcript and protein levels recovered to normal by days 6 and 7, respectively, and the changes in myofibril organization and myosin heavy chain isoform levels were reversed. Our data indicate that we can achieve transient N-RAP protein knockdown using the RNA interference technique and that alpha-actinin organization into myofibrils in cardiomyocytes is closely linked to N-RAP protein levels. Finally, N-RAP protein levels regulate the balance between nonmuscle myosin IIB and muscle myosin by post-trancriptional mechanisms.

Actinin↗
PubMed1989

Sound-generating (sonic) motor system in a teleost fish (Porichthys notatus): sexual polymorphism in the ultrastructure of myofibrils.

One mechanism used by teleost fishes to produce acoustic communication signals involves the contraction of sonic "drum" muscles that appose the lateral walls of the swimbladder. In one marine species, the midshipman (Porichthys notatus), there is a sex difference in the overall size of the swimbladder as well as in the ultrastructural properties of its myofibrils. Additionally, there are two classes of sexually mature males referred to as Type I and Type II. The peripheral sonic motor system of Type I males differs from that of Type II males and females (which resemble each other) in a number of ways: (1) the mass of their swimbladder and associated sonic muscles is 50% greater, (2) their muscle fibers are several times larger and have a characteristically large volume of sarcoplasm that surrounds the myofibrils and is densely filled with mitochondria, (3) the length of z-lines of their myofibrils is about 20-fold greater, and (4) their sarcoplasmic reticulum is more highly branched. The ultrastructure of the myofibrils of Type II males and females resembles that found in the sonic muscle of males and females in other related species. The larger mass and specializations of the sonic muscle in Type I males are considered to be adaptations related to their known role in sound production and the unique long duration "humming" sounds that they generate during the breeding season. The similarity in the sonic motor system between females and Type II males is considered to be related to the utilization of an "alternative mating strategy" by Type II males. To our knowledge, this is the first documentation of a sex difference or, for that matter, a sexual polymorphism in the ultrastructural features of a vertebrate myofibril.

Animals↗
PubMed2006

Obscurin is required for the lateral alignment of striated myofibrils in zebrafish.

Obscurin/obscurin-MLCK is a giant sarcomere-associated protein with multiple isoforms whose interactions with titin and small ankyrin-1 suggest that it has an important role in myofibril assembly, structural support, and the sarcomeric alignment of the sarcoplasmic reticulum. In this study, we characterized the zebrafish orthologue of obscurin and examined its role in striated myofibril assembly. Zebrafish obscurin was expressed in the somites and central nervous system by 24 hours post-fertilization (hpf) and in the heart by 48 hpf. Depletion of obscurin using two independent morpholino antisense oligonucleotides resulted in diminished numbers and marked disarray of skeletal myofibrils, impaired lateral alignment of adjacent myofibrils, disorganization of the sarcoplasmic reticulum, somite segmentation defects, and abnormalities of cardiac structure and function. This is the first demonstration that obscurin is required for vertebrate cardiac and skeletal muscle development. The diminished capacity to generate and organize new myofibrils in response to obscurin depletion suggests that it may have a vital role in the causation of or adaptation to cardiac and skeletal myopathies.

Amino Acid Sequence↗
PubMed1993

Incorporation of microinjected biotin-labelled actin into nascent myofibrils of cardiac myocytes: an immunoelectron microscopic study.

Incorporation of microinjected biotin-labelled actin into nascent myofibrils of cultured cardiac muscle cells was investigated by immunogold electron microscopy. At the proximal parts of myofibrils, gold labelling was first found (at about 4 min after injection) around the A-band level. This observation suggests that polymerization of actin or the addition of newly-formed actin filaments occurs preferentially in association with myosin filaments to increase the myofibrillar girth. The distal terminals of developing myofibrils were also labelled at about 4 min after injection. This rapid incorporation of actin subunits at the myofibrillar ends suggests the continued reorganization and/or de novo formation of myofibrils at these positions. Along the extending direction of the myofibrillar terminals, gold particles were arranged in rows on the inner surface of the sarcolemma. These rows of particles continued to become longer with incubation. It appears that actin subunits are added at the membrane-associated ends of pre-existing actin filaments to increase the length of myofibrils.

Actins↗
PubMed1991

Regulation of ATP-stimulated releasable myofilaments from cardiac and skeletal muscle myofibrils.

The mechanism underlying the formation of easily releasable myofilaments, from myofibrils treated with an ATP-containing relaxing solution, was examined in this investigation. The proportion of releasable myofilaments purified from myofibrils of cardiac, fast- and slow-twitch muscles increased as the [ATP] was raised from 0 to 8.5 mM. The protein composition of the easily releasable myofilaments did not differ with increasing ATP concentrations as observed by 5-15% linear gradient SDS-PAGE. There is a nucleotide specificity to the release of myofilaments in the order of ATP greater than GTP much greater than UTP greater than CTP. Experiments with AMP-PNP and inorganic phosphate (Pi) showed that ATP hydrolysis and the build up of Pi are not requirements in the formation of the easily releasable myofilaments. The release of myofilaments was found to be insensitive to variations in pH from 6.5 to 7.5. The ATP stimulation of myofilaments release is ubiquitin-independent, since incubation of purified myofibrils with ubiquitin (1-100 micrograms/ml) at both 20 and 37 degrees C did not change the amount released. Modifying the free sulfhydryl group content by treatment of myofibrils with NEM (0.01-1 mM) or silver nitrate (0.1-10 mM) decreased the proportion of myofilaments that were releasable. Exclusion of 1 mM DTT from the preparation of myofibrils had similar results. These results indicate that the formation of easily releasable myofilaments can be mediated by metabolically related parameters such as the adenosine nucleotides and the reduction-oxidation status of the myofibrillar proteins of striated muscle.

Actin Cytoskeleton↗
PubMed1988

Spontaneous oscillatory contraction of sarcomeres in skeletal myofibrils.

We found that the lengths of all sarcomeres spontaneously oscillated in an isolated skeletal myofibril, when both ends were fixed, submillimolar to millimolar concentrations of ATP, ADP and inorganic phosphate (Pi) were present, and Ca2+ was removed. Narrowing and widening of an H-zone and an I-band were observed corresponding to the shortening and lengthening of a sarcomere, suggesting that thick and thin filaments slide past each other. The oscillation of each sarcomere was asymmetrical, consisting of a rapid lengthening phase and a slow shortening phase. The period of oscillation was about 3s; the peak-to-peak amplitude of oscillation reached as much as 30% of the average sarcomere length. The propagation of the sarcomere oscillation along the long axis of the myofibril was observed occasionally in single myofibrils and frequently in bundles of myofibrils. The 'state'-diagram showing the concentration range of ADP and Pi in which contraction, oscillation or relaxation of myofibrils occurs in the presence of ATP and the absence of Ca2+ suggested that the oscillation is a third state of skeletal muscle located in between the contracting and relaxing states.

Adenosine Diphosphate↗