Lowered extracellular pH is involved in the pathogenesis of skeletal muscle insulin resistance

Insulin resistance in the skeletal muscle is manifested by diminished insulin-stimulated glucose uptake and is a core factor in the pathogenesis of type 2 diabetes mellitus (DM), but the mechanism causing insulin resistance is still unknown. Our recent study has shown that pH of interstitial fluids was lowered in early developmental stage of insulin resistance in OLETF rats, a model of type 2 DM. Therefore, in the present study, we confirmed effects of the extracellular pH on the insulin signaling pathway in a rat skeletal muscle-derived cell line, L6 cell. The phosphorylation level (activation) of the insulin receptor was significantly diminished in low pH media. The phosphorylation level of Akt, which is a downstream target of the insulin signaling pathway, also decreased in low pH media. Moreover, the insulin binding to its receptor was reduced by lowering extracellular pH, while the expression of insulin receptors on the plasma membrane was not affected by the extracellular pH. Finally, insulin-stimulated 2-deoxyglucose uptake in L6 cells was diminished in low pH media. Our present study suggests that lowered extracellular pH conditions may produce the pathogenesis of insulin resistance in skeletal muscle cells.     

Interactions between dihydropyridine receptors and ryanodine receptors in striated muscle

Excitation-contraction coupling in both skeletal and cardiac muscle depends on structural and functional interactions between the voltage-sensing dihydropyridine receptor L-type Ca²⁺ channels in the surface/transverse tubular membrane and ryanodine receptor Ca²⁺ release channels in the sarcoplasmic reticulum membrane. The channels are targeted to either side of a narrow junctional gap that separates the external and internal membrane systems and are arranged so that bi-directional structural and functional coupling can occur between the proteins. There is strong evidence for a physical interaction between the two types of channel protein in skeletal muscle. This evidence is derived from studies of excitation–contraction coupling in intact myocytes and from experiments in isolated systems where fragments of the dihydropyridine receptor can bind to the ryanodine receptors in sarcoplasmic reticulum vesicles or in lipid bilayers and alter channel activity. Although micro-regions that participate in the functional interactions have been identified in each protein, the role of these regions and the molecular nature of the protein–protein interaction remain unknown. The trigger for Ca²⁺ release through ryanodine receptors in cardiac muscle is a Ca²⁺ influx through the L-type Ca²⁺ channel. The Ca²⁺ entering through the surface membrane Ca²⁺ channels flows directly onto underlying ryanodine receptors and activates the channels. This was thought to be a relatively simple system compared with that in skeletal muscle. However, complexities are emerging and evidence has now been obtained for a bi-directional physical coupling between the proteins in cardiac as well as skeletal muscle. The molecular nature of this coupling remains to be elucidated.     

Longitudinal fibre division in skeletal muscle: A light- and electronmicroscopic study

Summary Under conditions of overloading, muscle fibres have been reported to undergo a process of longitudinal division. It has been claimed that this process leads to an increase in cross-sectional area and therefore contributes to the force of contraction. Recent work, however has demonstrated that the division is of limited extent and apparently pathological in origin. Examination of material taken from the immediate vicinity of a crush lesion has shown that a similar picture is reproduced by gross trauma. An electronmicroscopic study of dividing fibres in both overloaded and traumatized muscles has confirmed their similarity and revealed that atrophic changes are present. This evidence is sufficient to suggest that the longitudinal division of fibres seen in overloaded muscles and possibly in dystrophic muscles follows damage to the fibre and that division in this manner may allow the rejection from it of degenerated portions.We wish to thank Professor J. Z. Young, F.R.S. for his advice and encouragement and Mr. A. Aldrich and Mr. D. Gunn for their assistance with the illustrations.     

Protocol for rat single muscle fiber isolation and culture

To attain a superior in vitro model of mature muscle fibers, we modified the established protocol for isolating single muscle fibers from rat skeletal muscle. Muscle fiber cultures with high viability were obtained using flexor digitorum brevis muscle and lasted for at least 7 days. We compared the expression levels of adult myosin heavy chain (MyHC) isoforms in these single muscle fibers with myotubes formed from myoblasts; isolated fibers contained markedly more abundant adult MyHC isoforms than myotubes. This muscle fiber model, therefore, will be useful for studying the various functions and cellular processes of mature muscles in vitro.     

