Chronic contractile activity upregulates the proteasome system in rabbit skeletal muscle.

Remodeling of skeletal muscle in response to altered patterns of contractile activity is achieved, in part, by the regulated degradation of cellular proteins. The ubiquitin-proteasome system is a dominant pathway for protein degradation in eukaryotic cells. To test the role of this pathway in contraction-induced remodeling of skeletal muscle, we used a well-established model of continuous motor nerve stimulation to activate tibialis anterior (TA) muscles of New Zealand White rabbits for periods up to 28 days. Western blot analysis revealed marked and coordinated increases in protein levels of the 20S proteasome and two of its regulatory proteins, PA700 and PA28. mRNA of a representative proteasome subunit also increased coordinately in contracting muscles. Chronic contractile activity of TA also increased total proteasome activity in extracts, as measured by the hydrolysis of a proteasome-specific peptide substrate, and the total capacity of the ubiquitin-proteasome pathway, as measured by the ATP-dependent hydrolysis of an exogenous protein substrate. These results support the potential role of the ubiquitin-proteasome pathway of protein degradation in the contraction-induced remodeling of skeletal muscle.     

Comparison of protein expression in human deltoideus and vastus lateralis muscles using two-dimensional gel electrophoresis

We have used two-dimensional gel electrophoresis (2-DE) and mass spectrometry (MS) to study the expression of contractile and regulatory proteins in human vastus lateralis and deltoideus muscles, in order to understand protein turnover and isoform switching in muscles with the same fiber-type composition but different functional properties. We demonstrate a two- to six-fold overexpression of enzymes associated with glycolysis, the tricarboxylic acid cycle, oxidative phosphorylation, and substrate transport in vastus lateralis compared to deltoideus. Expression levels of contractile protein isoforms correlated to the proportion of slow-twitch fibers in deltoideus compared to vastus lateralis are consistent with the different contractile properties of the two muscles. Two proteins involved in free radical homeostasis were differentially expressed, suggesting a direct relationship between radical scavenging and the muscle function. The application of 2-DE and MS to studies of muscle physiology thus offers a more comprehensive assessment of the molecular determinants of muscle function than traditional approaches.     

The effect of glucocorticoids on the myosin heavy chain isoforms' turnover in skeletal muscle

The purpose of this study was to find the effect of dexamethasone on the myosin heavy chain (MyHC) isoforms' composition in different skeletal muscles and glycolytic (G) fibres in relation with their synthesis rate and degradation of MyHC isoforms by alkaline proteinases. Eighteen-week-old male rats of the Wistar strain were treated with dexamethasone (100 microg/100 g bwt) during 10 days. The forelimb strength decreased from 9.52 to 6.19 N (P<0.001) and hindlimb strength from 15.54 to 8.55 N (P<0.001). Daily motor activity decreased (total activity from 933 to 559 and ambulatory activity from 482 to 226 movements/h, P<0.001). The degradation rate of muscle contractile proteins increased from 2.0 to 5.9% per day (P<0.001), as well as the myosin heavy chain IIB isoform degradation with alkaline proteinase in fast-twitch (F-T) muscles (12 +/- 0.9%; P<0.05) and glycolytic muscle fibres (15 +/- 1.1%; P<0.001). The synthesis rate of MyHC type II isoforms decreased in Pla muscles (P<0.05) and MyHC IIA (P<0.05) and IIB in EDL muscle and G fibres (P<0.001). The relative content of MyHC IIB isoform decreased in F-T muscles (P<0.001) and in G fibres (P<0.01), and the relative content of IIA and IID isoforms increased simultaneously. Dexamethasone decreased the MyHC IIB isoform synthesis rate and increased the sensibility of MyHC IIB isoform to alkaline proteinase, which in its turn led to the decrease of MyHC IIB isoform relative content in F-T muscles with low oxidative potential and G muscle fibres.     

Characterization of contractile function and expression of muscarinic receptors,G proteins and adenylate cyclase in cultured tracheal smooth muscle of Swine

Smooth muscle cells lose their contractile function and phenotype very rapidly when placed in culture. During organ culture of smooth muscle strips, phenotype is lost more slowly. In the present studies, we established an organ culture model to study contractile function and expression of muscarinic receptors, G proteins and adenylyl cyclase in different serum concentrations in tracheal smooth muscle from swine. The results show that contractile function and the amounts of M(3) receptors, G proteins and adenylyl cyclase were maintained for up to 5 days in culture. The expression of M(2) receptors was significantly decreased in culture when compared to freshly isolated muscles. Maximal isometric tension was significantly increased in cultured muscles compared with freshly isolated muscles. Different serum concentrations did not significantly affect contractile function and expression of muscarinic receptors, G proteins and adenylyl cyclase. In conclusion, our studies suggest that cultured smooth muscle might be used as a model to study the regulation of contractile function of smooth muscle by various signal transduction pathways.     

