Myosin heavy chain isoform composition in striated muscle after denervation and self-reinnervation

The total content of myosin heavy chains (MHC) and their isoform pattern were studied by biochemical methods in the slow-twitch (soleus) and fast-twitch (extensor digitorum longus) muscles of adult rat during atrophy after denervation and recovery after self-reinnervation. The pattern of fibre types, in terms of ultrastructure, was studied in parallel. After denervation, total MHC content decreased sooner in the slow-twitch muscle than in the fast-twitch. The ratio of MHC-1 and the MHC-2B isoforms to the MHC-2A isoform decreased in the slow and the fast denervated muscles, respectively. After reinnervation of the slow muscle, the normal pattern of MHC recovered within 10 days and the type 1 isoform increased above the normal. In the reinnervated fast muscle, the 2B/2A isoform ratio continued to decrease. Traces of the embryonic MHC isoform, identified by immunochemistry, were found in both denervated and reinnervated slow and fast muscles. A shift in fibre types was similar to that found in the MHC isoforms. Within 2 months of recovery a tendency to normalization was observed. The results show that (a) MHC-2B isoform and the morphological characteristics of the 2B-type muscle fibres are susceptible to lack of innervation, similar to those of type 1, (b) during muscle recovery induced by reinnervation the MHC isoforms and muscle fibres shift transiently to type 1 in the soleus and to type 2A in the extensor digitorum longus muscles, and (c) the embryonic isoform of MHC may appear in the adult skeletal muscles if innervation is disturbed.     

Slow myosin in developing rat skeletal muscle

Through S1 nuclease mapping using a specific cDNA probe, we demonstrate that the slow myosin heavy-chain (MHC) gene, characteristic of adult soleus, is expressed in bulk hind limb muscle obtained from the 18-d rat fetus. We support these results by use of a monoclonal antibody (mAb) which is highly specific to the adult slow MHC. Immunoblots of MHC peptide maps show the same peptides, uniquely recognized by this antibody in adult soleus, are also identified in 18-d fetal limb muscle. Thus synthesis of slow myosin is an early event in skeletal myogenesis and is expressed concurrently with embryonic myosin. By immunofluorescence we demonstrate that in the 16-d fetus all primary myotubes in future fast and future slow muscles homogeneously express slow as well as embryonic myosin. Fiber heterogeneity arises owing to a developmentally regulated inhibition of slow MHC accumulation as muscles are progressively assembled from successive orders of cells. Assembly involves addition of new, superficial areas of the anterior tibial muscle (AT) and extensor digitorum longus muscle (EDL) in which primary cells initially stain weakly or are unstained with the slow mAb. In the developing AT and EDL, expression of slow myosin is unstable and is progressively restricted as these muscles specialize more and more towards the fast phenotype. Slow fibers persisting in deep portions of the adult EDL and AT are interpreted as vestiges of the original muscle primordium. A comparable inhibition of slow MHC accumulation occurs in the developing soleus but involves secondary, not primary, cells. Our results show that the fate of secondary cells is flexible and is spatially determined. By RIA we show that the relative proportions of slow MHC are fivefold greater in the soleus than in the EDL or AT at birth. After neonatal denervation, concentrations of slow MHC in the soleus rapidly decline, and we hypothesize that, in this muscle, the nerve protects and amplifies initial programs of slow MHC synthesis. Conversely, the content of slow MHC rises in the neonatally denervated EDL. This suggests that as the nerve amplifies fast MHC accumulation in the developing EDL, accumulation of slow MHC is inhibited in an antithetic fashion. Studies with phenylthiouracil-induced hypothyroidism indicate that inhibition of slow MHC accumulation in the EDL and AT is not initially under thyroid regulation. At later stages, the development of thyroid function plays a role in inhibiting slow MHC accumulation in the differentiating EDL and AT.(ABSTRACT TRUNCATED AT 400 WORDS)     

The Effect of Passive Movement on Denervated Soleus Highlights a Differential Nerve Control on SERCA and MyHC Isoforms

