Localization of paratropomyosin in skeletal muscle myofibrils and its translocation during postmortem storage of muscles

Using polyclonal antibodies against paratropomyosin, which is believed to modify the actin-myosin interaction in postrigor skeletal muscles, we studied the localization of paratropomyosin in chicken breast muscle myofibrils. Intact myofibrils stained with fluorescent antibodies showed that paratropomyosin was exclusively located at the A-I junction region of sarcomeres. In stretched myofibrils (3.7 micron in sarcomere length), the approximate width of the fluorescent stripes and their relation to the A band remained constant. Removal of the A band from myofibrils led to loss of stainability. During postmortem storage of muscles, on the other hand, paratropomyosin was translocated from its original position at the A-I junction region onto thin filaments. The translocation of paratropomyosin was successfully induced with a calcium ion concentration of 10(-4) M in the presence of protease inhibitors. We therefore conclude that in postrigor muscles, paratropomyosin is released from the A-I junction region following the increase in the sarcoplasmic calcium ion concentration to 10(-4) M, and then binds to thin filaments, which results in weakening of rigor linkages formed between actin and myosin.     

Paratropomyosin, a new myofibrillar protein, weakens rigor linkages formed between actin and myosin

We have used an enzymatic technique to determine the weakening effect of paratropomyosin, a new myofibrillar protein, on rigor linkages formed between actin and myosin, and to clarify the distinct function of paratropomyosin, as to that of tropomyosin. Paratropomyosin inhibited the Mg-ATPase activity and enhanced the K-ATPase activity of reconstituted actomyosin stoichiometrically, and its maximal binding to actin was estimated to occur at a molar ratio of 1: 12.5. Paratropomyosin also inhibited the myofibrillar Mg-ATPase activity by 49% and enhanced the myofibrillar K-ATPase activity to 126%, while tropomyosin had no effect on these ATPases. These results indicate that paratropomyosin is able to bind to thin filaments of myofibrils, because the binding site for paratropomyosin on F-actin is different from that for tropomyosin, and that, due to its greater affinity for the myosin binding site on actin, paratropomyosin competes for the binding site and helps weaken rigor linkages.     

Differences in the Charge Distribution of Glycerol-Extracted Muscle Fibers in Rigor, Relaxation, and Contraction

Glycerol-extracted rabbit psoas muscle fibers were impaled with KCl-filled glass microelectrodes. For fibers at rest-length, the potentials were significantly more negative in solutions producing relaxation than in solutions producing either rigor or contraction; further the potentials in the latter two cases were not significantly different. For stretched fibers, with no overlap between thick and thin filaments, the potentials did not differ in the rigor, the relaxation, or the contraction solutions. The potentials measured from fibers in rigor did not vary significantly with the sarcomere length. For relaxed fibers, however, the potential magnitude decreased with increasing sarcomere length. The difference between the potentials measured for rigor and relaxed fibers exhibited a nonlinear relationship with sarcomere length. The potentials from calcium-insensitive fibers were less negative in both the rigor and the relaxation solutions than those from normal fibers. When calcium-insensitive fibers had been incubated in Hasselbach and Schneider's solution plus MgCl₂ or Guba-Straub's solution plus MgATP the potentials recorded upon impalement were similar in the rigor and the relaxation solution to those obtained from normal fibers in the relaxed state. It is concluded that the increase in the negative potential as the glycerinated fiber goes from rigor to relaxation may be due to an alteration in the conformation of the contractile proteins in the relaxed state.     

Interaction of paratropomyosin with beta-connectin and its 400-kilodalton fragment from chicken skeletal muscle as influenced by the calcium ion concentration.

The binding of paratropomyosin to beta-connectin, which has been suggested to interact at the A-I junction of a sarcomere, was confirmed by measuring the changes in turbidity of a mixture with changing NaCl concentration, pH and free calcium ions, and by morphological observation and a coprecipitation assay of the aggregates formed in the mixture. Paratropomyosin also bound to the 400-kDa fragment which is the N-terminal portion of beta-connectin and contains the A-I junction region. Moreover, the interaction of paratropomyosin with the 400-kDa fragment was enhanced by a calcium ion concentration from 10(-7) M to 10(-5) M and markedly suppressed above 10(-4) M calcium ions. We conclude that paratropomyosin probably binds to the 400-kDa fragment of beta-connectin in the A-I junction region in living and pre-rigor skeletal muscle. In postmortem skeletal muscle paratropomyosin may be released from the 400-kDa portion of the connectin filament by increased calcium ion concentration and translocated on to thin filaments to induce meat tenderization.     