Proteome dynamics during contractile and metabolic differentiation of bovine foetal muscle

Contractile and metabolic properties of bovine muscles play an important role in meat sensorial quality, particularly tenderness. Earlier studies based on Myosin heavy chain isoforms analyses and measurements of glycolytic and oxidative enzyme activities have demonstrated that the third trimester of foetal life in bovine is characterized by contractile and metabolic differentiation. In order to complete this data and to obtain a precise view of this phase and its regulation, we performed a proteomic analysis of Semitendinosus muscle from Charolais foetuses analysed at three stages of the third trimester of gestation (180, 210 and 260 days). The results complete the knowledge of important changes in the profiles of proteins from metabolic and contractile pathways. They provide new insights about proteins such as Aldehyde dehydrogenase family, Enolase, Dihydrolipoyl dehydrogenase, Troponin T or Myosin light chains isoforms. These data have agronomical applications not only for the management of beef sensorial quality but also in medical context, as bovine myogenesis appears very similar to human one.     

Fiber type-specific distribution of M-band proteins in chicken muscle

The functions of two myofibrillar proteins, myomesin (Mr 185,000) and M-protein (Mr 165,000), associated with the M-band are as yet unknown. To extend our knowledge of these proteins, we have examined chicken striated muscles with fast and slow contractile properties, e.g., pectoralis major, PLD, ALD, medial adductor, and lateral adductor, to determine the expression and isoform composition of myomesin and M-protein in various muscles and fiber types. The high molecular weight M-band proteins were characterized and quantitated using monoclonal antibodies in immunoblotting and double-antibody sandwich ELISA. Fiber specificity was determined by immuno- and enzyme histochemistry. In addition to the previously reported Mr 195,000 and 190,000 isoforms of myomesin in heart [Grove et al. (1985): J Cell Biol 101:1431], the Mr 185,000 myomesin in skeletal muscles may represent different isoforms in fast and slow muscles on the basis of distinctive degradation patterns. M-protein has the same molecular weight in striated chicken muscles and degradation patterns indicate only one isoform. The low quantities of M-protein in slow muscles were shown to be due to the absence of M-protein in two of the generally recognized slow fiber types, types I and III. Thus, M-protein was present only in fast type II fibers, whereas myomesin was ubiquitous in all fiber types. Whatever the causal relationship, M-protein appears to function in fast motor units composed of type II fibers.     

PKB signaling and atrogene expression in skeletal muscle of aged mice

The purpose of this study was to determine if PKB signaling is decreased and contractile protein degradation is increased in extensor digitorum longus (EDL) and soleus (SOL) muscles from middle-aged (MA) and aged (AG) mice. We also examined the effect of age on atrogene expression in quadriceps muscle. PKB activity, as assessed by Thr(308) and Ser(473) phosphorylation, was significantly higher in EDL and SOL muscles from AG than MA mice. The age-related increase in PKB activity appears to be due to an increase in expression of the kinase, as PKB-α and PKB-β levels were significantly higher in EDL and SOL muscles from AG than MA mice. The phosphorylation of forkhead box 3a (FOXO3a) on Thr(32), a PKB target, was significantly higher in EDL muscles from AG than MA mice. The rate of contractile protein degradation was similar in EDL and SOL muscles from AG and MA mice. Atrogin-1 and muscle-specific RING finger protein 1 (MuRF-1) mRNA levels did not change in muscles from AG compared with MA mice, indicating that ubiquitin-proteasome proteolysis does not contribute to sarcopenia. A significant decrease in Bcl-2 and 19-kDa interacting protein 3 (Bnip3) and GABA receptor-associated protein 1 (Gabarap1) mRNA was observed in muscles from AG compared with MA mice, which may contribute to age-related contractile dysfunction. In conclusion, the mechanisms responsible for sarcopenia are distinct from experimental models of atrophy and do not involve atrogin-1 and MuRF-1 or enhanced proteolysis. Finally, a decline in autophagy-related gene expression may provide a novel mechanism for impaired contractile function and muscle metabolism with advancing age.     