The sarco-endoplasmic reticulum Ca²⁺ ATP-ase (SERCA) and myosin heavy chain (MyHC) levels were measured in hindlimb-denervated and selectively denervated rat soleus muscles. Selective denervation allowed passive movement of the soleus, whereas hindlimb denervation rendered it to passivity. To minimize chronic effects, we followed the changes only for 2 weeks. Selective denervation resulted in less muscle atrophy, a faster slow-to-fast transition of MyHC isoforms, and less coordinated expressions of the slow vs fast isoforms of MyHC and SERCA. Generally, expression of the slow-twitch type SERCA2a was found to be less dependent, whereas the slow-twitch type MyHC1 was the most dependent on innervation. Our study shows that passive movement is able to ameliorate denervation-induced atrophy of the soleus and that it also accentuates the dyscoordination in the expression of the corresponding slow and fast isoforms of MyHC and SERCA. (J Histochem Cytochem 56:1013–1022, 2008)     

Altered expression of skeletal muscle myosin isoforms in cancer cachexia

Cachexia is commonly seen in cancer and ischaracterized by severe muscle wasting, but little is known about theeffect of cancer cachexia on expression of contractile protein isoforms such as myosin. Other causes of muscle atrophy shift expression ofmyosin isoforms toward increased fast (type II) isoform expression. Weinjected mice with murine C-26 adenocarcinoma cells, a tumor cell linethat has been shown to cause muscle wasting. Mice were killed 21 daysafter tumor injection, and hindlimb muscles were removed. Myosin heavychain (MHC) and myosin light chain (MLC) content was determined inmuscle homogenates by SDS-PAGE. Body weight was significantly lower intumor-bearing (T) mice. There was a significant decrease in muscle massin all three muscles tested compared with control, with the largestdecrease occurring in the soleus. Although no type IIb MHC was detectedin the soleus samples from control mice, type IIb comprised 19% of thetotal MHC in T soleus. Type I MHC was significantly decreased in T vs. control soleus muscle. MHC isoform content was not significantly different from control in plantaris and gastrocnemius muscles. Thesedata are the first to show a change in myosin isoform expression accompanying muscle atrophy during cancer cachexia.

     

Disuse and passive stretch cause rapid alterations in expression of developmental and adult contractile protein genes in skeletal muscle

Contractile proteins exist as a number of isoforms that show a developmental and tissue-specific pattern of expression. Using gene-specific cDNA probes, the expression of the sarcomeric myosin heavy chain (MHC) multi-gene family and of cardiac (foetal) alpha-actin was analysed in three different rat hindlimb muscles immobilised for 5 days in either the shortened or lengthened positions. For each of the MHC genes normally expressed in adult muscle (slow, IIA and IIB), the effect of disuse alone (immobilisation in the shortened position) upon expression was markedly different to that of passive stretch (immobilisation in the lengthened position) in each of the three muscles. However, the same adult sarcomeric myosin heavy chain gene can be affected in a different, or even opposite, manner by either disuse or passive stretch depending on the muscle in which it is being expressed. The fast IIB MHC gene, for example, exhibits a rapid induction in the slow postural soleus muscle, in response to disuse but no such induction occurs in the faster plantaris and gastrocnemius muscles. Furthermore, the induction of this gene in the soleus was prevented by passive stretch. The MHC gene, normally only expressed in embryonic skeletal muscle, showed a similar response in all three muscles and was reinduced in adult muscle in response to passive stretch but not by disuse alone. In contrast, the isoform of alpha-actin which is normally only present in significant quantities in embryonic skeletal muscle and which is reduced postnatally, is not reinduced by passive stretch but is reduced still further by immobilisation in the shortened position.     

Unlike effects of denervation on the rate of entry of inorganic phosphate into rat slow and fast muscles.

The increased inorganic phosphate flow, characteristic of denervated gastrocnemius muscle is shown to be present in additional denervated fast muscles, i.e. the plantaris, tibialis anterior and extensor digitorum longus muscles. The response of the soleus, a slow muscle, to denervation is biphasic. After an initial decrease of the phosphate flow at the end of the first postoperative day, there is a secondary rise which has the same general characteristics as the rise observed in fast muscles i.e. an exponential or hyperbolic increase to an asymptotic value reached after thirty days. The denervated fast and slow muscles are not converging to an intermediate metabolic pattern. The changes in phosphate flow induced by denervation are reversible in the soleus as well as in the gastrocnemius muscles.     

