Effect of neonatal denervation on the distribution of fiber types in a mouse fast-twitch skeletal muscle

This study was designed to assess the changes in fiber-type distribution of the extensor digitorum longus (EDL) muscle of the mouse during the first 21 days of age following neonatal sciatic neurectomy. Denervated and normal muscles were compared at 7, 14, and 21 days of age and the normal EDL was also studied at 1 day of age. Frozen sections of the EDL were treated histochemically to detect NADH-tetrazolium reductase and myosin ATPase reactions. Quantitative assessment included measurements of cross-sectional areas and fiber counting. Denervation resulted in muscle atrophy which was due primarily to a decrease in individual fiber area as opposed to fiber loss. Histochemical maturation of the EDL was severely affected by neonatal denervation during the first three postnatal weeks. By 21 days, two extrafusal fiber types which were both oxidative could be distinguished. One type was highly atrophied and resembled an immature fiber exhibiting myosin ATPase staining at both acid and alkaline preincubation conditions, whereas another type was less atrophied and showed myosin ATPase staining resembling fast-twitch (type IIA) fibers. These findings emphasize the importance of an intact nerve supply in determining the phenotypic expression of skeletal muscle, and point to the early postnatal period as a critical stage in fiber type differentiation.     

Effect of denervation on the expression of glycogen phosphorylase in mouse skeletal muscle.

After sciatectomy of the left hind-limb of C57BL/J mice, a denervation-induced muscular atrophy ensued and was accompanied by a decrease in the specific activity of glycogen phosphorylase to approx. 25% of control values. The cofactor of phosphorylase, pyridoxal 5'-phosphate, was used as a specific label in the determination of the degradation rate of the enzyme following nerve section. After a delay of 3-4 days, phosphorylase was degraded approx, twice as rapidly in the denervated gastrocnemius (0.20 day-1) as in the control muscle (0.12 day-1). The effect of denervation on phosphorylase mRNA was measured by quantitative Northern-blot analysis using a rat skeletal-muscle phosphorylase cDNA probe. After an initial rapid decline, phosphorylase mRNA levels stabilized in denervated muscle at 50% of the value measured in the contralateral control muscle.     

Elementary steps of the cross-bridge cycle in fast-twitch fiber types from rabbit skeletal muscles

To understand the molecular mechanism underlying the diversity of mammalian skeletal muscle fibers, the elementary steps of the cross-bridge cycle were investigated in three fast-twitch fiber types from rabbit limb muscles. Skinned fibers were maximally Ca(2+)-activated at 20 degrees C and the effects of MgATP, phosphate (P, P(i)), and MgADP were studied on three exponential processes by sinusoidal analysis. The fiber types (IIA, IID, and IIB) were determined by analyzing the myosin heavy-chain isoforms after mechanical experiments using high-resolution SDS-PAGE. The results were consistent with the following cross-bridge scheme: where A is actin, M is myosin, D is MgADP, and S is MgATP. All states except for those in brackets are strongly bound states. All rate constants of elementary steps (k(2), 198-526 s(-1); k(-2), 51-328 s(-1); k(4), 13.6-143 s(-1); k(-4), 13.6-81 s(-1)) were progressively larger in the order of type IIA, type IID, and type IIB fibers. The rate constants of a transition from a weakly bound state to a strongly bound state (k(-2), k(4)) varied more among fiber types than their reversals (k(2), k(-4)). The equilibrium constants K(1) (MgATP affinity) and K(2) (=k(2)/k(-2), ATP isomerization) were progressively less in the order IIA, IID, and IIB. K(4) (=k(4)/k(-4), force generation) and K(5) (P(i) affinity) were larger in IIB than IIA and IID fibers. K(1) showed the largest variation indicating that the myosin head binds MgATP more tightly in the order IIA (8.7 mM(-1)), IID (4.9 mM(-1)), and IIB (0.84 mM(-1)). Similarly, the MgADP affinity (K(0)) was larger in type IID fibers than in type IIB fibers.     

