[Hyperthyreosis inhibits the ability of actin to form strong-binding with myosin].

The effect of hyperthyreosis development induced by the increase in thyroid hormones in rats (during 2-4 weeks) on the orientation and mobility of fluorescent probe N-(iodoacetyl)-(1-naphtyl-5-sulpho-ethylenediamine) specifically bound to Cys 374 of actin in ghost muscle fibers isolated from fast (EDL) and slow (SOL) rat muscles was studied. It was found that the binding of myosin subfragment-1 (S1) to F-actin induced the typical for the formation of strong binding actomyosin decrease in mobility of actin subdomain 1 and its rotation towards thin filament periphery. Development of hyperthyreosis markedly inhibited these phenomena. The maximal effect was observed after 21 days of disease development. It is suggested that one of the reasons of the contractile deficit of muscle in hyperthyreosis is inhibition of the strong binding between actin and myosin during ATPase cycle.

[Effect of hypothyreosis on actin subdomain-1 movement induced by myosin subfragment 1-binding in fast and slow rat skeletal muscles].

Orientation and mobility of fluorescent probe N-((iodoacetyl)-(1-naphtyl-5-sulpho-ethylenediamine)(1.5-IAEDANS)) specifically bound to Cys-374 of actin in ghost muscle fibers isolated from fast and slow rat muscles were studied by polarized fluorimetry in the absence and presence of myosin subfragment-1 (S1) in intact rats and in the animals with gradual (during 2-5 weeks) reduction of thyroid hormones synthesis (hypothyreosis development). S1 binding to F-actin of ghost muscle fibers was shown to induce changes in orientation of the dipoles of the fluorescent probe 1.5-IAEDANS and in the relative amount of the randomly oriented fluorophores that indicated changes in actin subdomain-1 orientation and mobility resulting from the formation of its strong binding with S1. This effect is markedly inhibited by hypothyreosis development. The maximal effect of hypothyreosis is observed after 34 days of disease development. It is suggested that the change of thyroid status in the muscle inhibits the ability of F-actin to form strong binding with myosin which is essential for force generation.

[The influence of caldesmon on strong binding of myosin with actin in denervated rat skeletal muscles].

The effect of caldesmon (CaD) on conformational changes in F-actin modified by fluorescent probe TRITC-phalloidin was investigated by polarized fluorimetry. Changes were induced by a subfragment-1 (S-1) of myosin in the absence or presence of CaD in ghost muscle fibers obtained from intact and denervated slow (SOL) and fast (EDL) skeletal muscles of rats. S-1 binding to actin of both SOL and EDL muscles was shown to cause changes in polarized parameters of TRITC-phalloidin typical for a strong actin-myosin binding as well as of transition ofactin subunits from "off" to "on" state. CaD inhibits this significantly. Denervation atrophy inhibits the effect of S-1 as well but does not affect the capability of CaD decreasing the formation of strong binding in actomyosin complex. It is supposed that CaD "freezes" F-actin structure in "off" state. The denervation atrophy has no effect on CaD responsibility to bind thin filaments and to switch "off" actin monomers.

Effect of denervation, reinnervation and hypertrophy on the state of actin filaments in skeletal muscle fibres.

The conformational state of actin filaments was studied in the rat soleus muscle atrophying after denervation, recovering following reinnervation, hypertrophying following tenotomy of synergists and in intact muscle. Intrinsic (tryptophan residues of F-actin) and extrinsic (rhodamine-phalloidin or 1,5-IAEDANS attached to F-actin) polarized fluorescence was measured. In parallel, the influence of ATP or NEM on the state of F-actin was studied. The results show that the conformational state of F-actin is changed in all experimental muscles. These changes of the denervated muscle differ from those of the reinnervated and hypertrophying muscles. In the reinnervated muscle, beginning with the first days of recovery, the structure of F-actin seems to "recover" to the state in intact muscle. In the later stage of muscle recovery, the state of F-actin is similar to that in hypertrophying muscle. Differences between the mentioned muscles in the conformational state of actin monomers, in the orientation of monomers and in the flexibility of thin filaments are discussed.

Degradation of alpha-actinin during Ca2+-sensitive proteolysis of myofibrils.

The noted loss of alpha-actinin from the Z-line of myofibrils during post-mortem autolysis, probably following the action of calcium-activated protease, has previously been attributed to its release without degradation. This report shows that in isolated myofibrils alpha-actinin is proteolysed in a Ca2+-sensitive manner presumably via the action of calcium-activated protease.

