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PubMed2003

Sheep heart RNA stimulates myofibril formation and beating in cardiac mutant axolotl hearts in organ culture.

In the Mexican axolotl, Ambystoma mexicanum, recessive mutant gene c, when homozygous, results in a failure of the heart to form sarcomeric myofibrils and contract normally. Previous studies have shown that purified RNA from normal anterior endoderm or from medium conditioned with anterior endoderm/pre-cardiac mesoderm has the capacity to rescue mutant hearts in organ culture. In the present study, RNA extracted from adult sheep heart was tested for its capacity to promote differentiation in the mutant axolotl hearts. Mutant hearts cultured in the presence of the sheep heart RNA in Steinberg's solution for 48 h displayed rhythmic contractions. Ultrastructural studies showed that the rescued mutant axolotl ventricular myocardial cells contained myofibrils of normal morphology. Mutant hearts cultured in Steinberg's solution alone did not beat throughout their lengths and myofibrils were not observable in the ventricles. Confocal microscopy confirmed the increase of Tropomyosin expression and formation of myofibrils in mutant hearts treated by sheep heart RNA. Thus, sheep heart RNA promotes myofibrillogenesis and the development of contractile function in embryonic cardiac mutant axolotl hearts.

Ambystoma mexicanum↗
PubMed2004

Passive stiffness of Drosophila IFM myofibrils: a novel, high accuracy measurement method.

As the smallest muscle-cell substructure that retains the intact contractile apparatus, the single myofibril is considered the optimal specimen for muscle mechanics, although its small size also poses some technical difficulties. Myofibrils from Drosophila indirect flight muscle (IFM) are particularly difficult to study because their high passive stiffness makes them hard to handle, and too resistant to stretch to produce enough elongation for the accurate measurement of sarcomere length change. In this study, we devised a novel method for accurate stiffness measurement of single relaxed myofibrils using microfabricated cantilevers and phase contrast microscopy. A special experimental protocol was developed to minimize errors, and some data analysis strategies were used to identify and exclude spurious data. Remarkably consistent results were obtained from Drosophila IFM myofibrils. This novel, high accuracy method is potentially an effective tool for detecting small passive stiffness change in muscle mutants.

Animals↗
PubMed2005

How to build a myofibril.

Building a myofibril from its component proteins requires the interactions of many different proteins in a process whose details are not understood. Several models have been proposed to provide a framework for understanding the increasing data on new myofibrillar proteins and their localizations during muscle development. In this article we discuss four current models that seek to explain how the assembly occurs in vertebrate cross-striated muscles. The models hypothesize: (a) stress fiber-like structures as templates for the assembly of myofibrils, (b) assembly in which the actin filaments and Z-bands form subunits independently from A-band subunits, with the two subsequently joined together to form a myofibril, (c) premyofibrils as precursors of myofibrils, or (d) assembly occurring without any intermediary structures. The premyofibril model, proposed by the authors, is discussed in more detail as it could explain myofibrillogenesis under a variety of different conditions: in ovo, in explants, and in tissue culture studies on cardiac and skeletal muscles.

Actinin↗
PubMed1984

Binding and location of AMP deaminase in rabbit psoas muscle myofibrils.

It is shown that an interaction exists between AMP deaminase (EC 3.5.4.6) and myofibrils that is sufficiently strong (Kd congruent to 10(-10) M) for more than 99% of the binding sites for the enzyme to be filled in vivo. The binding is not strong enough, however, to stop removal of the enzyme during the extensive washing normally used in the preparation of myofibrils. Fluorescent antibodies to the enzyme label myofibrils close to the junction of the A- and I-bands. The invariance of the position of the antibody stripes at this site, over a range of sarcomere lengths, indicates that the enzyme is attached to the A-band. The intensity of the fluorescence declines in parallel with dissociation of the enzyme. In this muscle, the number of AMP deaminase binding sites per thick filament is approximately six, suggesting that the enzyme is located at a single axial position in each half A-band. Electron microscopy of negatively stained, antibody-labelled myofibrils reveals the distance between the AMP deaminase sites at opposite ends of an A-band to be 1.69(+/- 0.02 micron). Since the length of the A-band is 1.57 micron, the binding site for the enzyme must be significantly beyond where thick filaments have previously been thought to end.

AMP Deaminase↗
PubMed1994

A latent form of protein phosphatase 1 alpha associated with bovine heart myofibrils.