Calcium currents in a fast-twitch skeletal muscle of the rat

Slow ionic currents were measured in the rat omohyoid muscle with the three-microelectrode voltage-clamp technique. Sodium and delayed rectifier potassium currents were blocked pharmacologically. Under these conditions, depolarizing test pulses elicited an early outward current, followed by a transient slow inward current, followed in turn by a late outward current. The early outward current appeared to be a residual delayed rectifier current. The slow inward current was identified as a calcium current on the basis that (a) its magnitude depended on extracellular calcium concentration, (b) it was blocked by the addition of the divalent cations cadmium or nickel, and reduced in magnitude by the addition of manganese or cobalt, and (c) barium was able to replace calcium as an inward current carrier. The threshold potential for inward calcium current was around -20 mV in 10mM extracellular calcium and about -35 mV in 2 mM calcium. Currents were net inward over part of their time course for potentials up to at least +30 mV. At temperatures of 20-26 degrees C, the peak inward current (at approximately 0 mV) was 139 +/- 14 microA/cm2 (mean +/- SD), increasing to 226 +/- 28 microA/cm2 at temperatures of 27-37 degrees C. The late outward current exhibited considerable fiber-to-fiber variability. In some fibers it was primarily a time-independent, nonlinear leakage current. In other fibers it was primarily a time-independent, nonlinear leakage current. In other fibers it appeared to be the sum of both leak and a slowly activated outward current. The rate of activation of inward calcium current was strongly temperature dependent. For example, in a representative fiber, the time-to-peak inward current for a +10-mV test pulse decreased from approximately 250 ms at 20 degrees C to 100 ms at 30 degrees C. At 37 degrees C, the time-to-peak current was typically approximately 25 ms. The earliest phase of activation was difficult to quantify because the ionic current was partially obscured by nonlinear charge movement. Nonetheless, at physiological temperatures, the rate of calcium channel activation in rat skeletal muscle is about five times faster than activation of calcium channels in frog muscle. This pathway may be an important source of calcium entry in mammalian muscle.     

Effect of denervation on loss of ions in chick skeletal muscle

Leaching behaviour of chick gastrocnemius muscle has been studied with respect to the loss of Na+, K+ and Ca2+ ions in a donor-solvent (muscle-water) system under normal as well as denervated conditions. An attempt has been made to explain the alterations in the rate of electrolytic loss in terms of membrane dysfunctions induced as a result of loss of neural control.     

The effect of taurine depletion on the contractile properties and fatigue in fast-twitch skeletal muscle of the mouse

Summary. Taurine as well as tauret (retinyliden taurine) levels were measured in locust Locusta migratoria compound eyes. HPLC measurements revealed relatively low taurine levels (1.9 ± 0.16 mM) in dark-adapted eyes. Glutamate, aspartate and glycine levels were 2.0 ± 0.2, 2.7 ± 0.4 and 3.0 ± 0.37 mM, respectively, while GABA was present only in trace amounts. After about 4 h of light adaptation at 1500–2000 lx, amino acid levels in the compound eye were as follows: taurine, 1.8 ± 0.17 mM; glutamate, no change at 2.1 ± 0.2 mM; aspartate sharply increased to 4.7 ± 0.7 mM; glycine slightly decreased to 2.8 ± 0.3 mM; and GABA trace levels. In the compound eye of locust Locusta migratoria, the existence of endogenous tauret in micro-molar range was established. In the dark, levels were several times higher compared with compound eye after light adaptation 1500 lx for 3 h, as estimated by TLC in combination with spectral measurements. Existence of tauret in compound eye is of special interest because in the compound eye, rhodopsin regeneration is based on photoregeneration.     

Protein kinase C signaling controls skeletal muscle fiber types

Slow myosin heavy chain 2 (MyHC2) gene expression in fetal avian skeletal muscle fibers is regulated by innervation and protein kinase C (PKC) activity. Fetal chick muscle fibers derived from the slow twitch medial adductor (MA) muscle express slow MyHC2 when innervated in vitro. The same pattern of slow MyHC2 regulation occurs in MA muscle fibers in which PKC activity is inhibited by staurosporine. To further test the function of PKC activity in the regulation of slow MyHC2 expression, wild-type and dominant-negative mutations of PKCalpha and PKCtheta were overexpressed in MA muscle fibers in vitro. Overexpression of wild-type PKCalpha and PKCtheta cDNAs resulted in increased PKC activities in muscle fibers and concomitant repression of slow MyHC2 expression under conditions that normally induced gene expression. Point mutations leading to single amino acid substitutions were generated in the ATP binding domains of PKCalpha and PKCtheta. Overexpression of CMVPKCalphaR368 and CMVPKCthetaR409 resulted in decreased PKC activities in transfected MA muscle fibers. Furthermore, transfection of CMVPKCalphaR368 and CMVPKCthetaR409 mutant constructs into MA muscle fibers did not repress the capacity of these fibers to express slow MyHC2 when cultured in medium containing staurosporine or when innervated. These results indicate that PKC activity represses slow MyHC2 expression and that PKC down-regulation, possibly in response to innervation, is required but not sufficient for slow MyHC2 expression.     

