Rigor Linkages Research Articles

Structural differences between myofibrillar protein, paratropomyosin, and tropomyosin as revealed by high-performance liquid chromatography.

Paratropomyosin (PTM) composes myofibril functions to weaken the rigor linkages formed between actin and myosin during postmortem aging of muscles. PTM has the similar physico-chemical properties as tropomyosin (TM) that is a regulatory protein of myofibrils. So far, it is unclear whether PTM is definitely different from TM, because the primary structure of PTM has not been determined yet. The aim of this study was to clarify structural difference of PTM from TM. PTM was prepared by column chromatography immediately after slaughter from broiler breast muscle, and purified by high-performance liquid chromatography (HPLC). Purified PTM was successfully separated from TM, and the recovered PTM molecule was reduced with dithiothreitol to separate again by HPLC. Two subunits were obtained and peptides from each digested subunit by V8 protease were recovered by HPLC, and then amino acid sequences of the peptides were analyzed by protein sequencing. As a result, some amino acid residues were replaced from that of TMα1 isoform which is the major isoform of TM, and also was different between the two subunits. Therefore, it is concluded that PTM clearly differs from TM and it is suggested that functional difference in PTM from TM is attributed to amino acid replacements in subunits composing PTM.

Effect of Antibodies to Myosin Head on the Development of Rigor Tension and Stiffness in Skinned Muscle Fibers

Using three antibodies to myosin head, attaching to (1) distal region and (2) proximal region of myosin head catalytic domain, and (3) to myosin head lever arm domain, respectively, we have shown definite differences between in vitro actin-myosin sliding and muscle contraction. In the present study, we studied the effect of these antibodies on the development of rigor tension and stiffness in single skinned muscle fibers at pCa>9. To form rigor actin-myosin linkages, myosin heads should override tropomyosin, covering myosin-binding sites on actin, and to detach antibodies from them. Despite their different attachment sites in myosin head, all these antibodies slowed down development of rigor tension and stiffness with or without changing their peak values. The rigor tension versus stiffness relation was highly variable, suggesting that the rigor tension reflects the sum of tension in individual rigor linkages, while rigor stiffness represents the total number of rigor linkages. Dummy antibody had no effect on the development of rigor state. These results indicate that the action of myosin heads overriding tropomyosin is inhibited by the antibodies, so that development of rigor state is slowed down due to gradual detachment of the antibodies from individual myosin heads. Highlights The effect of three antibodies, attaching to different regions in myosin heads on the development of rigor state was examined at pCa >9, using single skinned muscle fibers. Despite their different binding sites on myosin, all the antibodies slowed down development of rigor tension and stiffness with or without changes in their peak values. The rigor tension versus stiffness relation was highly variable, suggesting that rigor tension reflects the sum of tension generated by individual myosin heads, while stiffness serves as a measure of total number of rigor linkages. These results indicate that the antibodies inhibit myosin head movement to override tropomyosin, and detachment of the antibodies from myosin heads is necessary prerequisite for rigor linkage formation.

Evidence that Actin-Myosy Cycling in Muscle may not Pass through Rigor Configuration

In 1989, Sutoh, Tokunaga and Wakabayashi (J Mol Biol 206:357) prepared a monoclonal antibody (IgG) directed to junctional peptide between 50- and 20-kDa segments of myosin heavy chain, and showed that the antibody attaches around the tip of myosin heads facing actin filaments in muscle. using the same antibody, we successfuly position-mark the tip of myosin heads in synthetic myosin filaments kept in hydrated state in the gas environmental chamber, and succeeded in recording ATP-induced myosin head movement electron microscopically (Sugi et al. PNAS 105: 17396, 2008). We have examined the effect of this antibody on both ATP-dependent in vitro actin-myosin sliding and Ca-activated contraction of permealized rabbit psoas muscle fibers. Unexpectedly, the antibody (concentration, up to 2mg/ml) had no appreciable effect on the velocity profile of actin-myosin sliding and the contraction characteristics of muscle fibers, including magnitude of the maximum Ca-activated isometric force, the maximum unloaded shortening velocity, ans MgATPase activity during contraction. Since the junctional peptide is located between the two main actin- binding sites of myosin heads during formation of rigor linkages, attachment of massive antibodies to myosin heads completely covers their actin- binding sites. The possibility that the antibody does not diffuse into muscle fibers is excluded by the fact that other kinds of antibody diffuse into muscle fibers to inhibit Ca-activated force generation (e.g., Sugi et al. PNAS 89: 6134, 1992). Our work, therefore, suggests strongly that attachment-detachment cycle between actin and myosin in muscle may not pass through rigor configuration. It seems possible that so-called strong actin-myosin binding might be due to some electrostatic mechanism.

