Toll-like receptors differentially regulate CC and CXC chemokines in skeletal muscle via NF-kappaB and calcineurin

Immunologically active molecules such as cytokines and chemokines have been implicated in skeletal muscle weakness during sepsis as well as recovery from muscle injury. In sepsis, Toll-like receptors (TLRs) act as key sentinel molecules of the innate immune system. Here we determined skeletal muscle cell responses of two prototypical CC and CXC chemokine genes (monocyte chemoattractant protein 1 [MCP-1] and KC, respectively), to stimulation with specific TLR ligands. In addition, we examined whether NF-kappaB and calcineurin signaling are involved in these responses. Differentiated myotubes and intact whole muscles expressed TLR2, TLR4, TLR5, and TLR9. Stimulation with ligands for TLR2 (peptidoglycan) or TLR4 (LPS) elicited robust and equivalent levels of MCP-1 and KC mRNA expression, whereas stimulation of TLR5 (by flagellin) required gamma interferon priming to induce similar effects. Although both TLR2 and TLR4 ligands activated the NF-kappaB pathway, NF-kappaB reporter activity was approximately 20-fold greater after TLR4 stimulation than after TLR2 stimulation. Inhibitory effects of NF-kappaB blockade on TLR-mediated chemokine gene expression, by either pharmacological (pyrrolidine dithiocarbamate) or molecular (IKKbeta dominant-negative transfection) methods, were also more pronounced during TLR4 stimulation. In contrast, inhibitory effects on TLR-mediated chemokine expression of calcineurin blockade (by FK506) were greater for TLR2 than for TLR4 stimulation. MCP-1 and KC mRNA levels also demonstrated differential responses to NF-kappaB and calcineurin blockade during stimulation with specific TLR ligands. We conclude that skeletal muscle cells differentially utilize the NF-kappaB and calcineurin pathways in a TLR-specific manner to enable complex regulation of CC and CXC chemokine gene expression.     

Morphofunctional responses to anaemia in rat skeletal muscle

Adult male Sprague-Dawley rats were randomly assigned to two groups: control and anaemic. Anaemia was induced by periodical blood withdrawal. Extensor digitorum longus and soleus muscles were excised under pentobarbital sodium total anaesthesia and processed for transmission electron microscopy, histochemical and biochemical analyses. Mitochondrial volume was determined by transmission electron microscopy in three different regions of each muscle fibre: pericapillary, sarcolemmal and sarcoplasmatic. Muscle samples sections were also stained with histochemical methods (SDH and m-ATPase) to reveal the oxidative capacity and shortening velocity of each muscle fibre. Determinations of fibre and capillary densities and fibre type composition were made from micrographs of different fixed fields selected in the equatorial region of each rat muscle. Determination of metabolites (ATP, inorganic phosphate, creatine, creatine phosphate and lactate) was done using established enzymatic methods and spectrophotometric detection. Significant differences in mitochondrial volumes were found between pericapillary, sarcolemmal and sarcoplasmic regions when data from animal groups were tested independently. Moreover, it was verified that anaemic rats had significantly lower values than control animals in all the sampled regions of both muscles. These changes were associated with a significantly higher proportion of fast fibres in anaemic rat soleus muscles (slow oxidative group = 63.8%; fast glycolytic group = 8.2%; fast oxidative glycolytic group = 27.4%) than in the controls (slow oxidative group = 79.0%; fast glycolytic group = 3.9%; fast oxidative glycolytic group = 17.1%). No significant changes were detected in the extensor digitorum longus muscle. A significant increase was found in metabolite concentration in both the extensor digitorum longus and soleus muscles of the anaemic animals as compared to the control group. In conclusion, hypoxaemic hypoxia causes a reduction in mitochondrial volumes of pericapillary, sarcolemmal, and sarcoplasmic regions. However, a common proportional pattern of the zonal distribution of mitochondria was maintained within the fibres. A significant increment was found in the concentration of some metabolites and in the proportion of fast fibres in the more oxidative soleus muscle in contrast to the predominantly anaerobic extensor digitorum longus.     