Proteomic analysis of slow- and fast-twitch skeletal muscles

Skeletal muscles are composed of slow- and fast-twitch muscle fibers, which have high potential in aerobic and anaerobic ATP production, respectively. To investigate the molecular basis of the difference in their functions, we examined protein profiles of skeletal muscles using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and two-dimensional gel electrophoresis with pH 4-7 and 6-11 isoelectric focusing gels. A comparison between rat soleus and extensol digitorum longus (EDL) muscles that are predominantly slow- and fast-twitch fibers, respectively, showed that the EDL muscle had higher levels of glycogen phosphorylase, most glycolytic enzymes, glycerol 3-phosphate dehydrogenase, and creatine kinase; while the soleus muscle had higher levels of myoglobin, TCA cycle enzymes, electron transfer flavoprotein, and carbonic anhydrase III. The two muscles also expressed different isoforms of contractile proteins including myosin heavy and light chains. These protein patterns were further compared with those of red and white gastrochnemius as well as red and white quadriceps muscles. It was found that metabolic enzymes showed a concerted regulation dependent on muscle fiber types. On the other hand, expression of contractile proteins seemed to be independent of the metabolic characteristics of muscle fibers. These results suggest that metabolic enzymes and contractile proteins show different expression patterns in skeletal muscles.     

Control of adaptations in protein levels in response to exercise

The nature of the contractile stimuli to which a skeletal muscle is subjected determines which proteins will increase in skeletal muscle. Rates of muscle protein synthesis decrease during an exercise bout for durations of less than 30 min. Synthesis has been reported to increase, remain unchanged, or decrease during exercise bouts lasting from 30 min to 7 h. Protein synthesis rates apparently increase when exercise exceeds 7 h. After short bouts of exercise, protein synthesis rates in muscles appear to decrease in the first hour after exercise, but in the second hour after exercise increase to levels greater than normal. We hypothesize that decreases in ATP and pH levels in muscle during contractile activity may dampen a calcium-mediated stimulation of translation of RNA. That the content of alpha-actin mRNA in muscles of immobilized limbs is unchanged when actin synthesis initially decreases suggests that a decrease in the translation of alpha-actin mRNA is the facilitating step in the decrease in actin synthesis. Rates of muscle protein degradation decrease during exercise if exercise duration is less than 12 h, but increase when exercise is continuous for a day. After intense exercise, rates of protein degradation in skeletal muscle may be increased. An increased ratio of NAD(P)H:NAD(P) in muscle during short-term exercise may decrease degradation. Increased lysosomal enzyme activity in muscle occurs during the postexercise period.     

Evolution of skeletal type e-c coupling: a novel means of controlling calcium delivery

The functional separation between skeletal and cardiac muscles, which occurs at the threshold between vertebrates and invertebrates, involves the evolution of separate contractile and control proteins for the two types of striated muscles, as well as separate mechanisms of contractile activation. The functional link between electrical excitation of the surface membrane and activation of the contractile material (known as excitation-contraction [e-c] coupling) requires the interaction between a voltage sensor in the surface membrane, the dihydropyridine receptor (DHPR), and a calcium release channel in the sarcoplasmic reticulum, the ryanodine receptor (RyR). Skeletal and cardiac muscles have different isoforms of the two proteins and present two structurally and functionally distinct modes of interaction.We use structural clues to trace the evolution of the dichotomy from a single, generic type of e-c coupling to a diversified system involving a novel mechanism for skeletal muscle activation. Our results show that a significant structural transition marks the protochordate to the Craniate evolutionary step, with the appearance of skeletal muscle-specific RyR and DHPR isoforms.     

Metabolic and contractile differentiation of rabbit muscles during growth

• 1.1. A study was carried out of post-natal evolution of the oxidative, glycolytic and contractile capacities in various types of rabbit muscle.
• 2.2. At birth, muscles are non-differentiated and present very limited metabolic and contractile activity, metabolism is mainly oxidative in all muscles.
• 3.3. Although muscular discrimination is manifest from the sixth week after birth, the glycolytic metabolism reaches its maximum capacity only after six to eight weeks.
• 4.4. Subsequently, oxidative metabolic capacity steadily decreases until adulthood.

     

Developmental regulation of the aldolase A muscle-specific promoter during in vivo muscle maturation is controlled by a nuclear receptor binding element.