Effect of thyroid hormone on the myosin heavy chain isoforms in slow and fast muscles of the rat.

The myosin heavy chain (MHC) was studied by biochemical methods in the slow-twitch (soleus) and two fast-twitch leg muscles of the triiodothyronine treated (hyperthyroid), thyroidectomized (hypothyroid) and euthyroid (control) rats. The changes in the contents of individual MHC isoforms(MHC-1, MHC-2A, MHC-2B and MHC-2X) were evaluated in relation to the muscle mass and the total MHC content. The MHC-1 content decreased in hyperthyreosis, while it increased in hypothyreosis in the soleus and in the fast muscles. The MHC-2A content increased in hyperthyreosis and it decreased in hypothyreosis in the soleus muscle. In the fast muscles hyperthyreosis did not affect the MHC-2A content, whereas hypothyreosis caused an increase in this MHC isoform content. The MHC-2X, present only in traces or undetected in the control soleus muscle, was synthesised in considerable amount in hyperthyreosis; in hypothyreosis the MHC-2X was not detected in the soleus. In the fast muscles the content of MHC-2X was not affected by any changes in the thyroid hormone level. The MHC-2B seemed to be not influenced by hyperthyreosis in the fast muscles, whereas the hypothyreosis caused a decrease of its content. In the soleus muscle the MHC-2B was not detected in any groups of rats. The results suggest that the amount of each of the four MHC isoforms expressed in the mature rat leg muscles is influenced by the thyroid hormone in a different way. The MHC-2A and the MHC-2X are differently regulated in the soleus and in the fast muscles; thyroid hormone seems to be necessary for expression of those isoforms in the soleus muscle.

Contents of myosin heavy chains in denervated slow and fast rat leg muscles.

The total content of myosin heavy chain (MHC) and individual MHC isoforms were studied in 14-day denervated rat leg muscles: the slow-twitch (soleus) and fast-twitch (extensor digitorum longus and gastrocnemius) by biochemical methods. The weight of the denervated muscles decreased by about 50%, as compared to the control muscles. In all denervated muscles the total content of MHCs decreased, more so in the slow than in the fast muscles. We have observed that the proportion among the MHC isoforms changed: while MHC-1 and MHC-2B decreased, MHC-2A and MHC-2X increased. Taking into account muscle atrophy, the loss of MHC total content and the shift in pattern of MHC isoforms, the total net changes of the particular MHC isoforms were evaluated. It was found that the muscle content of each of the MHCs decreased after denervation, but their tissue concentration changed variously. The concentration of the MHC-1 and MHC-2B decreased in all denervated muscles, but that of the MHC-2A and MHC-2X changed variously, depending on the muscle. The concentration of MHC-2A decreased in the soleus and increased in the fast muscles, whereas the concentration of the MHC-2X changed inversely. In the denervated soleus a considerable amount of MHC-2X was expressed, while in the contralateral muscles this isoform was undetectable or appeared at trace levels.

Ultrastructure of the contractile apparatus of rat skeletal muscle embedded in an aqueous medium.

The method of tissue embedding in melamine resin was applied to rat skeletal muscle. This method does not require tissue dehydration with organic solvents; only aqueous solutions are used. Electron micrographs of muscles embedded in melamine differ from those embedded in the conventional epoxy resin. In melamine-embedded muscles the actin and myosin filaments appear larger in diameter and subunits can be recognized in cross-sectioned myosin filaments. Within the Z-line, the characteristic patterns described for muscles embedded in epoxy resin are not visible; the spaces between the actin filaments are filled with electron-dense material. This suggests that the Z-line is more compact than could be concluded from epoxy resin-embedded muscle specimens. The M-line appears to be different from what is observed in epoxy-embedded muscle. The membranes appear as several clearly delineated layers. Dehydration rather than the action of the organic solvents per se is the main reason for the differences in the structure of the contractile apparatus between melamine- and epoxy-embedded muscles.

Ultrastructure of mechanically tenderised pork muscle.

Porcine biceps femoris muscles were mechanically tenderised by the use of a meat activator. The kind and degree of damage of muscle tissue were then examined under an electron microscope. It was observed that several changes, known from the studies of the post mortem muscles, were much more frequent in tenderised than in intact muscles. Additional changes were found as: disruption of the contractile system or its expansion till the A- and I-bands disconnected. Thus we suggest that mechanical tenderisation, by destroying several linkages between muscle fibres, between myofibrils and within myofibrils, may be responsible for lattice expansion and increase of brine uptake and overcoming the myofibrillar and connective tissue toughness of porcine meat.