Cellular mechanisms and local progenitor activation to regulate skeletal muscle mass

Skeletal muscle hypertrophy is a result of increased load, such as functional and stretch-overload. Activation of satellite cells and proliferation, differentiation and fusion are required for hypertrophy of overloaded skeletal muscles. On the contrary, a dramatic loss of skeletal muscle mass determines atrophy settings. The epigenetic changes involved in gene regulation at DNA and chromatin level are critical for the opposing phenomena, muscle growth and atrophy. Physiological properties of skeletal muscle tissue play a fundamental role in health and disease since it is the most abundant tissue in mammals. In fact, protein synthesis and degradation are finely modulated to maintain an appropriate muscle mass. When the molecular signaling is altered muscle wasting and weakness occurred, and this happened in most common inherited and acquired disorders such as muscular dystrophies, cachexia, and age-related wasting. To date, there is no accepted treatment to improve muscle size and strength, and these conditions pose a considerable anxiety to patients as well as to public health. Several molecules, including Magic-F1, myostatin inhibitor, IGF, glucocorticoids and microRNAs are currently investigated to interfere positively in the blueprint of skeletal muscle growth and regeneration.     

Adaptive responses of human skeletal muscle to vibration exposure.

The aim of this study was to investigate the effects of whole-body vibrations (WBV) on the mechanical behaviour of human skeletal muscle. For this purpose, six female volleyball players at national level were recruited voluntarily. They were tested with maximal dynamic leg press exercise on a slide machine with extra loads of 70, 90, 110 and 130 kg. After the testing, one leg was randomly assigned to the control treatment (C) and the other to the experimental treatment (E) consisting of vibrations. The subjects were then retested at the end of the treatment using the leg press. Results showed remarkable and statistically significant enhancement of the experimental treatment in average velocity (AV), average force (AF) and average power (AP) (P < 0.05-0.005). Consequently, the velocity-force and power-force relationship shifted to the right after the treatment. In conclusion, it was affirmed that the enhancement could be caused by neural factors, as athletes were well accustomed to the leg press exercise and the learning effect was minimized.     

Dystrophin is expressed in mdx skeletal muscle fibers after normal myoblast implantation.

In mdx mice, the dystrophin gene of the X chromosome is defective and, as a result, immunoreactive dystrophin is undetectable in all muscle fibers of all animals of this highly inbred strain. This study showed that implantation of suspensions of clonal cultures of normal human myoblasts into different regions of quadriceps muscles of 6-to-10-day-old mdx mice or 60-day-old mdx mice (whose muscles have been crushed 4 days before implantation) results in the appearance of scattered fiber segments containing microscopically demonstrable immunoreactive dystrophin. In the animals that received the normal myoblast implantation in the prenecrotic stage of the disease (6 to 10 days of age), the dystrophin-positive fiber segments (demonstrated at ages 35, 45, and 60 days) escaped necrosis. This was determined by the absence of the characteristic chains of central nuclei, a reliable marker of prior necrosis in mdx muscle fibers. By heavy labeling of the nuclear DNA of the transplantable human myoblasts with H3-thymidine during culturing, and by sequential performance of an immunocytochemical staining for dystrophin and autoradiography on the same sections, some dystrophin-positive fiber segments were shown to contain radiolabeled myonuclei. It was concluded that nondystrophic myoblasts fused with host muscle fibers to form mosaic muscle fibers in which the normal dystrophin gene of the implanted myoblasts was expressed. This approach may be employed for the mitigation of the deleterious consequences of a gene defect in recessively inherited human muscle diseases such as Duchenne dystrophy.     

Postischemic skeletal muscle injury: patterns of injury in relation to adequacy of reperfusion

Ischemia-induced skeletal muscle injury was studied in rats subjected to tourniquet hindlimb ischemia for 3, 4, or 5 hr. The extent of injury was evaluated morphologically after 5 or 20 hr of reperfusion by a dye exclusion test using Evans blue/albumin (EBA) and a histochemical stain for calcium, Alizarin red S (ARS). In addition, the distribution of Evans blue in the postischemic muscle served as a means for detecting areas of defect reperfusion--no reflow. The combination of EBA and ARS allowed easy and reproducible quantification of induced injury in adequately reperfused areas. In areas of no reflow none of the markers used were useful. Muscle cells in these areas appeared structurally well preserved even 20 hr after release of the tourniquet and no cytoplasmic calcium accumulation could be demonstrated in muscle cells by staining with ARS. In many of these areas, however, ARS-positive structures were found between muscle cells, distributed in a pattern corresponding to the capillary network. The nature of these structures, constituting a morphological correlate to the no-reflow phenomenon, is unclear. It is concluded that for the proper evaluation of postischemic skeletal muscle injury, a suitable marker allowing estimation of the adequacy of the reperfusion must be used.     

Human and rat skeletal muscle adaptations to spinal cord injury.