Effect of hindlimb suspension on the functional properties of slow and fast soleus fibers from three strains of mice.

Cross-sectional area (CSA), peak Ca2+-activated force (Po), fiber specific force (Po/CSA), and unloaded shortening velocity (Vo) were measured in slow-twitch [containing type I myosin heavy chain (MHC)] and fast-twitch (containing type II MHC) chemically skinned soleus muscle fiber segments obtained from three strains of weight-bearing and 7-day hindlimb-suspended (HS) mice. HS reduced soleus slow MHC content (from approximately 50 to approximately 33%) in CBA/J and ICR strains without affecting slow MHC content in C57BL/6 mice ( approximately 20% of total MHC). Two-way ANOVA revealed HS-induced reductions in CSA, Po, and Po/CSA of slow and fast fibers from all strains. Fiber Vo was elevated post-HS, but not consistently across strains. No MHC x HS treatment interactions were observed for any variable for C57BL/6 and CBA/J mice, and the two significant interactions found for the ICR strain (CSA, Po) appeared related to inherent pre-HS differences in slow vs. fast fiber CSA. In the mouse HS models studied here, fiber atrophy and contractile dysfunction were partially dependent on animal strain and generally independent of fiber MHC isoform content.     

Interaction of hyperthyroidism and hindlimb suspension on skeletal myosin heavy chain expression

We examined the novel interaction ofhyperthyroidism and hindlimb suspension on the pattern of myosin heavychain (MHC) expression (mRNA and protein) in skeletal muscles. FemaleSprague-Dawley rats were assigned to four groups:1) normal control (Con);2) thyroid hormone treated[150 µg 3,5,3'-triiodothyronine(T₃) · kg¹ · day¹](T₃);3) hindlimb suspension (HS); or4)T₃-treated and HS(T₃ + HS). Results show for thefirst time the novel observation that the combinationT₃ + HS induces a rapid andsustained, marked (80-90%) downregulation of type I MHC geneexpression that is mirrored temporally by concomitant markedupregulation of type IIb MHC gene expression, as evidenced by the denovo synthesis of type IIb MHC protein in the soleus. The fast type IIxMHC isoform showed a differential response among the experimentalgroups, generally increasing with the separate and combined treatments in both the soleus and vastus intermedius muscles while decreasing inthe plantaris muscles. The fast type IIa MHC was the least responsiveto suspension of the MHCs and reflected its greatest responsiveness toT₃ treatment while also undergoingdifferential adaptations in slow vs. fast muscle (increases vs.decreases, respectively). These results confirm previous findings thatall four adult MHC genes are sensitive toT₃ and suspension in amuscle-specific manner. In addition, we show thatT₃ + HS can interactsynergistically to create novel adaptations in MHC expression thatcould not be observed when each factor was imposed separately.

     

Novel transitions in MHC isoforms: separate and combined effects of thyroid hormone and mechanical unloading

Single-fiber(n = 3,818 fibers) electrophoreticanalyses were used to delineate the separate and combined effects ofhyperthyroidism (T₃) andhindlimb suspension (HS) on the myosin heavy chain (MHC) isoformcomposition (1-, 2-, and 4-wk time points) of the rat soleus muscle.The key findings of this study are as follows. First,T₃ and HS both altered thedistribution of MHC isoforms at the single-fiber level; however, thepopulations of fibers produced by these two interventions were clearlydifferent from one another. Second,T₃ + HS rapidly converted thesoleus into a fast muscle, producing large increases in the relativecontents of the fast type IIx and IIb MHC isoforms which were primarily expressed in several populations of hybrid fibers (e.g., types I/IIa/IIx, I/IIx/IIb, I/IIa/IIx/IIb). Finally,T₃ + HS produced uniquepopulations of hybrid fibers that did not adhere to the IIIaIIxIIb sequential scheme of MHC plasticity. Collectively, the findings of this study demonstrate that the intervention of T₃ + HS is a powerful model formanipulating and studying MHC isoform plasticity in slow skeletal muscle.