Structural weakening of skeletal muscle tissue during post-mortem ageing of meat: the non-enzymatic mechanism of meat tenderization

Structural weakening of the myofibrils, the intermediate filaments and the intramuscular connective tissues the endomysium and perimysium contribute to tenderization of meat during post-mortem ageing. We have found four phenomena which effect the structural weakening of myofibrils: weakening of Z-disks, weakening of rigor linkages formed between actin and myosin, splitting of connectin (titin) filaments, and fragmentation of nebulin filaments. These reactions are specific to 0.1 mM calcium ions regardless of proteolysis. Desmin molecules which form intermediate filaments are also fragmented by 0.1 mM calcium ions nonenzymatically. The fragmentation of desmin molecules suggest depolymerization of intermediate filaments under non-physiological conditions. These facts are the basis of ‘the calcium theory of meat tenderization’ which I have proposed. The structural weakening of the endomysium and perimysium shows the effect on tenderization of extended ageing of meat, and is closely related to degradation of proteoglycans which link collagen fibrils and stabilize collagen fibres.

Relationship between the translocation of paratropomyosin and the restoration of rigor-shortened sarcomeres during post-mortem ageing of meat—A molecular mechanism of meat tenderization

The weakening effect of paratropomyosin on rigor linkages formed between actin and myosin was determined by measuring the restoration of rigor-shortened sarcomeres of chicken, pork and beef. We observed the rate of the post-mortem translocation of paratropomyosin from the A-I junction region of sarcomeres onto the thin filaments in the A-band, where rigor linkages had been formed; this agreed well with the rate of increase in length of rigor-shortened sarcomeres. The sarcomere lengths were found to be maximum at 1, 7 and 10 days post-mortem in chicken, pork and beef, respectively. Thus, translocated paratropomyosin weakens rigor linkages and brings about the recovery in the length of rigor-shortened sarcomeres. Paratropomyosin stimulates the resolution of rigor mortis, and is a key factor in meat tenderization during post-rigor ageing. These results powerfully support the ‘Calcium theory of meat tenderization’ which we have proposed.

Subsarcomeric distribution of calcium in demembranated fibers of rabbit psoas muscle

Direct measurements were made of the Ca distribution within sarcomeres of glycerinated rabbit psoas muscle fibers in rigor using electron probe x-ray microanalysis. Both analogue raster analysis and digital x-ray imaging were used to quantitate the Ca distribution along thick and thin filaments as a function of the concentration of free Ca2+. Even when corrected for the estimated contribution of Ca bound to thick filaments, the Ca measured in the region of overlap between thick and thin filaments significantly exceeded the Ca in the I-band at subsaturating concentrations of free Ca2+. At saturating levels of free Ca2+, the excess Ca in the overlap region was diminished but still statistically significant. The data thus suggest that the formation of rigor linkages exerts multiple effects on the binding of Ca2+ to thin filaments in the overlap region by increasing the affinity of troponin C for Ca2+ and possibly by unmasking additional Ca2+ binding sites. The data also show that the cooperativity invested in the thin filaments is insufficient to permit the effects of rigor cross-bridge formation on Ca2+ binding to propagate far along the thin filaments into the I-band.

Cellular and Mechanical Effects of Myocardial Ischemia

Summary Myocardial pump failure during ischemia and hypoxia is often a reversible dysfunction. Contractile changes can occur in several different ways: during acute ischute ischemia, during chronic ischemia as an adaptive process, and during reperfusion following ischemia. Both systolic and diastolic contractile properties are impaired. Although a number of processes, e.g., the failure of the action potential generation mechanisms and reduced energy supply, have been suspected as causes of systolic impairment, the most likely factors appear to be H+ and inorganic phosphate concentrations. The increase in diastolic stiffness is most likely due to an increase in intracellular Ca2+ and, in severe instances, to rigor linkages between actin and myosin filaments. The extent of diastolic dysfunction depends on the nature of ischemia, being more marked in demand ischemia as compared with supply ischemia. Useful criteria to delineate the transition from reversible to irreversible ischemic damage are lacking. Nevertheless, assessment of the reversibility of ischemic contractile changes by nitroglycerin, inotropes, and postextrasystolic potentiation before surgical correction of the flow obstruction may have value in predicting outcome. Several adaptive biochemical changes are known to occur after stress. Hyperthermia produces a family of heat stress proteins that have been found to offer protection against ischemic cardiac damage. Future studies need to be performed to understand the mechanism of the regulation of production of these substances and how they afford protection to the ischemic myocardium. Furthermore, improvements in methods for imaging cardiac metabolic and perfusion status and the development of newer pharmacologic tools to alter intracellular pH regulation are expected to improve our understanding of the mechanism of ischemic cardiac damage and offer better possibilities of therapeutic management.