Results of studies of rodent skeletal muscle plasticity are often extrapolated to humans. However, responses to "disuse" may be species specific, in part because of different inherent properties of anatomically similar muscles. Thus, this study quantified human and rat m. vastus lateralis (VL) fiber adaptations to 11 weeks of spinal cord injury (SCI). The m. VL was taken from 8 young (54 d) male Charles River rats after T-9 laminectomy (n = 4) or sham surgery (n = 4). In addition, the m. VL was biopsied in 7 able-bodied and in 7 SCI humans (31.3 +/- 4.7 years, mean +/- SE). Samples were sectioned and fibers were analyzed for type (I, IIa, IIb/x), cross-sectional area (CSA), succinate dehydrogenase (SDH), alpha-glycerol-phosphate dehydrogenase (GPDH), and actomyosin adenosine triphosphatase (qATPase) activities. Rat fibers had 1.5- to 2-fold greater SDH and GPDH activities while their fibers were 60% the size of those in humans. The most striking differences, however, were the absence of slow fibers in the rat and its four-fold greater proportion of IIb/x fibers (80% vs. 16% of the CSA) compared to humans. SCI decreased SDH activity more in rats whereas atrophy and IIa to IIb/x fiber shift occurred to a greater extent in humans. It is suggested that the rat is a reasonable model for studying the predominant response to SCI, atrophy. However, its high proportion of IIb/x fibers limits evaluation of the mechanical consequences of shifting to "faster" contractile machinery after SCI.     

CC chemokines induce neutrophils to chemotaxis, degranulation, and alpha-defensin release

We have previously shown that a Taiwanese cohort of HIV-uninfected individuals was associated with the significantly elevated levels of serum beta-chemokines, macrophage inflammatory protein (MIP-1)-alpha and MIP-beta, and RANTES. In the present study, we report that the members of this cohort have significantly greater numbers of lower buoyant-density neutrophils in their blood, which leads to further investigation of the effects of beta-chemokines on neutrophils. By electron and confocal microscopic techniques and FACScan, the results demonstrated that MIP-1alpha, MIP-beta, and/or RANTES readily activated the cells to release a large quantity of alpha-defensins in vitro through the degranulation process, which was the cause of low-buoyant-density neutrophil production. The purified neutrophils underwent chemotaxis and increased phagocytic capability when beta-chemokines were present. Only when using all 3 neutralizing antibodies for CCR1, CCR3, and CCR5 could the chemotaxis of neutrophils be inhibited completely, suggesting that these receptors are involved in transducing activating signals. Because neutrophils are the most abundant white blood cells that can be activated simultaneously to release alpha-defensins and because these proteins are antiviral, including anti-HIV, our results support the hypothesis that in addition to beta-chemokines, the innate immunity of the cohort plays a role in inhibiting the transmission of HIV.     

The changing AMPK expression profile in differentiating mouse skeletal muscle myoblast cells helps confer increasing resistance to apoptosis

AMP-activated protein kinase (AMPK) functions as a alpha/beta/gamma heterotrimer to preserve ATP levels and so cell viability during stressful conditions. However, its role in aiding survival of adult skeletal muscle precursor cells is unclear. Using the differentiating mouse C2C12 postnatal skeletal muscle myoblast cell line, we have determined that proteins for the AMPK subunit isoforms alpha2 and gamma2 are constitutively expressed, while those for alpha1, beta1 and beta2 are undetectable in undifferentiated myoblasts but increasingly expressed with differentiation to myotubes. Although the gamma3 subunit is expressed at a low level in myoblasts, it too is expressed increasingly with differentiation to myotubes. The p50 but not the p72 isoform of the embryonic alpha subunit homologue MELK is expressed only in proliferating myoblasts, while the ARK5 alpha subunit homologue is increasingly expressed with differentiation. Myotubes displayed higher basal and stimulated alpha1/alpha2 AMPK activation than myoblasts. Furthermore, serum starvation resulted in less apoptosis of differentiated myotubes than of undifferentiated myoblasts. This reflects, in part, the increased expression of functional AMPK in the myotubes, since specific inhibition of AMPK activity with 6-[4-(2-piperidin-1-ylethoxy)-phenyl]-3-pyridin-4-ylpyrazolo[1,5-alpha] pyrimidine (Compound C) exacerbated the apoptosis resulting from serum withdrawal. If these in vitro events can also occur in vivo, they could have implications for pathologies such as muscle wasting, in which undifferentiated satellite stem cells may be easier apoptotic targets than their differentiated counterparts. Furthermore, these results suggest that when interpreting results from in vitro or in vivo experiments on AMPK, the subunit expression profile should be taken into account.     