The catalytic subunit of the major protein phosphatase associated with bovine cardiac myofibrils was purified to homogeneity. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of the enzyme revealed only one band with an apparent molecular weight of 37,000. On gel filtration chromatography, the phosphatase activity and the protein co-eluted as a single peak with an apparent molecular weight of 37,000. The purified enzyme was identified as the catalytic subunit of protein phosphatase 1, as determined by sensitivity to inhibitor 1, inhibitor 2, okadaic acid and by specific immunostaining. Evidence obtained with specific antipeptide antibodies demonstrated that this myofibril protein phosphatase was predominantly the alpha isoform of protein phosphatase 1. The purified catalytic subunit was completely inactive. It was activated by pretreatment with Co2+/trypsin in the presence of high ionic strength. Treatment with trypsin alone did not activate the latent enzyme. The enzyme was also activated by Co2+ or Mn2+ alone but not by Ca2+, Mg2+, Ni2+, Cu2+ or Zn2+. Activation of the enzyme was not reversed by removal of Co2+, but Mn(2+)-activated phosphatase activity was partially reversed when Mn2+ was removed. The catalytic subunit could form a 1:1 complex with inhibitor 2 in vitro. The resulting holoenzyme was also activated by pretreatment with Co2+. Since phosphatase 1 alpha is the major phosphatase associated with cardiac myofibril, it is suggested that it is responsible for the dephosphorylation of myosin and other myofibril phosphoproteins.

Amino Acid Sequence↗
PubMed1991

The effect of Mg2+ on the Ca2+ binding to troponin C in rabbit fast skeletal myofibrils.

The effect of Mg2+ on the Ca2+ binding to rabbit fast skeletal troponin C and the CA2+ dependence of myofibrillar ATPase activity was studied in the physiological state where troponin C was incorporated into myofibrils. The Ca2+ binding to troponin C in myofibrils was measured directly by 45Ca using the CDTA-treated myofibrils as previously reported (Morimoto, S. and Ohtsuki, I. (1989) J. Biochem. 105, 435-439). It was found that the Ca2+ binding to the low and high affinity sites of troponin C in myofibrils was affected by Mg2+ competitively and the Ca2(+)- and Mg2(+)-binding constants were 6.20 x 10(6) and 1.94 x 10(2) M-1, respectively, for the low affinity sites, and 1.58 x 10(8) and 1.33 x 10(3) M-1, respectively, for the high affinity sites. The Ca2+ dependence of myofibrillar ATPase was also affected by Mg2+, with the apparent Ca2(+)- and Mg2(+)-binding constants of 1.46 x 10(6) and 276 x 10(2) M-1, respectively, suggesting that the myofibrillar ATPase was modulated through a competitive action of Mg2+ on Ca2+ binding to the low affinity sites, though the Ca2+ binding to the low affinity sites was not simply related to the myofibrillar ATPase.

Adenosine Triphosphatases↗
PubMed2006

Voltage-dependent calcium influx mediates maturation of myofibril arrangement in ascidian larval muscle.

Calcium signaling is important for multiple events during embryonic development. However, roles of calcium influx during embryogenesis have not been fully understood since routes of calcium influx are often redundant. To define roles of voltage-gated calcium channel (Cav) during embryogenesis, we have isolated an ascidian Cav beta subunit gene (TuCavbeta) and performed gene knockdown using the morpholino antisense oligonucleotide (MO). The suppression of Cav activity by TuCavbetaMO remarkably perturbed gastrulation and tail elongation. Further, larvae with normal morphology also failed to exhibit motility. Phalloidin-staining showed that arrangement of myofibrils was uncoordinated in muscle cells of TuCavbetaMO-injected larvae with normal tail. To further understand the roles of Cav activity in myofibrillogenesis, we tested pharmacological inhibitions with ryanodine, curare, and N-benzyl-p-toluensulphonamide (BTS). The treatment with ryanodine, an intracellular calcium release blocker, did not significantly affect the motility and establishment of the myofibril orientation. However, treatment with curare, an acetylcholine receptor blocker, and BTS, an actomyosin ATPase specific inhibitor, led to abnormal motility and irregular orientation of myofibrils that was similar to those of TuCavbetaMO-injected larvae. Our results suggest that contractile activation regulated by voltage-dependent calcium influx but not by intracellular calcium release is required for proper arrangement of myofibrils.

Animals↗
PubMed2005

Proteasomes are tightly associated to myofibrils in mature skeletal muscle.

