Effects of β-guanidinopropionic acid-feeding on the patterns of myosin isoforms in rat fast-twitch muscle

Administration of -guanidinopropionic acid (-GPA) to rats as 1% of their diet for 6 weeks led to an accumulation of -GPA and -GPA-phosphate and to a depletion of creatine and phosphocreatine in the fast-twitch plantaris muscle. Adenosine triphosphate concentration was also decreased. Electrophoretic analyses were performed to investigate the effects of -GPA on the patterns of fast (FM) and slow (SM) isomyosins, myosin heavy chain (HC) isoforms and myosin light chain (LC) isoforms. The relative concentrations of fast isomyosins FM1 and FM2 decreased, whereas slow isomyosin SM increased. The increase in slow isomyosin corresponded to an increase in the relative concentration of the slow myosin HCI. The changes of the myosin light chain pattern consisted of increases in the relative concentrations of the two slow isoforms, LC1sb and LC2s, and decreases in the fast isoforms LC2f and LC3f. These results demonstrate that -GPA administration, leading to a depletion in energy-rich phosphates and a reduced phosphorylation potential, has an impact on myosin isoform expression in rat fast-twitch skeletal muscle.     

Effects of clenbuterol and ICI118551, a selective β2-antagonist,on the growth of skeletal muscle of suckling rats

The ₂-adrenergic agonist, clenbuterol, was administered to lactating rats (4 mg/kg diet) from post-partum day 1 to day 19, or directly injected into neonate rats (0.1 and 1.0 mg/kg body weight) from post-partum day 3 until day 15. Changes in body weight and the skeletal muscles soleus (SOL) and extensor digitorum longus (EDL) were studied in both dams and suckling offspring. Drug treatment consistently increased body weight in dams whilst significantly reducing the growth of their suckling pups. In dams treated with clenbuterol (4 mg/kg of diet) muscle weights and protein contents were significantly increased. Total protein content increased by 16% in SOL and 47% in EDL after 19 days of treatment. In contrast, in their suckling pups, there was a 22% and 26% reduction in protein content of SOL and EDL respectively. Administration of the ₂-antagonist ICI1 18551 to these pups failed to prevent these reductions in body and muscle weights. Hence, if clenbuterol did reach the pups via the milk from treated mothers it did not act via conventional ₂-receptors. Injection of pups with clenbuterol (1.0 mg/kg every 12 h) from litters suckling from untreated dams also resulted in significant reductions in muscle weights and protein contents. Protein content was reduced by 10% in SOL and 13% in EDL after 12 days of treatment. No alteration in fibre type proportions in SOL or EDL resulted from this treatment. Further work is required to determine whether the growth suppression in the two situations occurs via the same mechanism.     

Molecular size-dependent leakage of intracellular molecules from frog skeletal muscle fibers permeabilized with β-escin

The permeability of -escin-treated cell membrane was characterized in terms of the permeant molecular size, by monitoring the leak of cytoplasmic molecules in frog skeletal muscle fibers. With a low concentration of -escin (5 M), most of the cellular ATP was lost within 30–40 min (as revealed by rigor force generation), whereas a fluorescence-labeled dextran injected into the cytoplasm (10 kDa) and cytoplasmic proteins (14–80 kDa) slowly leaked out of the cell. A high concentration of -escin (50–100 M) accelerated the leak of large molecules. Therefore, low concentrations of -escin may be used as a means of permeabilizing the cell membrane to relatively small molecules, while retaining a major fraction of the cellular macromolecules.Abbreviations MOPS 3-[N-morpholino]propanesulfonic acid - KMS potassium methanesulphonate - PIPES piperazine-N,N-bis[2-ethanesulfonic acid] - EGTA ethylene glycol-bis( -amino-ethyl ether)N,N,N,N-tetraacetic acid - SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis     

Effects of S-glutathionylation on the passive force–length relationship in skeletal muscle fibres of rats and humans

Journal of Muscle Research and Cell Motility - This study investigated the effect of S-glutathionylation on passive force in skeletal muscle fibres, to determine whether activity-related redox...     