SMHS1 is involved in oxidative/glycolytic-energy metabolism balance of muscle fibers

With the aim of finding important mediators of muscle atrophy, we cloned SMHS1, a novel gene that was found to be upregulated in rat soleus muscle atrophied by restriction of activity. The SMHS1 amino acid sequence shares 65% similarity with RTP801-which is a cellular stress response protein regulated by HIF-1-but SMHS1 expression was demonstrated to be independent of HIF-1. SMHS1 was found to be mainly expressed in skeletal muscle, and comparisons of its expression in atrophied versus hypertrophied muscles and in oxidative versus glycolytic muscles suggested that SMHS1 contributes to the muscle energy metabolism phenotypes.     

Heart and craniofacial muscle development: A new developmental theme of distinct myogenic fields

Head muscle development has been studied less intensively than myogenesis in the trunk, although this situation is gradually changing, as embryological and genetic insights accumulate. This review focuses on novel studies of the origins, composition and evolution of distinct craniofacial muscles. Cellular and molecular parallels are drawn between cardiac and branchiomeric muscle developmental programs, both of which utilize multiple lineages with distinct developmental histories, and argue for the tissues' common evolutionary origin. In addition, there is increasing evidence that the specification of skeletal muscles in the head appears to be distinct from that operating in the trunk: considerable variation among the different craniofacial muscle groups is seen, in a manner resembling myogenic specification in lower organisms.     

Redistribution of cell membrane probes following contraction-induced injury of mouse soleus muscle

Our aim was to study how mouse skeletal muscle membranes are altered by eccentric and isometric contractions. A fluorescent dialkyl carbocyanine dye (DiOC₁₈(3)) was used to label muscle membranes, and the membranes accessible to the dye were observed by confocal laser scanning microscopy. Experiments were done on normal mouse soleus muscles and soleus muscles injured by 20 eccentric or 20 isometric contractions. Longitudinal optical sections of control muscle fibers revealed DiOC₁₈(3) staining of the plasmalemma and regularly spaced transverse bands corresponding in location to the T-tubular system. Transverse optical sections showed an extensive reticular network with the DiOC₁₈(3) staining. Injured muscle fibers showed distinctively different staining patterns in both longitudinal and transverse optical sections. Longitudinal optical sections of the injured fibers revealed staining in a longitudinally-oriented pattern. No correlations were found between the abnormal DiOC₁₈(3) staining and the reductions in maximal isometric tetanic force or release of lactate dehydrogenase (P0.32). Additionally, no difference in the extent of abnormal staining was found between muscles performing eccentric contractions and those performing the less damaging isometric contractions. However, many fibers in muscles injured by eccentric contractions showed swollen regions with marked loss of membrane integrity and an elevated free cytosolic calcium concentration as observed in Fluo-3 images. In conclusion, a loss of cell membrane integrity results from contractile activity, enabling DiOC₁₈(3) staining of internal membranes. The resulting staining pattern is striking and fibers with damaged cell membranes are easily distinguished from uninjured ones.     

Somatic and intramuscular distribution of muscle spindles and their relation to muscular angiotypes

The distribution pattern of muscle spindles in the skeletal musculature has been reviewed in a large number of muscles (using the literature data especially from cat and man), and the relation of spindle content to muscle mass was quantitatively examined in 36 cat and 140 human muscles. In both species, the number of spindles increases with increasing muscle mass in a power law fashion of the form y=bx+a, whereby y denotes the logarithm of spindle content within a muscle, and x is the logarithm of muscle mass. For the cat, slope b and intercept a were estimated as 0.39 and 1.53, and for man as 0.48 and 1.33, respectively. The results show that the spindle content of a muscle may be related to its mass, confirming a similar analysis made previously by Banks and Stacey (Mechano receptors, Plenum Press, New York, 1988, pp. 263-269) in a different data set. With regard to the histological profile of muscle fibers, (as it is already well documented by many groups) muscle spindles tend to be located in deeper muscle regions where oxidative fibers predominate, and are far scarcer in superficial and flat muscle regions where glycolytic fibers predominate. These discrete muscle regions differ also in the properties of the vessel tree supplying them, for which the term oxidative and glycolytic "angiotype" has been used. The results from these three aspects of analysis (relation to muscle mass, relation to muscle regions with high oxidative index and relation to muscle regions with dense vascular supply) were combined with histological findings showing that spindles may be in systematic anatomical contact to intramuscular vessels. Based on these data a hypothesis is proposed according to which, both the number and intramuscular placement of muscle spindles are related to the oxidative angiotype supplying the muscle territories rich in oxidative fibers. The hypothesis is discussed.     

