Aggregation of isolated myofibrils stimulated by their contraction. I. Origin of the second phase of optical changes during myofibril contraction

Aggregation of contracted myofibrils was shown to be responsible for spontaneous: slow reduction of optical density of myofibril suspension on the final stage of their contraction. This effect faded with an increase in light wavelength, with increased angle of view of the photocell and diminished sizes of myofibrils.     

Effect of Trypsin Treatment on the ATPase Activity of Myofibrils from the Stored Rabbit Muscle

The extent of activation of myofibrillar ATPase activity by trypsin treatment has been measured.

When myofibril (5 mg/ml) was treated with a low concentration of trypsin (2.5 μg/ml), the Mg-modified ATPase activity of myofibrils at a low ionic strength increased appreciably, while the EDTA-enhanced ATPase activity of myofibrils at a high ionic strength did not change with the progress of trypsin digestion.

The dependence of myofibrillar ATPase activity on KCl concentration also became greater with the progress of trypsin digestion.

Trypsin treatment caused 5-fold increase in the Mg-modified ATPase activity of 0-myofibril, when treated with trypsin in a ratio of 1 to 2000 myofibril for 80 min. Under the same condition, the ATPase activity of 1-myofibril increased by about 150%, whereas that of 8-myofibril increased by approximately 50%.

When myofibrils were treated with trypsin in a ratio of 1 to 200 myofibril, the Mg-ATPase activity of 8-myofibril decreased earlier than that of 1-myofibril did by about 20 min.

Experimental results obtained in this study were enough to confirm that the myofibrils from the aged muscle are more susceptible to tryptic action.

An assumption was made that the structural alteration of myofibrils during aging might be attributed to the change in thin filament of myofibrils, including Z-lines, which are mainly due to the change in the native tropomyosin of thin filaments.     

Kinetic mechanism of myofibril ATPase.

The kinetic mechanism of myofibril ATPase was investigated using psoas and mixed back muscle over a range of ionic strengths. Myofibrils were labeled with pyrene iodoacetamide to measure the rate constants for the binding of ATP and formation of the weakly attached state. The velocity of shortening was measured by stopping the contraction at various times by mixing with pH 4.5 buffer. The transient and steady-state rates of ATP hydrolysis were measured by the quench flow method. The results fitted the kinetic scheme [formula: see text] The rate constants (or equilibrium constants for steps 1 and 6) were obtained for the six steps. k5 was calculated from the KM for shortening velocity, K1, and k2. The rate constants were essentially equal for myofibrils and acto-S-1 at low ionic strength. Increasing the ionic strength up to 100 mM in NaCl increased the rate of the hydrolysis step and the size of the phosphate burst and the effective rate of product release became the rate-limiting step. The step calculated from the velocity of shortening, k5, and k2 is 15 nm, based on a model in which step 4 is the force-generating step.     

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.     

Studies on Myofibrils from the Stored Muscle

Adenosine triphosphatase (ATPase) activity of myofibrils isolated from fresh muscle and the muscle stored at 4°C have been measured.

An increase in Mg-activated ATPase activity of myofibrils was caused by lengthened homogenization.

With the progress of aging of muscle, Mg-activated ATPase activity of myofibrils increased remarkably.

When myofibrils from pre-rigor and rigor muscle in 0.16 m KCl were treated with 0.6 m KCl-18 mm Tris-maleate solution (pH 7.0), Mg-activated ATPase activity of myofibrils at low ionic strength increased markedly. However, the Mg-activated ATPase activity of the myofibril isolated from the muscle stored at 4°C for 8 days (8-myofibril) increased slightly after the similar treatment.

The dependence of myofibrillar ATPase activity on KCl concentration became greater with the progress of aging of muscle.

These results may show that, as long as ATPase activity and the dependence of ATPase activity on KCl concentration are concerned, 8-myofibril is the most similar to the isolated actomyosin among myofibrils, although actomyosin in muscle may exist in a different form from that in solution. It is suggested that, with the progress of aging, the structural alteration of myofibril occurred and the myofibril became more susceptible to ATP-induced transformation.     