Paratropomyosin: a new myofibrillar protein that modifies the actin-myosin interaction in postrigor skeletal muscle. II. Distinct function from tropomyosin

The functional differences between paratropomyosin and tropomyosin were studied. The weight ratio of maximally bound paratropomyosin to F-actin was 1 : 7.6, whereas that of tropomyosin was 1 : 3.9. The actin-myosin interaction was markedly depressed by paratropomyosin under conditions where it was promoted by tropomyosin. Paratropomyosin had sensitized the actin-myosin-troponin system to Ca2+, but was much less effective than tropomyosin. Paratropomyosin showed an additive effect on the actomyosin systems containing tropomyosin, indicating that the binding site of paratropomyosin on F-actin is distinct from that of tropomyosin. Probably, due to greater affinity for myosin binding site on F-actin, paratropomyosin competes for the binding site and thus modifies the actin-myosin interaction. The role of paratropomyosin in tenderization of meat during postmortem ageing is also discussed.     

On the relation between filament overlap and the number of calcium-binding sites on glycerinated muscle fibers.

The formation of rigor complexes between the thick and thin filaments of glycerinated rabbit psoas muscle fibers causes the fibers to bind more calcium at any given level of free calcium. I studied the maximum amount of calcium bound as a function of filament overlap under rigor conditions. Fibers stretched to zero filament overlap (sarcomere length greater than 3.8 micron) bound exactly 75% as much calcium as fibers with maximum overlap. Between these extremes a linear relationship was found between maximum bound calcium and the length of the overlap zone. The results support the hypothesis that in the intact filament lattice one of the four calcium-binding sites of troponin depends for its existence on attachment between myosin and actin. In addition, the linear relation between maximum bound calcium and filament overlap is consistent with the assumption that the cooperative effect of rigor complex formation on calcium binding is limited to the binding site in the immediate vicinity of the rigor complex.     

Radial stiffness of frog skinned muscle fibers in relaxed and rigor conditions.

Radial stiffness in various conditions of mechanically skinned fibers of semitendinosus muscle of Rana catesbeiana was determined by compressing the fiber with polyvinylpyrrolidone (PVP K-30, Mr = 40,000) in incubating solution. The change in width (D) of fibers with increasing and decreasing PVP concentrations was highly reproducible at a range 0-6% PVP. Radial stiffness of relaxed fibers was almost independent of the sarcomere length. On the other hand, radial stiffness of rigor fibers showed a linear relation against the sarcomere length. These results indicate that cross-bridge attachment would be a major factor in the increase of the radial stiffness. Radial stiffness of relaxed and rigor fibers was (2.14 +/- 0.52) X 10(4) N/m2 (mean +/- SD) and (8.76 +/- 2.04) X 10(4) N/m2, respectively, at the relative fiber width (D/D0) of 0.92, where D0 denotes the fiber width in the rigor solution at 0% PVP. Radial stiffness of a fiber in a rigor solution containing pyrophosphate (PPi) was between those of relaxed and rigor fibers, i.e., (4.76 +/- 0.86) X 10(4) N/m2 at D/Do of 0.92. In PPi and rigor solutions, radial stiffness reversibly increased to around 150 and 130%, respectively, in the presence of 10(-6) M Ca2+. To explain these results, especially the Ca2+-induced change in the radial stiffness, some factor in addition to the number of attached cross-bridges has to be taken into account. The variation of radial stiffness under various conditions will be discussed in relation to the possible manner of cross-bridge attachment.     