Temperature adaptation and the contractile properties of live muscle fibres from teleost fish

Summary The contractile properties of swimming muscles have been investigated in marine teleosts from Antarctic (Trematomus lepidorhinus, Pseudochaenichthys georgianus), temperate (Pollachius virens, Limanda limanda, Agonis cataphractus, Callionymus lyra), and tropical (Abudefduf abdominalis, Thalassoma duperreyi) latitudes. Small bundles of fast twitch fibres were isolated from anterior myotomes and/or the pectoral fin adductor profundis muscle (m. add. p). Live fibre preparations were viable for several days at in vivo temperatures, but became progressively inexcitable at higher or lower temperatures. The stimulation frequency required to produce fused isometric tetani increased from 50 Hz in Antarctic species at 0°C to around 400 Hz in tropical species at 25°C. Maximum isometric tension (P_o) was produced at the normal body temperature (NBT) of each species (Antarctic, 0–2°C; North Sea and Atlantic, 8–10°C; Indo-West Pacific, 23–25°C). P₀ values at physiological temperatures (200–300 kN·m^–2) were similar for Antarctic, temperate, and tropical species. A temperature induced tension hysteresis was observed in muscle fibres from some species. Exposure to <0°C in Antarctic and <2°C in temperate fish resulted in the temporary depression of tension over the whole experimental range, an effect reversed by incubation at higher temperatures. At normal body temperatures the half-times for activation and relaxation of twitch and tetanic tension increased in the order Antarctic>temperate>tropical species. Relaxation was generally much slower at temperatures <10°C in fibres from tropical than temperate fish. Q₁₀ values for these parameters at NBTs were 1.3 2.1 for tropical species, 1.7–2.6 for temperate species, and 1.6–3.5 for Antarctic species. The forcevelocity (P-V) relationship was studied in selected species using iso-velocity releases and the data below 0.8 P₀ iteratively fitted to Hill's equation. The P-V relation at NBT was found to be significantly less curved in Antarctic than temperate species. The unloaded contraction velocity (V_max) of fibres was positively correlated with NBT increasing from about 1 muscle fibre length·s^–;1 in an Antarctic fish (Trematomus lepidorhinus) at 1°C to around 16 muscle fibre lengths·s^–1 in a tropical species (Thalassoma duperreyi) at 24°C. It is concluded that although muscle contraction in Antarctic fish shows adaptations for low temperature function, the degree of compensation achieved in shortening speed and twitch kinetics is relatively modest.Abbreviations ET environmental temperature - m. add. p major adductor profundis - m. add. s. major adductor superficialis - NBT normal body temperature - P ₀ maximum isometric tension - P-V force velocity - SR sarcoplasmic reticulum - T 1/2 a half activation time - T 1/2 r half relaxation time - V _max unloaded contraction     

Assessment of skeletal muscle damage in successive biopsies from strength-trained and untrained men and women

The effects of repeated biopsy sampling on muscle morphology was qualitatively and quantitatively assessed in strength-trained and untrained men and women. College-age men (13) and women (8) resistance trained twice a week for 8 weeks. A progressive resistance-training program was performed consisting of squats, leg presses, and leg extensions. Nontraining men (7) and women (5) served as controls. Muscle biopsy specimens and fasting bloods were obtained at the beginning and every 2 weeks and histochemical, biochemical, and ultrastructural methods were employed to assess the type and amount of damage. Except for a few scattered atrophic fibers in 2 of the 33 biopsy samples, all initial specimens were normal. In contrast, many of the subsequent biopsy samples from both untrained and resistance-trained men and women contained evidence of damage. Ultrastructural analysis confirmed that degenerative-regenerative processes were occurring in both groups. However, training subjects had a four-fold greater number of damaged fibers than nontraining subjects (8.53% vs 2.08%). In addition, only biopsy samples from training individuals contained fibers with internal disorganization (e.g., Z-line streaming, myofibrillar disruption). Calpain II levels in the biopsy samples and serum creatine kinase activity were not significantly affected supporting the light and electron microscopic observations that most of the damaged fibers were normal in appearance except for their small diameter. In summary, focal damage induced by the biopsy procedure is not completely repaired after 2 weeks and could affect the results, particularly cross-sectional area measurements. Moreover, resistance training appears to cause additional damage to the muscle and may delay repair of the biopsied region.     