The site of paramyosin in insect flight muscle and the presence of an unidentified protein between myosin filaments and Z-line.

The position of paramyosin in insect flight muscle was determined by labelling myofibrils with antibody to paramyosin and examining them by fluorescent and electron microscopy.Antiserum to dung beetle paramyosin had antibodies to another protein as well as to paramyosin. Specific anti-paramyosin bound to the H-zone of Lethocerus myofibrils showing paramyosin was exposed only in that region. Antibodies to the other protein bound at the ends of the A-band.The exposure of antigenic sites in the two regions of the myofibril depended on the extent of contraction in the myofibril: the sites at the end of the A-band were most exposed in rest-length myofibrils and those at the H-zone in shortened ones.Antibody-labelling in stretched bee muscle showed that the protein at the ends of the sarcomere extended from myosin filaments to Z-line.The high resting elasticity of insect flight muscle and hence its capacity for oscillatory contraction may be due to the protein between myosin filaments and Z-line.     

Does actin bind to the ends of thin filaments in skeletal muscle?

We examined whether or not purified actin binds to the ends of thin filaments in rabbit skeletal myofibrils. Phase-contrast, fluorescence, and electron microscopic observations revealed that actin does not bind to the ends of thin filaments of intact myofibrils. However, in I-Z-I brushes prepared by dissolving thick filaments at high ionic strength, marked binding of actin to the free ends, i.e., the pointed ends, of thin filaments was observed when actin was added at an early phase of polymerization. As the polymerization of actin proceeded, the binding efficiency decreased. The critical actin concentration for this binding was higher than that for polymerization in solution. The binding of G-actin was not observed at low ionic strength. On the basis of these results, we suggest that a particular structure suppressing the binding of actin is present at the free ends of thin filaments in intact myofibrils and that a part of the end structure population is eliminated or modified at high ionic strength so that further binding of actin becomes possible. The myofibril and I-Z-I brush appear to be useful systems for studies aimed at elucidating the organizational mechanisms of actin filaments in vivo.     

Localization of creatine kinase isoenzymes in myofibrils. I. Chicken skeletal muscle

Purified, repeatedly washed, skeletal muscle myofibrils contain approx. 0.2 U of creatine kinase (CK) activity (equivalent to 2.5 micrograms CK) per milligram dry weight; this firmly bound CK activity is estimated to represent 3-5% of the total cellular CK. It had been shown previously that the myofibrillar CK, which can be quantitatively extracted at low ionic strength and purified to homogeneity, is very similar, if not identical, to the bulk MM-CK. It is shown that the two protein preparations also have the same peptide pattern after cyanogen bromide fractionation and very similar specific activities, confirming their identity. The earlier demonstration that the bound CK is specifically located at the M-lines of isolated myofibrils has been confirmed by immunofluorescence. Antibodies directed against purified MM- and BB-CK were used in the indirect fluorescent antibody technique to study the specificity of myofibril binding sites for different forms of CK. With myofibrils from adult muscle, which has only MM-CK, as well as from early developmental stages in which BB-CK is the predominant isoenzyme, M-type CK was localized exclusively at the M-line, while greater or lesser amounts of B-type CK were found at the Z-line. The data provide strong evidence that the MM-CK at the M-lines in skeletal myofibrils is not adventitiously bound but is rather an integral element in the M-line structure. The amount of CK bound is reasonably consistent with the earlier proposal that the CK molecules might be the transverse M-bridges and appears to be sufficient to regenerate all of the ATP hydrolyzed during muscle contraction.     

