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PubMed2000

Identification of a myofibril-bound serine proteinase (MBSP) in the skeletal muscle of lizard fish Saurida wanieso which specifically cleaves the arginine site.

A myofibril-bound serine proteinase (MBSP) from the skeletal muscle of lizard fish (Saurida wanieso) was purified to homogeneity by a heating treatment followed by a series of column chromatographies on DEAE-Sephacel, Sephacryl S-200, Q-Sepharose, Hydroxyapatite and Benzamidine-Sepharose 6B, and characterized enzymatically. On SDS-poly-acrylamide gel electrophoresis (SDS-PAGE), the purified enzyme showed a band with molecular mass of approximately 29 kDa under reducing conditions, while 60 kDa under non-reducing conditions. The optimum temperature of the enzyme was 50 degrees C using t-butyloxycarbonyl-Phe-Ser-Arg-4-methylcoumaryl-7-amide (Boc-Phe-Ser-Arg-MCA) as a substrate. Substrate specificity analysis both using MCA-substrates and peptides showed that MBSP specifically cleaved at the carboxyl side of the arginine residue. Inhibitor susceptibility analysis revealed that MBSP was inhibited effectively by Pefabloc SC, soybean trypsin inhibitor (STI) and aprotinin, indicating the characteristic of a serine proteinase. When myofibril was incubated with the enzyme, it optically degraded myosin heavy chain at 55-60 degrees C, while alpha-actinin and actin were not at all hydrolyzed as detected by immunoblotting. The N-terminal amino acid sequence of MBSP was partially determined as IVGGAEXVPY- and was very homologous to other serine proteases.

Amino Acid Motifs↗
PubMed1994

The contraction state of myofibrils during global ischemia and after reperfusion following different forms of cardiac arrest. Correlation with metabolic parameters in the canine heart.

This study was undertaken in order to obtain information on the mode of reaction of the contractile apparatus after different forms of cardiac arrest, global ischemia and reperfusion, as well as on possible correlations between the contraction state of myofibrils and biochemical parameters. During the survival time, before the level of 3 mumol/gww creatine phosphate (CP) is reached, the contraction state shows only minor changes. During the revival time in which ATP tissue concentrations decay to 4 mumol/gww, the contribution of ATP, lactate, anorganic phosphate (Pa) and acidosis to the degree of relaxation depends on the method of cardiac arrest. At defined biochemical values, the degree of relaxation is comparable after aortic cross clamping (ACC) and St. Thomas perfusion, but significantly different compared to HTK perfusion. Thus, during the revival time, the relaxation of sarcomeres depends predominantly on the composition of the solutions used for cardiac arrest. The re-entry of contraction below 3 mumol/gww ATP is correlated with the ATP concentration, independent of the form of cardiac arrest. Reperfusion after HTK or St. Thomas cardioplegia and reversible ischemia leads to the focal formation of contraction bands, which do not occur during ischemia. This contraction state is significantly more pronounced after reperfusion of St. Thomas arrested hearts. Thus, the contraction state of myofibrils is influenced not only by alterations in metabolite concentrations, but also by the composition of cardioplegic solutions and by the characteristic conditions (sufficient energy, oxygen and Calcium) during reperfusion.

Adenosine Triphosphate↗
PubMed2000

To the heart of myofibril assembly.

One of the most fascinating examples of cytoskeletal assembly is the myofibril, the contractile structure of striated (i.e. skeletal and cardiac) muscle. Myofibrils are composed of repeating contractile units known as sarcomeres, perhaps the most highly ordered macromolecular structures in eukaryotic cells. When skeletal and cardiac muscle cells differentiate, thousands of structural and regulatory molecules assemble into the semicrystalline sarcomeric contractile units. As a consequence of this precise assembly, many different classes of proteins function together to convert the molecular interactions of actin and myosin efficiently into the macroscopic movements of contractile activity.

Animals↗
PubMed1975

3H leucine incorporation into myofibrils of normal and dystrophic mouse skeletal muscle.

The study of 3H leucine incorporation into skeletal muscle of mouse muscular dystrophy (129 ReJ/dy Bar Harbour strain) shows the uptake of isotope into myofibrils. The techniques employed were light and EM autoradiography before and after glycerination (Szent-Gyorgyi 1947). The results indicate a marked drop in uptake of the 3H-Leucine into myofibrils in the dystrophic animals, supporting the contention of Nihei et al (1971) that reduced myosin synthesis occurs in mouse muscular dystrophy.