Birefringence changes in vertebrate striated muscle

The changes in birefringence in the rigor to relax transition of single Triton-extracted rabbit psoas muscle fibers have been investigated. The total birefringence of rigor muscle fibers was dependent on sarcomere length and ranged from (1.46 ± 0.08) × 10⁻³ to (1.60 ± 0.06) ± 10⁻³ at sarcomere lengths from 2.70 μm to 3.40 μm. An increase in total birefringence was measured dependent on sarcomere length when 55 single fibers were relaxed from the rigor state with Mg-ATP. Pyrophosphate relaxation produced a smaller increase in retardation when compared to Mg-ATP. The expected change in intrinsic birefringence during the rigor to relax transition was calculated assuming a hinge function of the subfragment 2 moiety of myosin. The changes in birefringence during isometric contraction and relaxation have been discussed in relation to possible structural changes.     

Fluorescent probes of the orientation of myosin regulatory light chains in relaxed, rigor, and contracting muscle.

The orientation of the light-chain region of myosin heads in relaxed, rigor, and isometrically contracting fibers from rabbit psoas muscle was studied by fluorescence polarization. Cysteine 108 of chicken gizzard myosin regulatory light chain (cgRLC) was covalently modified with iodoacetamidotetramethylrhodamine (iodo-ATR). Native RLC of single glycerinated muscle fibers was exchanged for labeled cgRLC in a low [Mg2+] rigor solution at 30 degrees C. Troponin and troponin C removed in this procedure were replaced. RLC exchange had little effect on active force production. X-ray diffraction showed normal structure in rigor after RLC exchange, but loss of axial and helical order in relaxation. In isolated myofibrils labeled cgRLC was confined to the regions of the sarcomere containing myosin heads. The ATR dipoles showed a preference for orientations perpendicular to the fiber axis, combined with limited nanosecond rotational motion, in all conditions studied. The perpendicular orientation preference was more marked in rigor than in either relaxation or active contraction. Stretching relaxed fibers to sarcomere length 4 microns to eliminate overlap between actin- and myosin-containing filaments had little effect on the orientation preference. There was no change in orientation preference when fibers were put into rigor at sarcomere length 4.0 microns. Qualitatively similar results were obtained with ATR-labeled rabbit skeletal RLC.     

Unusual thick and thin filament packing in a crustacean muscle

The proximal accessory flexor (PAF) of the myochordotonal organ (MCO) in the meropodite of crayfish walking legs contains two populations of muscle fibers which are distinguishable by their diameters. The large accessory (LA) fibers are 40-80 micrometer in diam and are similar in ultrastructure to other slow crustacean fibers. The small accessory (SA) fibers are 1-12 micrometer in diam and have a unique myofilament distribution at normal body lengths. There is extensive double overlap of thin filaments at these lengths, and some of them form bundles that may extend the length of the sarcomere. In the middle of the sarcomeres, thick and thin filaments are totally segregated from each other. When the fibers are stretched to lengths beyond double overlap length, the myofilament patterns are conventional. The segregated pattern is reestablished when stretched fibers are allowed to shorten passively. The length-tension relationship of the SA fibers is described by a linear ascending branch, a plateau, and a linear descending branch. The ascending branch encompasses normal body lengths from slack length (Ls) with maximum double overlap to the length at which double overlap ceases (1.8 X Ls). The descending phase is comparable to that of other skeletal muscles. That is, tension decreases in proportion with the reduction in thick-thin filament interdigitation (2 X Ls to 3 X Ls).     

Extensibility of the myofilaments in vertebrate skeletal muscle as revealed by stretching rigor muscle fibers

The extensibility of the myofilaments in vertebrate skeletal muscle was studied by stretching glycerinated rabbit psoas muscle fibers in rigor state and examining the resulting extension of sarcomere structures under an electron microscope. Although stretches applied to rigor fibers produced a successive yielding of the weakest sarcomeres, the length of the remaining intact sarcomeres in many myofibrils was fairly uniform, being definitely longer than the sarcomeres in the control, nonstretched part of rigor fibers. The stretch-induced increase in sarcomere length was found to be taken up by the extension of the H zone and the I band, whereas the amount of overlap between the thick and thin filaments did not change appreciably with stretches of 10-20%. The thick filament extension in the H zone was localized in the bare regions, whereas the thin filament extension in the I band appeared to take place uniformly along the filament length. No marked increase in the Z-line width was observed even with stretches of 20-30%. These results clearly demonstrate the extensibility of the thick and thin filaments. The possible contribution of the myofilament compliance to the series elastic component (SEC) in vertebrate skeletal muscle fibers is discussed on the basis of the electron microscopic data and the force-extension curve of the SEC in rigor fibers.     