Opposing responses of apoptosis and autophagy to moderate compression in skeletal muscle

Aim: The molecular mechanism that contributes to the pathogenesis of deep pressure ulcer remains to be elucidated. This study tested the hypotheses that: (1) apoptosis and autophagy are activated in compression‐induced muscle pathology and (2) apoptotic and autophagic changes precede pathohistological changes in skeletal muscle in response to prolonged moderate compression. Methods: Adult Sprague–Dawley rats were subjected to an experimental model of pressure‐induced deep tissue injury. Static pressure of 100 mmHg was applied to an area of 1.5 cm² over the mid‐tibialis region of right limb of rats for one single session of 6‐h compression (1D) or two sessions of 6‐h compression over two consecutive days with rats sacrificed one day (2D) or immediately after (2D‐IM) the compression. The left uncompressed limb served as the intra‐animal control. Muscle tissues underneath compression region were collected for analysis. Results: Our histological analysis indicated that pathohistological characteristics including rounding contour of myofibres and massive nuclei accumulation were apparently demonstrated in muscles of 2D and 2D‐IM. In contrast, these pathohistological changes were generally not found in muscle following 1D. Apoptotic DNA fragmentation, terminal dUTP nick‐end labelling index and caspase‐3 protease activity were significantly elevated in compressed muscles of all groups. Caspase‐9 enzymatic activity was found to be significantly increased in compressed muscles of 2D and 2D‐IM whereas increase in caspase‐8 activity was exclusively found in compressed muscle of 1D. According to our immunoblot analysis, FoxO3 was significantly reduced in compressed muscles of all groups whereas Beclin‐1 was decreased only in 2D. LC3‐I was significantly reduced in compressed muscles of all groups while LC3‐II was decreased in 2D and 1D. No significant differences were found in the protein abundance of Akt and phospho‐Akt in muscles among all groups. Conclusion: These data demonstrate the opposing responses of apoptosis and autophagy to moderate compression in muscle. Moreover, our findings suggest that cellular changes in apoptosis and autophagy have already taken place in the very early stage in which apparent histopathology has yet to develop in the process of compression‐induced muscle pathology.     

Force responses to fast ramp stretches in stimulated frog skeletal muscle fibres

Force responses to fast ramp stretches at various velocities were recorded from single muscle fibres isolated from either lumbricalis digiti IV or tibialis anterior muscle of the frog (Rana esculenta) at sarcomere length between 2.15 and 3.25 μm at 15° C. Stretches were applied at rest, at tetanus plateau and during the tetanus rise. Stretches with the same velocity but different accelerations were imposed to the fibre to evaluate the effect of fibre inertia on the force responses. Length changes were measured at sarcomere level with either a laser diffractometer or a striation follower apparatus. The force response to a fast ramp stretch could be divided into two phases. The initial fast one (phase 1) lasts for the acceleration period during which the stretching velocity rises up to the steady state. The second slower phase (phase 2) lasts for the remainder of the stretch and corresponds to the well-known elastic response of the fibre. Most of this paper is concerned with phase 1. The amplitude of the initial fast phase was proportional to the stretching velocity as expected from a viscous response. This viscosity was associated with a very short (about 10 μs) relaxation time. The amplitude of the fast phase increased progressively with tension during the tetanus rise and scaled down with sarcomere length approximately in the same way as tetanic tension and fibre stiffness. These data suggest that activated fibres have a significant internal viscosity which may arise from crossbridge interactionThis revised version was published online in July 2005 with corrections to the Cover Date.     

The myogenic factor Myf5 supports efficient skeletal muscle regeneration by enabling transient myoblast amplification

The myogenic factor Myf5 defines the onset of myogenesis in mammals during development. Mice lacking both Myf5 and MyoD fail to form myoblasts and are characterized by a complete absence of skeletal muscle at birth. To investigate the function of Myf5 in adult skeletal muscle, we generated Myf5 and mdx compound mutants, which are characterized by constant regeneration. Double mutant mice show an increase of dystrophic changes in the musculature, although these mice were viable and the degree of myopathy was modest. Myf5 mutant muscles show a small decrease in the number of muscle satellite cells, which was within the range of physiological variations. We also observed a significant delay in the regeneration of Myf5 deficient skeletal muscles after injury. Interestingly, Myf5 deficient skeletal muscles were able to even out this flaw during the course of regeneration, generating intact muscles 4 weeks after injury. Although we did not detect a striking reduction of MyoD positive activated myoblasts or of Myf5-LacZ positive cells in regenerating muscles, a clear decrease in the proliferation rate of satellite cell-derived myoblasts was apparent in satellite cell-derived cultures. The reduction of the proliferation rate of Myf5 mutant myoblasts was also reflected by a delayed transition from proliferation to differentiation, resulting in a reduced number of myotube nuclei after 6 and 7 days of culture. We reason that Myf5 supports efficient skeletal muscle regeneration by enabling transient myoblast amplification. Disclosure of potential conflicts of interest is found at the end of this article.     