During the post-natal period, skeletal muscles undergo important modifications leading to the appearance of different types of myofibers which exhibit distinct contractile and metabolic properties. This maturation process results from the activation of the expression of different sets of contractile proteins and metabolic enzymes, which are specific to the different types of myofibers. The muscle-specific promoter of the aldolase A gene (pM) is expressed mainly in fast-twitch glycolytic fibers in adult body muscles. We investigate here how pM is regulated during the post-natal development of different types of skeletal muscles (slow or fast-twitch muscles, head or body muscles). We show that pM is expressed preferentially in prospective fast-twitch muscles soon after birth; pM is up-regulated specifically in body muscles only later in development. This activation pattern is mimicked by a transgene which comprises only the 355 most proximal sequences of pM. Within this region, we identify a DNA element which is required for the up-regulation of the transgene during post-natal development in body muscles. Comparison of nuclear M1-binding proteins from young or adult body muscles show no qualitative differences. Distinct M1-binding proteins are present in both young and adult tongue nuclear extracts, compared to that present in gastrocnemius extracts.     

Differential expression of sarcoplasmic proteins in four heterogeneous ovine skeletal muscles

Fiber-type distribution is known to vary widely within and between muscles according to differences in muscle functions. 2-DE and MALDI-MS were used to investigate the molecular basis of muscle fiber type-related variability. We compared four lamb skeletal muscles with heterogeneous fiber-type composition that are relatively rich in fast-twitch fiber types, i.e., the semimembranosus, vastus medialis, longissimus dorsi, and tensor fasciae latae (TL). Our results clearly showed that none of the glycolytic metabolism enzymes detected, including TL which was most strongly glycolytic, made intermuscular differentiation possible. Muscle differentiation was based on the differential expression of proteins involved in oxidative metabolism, including not only citric acid cycle enzymes but also other classes of proteins with functions related to oxidative metabolism, oxidative stress, and probably to higher protein turnover. Detected proteins were involved in transport (carbonate dehydratase, myoglobin, fatty acid-binding protein), repair of misfolding damage (heat shock protein (HSP) 60 kDa, HSP-27 kDa, alpha-crystallin beta subunit, DJ1, stress-induced phosphoprotein), detoxification or degradation of impaired proteins (GST-Pi, aldehyde dehydrogenase, peroxiredoxin, ubiquitin), and protein synthesis (tRNA-synthetase). The fractionating method led to the detection of proteins involved in different functions related to oxidative metabolism that have not previously been shown concomitancy.     

Making muscle elastic: the structural basis of myomesin stretching

Skeletal and cardiac muscles are remarkable biological machines that support and move our bodies and power the rhythmic work of our lungs and hearts. As well as producing active contractile force, muscles are also passively elastic, which is essential to their performance. The origins of both active contractile and passive elastic forces can be traced to the individual proteins that make up the highly ordered structure of muscle. In this Primer, we describe the organization of sarcomeres--the structural units that produce contraction--and the nature of the proteins that make muscle elastic. In particular, we focus on an elastic protein called myomesin, whose novel modular architecture helps explain elasticity.     

Signal transduction and protein phosphorylation in smooth muscle contraction

Smooth muscles are important constituents of vertebrate organisms that provide for contractile activity of internal organs and blood vessels. Basic molecular mechanism of both smooth and striated muscle contractility is the force-producing ATP-dependent interaction of the major contractile proteins, actin and myosin II molecular motor, activated upon elevation of the free intracellular Ca²⁺ concentration ([Ca²⁺]_i). However, whereas striated muscles display a proportionality of generated force to the [Ca²⁺]_i level, smooth muscles feature molecular mechanisms that modulate sensitivity of contractile machinery to [Ca²⁺]_i. Phosphorylation of proteins that regulate functional activity of actomyosin plays an essential role in these modulatory mechanisms. This provides an ability for smooth muscle to contract and maintain tension within a broad range of [Ca²⁺]_i and with a low energy cost, unavailable to a striated muscle. Detailed exploration of these mechanisms is required to understand the molecular organization and functioning of vertebrate contractile systems and for development of novel advances for treating cardiovascular and many other disorders. This review summarizes the currently known and hypothetical mechanisms involved in regulation of smooth muscle Ca²⁺-sensitivity with a special reference to phosphorylation of regulatory proteins of the contractile machinery as a means to modulate their activity.     

Concentration of macroergic phosphates and oxidative potential of skeletal muscles

It is known that a long-duration decline of high-energy phosphate (HP) level in skeletal muscles, induced by administration of beta-guanidinpropionic acid (beta-GPA), is followed by an increase in mitochondrial enzyme activities (MEA). The same increase in MEA was observed in the course of physical exercise training. Under gravitational inloading decrease in MEA and increase in the level of high-energy phosphates occurred. If changes in (HP) level are believed to trigger the alterations in MEA, the increase in high-energy phosphate levels in muscles is to lead to a decline in MEA as well. The present work was purposed to reveal if changes in HP level under different contractile activity levels may be associated with changes in oxidative potential in the skeletal muscles.     