Animals↗
PubMed2001

Ultrastructural and biochemical localization of N-RAP at the interface between myofibrils and intercalated disks in the mouse heart.

N-RAP is a recently discovered muscle-specific protein found at cardiac intercalated disks. Double immunogold labeling of mouse cardiac muscle reveals that vinculin is located immediately adjacent to the fascia adherens region of the intercalated disk membrane, while N-RAP extends approximately 100 nm further toward the interior of the cell. We partially purified cardiac intercalated disks using low- and high-salt extractions followed by density gradient centrifugation. Immunoblots show that this preparation is highly enriched in desmin and junctional proteins, including N-RAP, talin, vinculin, beta1-integrin, N-cadherin, and connexin 43. Electron microscopy and immunolabeling demonstrate that N-RAP and vinculin are associated with the large fragments of intercalated disks that are present in this preparation, which also contains numerous membrane vesicles. Detergent treatment of the partially purified intercalated disks removed the membrane vesicles and extracted vinculin and beta1-integrin. Further separation on a sucrose gradient removed residual actin and myosin and yielded a fraction morphologically similar to fasciae adherentes that was highly enriched in N-RAP, N-cadherin, connexin 43, talin, desmin, and alpha-actinin. The finding that N-RAP copurifies with detergent-extracted intercalated disk fragments even though beta-integrin and vinculin have been completely removed suggests that N-RAP association with the adherens junction region is mediated by the cadherin system. Consistent with this hypothesis, we found that recombinant N-RAP fragments bind alpha-actinin in a gel overlay assay. In addition, immunofluorescence shows that N-RAP remains bound at the ends of isolated, detergent-treated cardiac myofibrils. These results demonstrate that N-RAP remains tightly bound to myofibrils and fasciae adherentes during biochemical purification and may be a key constituent in the mechanical link between these two structures.

Animals↗
PubMed2005

Enzyme-aided modification of chicken-breast myofibril proteins: effect of laccase and transglutaminase on gelation and thermal stability.

The effect of laccase and transglutaminase (TG) on cross-linking, gelation, and thermal stability of salt-soluble chicken-breast myofibril proteins was investigated at pH 6. Both enzymes modified the protein pattern detected by SDS-PAGE. Identification of proteins by peptide mass mapping showed that myosin heavy chain (MHC) and troponin T were the most affected proteins. These proteins faded or disappeared as a function of the incubation time with both enzymes on SDS-PAGE. The molecular weight of actin was not, however, affected by either enzyme. The effects that the enzymes had on the gel formation of chicken-breast myofibrils were studied in 0.35 and 0.60 M NaCl solutions at 3% protein content and a constant temperature of 40 degrees C by using a small deformation viscoelastic measurement. TG substantially increased the storage modulus (G') of 3% protein in 0.35 M NaCl. Without the enzymes, gelation was insignificant in 0.35 M NaCl. The increased solubility of the proteins at 0.60 M NaCl intensified gelation with TG. G' increased 32 and 64% at dosages of 10 and 100 nkat of TG, respectively. Also, laccase increased G' of the gel in 0.60 M salt concentration. However, a high laccase dosage decreased the magnitude of G' below the control level. Differential scanning calorimetric (DSC) measurements indicated slightly reduced myosin heat stability after TG pretreatment and increased actin heat stability with both enzymes. Maximum transition temperatures did not alter with either enzyme.

Animals↗
PubMed1998

Expression of sarcomeric proteins and assembly of myofibrils in the putative myofibroblast cell line BHK-21/C13.

The expression and organization patterns of several myofibrillar proteins were analysed in the putative myofibroblast cell line BHK-21/C13. Although this cell line originates from renal tissue, the majority of the cells express titin. In these cells, titin is, under standard culture conditions, detected in myofibril-like structures (MLSs), where it alternates with non-muscle myosin (NMM). Expression of sarcomeric myosin heavy chain (sMyHC) is observed in a small minority of cells, while other sarcomeric proteins, such as nebulin, myosin binding protein C (MyBP-C), myomesin and M-protein are not expressed at all. By changing the culture conditions in a way equal to conditions that induce differentiation of skeletal muscle cells, a process reminiscent of sarcomerogenesis in vitro is induced. Within one day after the switch to a low-nutrition medium, myofibrillar proteins can be detected in a subset of cells, and after two to five days, all myofibrillar proteins examined are organized in typical sarcomeric patterns. Frequently, cross-striations are visible with phase contrast optics. Transfection of these cells with truncated myomesin fragments showed that a specific part of the myomesin molecule, known to contain a titin-binding site, binds to MLSs, whereas other parts do not. These results demonstrate that this cell line could serve as a powerful model to study the assembly of myofibrils. At the same time, its transfectability offers an invaluable tool for in vivo studies concerning binding properties of sarcomeric proteins.