Irradiations of rabbit myofibrils with an ultraviolet microbeam. I. Effects of ultraviolet light on the myofibril components necessary for contraction

Glycerinated rabbit psoas myofibrils, F-actin, and myofibril ghosts were irradiated with ultraviolet light (UV) to investigate how UV blocks myofibril contraction. Myofibril contraction is most sensitive to 270- and 290-nm wavelength light. We irradiated I and A bands separately with 270- and 290-nm wavelength light using a UV microbeam and constructed dose-response curves for blocking sarcomere contraction. For both wavelengths, irradiations of A bands required less energy per area to block contraction than did irradiations of I bands, suggesting that the primary effects of both 270- and 290-nm wavelength light in stopping myofibril contraction are on myosin. We investigated whether the primary effect of UV in blocking I-band contraction is the depolymerization of actin by comparing the relative sensitivities of I-band contraction, F-actin depolymerization, and thin filament depolymerization to 270- and 290-nm light. We also compared the dose of UV required to depolymerize F-actin in solution with the dose needed to block I-band contraction and the dose required to alter thin filament structure in myofibril ghosts. The results confirm that UV blocks I-band contraction by depolymerizing actin. We discuss how the results might be relevant to the hypothesis that an actomyosin-based system is involved in chromosome movement.     

Mutation of the high affinity calcium binding sites in cardiac troponin C.

Fast skeletal and cardiac troponin C (TnC) contain two high affinity Ca2+/Mg2+ binding sites within the C-terminal domain that are thought to be important for association of TnC with the troponin complex of the thin filament. To test directly the function of these high affinity sites in cardiac TnC they were systematically altered by mutagenesis to generate proteins with a single inactive site III or IV (CBM-III and CBM-IV, respectively), or with both sites III and IV inactive (CBM-III-IV). Equilibrium dialysis indicated that the mutated sites did not bind Ca2+ at pCa 4. Both CBM-III and CBM-IV were similar to the wild type protein in their ability to regulate Ca(2+)-dependent contraction in slow skeletal muscle fibers, and Ca(2+)-dependent ATPase activity in fast skeletal and cardiac muscle myofibrils. The mutant CBM-III-IV is capable of regulating contraction in permeabilized slow muscle fibers but only if the fibers are maintained in a contraction solution containing a high concentration of the mutant protein. CBM-III-IV also regulates myofibril ATPase activity in fast skeletal and cardiac myofibrils but only at concentrations 10-100-fold greater than the normal protein. The pCa50 and Hill coefficient values for Ca(2+)-dependent activation of fast skeletal muscle myofibril ATPase activity by the normal protein and all three mutants are essentially the same. Competition between active and inactive forms of cardiac and slow TnC in a functional assay demonstrates that mutation of both sites III and IV greatly reduces the affinity of cardiac and slow TnC for its functionally relevant binding site in the myofibrils. The data indicate that although neither high affinity site is absolutely essential for regulation of muscle contraction in vitro, at least one active C-terminal site is required for tight association of cardiac troponin C with myofibrils. This requirement can be satisfied by either site III or IV.     

A new protein of the thick filaments of vertebrate skeletal myofibrils. Extractions, purification and characterization

A new protein component of skeletal myofibrils has been isolated and characterized. It is prepared from impure myosin preparations and corresponds to band C, the principal contaminant observed in sodium dodecyl sulphate polyacrylamide gel electrophoresis patterns of such preparations (Starr and Offer, 1971).The C-protein, as we term it, is deduced to be a component of the skeletal myofibril because (i) glycerinated or fresh myoflbrils contain a component with a mobility identical to C-protein on sodium dodecyl sulphate gels, (ii) this component is extracted from myofibrils by the same solvent which extracts C-protein and (iii) C-protein may be prepared from preparations of isolated myofibrils. It is presumed to be a component of the thick filaments because it binds strongly to myosin at low ionic strength; immunological evidence which confirms this view is presented elsewhere.The quantity of C-protein in the myofibril has been estimated to be 2.0% by densitometry of sodium dodecyl sulphate gels of glycerinated myofibrils using actin as an internal reference. About forty molecules of C-protein are present in a thick filament.The properties of C-protein distinguish it from the other well-characterized myoflbrillar proteins. The C-protein molecule contains a single polypeptide chain of molecular weight 140,000. The intrinsic viscosity of 13.6 ml/g suggests that the molecule is neither completely globular nor as elongated as molecules like paramyosin or tropomyosin. The α-helical content is very low and the proline content higher than the other myofibrillar proteins. The molecule associates at low ionic strength.C-protein has no ATPase activity, nor does it affect the ATPase of pure myosin. But it reduces the activity of the actin-activated myosin ATPase by about half, this inhibition being independent of the level of Ca²⁺. C-protein does not bind Ca²⁺ in the presence of Mg²⁺. Its possible location and function are discussed.     