The effects of β-adrenergic stimulation and exercise on NR4A3 protein expression in rat skeletal muscle

β-Adrenergic stimulation and exercise up-regulate the mRNA expression of nuclear receptor NR4A3, which is involved in the regulation of glucose and fatty acid utilization genes in skeletal muscle. The objective of our study was to examine the effects of β-adrenergic stimulation and exercise on the expression of NR4A3 protein in rat skeletal muscle. A single subcutaneous injection of clenbuterol, which is a β2-adrenergic receptor (β2-AR) agonist, increased NR4A3 mRNA and protein expression in the fast-twitch glycolytic triceps muscle. On the other hand, an acute 3-h session of either treadmill running or swimming did not increase the NR4A3 protein level in the exercised muscle, although both treadmill running and swimming increased NR4A3 mRNA. Finally, loss of postural contractile activity because of hindlimb immobilization reduced NR4A3 mRNA and protein in the slow-twitch oxidative soleus muscle. These results suggest that: β-adrenergic stimulation up-regulates not only NR4A3 mRNA but also NR4A3 protein in fast-twitch glycolytic muscle; exercise may increase NR4A3 mRNA but not NR4A3 protein in skeletal muscle; and local postural contractile activity plays a crucial role in maintaining NR4A3 protein expression level in postural muscle.     

Effects of concentric and repeated eccentric exercise on muscle damage and calpain–calpastatin gene expression in human skeletal muscle

The purpose of this study was to compare the responsiveness of changes in Ca²⁺-content and calpain–calpastatin gene expression to concentric and eccentric single-bout and repeated exercise. An exercise group (n = 14) performed two bouts of bench-stepping exercise with 8 weeks between exercise bouts, and was compared to a control-group (n = 6). Muscle strength and soreness and plasma creatine kinase and myoglobin were measured before and during 7 days following exercise bouts. Muscle biopsies were collected from m. vastus lateralis of both legs prior to and at 3, 24 h and 7 days after exercise and quantified for muscle Ca²⁺-content and mRNA levels for calpain isoforms and calpastatin. Exercise reduced muscle strength and increased muscle soreness predominantly in the eccentric leg (P < 0.05). These responses as well as plasma levels of creatine kinase and myoglobin were all attenuated after the repeated eccentric exercise bout (P < 0.05). Total muscle Ca²⁺-content did not differ between interventions. mRNA levels for calpain 2 and calpastatin were upregulated exclusively by eccentric exercise 24 h post-exercise (P < 0.05), with no alteration in expression between bouts. Calpain 1 and calpain 3 mRNA did not change at any specific time point post-exercise for either intervention. Our mRNA results suggest a regulation on the calpain–calpastatin expression response to muscle damaging eccentric exercise, but not concentric exercise. Although a repeated bout effect was demonstrated in terms of muscle function, no immediate support was provided to suggest that regulation of expression of specific system components is involved in the repeated bout adaptation. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Lymphocyte plasma membranes—V. Immunoglobulins on isolated plasma membranes of the thymic and splenic lymphocytes of the rat

The plasma membranes from thymic and splenic lymphocytes of inbred ACI rats were isolated and compared for immunoglobulin content before and after immunization of the animals with poly(Glu⁵²Lys³³Tyr¹⁵). The highly purified plasma membranes were solubilized in the nonionic detergent Triton X-100 and analyzed by double immunodiffusion with antisera specific for heavy chains of rat IgM and IgG immunoglobulins. Both the thymic and splenic lymphocyte plasma membranes from unimmunized and immunized animals had IgM and IgG immunoglobulins. The IgM content was lowest in thymocyte plasma membranes from unimmunized animals. Immunization resulted in an increase in the IgM content of the thymocyte plasma membranes only. Both thymic and splenic cell membranes appeared to have similar amounts of IgG before and after immunization. Semiquantitative estimates indicated that the content of IgG in thymocyte membranes was about 1% of the total membrane protein. Since our previous studies of rat thymocytes by surface radioiodination demonstrated IgM but no IgG immunoglobulin, the present findings suggest that IgG in rat thymocyte membranes may lie within the plasma membrane matrix.     

Differential distribution of dystrophin and β-spectrin at the sarcolemma of fast twitch skeletal muscle fibers

We used double label immunofluorescence and confocal microscopy to examine the organization of -spectrin and dystrophin at the sarcolemma of fast twitch myofibers in the Extensor Digitorum Longus (EDL) of the rat. Both -spectrin and dystrophin are concentrated in costameres, a rectilinear sarcolemmal array composed of longitudinal strands and transverse elements overlying Z and M lines. In contrast, intercostameric regions, lying between these linear structures, contain significant levels of dystrophin but little detectable -spectrin. The dystrophin-associated proteins, syntrophin and -dystroglycan, are also concentrated at costameres but, like dystrophin, are present in intercostameric regions as well. Dystrophin is present at costameres and intercostameric regions in fast twitch muscles of the mouse but is absent from all regions of the sarcolemma in the mdx mouse, which lacks dystrophin. Areas of the sarcolemma near myonuclei also contain dystrophin without -spectrin, consistent with the idea that the distribution of dystrophin at the sarcolemma is not dependent on -spectrin. We conclude that dystrophin is present under all areas of the sarcolemma. The increased fragility of the sarcolemma in patients with Duchennes muscular dystrophy may be explained in part by the absence of dystrophin not only from costameres, but also from intercostameric regions.     