Animals↗
PubMed2000

Factors contributing to troponin exchange in myofibrils and in solution.

The troponin complex in a muscle fiber can be replaced with exogenous troponin by using a gentle exchange procedure in which the actin-tropomyosin complex is never devoid of a full complement of troponin (Brenner et al. (1999) Biophys J 77: 2677-2691). The mechanism of this exchange process and the factors that influence this exchange are poorly understood. In this study, the exchange process has now been examined in myofibrils and in solution. In myofibrils under rigor conditions, troponin exchange occurred preferentially in the region of overlap between actin and myosin when the free Ca2+ concentration was low. At higher concentrations of Ca2+, the exchange occurred uniformly along the actin. Ca2+ also accelerated troponin exchange in solution but the effect of S1 could not be confirmed in solution experiments. The rate of exchange in solution was insensitive to moderate changes in pH or ionic strength. Increasing the temperature resulted in a two-fold increase in rate with each 10 degrees C increase in temperature. A sequential two step model of troponin binding to actin-tropomyosin could simulate the observed association and dissociation transients. In the absence of Ca2+ or rigor S1, the following rate constants could describe the binding process: k1 = 7.12 microM(-1) s(-1), k(-1) = 0.65 s(-1), k2 = 0.07 s(-1), k(-2) = 0.0014 s(-1). The slow rate of detachment of troponin from actin (k(-2)) limits the rate of exchange in solution and most likely contributes to the slow rate of exchange in fibers.

Actins↗
PubMed2000

Dynamics of actin and alpha-actinin in nascent myofibrils and stress fibers.

Actin labeled with fluorescein isothiocyanate (FITC) and alpha-actinin labeled with rhodamine (rh) were co-injected into chick embryonic cardiac myocytes and fibroblasts. In cardiomyocytes, FITC-actin was distributed in nonstriated lines, linearly arranged punctate structures with short intervals, and cross-striated bands with regular sarcomeric intervals. rh-alpha-Actinin was seen to be distributed in the same pattern in the former two portions, and in the center of each striation in the latter portion. Photobleaching of structures incorporated with these fluorescent analogs revealed that the fluorescent recovery rate of actin decreased in the order of nonstriated > punctated > striated portions, while that of alpha-actinin was low and stable at all portions. During the transition phase from punctate to regular sarcomere structures of these proteins, short spaced alpha-actinin dots adjoined each other and aligned with Z bands of neighboring myofibrils. It appears that both the difference in exchangeability between actin and alpha-actinin molecules and the movement of alpha-actinin dots during this phase of myofibrillogenesis are related to sarcomere lengthening and I-Z-I brush formation; adjoining dots of low-exchangeable alpha-actinin may provide favorable situations for exchangeable actin molecules in filaments to elongate and/or rearrange. In fibroblasts, both FITC-actin and rh-alpha-actinin formed nonstriated lines. In these cells, exchangeabilities of both proteins were high and similar in rate. This seems to indicate that stress fibers are constantly exchanging their components for motile and other vital functions of these cells. The high exchangeabilities of both proteins in stress fibers showthat these fibers are clearly different from nonstriated, stress-fiber like structures of nascent myofibrils.

Actinin↗
PubMed2002

Subcellular and molecular mechanisms of the effects of cardiac glycosides and angiotensin-converting enzyme inhibitors on contractile function and energy conversion in myocardial myofibrils under normal conditions and during acute cardiac insufficiency.