Regulation and dysregulation of fibrosis in skeletal muscle

In response to skeletal muscle injury, distinct cellular pathways are activated to repair the damaged tissue. Activation and restriction of these pathways must be temporally coordinated in a precise sequence as regeneration progresses if muscle integrity and homeostasis are to be restored. However, if tissue injury persists, as in severe muscular dystrophies, the repair process becomes uncontrolled leading to the substitution of myofibers by a non-functional mass of fibrotic tissue. In this review, we provide an overview of how muscle responds to damage and aging, with special emphasis on the cellular effectors and the regulatory and inflammatory pathways that can shift normal muscle repair to fibrosis development.     

Alix protein is substrate of Ozz-E3 ligase and modulates actin remodeling in skeletal muscle

Alix/AIP1 is a multifunctional adaptor protein that participates in basic cellular processes, including membrane trafficking and actin cytoskeleton assembly, by binding selectively to a variety of partner proteins. However, the mechanisms regulating Alix turnover, subcellular distribution, and function in muscle cells are unknown. We now report that Alix is expressed in skeletal muscle throughout myogenic differentiation. In myotubes, a specific pool of Alix colocalizes with Ozz, the substrate-binding component of the muscle-specific ubiquitin ligase complex Ozz-E3. We found that interaction of the two endogenous proteins in the differentiated muscle fibers changes Alix conformation and promotes its ubiquitination. This in turn regulates the levels of the protein in specific subcompartments, in particular the one containing the actin polymerization factor cortactin. In Ozz(-/-) myotubes, the levels of filamentous (F)-actin is perturbed, and Alix accumulates in large puncta positive for cortactin. In line with this observation, we show that the knockdown of Alix expression in C2C12 muscle cells affects the amount and distribution of F-actin, which consequently leads to changes in cell morphology, impaired formation of sarcolemmal protrusions, and defective cell motility. These findings suggest that the Ozz-E3 ligase regulates Alix at sites where the actin cytoskeleton undergoes remodeling.     

Expression of nestin,desmin and vimentin in intact and regenerating muscle spindles of rat hind limb skeletal muscles

We describe the expression and distribution patterns of nestin, desmin and vimentin in intact and regenerating muscle spindles of the rat hind limb skeletal muscles. Regeneration was induced by intramuscular isotransplantation of extensor digitorum longus (EDL) or soleus muscles from 15-day-old rats into the EDL muscle of adult female inbred Lewis rats. The host muscles with grafts were excised after 7-, 16-, 21- and 29-day survival and immunohistochemically stained. Nestin expression in intact spindles in host muscles was restricted to Schwann cells of sensory and motor nerves. In transplanted muscles, however, nestin expression was also found in regenerating “spindle fibers”, 7 and 16 days after grafting. From the 21st day onwards, the regenerated spindle fibers were devoid of nestin immunoreactivity. Desmin was detected in spindle fibers at all developmental stages in regenerating as well as in intact spindles. Vimentin was expressed in cells of the outer and inner capsules of all muscle spindles and in newly formed myoblasts and myotubes of regenerating spindles 7 days after grafting. Our results show that the expression pattern of these intermediate filaments in regenerating spindle fibers corresponds to that found in regenerating extrafusal fibers, which supports our earlier suggestion that they resemble small-diameter extrafusal fibers.     

Connexin 39.9 protein is necessary for coordinated activation of slow-twitch muscle and normal behavior in zebrafish

In many tissues and organs, connexin proteins assemble between neighboring cells to form gap junctions. These gap junctions facilitate direct intercellular communication between adjoining cells, allowing for the transmission of both chemical and electrical signals. In rodents, gap junctions are found in differentiating myoblasts and are important for myogenesis. Although gap junctions were once believed to be absent from differentiated skeletal muscle in mammals, recent studies in teleosts revealed that differentiated muscle does express connexins and is electrically coupled, at least at the larval stage. These findings raised questions regarding the functional significance of gap junctions in differentiated muscle. Our analysis of gap junctions in muscle began with the isolation of a zebrafish motor mutant that displayed weak coiling at day 1 of development, a behavior known to be driven by slow-twitch muscle (slow muscle). We identified a missense mutation in the gene encoding Connexin 39.9. In situ hybridization found connexin 39.9 to be expressed by slow muscle. Paired muscle recordings uncovered that wild-type slow muscles are electrically coupled, whereas mutant slow muscles are not. The further examination of cellular activity revealed aberrant, arrhythmic touch-evoked Ca(2+) transients in mutant slow muscle and a reduction in the number of muscle fibers contracting in response to touch in mutants. These results indicate that Connexin 39.9 facilitates the spreading of neuronal inputs, which is irregular during motor development, beyond the muscle cells and that gap junctions play an essential role in the efficient recruitment of slow muscle fibers.     

