Anna Jakubiec-Puka

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.

Endogenous proteinases in vertebrate skeletal muscle

1. 1. Activity of endogenous proteases in the muscles of various vertebrates was measured over a wide pH range. Three distinct regions of autolysis was found at acid, neutral and alkaline pH range. The activity in the latter pH was observed only in rat. mouse, and to a smaller extent in dog and human skeletal muscles. 2. 2. In the rat muscle the amount and size of peptides formed during autolysis as well as distribution of autolytic activity in the subcellular fractions was determined. 3. 3. Proteases working at acid pH range and localized in lyzosomes seem to degrade chiefly soluble sarcoplasmic and not myofibrillar proteins. 4. 4. Autolytic activity at alkaline pH is present in 0–600 g fractions. This protease degrades all myofibrillar proteins. Its activity is inhibited almost completely by diisopropyl-fluorophosphate and soy-bean trypsin inhibitor, partially by l-I-tosylamidophenylethylchloromethylketone and by injection of rats with the preparation “48/80”. All the results indicate that the alkaline protease is a chymotrypsin-like protease of mast-cell origin. 5. 5. The results suggest that at neutral pH range another proteolytic system is active in muscle. although its activity is rather low.

Loss of dystrophin and some dystrophin-associated proteins with concomitant signs of apoptosis in rat leg muscle overworked in extension.

Abstract This study investigated the basis for the high severity of damage to skeletal muscle due to eccentric exercise, i.e., to muscles generating force while lengthened. Fast and slow rat leg muscles maintained in an extended position were examined after 2–24 h of continuous stimulation. The treatment caused the injury to some regions of both muscles. Within the better preserved parts of the muscles, i.e., those without signs of necrotic processes, dystrophin, spectrin, and some of the dystrophin-associated proteins (β-dystroglycan, α-sarcoglycan, and γ-sarcoglycan) disappeared from sarcolemma of many fibers. The reduction or loss of dystrophin from the sarcolemma was more evident than that of other proteins examined, with sarcoglycans apparently being the most preserved. Several muscle fibers devoid of dystrophin contained apoptotic nuclei. Simultaneously, Bax, Bcl-2 and caspase-3 proteins appeared in many fibers. Our results indicate that a normal muscle overworking in an extended position undergoes the loss of several membrane skeletal proteins because of the excessive stress to the membrane cytoskeleton, which can lead to fiber death by either apoptosis or necrosis. This experimental model may represent a good model for mimicking the pathogenetic events in several muscular dystrophies.

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).

The contractile apparatus of striated muscle in the course of atrophy and regeneration

SummaryThe ultrastructure of the contractile apparatus of the rat soleus muscle during the course of denervation atrophy was investigated. It was found that the ratio of thin to thick filaments increased in myofibrils of atrophying muscle fibers. Elevation of the ratio was observed as early as the second day after denervation, and became more pronounced with the progress of atrophy. Parallel measurements of the amounts of actin and myosin in the myofibrils and in the muscle protein extracts revealed a lower proportion of myosin heavy chains to actin in the fractions from denervated muscles, compared with the control values. Both the electron-microscopic observations and the biochemical evaluation of the actin content of the muscle, suggests that the elevated ratio of thin to thick filaments seen in the course of the muscle atrophy appears as the result of an earlier and more intensive disappearance of thick filaments. Thin filaments disappeared more slowly, in parallel to the decrease in muscle weight.On the basis of the results presented a mechanism of progress of “simple atrophy” of muscle in suggested.

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

SummaryThe 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.

Degradation of α-actinin during Ca2+-sensitive proteolysis of myofibrils

The noted loss of α‐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 α‐actinin is proteolysed in a Ca2+‐sensitive manner presumably via the action of calcium‐activated protease.

Influence of locomotor training on the structure and myosin heavy chains of the denervated rat soleus muscle

Abstract Objective: Mechanism of denervation atrophy remains poorly understood. In particular, the question about irreversibility of the late atrophy is still open. Therefore, in the present study, we investigated whether and how a passive movement can affect a progress of atrophy in rat soleus muscle. To address this issue, a locomotor training on a treadmill was applied to rats with their right hindlimb muscles denervated. Methods: The hindlimb muscles were denervated by cutting the sciatic nerve. Starting either 7 days or 1 month after the surgery, the animals were trained on a treadmill. Two months after denervation, the soleus muscle was investigated using light and electron microscopy and biochemical methods. Control soleus muscles were obtained from non-trained animals: the untreated and the 2-month denervated. Results: Locomotor training caused slight increase in denervated rat soleus muscle weight and significant increase in its fiber diameter. The training positively affected some of the factors that were believed to be the reasons of atrophy irreversibility, because of significant increase in the number of capillary blood vessels and muscle fiber nuclei with the concomitant decrease in the number of severely damaged muscle fibers and amount of collagen. Morphology of the contractile apparatus was also improved as more regular organization of sarcomeres and the hexagonal arrangement of myosin filaments was evident. Moreover, the amount of myosin heavy chains (MHC) significantly increased after training. The effects were more evident in the animals with longer training. Conclusion: Passive movement seems to attenuate some of the pathologic processes within the denervated muscle.

Effects of denervation and muscle inactivity on the organization of F-actin

To discriminate between the influences of a motoneuron and muscle activity on the conformation of actin filaments, the extrinsic polarized fluorescence [of rhodamine–phalloidin and N‐(iodoacetylamine)‐1‐naphthylamine‐5‐sulfonic acid attached to F‐actin] was measured in “ghost” fibers from intact rat soleus muscles and atrophying muscles after denervation, immobilization, or tenotomy. The results show that the conformation of F‐actin changed in all the atrophying muscles, but differently. In the denervated muscle, the flexibility of the actin filaments decreased, whereas in the other experimental muscles it remained as in the intact muscle. In the denervated muscle, the mobility of the C‐terminus of the actin polypeptide increased. Attachment of myosin subfragment‐1 influenced the F‐actin conformation differently in the denervated muscle than in the other muscles studied. These results suggest that changes in the conformation of the actin filament are induced by the lack of connection with the motoneuron rather than by muscle inactivity.