Experiments on skinned and hybrid myocardial fibers isolated from normal dogs and animals subjected to 120-min occlusion of the anterior interventricular branch of the coronary artery showed that in contrast to cardiac glycosides, angiotensin-converting enzyme inhibitors suppress contractile ability of myocardial myofibrils in a dose-independent manner within the concentration range of 10(-12)-10(-4)M. This effect is accompanied by a decrease in fiber relaxation rate most pronounced in the presence of captopril. Actin, the major protein of fine filaments is the target for b-acetyldigoxin, K-strophanthin, captopril, enalapril, and trandolapril in myocardial myofibrils. During coronary occlusion, the inhibitors of angiotensin-converting enzyme induce structural and conformational changes in actin that decrease efficiency of contraction. The data obtained cast doubt on advisability of therapeutic use of angiotensin-converting enzyme inhibitors in the therapy of myocardial infarction, especially in its early period.

Actins↗
PubMed2001

Dimethyl sulphoxide enhances the effects of P(i) in myofibrils and inhibits the activity of rabbit skeletal muscle contractile proteins.

In the catalytic cycle of skeletal muscle, myosin alternates between strongly and weakly bound cross-bridges, with the latter contributing little to sustained tension. Here we describe the action of DMSO, an organic solvent that appears to increase the population of weakly bound cross-bridges that accumulate after the binding of ATP, but before P(i) release. DMSO (5-30%, v/v) reversibly inhibits tension and ATP hydrolysis in vertebrate skeletal muscle myofibrils, and decreases the speed of unregulated F-actin in an in vitro motility assay with heavy meromyosin. In solution, controls for enzyme activity and intrinsic tryptophan fluorescence of myosin subfragment 1 (S1) in the presence of different cations indicate that structural changes attributable to DMSO are small and reversible, and do not involve unfolding. Since DMSO depresses S1 and acto-S1 MgATPase activities in the same proportions, without altering acto-S1 affinity, the principal DMSO target apparently lies within the catalytic cycle rather than with actin-myosin binding. Inhibition by DMSO in myofibrils is the same in the presence or the absence of Ca(2+) and regulatory proteins, in contrast with the effects of ethylene glycol, and the Ca(2+) sensitivity of isometric tension is slightly decreased by DMSO. The apparent affinity for P(i) is enhanced markedly by DMSO (and to a lesser extent by ethylene glycol) in skinned fibres, suggesting that DMSO stabilizes cross-bridges that have ADP.P(i) or ATP bound to them.

Adenosine Triphosphate↗
PubMed1966

Observations on the actin content of the rabbit myofibril.

1. On extraction of whole muscle by the procedure of Hasselbach & Schneider (1951), the amount of actin that passes into solution seems to account for little more than 10% of the protein content of the myofibrils. 2. Extraction of isolated myofibrils with suitable media that allow identification and estimation of dissolved proteins seems to give about the same yield of actin (10-13% of the total). 3. A comparatively large residue of myofibrillar components remains after extraction. The amount of actin present in the residue can be only hypothetical.

Animals↗
PubMed1971

The regulatory proteins of the myofibril. Characterization and properties of the inhibitory factor (troponin B).

1. Gel-filtration results indicate that the major component of inhibitory-factor preparations isolated by dissociation of the troponin complex consisted of a protein of subunit weight 23000 daltons. By the same procedure a molecular weight of 18000 was obtained for the calcium-sensitizing factor. 2. The inhibitory factor is specific for the actomyosin type of ATPase and ITPase. It is effective on desensitized actomyosin, natural actomyosin and intact myofibrils. 3. For inhibition, the actomyosin ATPase must be stimulated by Mg(2+), Ca(2+) or Mn(2+). The Co(2+)-, Cd(2+)- or Zn(2+)-stimulated ATPases are not affected. 4. Biological activity is stable to treatment with dissociating agents, heat, pH11, pH1 and carboxymethylation. 5. Increasing amounts of actin, but not myosin or tropomyosin, progressively neutralize the inhibitory activity when added to desensitized actomyosin or myofibrils.

Actins↗
PubMed1972

The regulatory proteins of the myofibril. Separation and biological activity of the components of inhibitory-factor preparations.

1. Inhibitory-factor preparations isolated from myofibrils were shown to consist principally of proteins with molecular weights of 37000 and 23000. Under certain preparative procedures an additional component of molecular weight 14000 was present. 2. The 23000-dalton protein, the inhibitory factor, was the major active component. Its activity was enhanced by tropomyosin. 3. The 14000-dalton component also possessed inhibitory activity, although less than that of the 23000-dalton component when compared on a molar basis. Its activity was not always enhanced by tropomyosin. The 14000-dalton component could not be detected in whole fresh myofibrils and the limited evidence available is compatible with its formation during the preparation of the troponin complex. 4. The 37000-dalton component could not replace the inhibitory factor, calcium-sensitizing factor or tropomyosin as components of the relaxing-protein system. 5. All three components had distinctive amino acid compositions, particularly in their cysteine content.