     

Slow myosin heavy chain expression in the absence of muscle activity

Innervation has been generally accepted to be a major factor involved in both triggering and maintaining the expression of slow myosin heavy chain (MHC-1) in skeletal muscle. However, previous findings from our laboratory have suggested that, in the mouse, this is not always the case (30). Based on these results, we hypothesized that neurotomy would not markedly reduced the expression of MHC-1 protein in the mouse soleus muscles. In addition, other cellular, biochemical, and functional parameters were also studied in these denervated soleus muscles to complete our study. Our results show that denervation reduced neither the relative amount of MHC-1 protein, nor the percentage of muscle fibers expressing MHC-1 protein (P > 0.05). The fact that MHC-1 protein did not respond to muscle inactivity was confirmed in three different mouse strains (129/SV, C57BL/6, and CD1). In contrast, all of the other histological, biochemical, and functional muscle parameters were markedly altered by denervation. Cross-sectional area (CSA) of muscle fibers, maximal tetanic isometric force, maximal velocity of shortening, maximal power, and citrate synthase activity were all reduced in denervated muscles compared with innervated muscles (P < 0.05). Contraction and one-half relaxation times of the twitch were also increased by denervation (P < 0.05). Addition of tenotomy to denervation had no further effect on the relative expression of MHC-1 protein (P > 0.05), despite a greater reduction in CSA and citrate synthase activity (P < 0.05). In conclusion, a deficit in neural input leads to marked atrophy and reduction in performance in mouse soleus muscles. However, the maintenance of the relative expression of slow MHC protein is independent of neuromuscular activity in mice.     

Effects of sensitization of the guinea pig slow muscle in disorders of the neurotrophic control]

The immunohistochemical profile of intact and denervated soleus muscle of guinea pigs after sensibilization was studied. It is shown, that intact soleus muscle consists of slow fibers, which have low ATP-ase activity and don't react with monoclonal antibodies against fast myosin heavy chain. No changes of immunohistochemical profile were found after denervation or sensibilization. At the same time, the fibers, reacting with monoclonal antibodies against fast myosin heavy chain and having low ATP-ase activity, were found in denervated muscles after sensibilization. It is concluded, that the synthesis of fast myosin is induced after sensibilization of denervated muscles. Validity of myosin ATP-ase histochemistry for muscle fibers typing is discussed.     

Response to denervation of rabbit soleus and gastrocnemius muscles. Time-course study of postnatal changes in myosin isoforms,fiber types,and contractile properties

Summary— In contrast to general belief, the response of rabbit muscles to denervation is maturation to slow-like type muscles [7]. We report now an investigation by biochemical, morphological, and mechanical studies of the time course effects of muscle denervation on the slow-type soleus and fast-type gastrocnemius to help clucidate the mechanism of maturation of rabbit denervated muscles to slow-like muscles. In both muscles, denervation induced selective progressive atrophy of most fast fibers and hypertrophy of many slow fibers which displayed wide Z-lines; this was accompanied by the appearance of hybrid LC1F- and LC1E-associated slow myosins. The percentage of slow myosins increased with age similarly in the contralateral and denervated soleus. On the other hand, the percentage of slow myosins remained low in the contralateral gastrocnemius, whereas it increased to 95% in the denervated gastrocnemius; in the denervated gastrocnemius, the percentage of slow myosins reached 50% at about 35 days postnatal. At this age, the maximal shortening velocity of the denervated gastrocnemius and its twitch contraction time were already those of a slow-type muscle. This suggests that in addition to myosin, other proteins contributed to the mechanical properties of the denervated gastrocnemius. Transformation of rabbit denervated muscles to slow-like type muscles, which are associated with a lower energy requirement and higher muscle endurance than fast-type muscles, may constitute an adequate model for human neuromuscular pathology.     