Proteasomes are the major actors of nonlysosomal cytoplasmic protein degradation. In particular, these large protein complexes (about 2500 kDa) are considered to be responsible for muscular degradation during skeletal muscle atrophy. Despite their unusual and important size, they are widely described as soluble and mobile in the cytoplasm. In mature skeletal muscle, we have previously observed a sarcomeric distribution of proteasomes, as revealed by the distribution of alpha1/p27K, a subunit of the 20S core-particle (prosome) of proteasome. Here, we extend these observations at the electron microscopic level in vivo. We also show that this sarcomeric pattern is dependent of the extension of the sarcomere. Using isolated myofibrils, we demonstrate that proteasomes are still attached to the myofibrils after the isolation procedure, and reproduce the observations made in vivo. In addition, the extraction of actin by gelsolin largely removes proteasomes from isolated myofibrils, but some of them are held in place after this extraction, showing a sarcomeric disposition in the absence of any detectable actin, and suggesting the existence of another molecular partner for these interactions. From these results, we conclude that most of detectable 20S proteasomes in skeletal muscle cells is tightly attached to the myofibrils.

Actins↗
PubMed2000

Morphology and transverse stiffness of Drosophila myofibrils measured by atomic force microscopy.

Atomic force microscopy was used to investigate the surface morphology and transverse stiffness of myofibrils from Drosophila indirect flight muscle exposed to different physiologic solutions. I- and A-bands were clearly observed, and thick filaments were resolved along the periphery of the myofibril. Interfilament spacings correlated well with estimates from previous x-ray diffraction studies. Transverse stiffness was measured by using a blunt tip to indent a small section of the myofibrillar surface in the region of myofilament overlap. At 10 nm indention, the effective transverse stiffness (K( perpendicular)) of myofibrils in rigor solution (ATP-free, pCa 4.5) was 10.3 +/- 5.0 pN nm(-1) (mean +/- SEM, n = 8); in activating solution (pCa 4.5), 5.9 +/- 3.1 pN nm(-1); and in relaxing solution (pCa 8), 4.4 +/- 2.0 pN nm(-1). The apparent transverse Young's modulus (E( perpendicular)) was 94 +/- 41 kPa in the rigor state and 40 +/- 17 kPa in the relaxed state. The value of E( perpendicular) for calcium-activated myofibrils (55 +/- 29 kPa) was approximately a tenth that of Young's modulus in the longitudinal direction, a difference that at least partly reflects the transverse flexibility of the myosin molecule.

Animals↗
PubMed1971

Correlations of length and volume measurements in myofibril suspensions.

Instrumentation has been developed for the rapid electronic sizing of large numbers of myofibrils. The response of myofibrils in the presence of ATP to changes in Ca(++) concentration was examined. Shortening of myofibrils upon addition of Ca(++) was accompanied by an increased protein effective volume of approximately 10-40%. Whereas ATPase activation and increased turbidity of myofibrils upon addition of Ca(++) were reversible upon subsequent addition of EGTA, the shortening and swelling were irreversible. It is proposed that the swelling may result from the breaking of hydrophobic bonds within myosin. The ATPase activity and turbidity are measures of the input, while the shortening and swelling are measures of the output of a coupled nonequilibrium process; failure of reversal of the output indicates an uncoupling under the experimental conditions.

Adenosine Triphosphatases↗
PubMed1996

Mechanical characterization of skeletal muscle myofibrils.

A new instrument, based on a technique described previously, is presented for studying mechanics of micron-scale preparations of two to three myofibrils or single myofibrils from muscle. Forces in the nanonewton to micronewton range are measurable with 0.5-ms time resolution. Programmed quick (200-microseconds) steps or ramp length changes are applied to contracting myofibrils to test their mechanical properties. Individual striations can be monitored during force production and shortening. The active isometric force, force-velocity relationship, and force transients after rapid length steps were obtained from bundles of two to three myofibrils from rabbit psoas muscle. Contrary to some earlier reports on myofibrillar mechanics, these properties are generally similar to expectations from studies on intact and skinned muscle fibers. Our experiments provide strong evidence that the mechanical properties of a fiber result from a simple summation of the myofibrillar force and shortening of independently contracting sarcomeres.

Animals↗
PubMed1997

Actin-titin interaction in cardiac myofibrils: probing a physiological role.