Atrophy of the soleus muscle by hindlimb unweighting

The unweighting model is a unique whole animal model that will permit the future delineation of the mechanism(s) by which gravity maintains contractile mass in postural (slow-twitch) skeletal muscle. Since the origination of the model of rodent hindlimb unweighting almost one decade ago, about half of the 59 refereed articles in which this model was utilized have been published in the Journal of Applied Physiology. Thus the purpose of this review is to provide, for those researchers with an interest in the hindlimb unweighting model, a summation of the data derived from this model to data and hopefully to stimulate research interest in aspects of the model for which data are lacking. The stress response of the animal to hindlimb unweighting is transient, minimal in magnitude, and somewhat variable. After 1 wk of unweighting, the animal exhibits no chronic signs of stress. The atrophy of the soleus muscle, a predominantly slow-twitch muscle, is emphasized because unweighting preferentially affects it compared with other calf muscles, which are mainly fast-twitch muscles. The review considers the following information about the unweighted soleus muscle: electromyogram activity, amount and type of protein lost, capillarization, oxidative capacity, glycolytic enzyme activities, fiber cross section, contractile properties, glucose uptake, sensitivity to insulin, protein synthesis and degradation rates, glucocorticoid receptor numbers, responses of specific mRNAs, and changes in metabolite concentrations.     

Effect of pyruvate, octanoate and glucose on leucine degradation in skeletal muscle from fed and fasted chicks

1. Pyruvate at 5 mM decreased the rate of leucine oxidative decarboxylation and increased the rate of 2-oxoisocaproate production in extensor digitorum communis (EDC) muscles from both fed and 24-hr fasted chicks. Pyruvate at 5 mM increased the net rate of leucine transamination in EDC muscle from fed chicks and had no effect in EDC muscles from 24-hr fasted chicks. 2. Octanoate at 0.2 and 1 mM markedly increased the rates of net leucine transamination, leucine oxidative decarboxylation and oxidation of decarboxylated leucine carbons 2-6 in EDC muscles from fed chicks, but had no effect on these parameters of leucine degradation in muscles from 24-hr fasted chicks. 3. Glucose at 5 and 12 mM decreased the rates of leucine oxidative decarboxylation and oxidation of decarboxylated leucine carbons 2-6, and increased the net rate of 2-oxoisocaproate production as compared to control (no glucose) group in muscles from fed chicks. Glucose had no effect on these parameters of leucine degradation in muscles from 24-hr fasted chicks.     

Myosin heavy chain and parvalbumin expression in swimming and feeding muscles of centrarchid fishes: the molecular basis of the scaling of contractile properties

In centrarchid fishes, such as bluegill (Lepomis macrochirus, Rafinesque) and largemouth bass (Micropterus salmoides, Lacepède), the contractile properties of feeding and swimming muscles show different scaling patterns. While the maximum shortening velocity (V(max)) and rate of relaxation from tetanus of swimming or myotomal muscle slow with growth, the feeding muscle shows distinctive scaling patterns. Cranial epaxial muscle, which is used to elevate the head during feeding strikes, retains fast contractile properties across a range of fish sizes in both species. In bass, the sternohyoideous muscle, which depresses the floor of the mouth during feeding strikes, shows faster contractile properties with growth. The objective of this study was to determine the molecular basis of these different scaling patterns. We examined the expression of two muscle proteins, myosin heavy chain (MyHC) and parvalbumin (PV), that affect contractile properties. We hypothesized that the relative contribution of slow and fast MyHC isoforms will modulate V(max) in these fishes, while the presence of PV in muscle will enhance rates of muscle relaxation. Myotomal muscle displays an increase in sMyHC expression with growth, in agreement with its physiological properties. Feeding muscles such as epaxial and sternohyoideus show no change or a decrease in sMyHC expression with growth, again as predicted from contractile properties. PV expression in myotomal muscle decreases with growth in both species, as has been seen in other fishes. The feeding muscles again show no change or an increase in PV expression with growth, contributing to faster contractile properties in these fishes. Both MyHC and PV appear to play important roles in modulating muscle contractile properties of swimming and feeding muscles in centrarchid fishes.     

Effect of chelators of bivalent metal ions on the reaction of myometrium actomoysin superprecipitation]

Kinetic regularities of the reaction of superprecipitation of myometrium actomyosin, as well as the effect of different concentrations of EGTA, EDTA and diphosphonic acids on this process have been studied. Results obtained are of interest from the viewpoint of possible practical use of diphosphonates as factors modifying interaction of the contractile proteins of the uterus smooth muscles under the pathology of contractile response.