Effect of ionic strength on the interaction between aldolase and actin-containing filaments

The effect of ionic strength on the adsorption of aldolase to synthetic thin filaments derived from rabbit skeletal muscle has been investigated by partition equilibrium experiments, the results being interpreted in terms of the intrinsic association constant for the interaction of four sites on aldolase with two sites per filament repeat unit. At physiological ionic strength, values of 10,000 and 2000 m^?1 were obtained for this equilibrium constant in the absence and presence, respectively, of calcium ions. Comparison of binding curves obtained with synthetic thin filaments and myofibrils indicated a lesser extent of enzyme adsorption to the myofibrillar system, a difference attributed to the covert nature of many of the potential binding sites on the filaments in the assembly of the myofibril. On the basis of the quantitative information on the effect of ionic strength on the adsorption of aldolase, a case is made for the probable occurrence of the enzyme-filament interaction as a physiologically significant phenomenon in skeletal muscle.     

Aggregation of poliovirus and reovirus by dilution in water.

Poliovirus and reovirus were found to aggregate into clumps of up to several hundred particles when diluted 10-fold into distilled water from a stock preparation of minimal aggregation in 0.05 M phosphate buffer, pH 7.2, plus 22 to 30% sucrose. Reovirus was also found to aggregate when diluted into phosphate-buffered saline. The aggregation was concentration dependent and did not occur when either virus was diluted into water 100-fold or greater. The aggregation of poliovirus was reversible by further addition of saline and produced a dispersed preparation of virus. Reovirus aggregation was not reversible. Both viruses aggregated when diluted into buffers at pH 5 and 3, and poliovirus aggregated at pH 6, and this aggregation of both viruses was reversible when returned to pH 7. Aggregation did not occur at alkaline pH values. Aggregation at low pH could be caused aggregation of either virus at pH 7. Calcium ions, however, were found to aggregate both viruses at a concentration of 0.01 M.     

Aggregation of poliovirus and reovirus by dilution in water.

Poliovirus and reovirus were found to aggregate into clumps of up to several hundred particles when diluted 10-fold into distilled water from a stock preparation of minimal aggregation in 0.05 M phosphate buffer, pH 7.2, plus 22 to 30% sucrose. Reovirus was also found to aggregate when diluted into phosphate-buffered saline. The aggregation was concentration dependent and did not occur when either virus was diluted into water 100-fold or greater. The aggregation of poliovirus was reversible by further addition of saline and produced a dispersed preparation of virus. Reovirus aggregation was not reversible. Both viruses aggregated when diluted into buffers at pH 5 and 3, and poliovirus aggregated at pH 6, and this aggregation of both viruses was reversible when returned to pH 7. Aggregation did not occur at alkaline pH values. Aggregation at low pH could be caused aggregation of either virus at pH 7. Calcium ions, however, were found to aggregate both viruses at a concentration of 0.01 M.     

Theory of light diffraction by single skeletal muscle fibers.

A theoretical discussion is presented describing the diffraction of laser light by a single fiber of striated muscle. The complete three-dimensional geometry of the fiber has been taken into consideration. The basic repeated unit is taken as the sarcomere of a single myofibril, including its cylindrical geometry. The single fiber is considered as the sum of myofibrils up to the fiber dimensions. When proper phasing is taken into account, three cases of interest are analyzed. (a) When the adjacent myofibrils are totally aligned with respect to their index of refraction regions (e.g., A and I bands), then the diffraction pattern reflects that of a larger striated cylinder with the dimensions of the fiber. (b) When a particular skew plane develops for the myofibril elements, additional Bragg reflection occurs at certain specific sarcomere lengths, and intensity asymmetry amongst the diffracted orders occurs. (c) When the myofibril phasing changes in a random fashion, while all sarcomeres remain at the same length, then intensity decrease is directly related to the phase deviation from a reference phase point. This condition may well describe a fiber undergoing active isometric contraction.     