The high stiffness of relaxed cardiac myofibrils is explainable mainly by the expression of a short-length titin (connectin), the giant elastic protein of the vertebrate myofibrillar cytoskeleton. However, additional molecular features could account for this high stiffness, such as interaction between titin and actin, which has previously been reported in vitro. To probe this finding for a possible physiological significance, isolated myofibrils from rat heart were subjected to selective removal of actin filaments by a calcium-independent gelsolin fragment, and the "passive" stiffness of the specimens was recorded. Upon actin extraction, stiffness decreased by nearly 60%, and to a similar degree after high-salt extraction of thick filaments. Thus actin-titin association indeed contributes to the stiffness of resting cardiac muscle. To identify possible sites of association, we employed a combination of different techniques. Immunofluorescence microscopy revealed that actin extraction increased the extensibility of the previously stiff Z-disc-flanking titin region. Actin-titin interaction within this region was confirmed in in vitro cosedimentation assays, in which multimodule recombinant titin fragments were tested for their ability to interact with F-actin. By contrast, such assays showed no actin-titin-binding propensity for sarcomeric regions outside the Z-disc comb. Accordingly, the results of mechanical measurements demonstrated that competition with native titin by recombinant titin fragments from Z-disc-remote, I-band or A-band regions did not affect passive myofibril stiffness. These results indicate that it is actin-titin association near the Z-disc, but not along the remainder of the sarcomere, that helps to anchor the titin molecule at its N-terminus and maintain a high stiffness of the relaxed cardiac myofibril.

Actins↗
PubMedDate not supplied

Identification of a myofibril-bound serine protease and its endogenous inhibitor in mouse skeletal muscle.

Myofibrillar proteins, like all other intracellular proteins, are in a dynamic state of continual degradation and resynthesis. The proteolytic system responsible for degrading myofibrillar proteins in skeletal muscle is not well defined. A proteolytic activity associated to myofibrils was found in mouse skeletal muscle, as show electrophoretic patterns, and denominated by us, as protease M. During incubation of whole myofibrils at 37 degrees C, myosin heavy chain, alpha actinin, actin and troponin T suffered degradation. These effects were inhibited selectively by serine protease inhibitors (soybean trypsin inhibitor, di-isopropyl phosphofluoridate, phenylmethanesulfonyl fluoride). Using myofibrils as protease M source, azocaseinolytic activity was also detected. Endogenous inhibitor and various compounds effects on protease M activity were also quantified by trichloroacetic acid soluble products formation, using radiolabeled myofibrils. An endogenous trypsin inhibitor isolated from the muscle cytoplasmic fraction could inhibit protease M activity on myofibrillar proteins and on azocasein. While K(+) increased protease M activity, the presence of Ca(2+) did not show any effect. Data presented in this study suggest that reported protease M may be implicated in myofibrillar degradation in vivo and isolated endogenous inhibitor may provide a mechanism to control its action in mouse skeletal muscle.

Actinin↗
PubMed1997

Imaging 'intact' myofibrils with a near-field scanning optical microscope.

Fluorescently labelled myofibrils were imaged in physiological salt solution by near-field scanning optical microscopy and shear-force microscopy. These myofibrils were imaged in vitro, naturally adhering to glass while retaining their ability to contract. The Z-line protein structure of the myofibrils was antibody labelled and easily identified in the near-field fluorescence images. The distinctive protein banding structure of the myofibril was also seen clearly in the shear-force images without any labelling requirement. With the microscope in the transmission mode, resolution of the fluorescence images was degraded significantly by excessive specimen thickness (> 1 micron), whereas the shear-force images were less affected by specimen thickness and more affected by poor adherence to the substrate. Although the exact mechanism generating contrast in the shear-force images is still unknown, shear-force imaging appears to be a promising new imaging modality.

Animals↗
PubMed1997

The C terminus of cardiac troponin I is essential for full inhibitory activity and Ca2+ sensitivity of rat myofibrils.

Although the C terminus of troponin I is known to be important in myofilament Ca2+ regulation in skeletal muscle, the regulatory function of this region of cardiac troponin I (cTnI) has not been defined. To address this question, the following recombinant proteins were expressed in Escherichia coli and purified: mouse wild-type cTnI (WT cTnI; 211 residues), cTnI-(1-199) (missing 12 residues), cTnI-(1-188) (missing 23 residues), and cTnI-(1-151) (missing 60 residues). The inhibitory activity of cTnI and the mutants was tested in myofibrils, from which cTnI.cTnC was extracted by exchanging endogenous cardiac troponin with exogenous cTnT causing the Ca2+ sensitivity of the myofibrils to be lost. Addition of increasing amounts of exogenous WT cTnI or cTnI-(1-199) to cTnT-treated myofibrils at pCa 8 caused a concentration-dependent inhibition of the maximum ATPase activity. However, cTnI-(1-188) and cTnI-(1-151) inhibited this activity to about 75% and 50% of that of the WT cTnI, respectively. We also formed a complex of either WT cTnI or each of the mutants with cTnC, reconstituted the complex into the cTnT-treated myofibrils, and measured the Mg2+-ATPase activity as a function of pCa. We found that the cTnI-(1-188).cTnC complex only partially restored Ca2+ sensitivity, whereas the cTnI-(1-151).cTnC complex did not restore any Ca2+ sensitivity. Each cTnI C-terminal deletion mutant was able to bind to cTnC, as shown by urea-polyacrylamide gel-shift analysis and size exclusion chromatography. Each mutant also co-sedimented with actin. Our results indicate that residues 152-199 (C-terminal to the inhibitory region) of cTnI are essential for full inhibitory activity and Ca2+ sensitivity of myofibrillar ATPase activity in the heart.