Chronic denervation of rat hemidiaphragm: maintenance of fiber heterogeneity with associated increasing uniformity of myosin isoforms

During several months of denervation, rat mixed muscles lose slow myosin, though with variability among animals. Immunocytochemical studies showed that all the denervated fibers of the hemidiaphragm reacted with anti-fast myosin, while many reacted with anti-slow myosin as well. This has left open the question as to whether multiple forms of myosin co-exist within individual fibers or a unique, possibly embryonic, myosin is present, which shares epitopes with fast and slow myosins. Furthermore, one can ask if the reappearance of embryonic myosin in chronically denervated muscle is related both to its re-expression in the pre-existing fibers and to cell regeneration. To answer these questions we studied the myosin heavy chains from individual fibers of the denervated hemidiaphragm by SDS PAGE and morphologically searched for regenerative events in the long term denervated muscle. 3 mo after denervation the severely atrophic fibers of the hemidiaphragm showed either fast or a mixture of fast and slow myosin heavy chains. Structural analysis of proteins sequentially extracted from muscle cryostat sections showed that slow myosin was still present 16 mo after denervation, in spite of the loss of the selective distribution of fast and slow features. Therefore muscle fibers can express adult fast myosin not only when denervated during their differentiation but also after the slow program has been expressed for a long time. Light and electron microscopy showed that the long-term denervated muscle maintained a steady-state atrophy for the rat's life span. Some of the morphological features indicate that aneural regeneration events continuously occur and significantly contribute to the increasing uniformity of the myosin gene expression in long-term denervated diaphragm.     

Neural regulation of acetylcholinesterase-associated collagen Q in rat skeletal muscles

Acetylcholinesterase-associated collagen Q is expressed also outside of neuromuscular junctions in the slow soleus muscle, but not in fast muscles. We examined the nerve dependence of muscle collagen Q expression and mechanisms responsible for these differences. Denervation decreased extrajunctional collagen Q mRNA levels in the soleus muscles and junctional levels in fast sternomastoid muscles to about one third. Cross-innervation of denervated soleus muscles by a fast muscle nerve, or electrical stimulation by 'fast' impulse pattern, reduced their extrajunctional collagen Q mRNA levels by 70–80%. In contrast, stimulation of fast muscles by 'slow' impulse pattern had no effect on collagen Q expression. Calcineurin inhibitors tacrolimus and cyclosporin A decreased collagen Q mRNA levels in the soleus muscles to about 35%, but did not affect collagen Q expression in denervated soleus muscles or the junctional expression in fast muscles. Therefore, high extrajunctional expression of collagen Q in the soleus muscle is maintained by its tonic nerve-induced activation pattern via the activated Ca²⁺-calcineurin signaling pathway. The extrajunctional collagen Q expression in fast muscles is independent of muscle activation pattern and seems irreversibly suppressed. The junctional expression of collagen Q in fast muscles is partly nerve-dependent, but does not encompass the Ca²⁺-calcineurin signaling pathway.     

Fibre-type specificity and effect of thyroid hormone on glucose 6-phosphate dehydrogenase activity in normal and denervated skeletal muscles of the rat

Summary Glucose-6-phosphate dehydrogenase activity increases following denervation of rat skeletal muscle. The specificity of this effect to muscle fibre type was studied. Basal activity of the dehydrogenase was higher in soleus, a muscle composed predominantly of type I fibres, than in extensor digitorum longus, a muscle composed predominantly of type IIa and b fibres. The enzymatic activity of the soleus was also greater than that of the red (RQ) and white (WQ) portions of quadriceps muscle (predominantly type IIa and type IIb fibres, respectively). Following denervation, glucose-6-phosphate dehydrogenase increased in extensor digitorum longus and RQ, but not in WQ or the soleus. Following chronic treatment of rats with 3,3,5-triiodothyronine, which converts type I muscle fibres to type II, the dehydrogenase activity increased in both denervated soleus and extensor digitorum longus. It is concluded that the effect of denervation on glucose-6-phosphate dehydrogenase activity is selective for type IIa (fast oxidative-glycolytic) muscle fibres.