Non-specific termination of simian virus 40 DNA replication.

Axial X-ray diffraction patterns have been studied from relaxed, contracted and rigor vertebrate striated muscles at different sarcomere lengths to determine which features of the patterns depend on the interaction of actin and myosin. The intensity of the myosin layer lines in a live, relaxed muscle is sometimes less in a stretched muscle than in the muscle at rest-length; the intensity depends not only on the sarcomere length but on the time that has elapsed since dissection of the muscle. The movement of cross-bridges giving rise to these intensity changes are not caused solely by the withdrawal of actin from the A-band.When a muscle contracts or passes into rigor many changes occur that are independent of the sarcomere length: the myosin layer lines decrease in intensity to about 30% of their initial value when the muscle contracts, and disappear completely when the muscle passes into rigor. Both in contracting and rigor muscles at all sarcomere lengths the spacings of the meridional reflections at 143 Å and 72 Å are 1% greater than from a live relaxed muscle at rest-length. It is deduced that the initial movement of cross-bridges from their positions in resting muscle does not depend on the interaction of each cross-bridge with actin, but on a conformational change in the backbone of the myosin filament: occurring as a result of activation. The possibility is discussed that the conformational change occurs because the myosin filament, like the actin filament, has an activation control mechanism. Finally, all the X-ray diffraction patterns are interpreted on a model in which the myosin filament can exist in one of two possible states: a relaxed state which gives a diffraction pattern with strong myosin layer lines and an axial spacing of 143.4 Å, and an activated state which gives no layer lines but a meridional spacing of 144.8 Å.     

The binding of calcium to glycerinated muscle fibers in rigor. The effect of filament overlap.

The binding of Ca2+ to glycerinated rabbit psoas fibers of varying sarcomere length was measured with a double isotope technique and ethyleneglycol-bis-(beta-aminoethylether)-N,N'-tetraacetic acid buffers. Experiments were carried out under rigor conditions with fiber bundles pre-set at different lengths prior to extraction with detergent and glycerol. These experiments were designed to test whether rigor complex formation, determined by the degree of filament overlap, enhances Ca2+-receptor affinity in the intact filament lattice, as it does in reconstituted actomyosin systems. The Ca2+-receptor affinity, as indicated by the free Ca2+ concentration at half-saturation and by the slopes of Scatchard plots, was found to be relatively unaffected by variations in filament overlap. However, the maximum bound Ca2+ was significantly reduced in stretched fibers. With maximum filament overlap the bound Ca2+ was equivalent to 4 mol per mol troponin. When stretched to zero overlap the fibers bound a maximum of 3 mol Ca2+ per mol troponin. When fibers with maximum overlap were incubated in the presence of 5 mM MgATP there was a reduction in the number of Ca2+-binding sites equivalent to that caused by stretching the fibers. These findings, taken together with other data in the literature, suggest that in the intact filament lattice at least one of the Ca2+-binding sites is present only when cross-bridge attachments are formed.     

The stiffness of skeletal muscle in isometric contraction and rigor: the fraction of myosin heads bound to actin.

Step changes in length (between -3 and +5 nm per half-sarcomere) were imposed on isolated muscle fibers at the plateau of an isometric tetanus (tension T0) and on the same fibers in rigor after permeabilization of the sarcolemma, to determine stiffness of the half-sarcomere in the two conditions. To identify the contribution of actin filaments to the total half-sarcomere compliance (C), measurements were made at sarcomere lengths between 2.00 and 2.15 microm, where the number of myosin cross-bridges in the region of overlap between the myosin filament and the actin filament remains constant, and only the length of the nonoverlapped region of the actin filament changes with sarcomere length. At 2.1 microm sarcomere length, C was 3.9 nm T0(-1) in active isometric contraction and 2.6 nm T0(-1) in rigor. The actin filament compliance, estimated from the slope of the relation between C and sarcomere length, was 2.3 nm microm(-1) T0(-1). Recent x-ray diffraction experiments suggest that the myosin filament compliance is 1.3 nm microm(-1) T0(-1). With these values for filament compliance, the difference in half-sarcomere compliance between isometric contraction and rigor indicates that the fraction of myosin cross-bridges attached to actin in isometric contraction is not larger than 0.43, assuming that cross-bridge elasticity is the same in isometric contraction and rigor.     