Glycogen synthase binds to sarcoplasmic reticulum and is phosphorylated by CaMKII in fast-twitch skeletal muscle

We investigated the subcellular localization of glycogen synthase (GS) in the adductor muscle of anesthetized rabbits injected intravenously with propranolol. Under these experimental conditions, glycogen content was about 10 mmol/kg of fresh tissue. Immunofluorescent and fractionation studies showed that GS associated with sarcoplasmic reticulum (SR) membranes. Glycogen and GS always co-sedimented, suggesting a predominant role of glycogen in targeting of GS to SR. SR-associated GS was phosphorylated in vitro by SR-bound Ca2+-calmodulin dependent protein kinase (CaMKII) and dephosphorylated by endogenous protein phosphatase 1 (PP1c). Based on measurements of GS activity ratio, in vitro phosphorylation of GS by CaMKII did not significantly affect GS activity per se. However, GS activity ratio was slightly reduced, when SR membranes were further incubated with ATP after prior phosphorylation by CaMKII, suggesting that CaMKII might act sinergistically with other protein kinases. We propose that SR-bound CaMKII plays a role in regulation of glycogen metabolism in skeletal muscle, when intracellular Ca2+ is raised.     

IP3 receptor blockade restores autophagy and mitochondrial function in skeletal muscle fibers of dystrophic mice

Duchenne muscular dystrophy (DMD) is characterized by a severe and progressive destruction of muscle fibers associated with altered Ca²⁺ homeostasis. We have previously shown that the IP₃ receptor (IP₃R) plays a role in elevating basal cytoplasmic Ca²⁺ and that pharmacological blockade of IP₃R restores muscle function. Moreover, we have shown that the IP₃R pathway negatively regulates autophagy by controlling mitochondrial Ca²⁺ levels. Nevertheless, it remains unclear whether IP₃R is involved in abnormal mitochondrial Ca²⁺ levels, mitochondrial dynamics, or autophagy and mitophagy observed in adult DMD skeletal muscle. Here, we show that the elevated basal autophagy and autophagic flux levels were normalized when IP₃R was downregulated in mdx fibers. Pharmacological blockade of IP₃R in mdx fibers restored both increased mitochondrial Ca²⁺ levels and mitochondrial membrane potential under resting conditions. Interestingly, mdx mitochondria changed from a fission to an elongated state after IP₃R knockdown, and the elevated mitophagy levels in mdx fibers were normalized. To our knowledge, this is the first study associating IP₃R1 activity with changes in autophagy, mitochondrial Ca²⁺ levels, mitochondrial membrane potential, mitochondrial dynamics, and mitophagy in adult mouse skeletal muscle. Moreover, these results suggest that increased IP₃R activity in mdx fibers plays an important role in the pathophysiology of DMD. Overall, these results lead us to propose the use of specific IP₃R blockers as a new pharmacological treatment for DMD, given their ability to restore both autophagy/mitophagy and mitochondrial function.     

Initiation and duration of muscle precursor replication after mild and severe injury to skeletal muscle of mice

Summary We test the proposal (McGeachie and Grounds 1985) that myogenesis following severe (crush) injury is prolonged compared with minor (cut) injury. Forty-four mice were injured with a cut and a crush lesion on different legs, and tritiated thymidine was injected at various times after injury (0 to 120 h), samples of regenerated muscle were taken 9d after injury and autoradiography was used to determine the initiation of muscle precursor replication, and duration of proliferation after the two different injuries.In both lesions replication of potential myoblasts was initiated 30 h after injury. Myogenesis was essentially completed in cut lesions by 96 h after injury, although the peak was finished by 60 h. In contrast, significant muscle precursor replication in crush lesions was still occurring 96 h after injury, and myogenesis was almost finished by 120 h. The pronounced difference in duration of myogenesis in different lesions strongly supports the original proposalThe extended duration of myogenesis in crush lesions, in conjunction with tritiated thymidine reutilisation, appears to account for conflicting experimental results in support of the concept of a circulating muscle precursor cell.