Lysosomes in skeletal muscle following denervation

Summary The in-vivo uptake of exogenously applied horseradish peroxidase and the activities of the lysosomal enzymes acid phosphatase and cathepsin D were studied histochemically and/or biochemically in innervated and 2–14 day-denervated tibialis anterior muscles of the mouse. The biochemically determined uptake of horseradish peroxidase showed a large increase already 4 days after denervation. The activities of the lysosomal enzymes increased in a more gradual fashion, and only cathepsin D showed an increase in activity when expressed as total activity per muscle. Histochemically horseradish peroxidase was found to be localized in muscle fibres in characteristic spindle-shaped segments after denervation. The main increase in the number of such segments per transverse section of the muscle occurred between 3 and 6 days after denervation. In serial sections these segments frequently showed positive staining also for acid phosphatase.It is concluded that exogenously applied horseradish peroxidase is taken up into the lysosomal system, which after denervation becomes organized into characteristic spindle-shaped segments in the muscle fibres. The endocytic activity of muscle fibres increases early after denervation. This is followed by a more gradual increase in activity of lysosomal enzymes and finally by an organization of the lysosomal system into characteristic spindle-shaped segments. The results are compatible with the working hypothesis that increased endocytosis may initiate lysosomal activation in denervated skeletal muscle.     

Diameter changes of muscle fiber types of the inferior oblique muscle of the rabbit following denervation]

Changes in fibre diameters of extraocular muscles of the rabbit were studied at different times after denervation. The whole inferior oblique muscle hypertrophied, while some of the muscle fibres hypertrophied and others showed atrophy, depending on the fibre type. Fibre types have been determined by their histochemical enzyme profile. In the central layer of the muscle the phasic muscle fibres, which are rich in mitochondria, exhibited a transient hypertrophy being maximal 4-5 weeks after denervation and afterwards they atrophied; other phasic muscle fibres, which are poor in mitochondria, atrophied without having shown any sign of hypertrophy. Special, putatively slow tonic muscle fibres, which have low enzyme activities, underwent small long-lasting increases of their diameters. In the superficial layer of extraocular muscle there are two types of extremely thin muscle fibres rich in mitochondira. Both these fibre types hypertrophied to the greatest degree and for a very long time. Comparable changes in fibre diameters as described here for the muscle fibre types of an extraocular muscle are known from special muscle fibres in other vertebrate     

Quantitative distribution of muscle fiber types in the scup Stenotomus chrysops

Because the mass-specific power generated by myotomal muscle during swimming varies along the length of the fish, a realistic assessment of total power generation by the musculature requires integrating the product of mass-specific power and muscle mass at each position over the length of the fish. As a first step toward this goal, we examined the distribution of red, pink, and white muscle along the length of Stenotomus chrysops (scup) using histochemical and image analysis techniques. The largest cross-sectional area of red fibers occurs at 60% of total fish length and declines both anteriorly and posteriorly. By contrast, white fibers have the largest cross-sectional area in the anterior and decline dramatically moving posteriorly. The proportion of the fishes' cross-section occupied by red fibers increases from 1.37% to 8.42% moving posteriorly along the length of the fish. In contrast, the proportion of cross-sectional area occupied by pink fibers is constant (1.19%), while the proportional cross-sectional area of white fibers falls from 82.5% to 66.3%. The red, pink, and white fibers comprise 2.09, 0.73, and 51.1%, respectively, of total fish weight. We also compared the distribution of muscle in 10°C-and 200°C-acclimated animals. The value for red fiber volume, though slightly higher (13%) in cold-acclimated fish, is not statistically different. No difference was found in pink or white fibers. Finally, the finding that most of the red muscle is in the posterior half of the fish further supports the notion that most power for steady swimming at moderate speeds comes from posterior rather than anterior musculature. © 1996 Wiley-Liss, Inc.     

Effect of sympathetic denervation on the rate of protein synthesis in rat skeletal muscle

Rates of protein synthesis were investigated in skeletal muscles from rats submitted to chemical and surgical sympathectomy. Three models of sympathetic denervation were used: 1) treatment with guanethidine (100 mg.kg(-1).day(-1) sc); 2) lumbar sympathetic denervation (surgical excision of the second and third lumbar ganglia of the sympathetic chain, from which arises the postganglionic fibers to the skeletal muscles of rat hindlimb); and 3) adrenodemedullation. Protein synthesis was estimated in isolated soleus muscle by the rate of incorporation of [(14)C]tyrosine (0.1 mM, 0.05 microCi/ml) into total protein. Soleus isolated after 2 and 4 days of chemical sympathectomy or after 3 days of lumbar denervation showed a 17-20% statistically significant decrease in in vitro rates of protein synthesis. These effects were reverted by addition of 10(-5) M isoproterenol or epinephrine in vitro. Neither clenbuterol nor isoproterenol (10(-7), 10(-6), or 10(-5) M) in vitro affected the rate of protein synthesis in soleus from normal rats. On the other hand, clenbuterol or epinephrine (10(-5) M) increased by 20% the rate of protein synthesis in soleus muscles from adrenodemedullated rats and prevented its decrease in muscles from fasted rats. The data suggest that the sympathetic nervous system stimulates protein synthesis in oxidative muscles, probably through the activation of beta(2)-adrenoceptors, especially in situations of hormonal or nutritional deficiency.     