Compliance of thin filaments in skinned fibers of rabbit skeletal muscle.

The mechanical compliance (reciprocal of stiffness) of thin filaments was estimated from the relative compliance of single, skinned muscle fibers in rigor at sarcomere lengths between 1.8 and 2.4 micron. The compliance of the fibers was calculated as the ratio of sarcomere length change to tension change during imposition of repetitive cycles of small stretches and releases. Fiber compliance decreased as the sarcomere length was decreased below 2.4 micron. The compliance of the thin filaments could be estimated from this decrement because in this range of lengths overlap between the thick and thin filaments is complete and all of the myosin heads bind to the thin filament in rigor. Thus, the compliance of the overlap region of the sarcomere is constant as length is changed and the decrease in fiber compliance is due to decrease of the nonoverlap length of the thin filaments (the I band). The compliance value obtained for the thin filaments implies that at 2.4-microns sarcomere length, the thin filaments contribute approximately 55% of the total sarcomere compliance. Considering that the sarcomeres are approximately 1.25-fold more compliant in active isometric contractions than in rigor, the thin filaments contribute approximately 44% to sarcomere compliance during isometric contraction.     

Radial forces within muscle fibers in rigor

Considering the widely accepted cross-bridge model of muscle contraction (Huxley. 1969. Science [Wash. D. C.]. 164:1356-1366), one would expect that attachment of angled cross-bridges would give rise to radial as well as longitudinal forces in the muscle fiber. These forces would tend, in most instances, to draw the myofilaments together and to cause the fiber to decrease in width. Using optical techniques, we have observed significant changes in the width of mechanically skinned frog muscle fibers when the fibers are put into rigor by deleting ATP from the bathing medium. Using a high molecular weight polymer polyvinylpyrrolidone (PVP-40; number average mol. wt. (Mn) = 40,000) in the bathing solution, we were able to estimate the magnitude of the radial forces by shrinking the relaxed fiber to the width observed with rigor induction. With rigor, fiber widths decreased up to approximately 10%, with shrinking being greater at shorter sarcomere spacing and at lower PVP concentrations. At higher PVP concentrations, some fibers actually swelled slightly. Radial pressures seen with rigor in 2 and 4% PVP ranged up to 8.9 x 10(3) N/m2. Upon rigor induction, fibers exerted a longitudinal force of approximately 1 x 10(5) N/m2 that was inhibited by high PVP concentrations (greater than or equal to 13%). In very high PVP concentrations (greater than or equal to 20%), fibers exerted an anomalous force, independent of ATP, which ranged up to 6 x 10(4) N/m2 at 60% PVP. Assuming that all the radial force is the result of cross- bridge attachment, we calculated that rigor cross-bridges exert a radial force of 0.2 x 1.2 x 10(-9) N per thick filament in sarcomeres near rest length. This force is of roughly the same order of magnitude as the longitudinal force per thick filament in rigor contraction or in maximal (calcium-activated) contraction of skinned fibers in ATP- containing solutions. Inasmuch as widths of fibers stretched well beyond overlap of thick and thin filaments decreased with rigor, other radially directed forces may be operating in parallel with cross-bridge forces.     

Polarization of fluorescence from single skinned glycerinated rabbit psoas fibers in rigor and relaxation