Amino Acid Sequence↗
PubMed1988

Posttranslational incorporation of contractile proteins into myofibrils in a cell-free system.

The incorporation of newly synthesized protein into myofibrils has been examined in a cell-free system. Myofibrils were added to a reticulocyte lysate after the in vitro translation of muscle-specific poly(A)+RNA. Only a small number of the many synthesized proteins were found to associate with the exogenously added myofibrils. These proteins were all identified as sarcomeric components and had subunit mobilities (Mr) of 200, 140, 95, 86, 43, 38, 35, 25, 23, 20, and 18 kD. The association was rapid (t1/2 less than 15 min) and, for most of the proteins, relatively temperature insensitive. Except for a 43-kD polypeptide, tentatively identified as beta-actin, none of the proteins encoded by brain poly(A)+RNA associated with the myofibrils. When filaments made from purified myosin or actin were used as the "capture" substrates, only thick or thin filament proteins, respectively, were incorporated. Incorporation was substantially reduced when cross-linked myosin filaments were used. These results are compatible with a model in which proteins of the sarcomere are in kinetic equilibrium with homologous proteins in a soluble pool.

Animals↗
PubMed1999

UNC-60B, an ADF/cofilin family protein, is required for proper assembly of actin into myofibrils in Caenorhabditis elegans body wall muscle.

The Caenorhabditis elegans unc-60 gene encodes two functionally distinct isoforms of ADF/cofilin that are implicated in myofibril assembly. Here, we show that one of the gene products, UNC-60B, is specifically required for proper assembly of actin into myofibrils. We found that all homozygous viable unc-60 mutations resided in the unc-60B coding region, indicating that UNC-60B is responsible for the Unc-60 phenotype. Wild-type UNC-60B had F-actin binding, partial actin depolymerizing, and weak F-actin severing activities in vitro. However, mutations in UNC-60B caused various alterations in these activities. Three missense mutations resulted in weaker F-actin binding and actin depolymerizing activities and complete loss of severing activity. The r398 mutation truncated three residues from the COOH terminus and resulted in the loss of severing activity and greater actin depolymerizing activity. The s1307 mutation in a putative actin-binding helix caused greater activity in actin-depolymerizing and severing. Using a specific antibody for UNC-60B, we found varying protein levels of UNC-60B in mutant animals, and that UNC-60B was expressed in embryonic muscles. Regardless of these various molecular phenotypes, actin was not properly assembled into embryonic myofibrils in all unc-60 mutants to similar extents. We conclude that precise control of actin filament dynamics by UNC-60B is required for proper integration of actin into myofibrils.

Actin Depolymerizing Factors↗
PubMed1962

Fine structural localization of adenosinetriphosphatase activity in heart muscle myofibrils.

Activity of myofibrillar adenosinetriphosphatase was demonstrated histochemically at a fine structural level in isolated, unfixed or hydroxyadipaldehyde-fixed cardiac myofibrils in the rat, using a lead precipitation technique and either Ca(++) or Mg(++) as activating ion. Activity in relaxed myofibrils was found in the A band, but not the H, I, or Z bands. Deposits of final product frequently exhibited an axial periodicity of near 365 A, and bore a close relationship to filaments within the A band. Several patterns of distribution occurred in contracted myofibrils. In myofibrils which had shortened to the point of disappearance of the I band, final product was distributed throughout the sarcomere, except for the unreactive Z band. A second type of distribution occurred in strongly contracted fibers in which there was intensification of activity in the center of the sarcomere. These findings are discussed in the light of the recent morphological evidence and it is suggested that the distribution of final product is consistent with localization of enzyme activity to the cross-bridges between the thick and thin filaments.

Actin Cytoskeleton↗