Actomyosin↗
PubMed1985

Phosphorylation and functional modifications of sarcoplasmic reticulum and myofibrils in isolated rabbit hearts stimulated with isoprenaline.

Isoprenaline stimulation of perfused rabbit hearts was associated with simultaneous phosphorylation of proteins in the myofilaments and phospholamban in the sarcoplasmic reticulum (SR). Hearts were perfused with Krebs-Henseleit buffer containing [32P]Pi, freeze-clamped in a control condition or at the peak of the inotropic response to isoprenaline, and myofibrils and SR were prepared from the same hearts. Stimulation of 32P incorporation in troponin I (TnI) and C-protein by isoprenaline was associated with a decrease in Ca2+-sensitivity of the myofibrillar Mg2+-dependent ATPase activity. Stimulation of 32P incorporation in SR by isoprenaline was associated with an increase in the initial rates of oxalate-facilitated Ca2+ transport, assayed with SR vesicles in either microsomal fractions or homogenates from the perfused hearts. These findings provide evidence that phosphorylation of TnI, C-protein and phospholamban in the intact cell is associated with functional alterations of the myofibrils and SR which may be responsible in part for the effects of catecholamines on the mammalian myocardium.

Animals↗
PubMed1993

Effects of different enzymic treatments on the release of titin fragments from rabbit skeletal myofibrils. Purification of an 800 kDa titin polypeptide.

In myofibrils, titin (also called connectin) molecules span from Z line to M line and constitute a third filament system containing an elastic domain in the I band. This giant protein is particularly sensitive to proteolysis in situ. Treatment of rabbit skeletal myofibrils with exogenous proteinases induces a release of titin fragments, which are detected in the soluble myofibrillar fraction. The cleavage of titin occurs at specific points localized at the proximity of Z line and could lead to a concomitant release of alpha-actinin.

Animals↗
PubMed2006

SmyD1, a histone methyltransferase, is required for myofibril organization and muscle contraction in zebrafish embryos.

Histone modification has emerged as a fundamental mechanism for control of gene expression and cell differentiation. Recent studies suggest that SmyD1, a novo SET domain-containing protein, may play a critical role in cardiac muscle differentiation. However, its role in skeletal muscle development and its mechanism of actions remains elusive. Here we report that SmyD1a and SmyD1b, generated by alternative splicing of SmyD1 gene, are histone methyltransferases that play a key role in skeletal and cardiac muscle contraction. SmyD1a and SmyD1b are specifically expressed in skeletal and cardiac muscles of zebrafish embryos. Knockdown of SmyD1a and SmyD1b expression by morpholino antisense oligos resulted in malfunction of skeletal and cardiac muscles. The SmyD1 morphant embryos (embryos injected with morpholino oligos) could not swim and had no heartbeat. Myofibril organization in the morphant embryos was severely disrupted. The affected myofibers appeared as immature fibers with centrally located nuclei. Together, these data indicate that SmyD1a and SmyD1b are histone methyltransferases and play a critical role in myofibril organization during myofiber maturation.

Alternative Splicing↗
PubMed1972

The sensitivity of developing cardiac myofibrils to cytochalasin-B (electron microscopy-polarized light-Z-bands-heartbeat).

Developing cardiac muscle cells of 11- to 13-somite chick embryos are sensitive to cytochalasin-B. In cultured chick embryos, ranging in development from 11 to 13 somites, hearts stop beating in the presence of this agent. Both polarized light and electron microscopic examination show that cytochalasin-B disrupts existing myofibrils and inhibits the formation of new ones. Discrete Z-bands are not present in treated heart cells and thick, presumably myosin, filaments are found in disarray. These effects are reversible; after cytochalasin-B is removed from the medium, heartbeat recovers and myofibrils with discrete Z-bands reappear. Fibrillar sensitivity appears to be a function of age since fibrils in hearts of embryos having from 22 to 28 pairs of somites are more resistant.

Animals↗