Influence of training on blood flow to different skeletal muscle fiber types

Blood flows to fast-twitch red (FTR), fast-twitch white (FTW), and slow-twitch red (STR) fiber sections of the gastrocnemius-soleus-plantaris muscle group of sedentary and trained rats were determined using radiolabeled microspheres during the 1st and 10th min of in situ contractions at frequencies ranging from 7.5 to 90 tetani/min. Treadmill training increased the cytochrome c content of both FTW (6.0 +/- 0.13 nmol/g to 12.2 +/- 0.27) and FTR (22.2 +/- 0.32 to 26.7 +/- 0.25) muscle. Loss of tension, evident at 15 tetani/min and above, was less (P less than 0.001) in trained animals. Although steady-state blood flows (10th min) to FTR and STR fibers were not altered by training, initial flows (1st min) to the trained FTR section were greater (P less than 0.025). Overall initial flows to both red fiber types were excessively high at the easier contraction conditions, but subsequently declined to values more reflective of the expected energy demands. This time-dependent relative hyperemia was not found in either sedentary or trained FTW muscle. However, training increased the maximal blood flow in the FTW sections [60 +/- 3.2 (n = 36) vs. 88 +/- 5.2 ml X min X 100 g-1 (n = 36)]. This 40-50% increase in FTW blood flow would produce only a modest 10% increase in blood flow to a whole mixed-fiber muscle, since the flow capacity of the FTW muscle is only one third to one fourth that of FTR muscle. This overall increase in blood flow, however, is similar to changes in VO2max found in trained rats.(ABSTRACT TRUNCATED AT 250 WORDS)     

Fasting-related autophagic response in slow- and fast-twitch skeletal muscle

This study investigated regulation of autophagy in slow-twitch soleus and fast-twitch plantaris muscles in fasting-related atrophy. Male Fischer-344 rats were subjected to fasting for 1, 2, or 3 days. Greater weight loss was observed in plantaris muscle than in soleus muscle in response to fasting. Western blot analysis demonstrated that LC3-II, a marker protein for macroautophagy, was expressed at a notably higher level in plantaris than in soleus muscle, and that the expression level was fasting duration-dependent. To identify factors related to LC3-II enhancement, autophagy-related signals were examined in both types of muscle. Phosphorylated mTOR was reduced in plantaris but not in soleus muscle. FOXO3a and ER stress signals were unchanged in both muscle types during fasting. These findings suggest that preferential atrophy of fast-twitch muscle is associated with induction of autophagy during fasting and that differences in autophagy regulation are attributable to differential signal regulation in soleus and plantaris muscle.     

Subcellular distribution of phospholipids in different types of skeletal muscle.

Subcellular distribution of choline and non-choline phosphatides has been studied in tetanic (fast-twitch) and tonic (slow-twitch) muscles of rabbits. The choline phosphatide content of the subcellular fraction including the sarcolemma was greater in the tetanic than in the tonic muscle. On the other hand, the choline phosphatide content of the mitochondria-free sarcoplasmic fraction was greater in the tonic than in the tetanic muscle. A greater amount of non-choline phosphatide was found in each subcellular fraction of the tonic muscle as compared with those of the tetanic one. There was more fatty aldehyde in the non-choline phosphatides of each subcellular fraction of the tetanic muscle, than in those of the tonic one, of this type being much smaller in the tetanic muscle. There is not such an expressed difference in the fatty aldehyde contents of choline phosphatides of the subcellular fractions between the two kinds of muscle.     

Effect of denervation and phentolamine on the thermogenic action of noradrenaline in rat skeletal muscle

Vascularly isolated skeletal muscle of the cold-acclimated (CA) rat was perfused with blood in situ or in vitro and the effect of denervation and an alpha-adrenolytic agent (phentolamine) on its oxygen consumption was studied in the resting state and after administering noradrenaline (NA). The resting metabolism of muscle in situ rose by 28% after denervation. The infusion of NA further raised the oxygen consumption of acutely denervated muscle perfused in situ of in vitro by 43%. The thermogenic effect of NA on muscle denervated two hours before the experiment was only transitory. Phentolamine raised the oxygen consumption of the innervated muscle in situ by 42%; the infusion of NA did not stimulate metabolism any further. Phentolamine reduced the vascular resistance of resting muscle, but did not inhibit the vasoconstriction during the infusion of NA. The results show that the thermogenic effect of infused NA in perfused muscle is inhibited not by acute denervation, but by a vasoconstriction, which cannot be prevented by the administration of an alpha-adrenolytic agent.