Reconstruction of the contractile apparatus of striated muscle. I. Muscle maintained in extension.

The ultrastructure of the contractile apparatus was observed in muscles maintained in excessive extension, i.e. in conditions in which an increase takes place in the number of sarcomeres. Rat leg muscles (soleus, extensor digitorum longus and gastrocnemius) were studied, at variable time intervals in the range 3-7 days. Several irregularities were found in the contractile structure. The most frequent were the variability of sarcomere length, the appearance of 'extra' sarcomeres, irregularities of the Z-line (including Z-band 'streaming') and A-bands of abnormal length. The character of these irregularities depended on the muscle fibre type. Variations of the Z-line were seen mostly within continuously working fibres, especially slow ones, while anomalies in the size of the A-band and variability of the sarcomere length were more pronounced in fast fibres. All these irregularities appearing in the muscles maintained in excessive extension were also occasionally found in control muscles. The reasons for these contractile structure irregularities, and their possible significance for contractile structure reorganization, are discussed.

Changes in myosin and actin filaments in fast skeletal muscle after denervation and self-reinnervation.

1. Myosin and actin filaments of the contractile apparatus of the denervated and self-reinnervated rat leg fast muscle were examined in ultrastructure. In parallel, the total contents of actin and of myosin heavy chains (MHC) were investigated. The results were compared with the corresponding ones in the slow muscle. 2. In the denervated-atrophying fast muscle the myosin filaments disappeared before the actin filaments. However, in contrast to the slow muscle, the local disproportion between the filaments was soon compensated, and their hexagonal arrangement was maintained for about one month after denervation. The contents of MHC and actin decreased, but their ratio remained similar to that in the controls. 3. In the later stage of atrophy the proportion of myosin to actin filaments and the ratio of the corresponding proteins decreased, and the hexagonal arrangement of filaments was disturbed. The denervated fast and slow muscles became similar (in the latter, such changes occurred during the initial weeks after denervation). 4. In the fast muscle recovering after reinnervation (on the third week after denervation) the numbers of myosin and actin filaments, and the contents of the corresponding proteins increased in parallel and the hexagonal arrangement of filaments was maintained (differently than those observed in the slow muscle).

Remodelling of the contractile apparatus of striated muscle stimulated electrically in a shortened position.

The aim of this study was to examine reorganisation of the contractile apparatus during adaptation to function when the length of a muscle is decreased. The rat soleus muscle was maintained in a shortened position and simultaneously stimulated electrically at a low frequency for 1-45 h. This experimental model decreased the length of the muscle and made the contractile apparatus irregular. The length of the sarcomeres decreased and became variable. The Z-line appeared wavy or fragmented. Foci, within which the sarcomeric organisation was lacking, often appeared within the contractile apparatus. These changes, occurring during the initial hours of stimulation, increased in number during the following hours. Their frequency and intensity depended on the degree of muscle shortening. In muscle moderately shortened during stimulation (up to 20%) the contractile apparatus recovered its normal appearance within 12 h of the experiment. In the muscle considerably shortened (by about 30%) the process of normalisation took much longer, in spite of recovery of sarcomere length. We conclude from these results that these changes are related to the accelerated work-induced reorganisation of the contractile apparatus whenever sarcomere number is reduced and/or when elimination of portions of the contractile apparatus occurs. These anomalies, occurring transiently in normal mature muscle when stimulated electrically in a shortened position, should be considered as adaptive phenomena. They resemble abnormalities appearing in the contractile apparatus of myopathic muscle.

Morphological changes in rat skeletal muscle following reinnervation.

Morphological changes appearing in the course of muscle regeneration after reinnervation of denervated M. soleus (slow) and M. tibialis anterior (fast) rat skeletal muscle were investigated. It was found that pathological changes typical for denervation atrophy (seen on the 10th day after crushing the sciatic nerve) and symptoms of regeneration (beginning about the 15th day) were much more pronounced in the soleus than in the tibialis muscle. Some stages of regeneration in the soleus muscle could be distinguished. The contractile material destructions were the first pathological changes that disappeared after the beginning of regeneration. In the second stage other denervation changes disappeared and intensive regeneration of muscle fibres was observed. In the next stage regeneration slowed down, and the reduction of the excess of muscle nuclei was visible. Four months after crushing the nerve, regeneration proceeded to completion with only some traces of the passed processes: in the soleus muscle, chains of sarcolemmal nuclei, satellite cells and newly formed muscle fibres were more often seen than in contralateral muscle; in the tibialis, collagen depots were present around the vessels and between muscle fascicles.