Decoy receptors: a strategy to regulate inflammatory cytokines and chemokines

The canonical concept of a receptor includes specific ligand recognition, usually with high affinity and specificity, and signaling. Decoy receptors recognize certain inflammatory cytokines with high affinity and specificity, but are structurally incapable of signaling or presenting the agonist to signaling receptor complexes. They act as a molecular trap for the agonist and for signaling receptor components. The interleukin-1 type II receptor (IL-1RII) was the first pure decoy to be identified. Decoy receptors have subsequently been identified for members of the tumor necrosis factor receptor and IL-1R families. Moreover, silent nonsignaling receptors could act as decoys for chemokines. Therefore, the use of decoy receptors is a general strategy to regulate the action of primary pro-inflammatory cytokines and chemokines.     

Early responses of skeletal muscle in recovery from hypothyroidism

Postnatal growth of skeletal muscle (m. gastrocnemius) was compared in rats under euthyroid, hypothyroid and hypothyroid-rehabilitated conditions. In normal (euthyroid) animals, gastrocnemius muscle grows significantly in terms of weight (150 x) from birth to the young adult and, in terms of total contractile myofibril protein (15 x) and myosin ATPase activity (10 x) between days 25 and 90. Rats made hypothyroid (with 0.1% w/v propylthiouracil, PTU) from birth show reduced growth. At 25 days (weaning), compared with euthyroid, muscle weight is only 25% of normal, and a similar reduction is found in total DNA, RNA, protein, myofibril protein, and myosin ATPase activity. These deficits, already significant by day 10, are more marked by day 50 due to the near arrest of growth. Hypothyroid rats allowed to recover by PTU withdrawal after day 25 (rehabilitated) undergo marked compensatory muscle growth. By day 90, muscle weight and protein content increase 50 x, DNA 7 x and RNA 17 x. Over this period, total myofibrillar protein and myosin ATPase increase 20-40 x, but are still below those of 90-day controls, suggesting that the severe growth retardation had not yet been fully compensated. Early thyroid deficiency drastically reduces the normal age-related growth of skeletal muscle and severely retards the development of contractile elements, affecting muscle hypertrophy (protein content) more than cell proliferation (DNA content). Rehabilitation compensates to a major degree for this growth retardation. These results underline the key role of thyroid hormones in regulating development and maturation of skeletal muscle throughout the preweaning and postweaning phases of growth.     

CTGF/CCN-2 over-expression can directly induce features of skeletal muscle dystrophy

Muscular dystrophies are diseases characterized by muscle weakness together with cycles of degeneration and regeneration of muscle fibres, resulting in a progressive decrease of muscle mass, diminished muscle force generation and an increase in fibrosis. Fibrotic disorders are the endpoint of many chronic diseases in different tissues, where accumulation of the extracellular matrix (ECM) occurs. Connective tissue growth factor CTGF/CCN2, which is over-expressed in muscular dystrophies, plays a major role in many progressive scarring conditions. To test the hypothesis that CTGF might not only contribute conversion of already damaged muscle into scar tissue, but that it could by itself also directly contribute to skeletal muscle deterioration, we evaluated the effect of CTGF over-expression in tibialis anterior muscle of wild-type mice, using an adenovirus containing the CTGF mouse sequence (Ad-mCTGF). CTGF over-expression induced extensive skeletal muscle damage, which was followed by a massive regeneration of the damaged muscle, as evidenced by increased embryonic myosin and fibres with centrally located nuclei. It also induced strong fibrosis with increased levels of fibronectin, collagen, decorin and α-smooth muscle actin (α-SMA). Moreover, CTGF over-expression caused a decrease of the specific isometric contractile force. Strikingly, when CTGF over-expression stopped, the entire phenotype proved to be reversible, in parallel with normalization of CTGF levels. Thus, CTGF not merely acts downstream of muscle injury but also contributes directly to the deterioration of skeletal muscle phenotype and function. Moreover, normalization of expression levels led to spontaneous reversal of the CTGF-induced phenotype and to full recovery of muscle structure. These observations underscore the importance of CTGF in the pathophysiology of muscular dystrophies and suggest that targeting CTGF might have significant potential in the development of novel therapies for Duchenne muscular dystrophy and related diseases.