Changes in the X-ray diffraction pattern from rigor muscles by application of external length changes

The effect of external force on the X-ray pattern from frog muscles in rigor was studied by a time-resolved diffraction technique. When sinusoidal length changes (1.5-3% of the muscle length, 5 Hz) were applied to the muscle, the 14.3 nm intensity decreased during the releasing phase and increased during the stretching phase. The intensity ratio of the equatorial 1.0 and 1.1 reflections did not change, nor were there any appreciable intensity changes in the 5.9 nm and 5.1 nm reflections during the length change. Experiments were also done with the relaxed muscles and no change was seen in any reflection, indicating that the rigor linkages are needed to produce the 14.3 nm intensity change. Thus the distinct effect of the length change was detected only on the 14.3 nm reflection. These results suggest no large conformational changes are induced in both the distal part of the myosin head attached to actin and the actin filament during the oscillation. It is therefore most probable that the proximal portion of myosin heads including S-2 contributes to the intensity change in response to the length change (see, also ref. 21). When the muscle was stretched beyond the filament overlap, the 14.3 nm intensity change was suppressed to less than 50% of that of the slack length. It was also found that the tension change delayed the intensity change during the length oscillation. However, this delay of the tension change as observed in the muscle at the slack length was lacking in the overstretched muscle, indicating that the 14.3 nm intensity change may arise partly from a portion other than the crossbridges.

Effect of paratropomyosin on the increase in sarcomere length of rigor-shortened skeletal muscles.

Paratropomyosin is a myofibrillar protein believed to weaken rigor linkages formed between actin and myosin. Using glycerinated fibers of rabbit psoas muscles, we studied the effect of paratropomyosin on the weakening of rigor linkages, which was followed in terms of the increase in sarcomere length of rigor-shortened muscles. The rigor tension developed was reduced to about 65% of the initial value within 10 min after the addition of purified paratropomyosin, whereas it remained constant for at least 3.5 h in control fibers. Paratropomyosin showed a stronger effect on the increase in sarcomere length of passively stretched fibers, which developed weaker rigor-tensions. The purpose of our research was to establish a rigor solution which would best permit the observation of the workings of paratropomyosin. The most successful rigor solution contained 0.2-0.25 M KCl, pH 5.5, at 5-10 degrees C. Under these conditions, the sarcomere length was easily increased from 2.4 to 3.6 micron, if rigor-contracted fibers were passively stretched after the addition of purified paratropomyosin. Because the experimental conditions coincide well with those of postmortem muscles, it is very probable that paratropomyosin plays an important role in restoration of the sarcomere length of rigor-shortened muscles, resulting in tenderization of meat during postrigor ageing.

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 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.

A myofibrillar component that modifies the actin-myosin interaction in postrigor skeletal muscle.

A protein component that modified the interaction between actin and myosin was released from myofibrils on Ca2+-treatment, accompanying the weakening of Z disks. For its release 10(-4) M Ca2+ was required. The presence of the component facilitated the dissociation of thin and thick filaments from myofibrils and delayed superprecipitation of reconstituted actomyosin, independently of Ca2+ concentration. The component is probably a constituent of Z disks. In postmortem muscle, it is possible that the component is released from Z disks with an increased concentration of Ca2+, and that it weakens rigor linkages formed between actin and myosin, giving rise to lengthening of rigor-shortened sarcomeres.

Mechanics of guinea pig taenia coli smooth muscle during anoxia and rigor.

Potassium-contracted guinea pig taenia coli relaxed when exposed to hypoxic glucose-free conditions, and this was followed by a second contraction. During this second contraction, which was not inhibited by ethylene glycol-his-(beta-aminoethylether)-N,N'-tetracetic acid (EGTA) the muscle was in rigor. After development of this contraction the muscle did not have an active state and was less extensible than a muscle normally contracted with KCl. Tension during rigor in an isometrically contracting muscle was short-lived in contrast to shortening during rigor development in a lightly loaded isotonically contracting muscle. These findings, together with the observation of irreversible lengthening of a muscle in rigor on stretching, suggest that rigor linkages can be broken. Glucose removal under aerobic conditions did not produce rigor, but oxygen removal in the presence of glucose resulted in relaxation followed by secondary contraction. This later contraction, unlike that accompanying rigor, was reversed by removal of calcium with EGTA. These data imply that in the absence of oxygen the removal of cytoplasmic free-Ca2+ is inhibited.