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.     

Sarcomere Length Nonuniformity and Force Regulation in Myofibrils and Sarcomeres

The smallest contractile unit in striated muscles is the sarcomere. Although some of the classic features of contraction assume a uniform behavior of sarcomeres within myofibrils, the occurrence of sarcomere length nonuniformities has been well recognized for years, but it is yet not well understood. In the past years, there has been a great advance in experiments using isolated myofibrils and sarcomeres that has allowed scientists to directly evaluate sarcomere length nonuniformity. This review will focus on studies conducted with these preparations to develop the hypotheses that 1) force production in myofibrils is largely altered and regulated by intersarcomere dynamics and that 2) the mechanical work of one sarcomere in a myofibril is transmitted to other sarcomeres in series. We evaluated studies looking into myofibril activation, relaxation, and force changes produced during activation. We conclude that force production in myofibrils is largely regulated by intersarcomere dynamics, which arises from the cooperative work of the contractile and elastic elements within a myofibril.     

Interaction of reduced glutathione with bovine brain tubulin

Incubation of phosphocellulose-purified tubulin with GSH at 30 degrees C results in an inhibition of colchicine binding activity. GSSG has a protective effect against the GSH-induced loss of colchicine-binding. Incubation of tubulin with GSH at 30 degrees C results in the formation of abnormal tubulin polymers which are insensitive to cold. Such aggregation is insensitive to antimicrotubular drugs. Aggregation is inhibited by GSSG but not by DTT or mercaptoethanol. GSH-induced aggregation is very sensitive to the ionic strength of the assembly medium; both the aggregation and colchicine binding inhibition induced by GSH are inhibited at higher ionic strength. These results indicate a very complex interaction of GSH with tubulin.     

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.     

Identification and functional characterization of an aggregation domain in long myosin light chain kinase

The functions of long smooth muscle myosin light chain kinase (L-MLCK), a molecule with multiple domains, are poorly understood. To examine the existence of further potentially functional domains in this molecule, we analyzed its amino acid sequence with a tango program and found a putative aggregation domain located at the 4Ig domain of the N-terminal extension. To verify its aggregation capability in vitro, expressible truncated L-MLCK variants driven by a cytomegalovirus promoter were transfected into cells. As anticipated, only the overexpression of the 4Ig fragment led to particle formation in Colon26 cells. These particles contained 4Ig polymers and actin. Analysis with detergents demonstrated that the particles shared features in common with aggregates. Thus, we conclude that the 4Ig domain has a potent aggregation ability. To further examine this aggregation domain in vivo, eight transgenic mouse lines expressing the 4Ig domain (4Ig lines) were generated. The results showed that the transgenic mice had typical aggregation in the thigh and diaphragm muscles. Histological examination showed that 7.70 +/- 1.86% of extensor digitorum longus myofibrils displayed aggregates with a 36.44% reduction in myofibril diameter, whereas 65.13 +/- 3.42% of diaphragm myofibrils displayed aggregates and the myofibril diameter was reduced by 43.08%. Electron microscopy examination suggested that the aggregates were deposited at the mitochondria, resulting in structural impairment. As a consequence, the oxygen consumption of mitochondria in the affected muscles was also reduced. Macrophenotypic analysis showed the presence of muscular degeneration characterized by a reduction in force development, faster fatigue, decreased myofibril diameters, and structural alterations. In summary, our study revealed the existence of a novel aggregation domain in L-MLCK and provided a direct link between L-MLCK and aggregation. The possible significance and mechanism underlying the aggregation-based pathological processes mediated by L-MLCK are also discussed.