Single skinned glycerinated muscle fibers were labelled with the fluorescent dye N-(iodoacetylamino)-1-naphthylamine-5-sulfonic acid (1,5-IAEDANS). The heavy chain of myosin (EC 3.6.1.3) was labelled predominantly when the reaction was carried out in relaxation at 0 °C. Mechanical properties of skinned fibers were little affected by labelling with the fluorophore. Rigor tension developed upon transferring native or labelled skinned fibers from relaxing to rigor solutions lacking Ca²⁺ was very small but could be enhanced by progressively increasing Ca²⁺ concentration; the rigor tension decreased with increasing sarcomere length.Polarization of fluorescence of skinned fibers reacted with 1,5-IAEDANS was measured along the line of excitation as well as at 90° to it. The mean values of parallel and perpendicular components of polarization of labelled fibers measured at 0° were close to the values obtained for native fibers irrigated with 1,5-IAEDANS-labelled heavy meromyosin, fiber “ghosts” irrigated with labelled heavy meromyosin, and oriented bundles of myofibrils reacted with the same fluorophore. Skinned fibers stretched above the rest length and then irrigated with 1,5-IAEDANS-labelled heavy meromyosin gave rise to polarized fluorescence close to the values theoretically predicted for an assembly of helically arranged fluorophores. Using 90° detection system a satisfactory fit to the theory could be obtained from single fibers labelled with 1,5-IAEDANS and measured in rigor. The angle between the fiber axis and the direction of the emission dipole of 1,5-IAEDANS attached to subfragment-1 was estimated to be near 40°.     

Paratropomyosin: a new myofibrillar protein that modifies the actin-myosin interaction in postrigor skeletal muscle. I. Preparation and characterization

A protein component which is released from skeletal-muscle myofibrils on the treatment with Ca2+ at concentrations above 10(-5) M and modifies the actin-myosin interaction was purified by a method involving column chromatography on Sephadex G-200 and DEAE-cellulose in succession. Although this protein resembles tropomyosin in some physicochemical properties, it has the same chain weight of 34,000 as the alpha-component of tropomyosin on SDS-polyacrylamide gel electrophoresis, and differed from tropomyosin not only in the amino acid composition but also in prolonging the clearing phase of superprecipitation of reconstituted actomyosin. We therefore concluded that this protein is a new myofibrillar one, and termed it "paratropomyosin." In postrigor muscle, it seems likely that paratropomyosin is released from its original locus with an increased concentration of Ca2+, and that it weakens rigor linkages formed between actin and myosin.     

Susceptibility to sarcomere injury induced by single stretches of maximally activated muscles of mdx mice.

The purpose was to investigate the contribution of mechanical damage to sarcomeres to the greater susceptibility of dystrophic muscle fibers to contraction-induced injury. Single stretches provide an effective method for studying mechanical factors that contribute to the initiation of contraction-induced injury. We hypothesized that, after single stretches, the deficits in isometric force would be greater for muscles of mdx than C57BL/10 mice, whereas membrane damage would be minimal for all muscles. Extensor digitorum longus (EDL) and soleus muscles of mice were removed under anesthesia with Avertin (tribromoethanol). During the plateau of a maximum isometric contraction in vitro, muscles were stretched through single strains of 20-60% fiber length. Isometric force was remeasured 1 min later, and muscles were then incubated in procion orange dye to identify fibers with membrane damage. Force deficits at 1 min were two- to threefold greater for EDL muscles of mdx compared with C57BL/10 mice, whereas no significant differences were observed between soleus muscles of mdx and C57BL/10 mice. For all muscles, membrane damage was minimal and not significantly increased by single stretches for either strain of mice. These data support a critical role of dystrophin maintaining sarcomere stability in EDL muscles, whereas soleus muscles retain abilities, in the absence of dystrophin, not different from control muscles to resist sarcomere damage.     

Stiffness of glycerinated rabbit psoas fibers in the rigor state. Filament-overlap relation

The stiffness of glycerinated rabbit psoas fibers in the rigor state was measured at various sarcomere lengths in order to determine the distribution of the sarcomere compliance between the cross-bridge and other structures. The stiffness was determined by measuring the tension increment at one end of a fiber segment while stretching the other end of the fiber. The contribution of the end compliance to the rigor segments was checked both by laser diffractometry of the sarcomere length change and by measuring the length dependence of the Young's modulus; the contribution was found to be small. The stiffness in the rigor state was constant at sarcomere lengths of 2.4 microns or less; at greater sarcomere lengths the stiffness, when corrected for the contribution of resting stiffness, scaled with the amount of overlap between the thick and thin filaments. These results suggest that the source of the sarcomere compliance of the rigor fiber at the full overlapping of filaments is mostly the cross-bridge compliance.