Effects of the PKA inhibitor H-89 on excitation–contraction coupling in skinned and intact skeletal muscle fibres

This study investigated the effects of the protein kinase A (PKA) inhibitor, H-89, in mechanically-skinned muscle fibres and intact muscle fibres, in order to determine whether PKA phosphorylation is essential for normal excitation–contraction (E–C) coupling. In skinned EDL fibres of the rat, force responses to depolarization (by ion substitution) were inhibited only slightly by 10M H-89, a concentration more than sufficient to fully inhibit PKA. Staurosporine (1 M), a potent non-specific kinase inhibitor, also had little if any effect on depolarization-induced responses. At 1–2 M, H-89 significantly slowed the repriming rate in rat skinned fibres, most likely due to it deleteriously affecting the T-system potential. With 100 M H-89, the force response to depolarization by ion substitution was completely abolished. This inhibitory effect was reversed by washout of H-89 and was not due to block of the Ca²⁺ release channel in the sarcoplasmic reticulum (SR). In intact single fibres of the flexor digitorum longus (FDB) muscle of the mouse, 1–3 M H-89 had no noticeable effect on action-potential-mediated Ca²⁺ transients. Higher concentrations (4–10 M) caused Ca²⁺ transient failure in fibres stimulated at 20 Hz in a manner indicative of action-potential failure. At 10–100 M, H-89 also inhibited net Ca²⁺ uptake by the SR and affected the Ca²⁺-sensitivity of the contractile apparatus in rat skinned fibres. All such effects were proportionately greater in toad muscle fibres. These results do not support the hypothesis that phosphorylation is essential for the Ca²⁺ release channel to open in response to voltage-sensor activation in skeletal muscle fibres.     

Chaperone activity of α-crystallin in relation to the sarcoplasmic reticulum Ca2+ pump of skeletal muscles in stress and adaptation

α-Crystallin, an endogenous low-molecular-weight protein with chaperone activity, exerted protective effects on membrane systems of Ca²⁺ transport into the sarcoplasmic reticulum of skeletal muscles. Protective action of α-crystallin depended on the body state. This effect was not observed in the control and after adaptation to stress, while after stress, especially against the background of adaptation, α-crystallin increased the rate of Ca²⁺ transport into the sarcoplasmic reticulum and thermal resistance of Ca²⁺ pump. The mechanisms of α-crystallin activation during stress are discussed. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 128, No. 9, pp. 279–282, September, 1999     

Mechanical and kinetic properties of β-cardiac/slow skeletal muscle myosin

We aimed to establish reference parameters to identify functional effects of familial hypertrophic cardiomyopathy-related point mutations in the ??-cardiac/slow skeletal muscle myosin heavy chain (??-cardiac/MyHC-1). We determined mechanical and kinetic parameters of the ??-cardiac/MyHC-1 using human soleus muscle fibers that express the same myosin heavy chain (MyHC-1) as ventricular myocardium (??-cardiac). The observed parameters are compared to previously reported data for rabbit psoas muscle fibers. We found all of the examined kinetic parameters to be slower in soleus fibers than in rabbit psoas muscle. Somewhat surprisingly, however, we also found that the stiffness of the ??-cardiac/MyHC-1 head domain is more than 3-fold lower than the stiffness of the fast isoform of psoas fibers. Furthermore, and different from rabbit psoas muscle, in human soleus fibers both the occupancy of force-generating cross-bridge states as well as the elastic extension of force-generating heads increase with temperature. Thus, a myosin head in the force generating states makes an increasing contribution to force with temperature. We support some of our fiber data by data from in vitro motility and optical trapping assays. Initial findings with FHC-related point mutations in the converter imply that the differences in stiffness of the head domain between the slow and fast isoform may well be due to particular differences in the amino acid sequence of the converter. We show that the slower kinetics may be linked to a larger flexibility of the ??-cardiac/MyHC-1 isoform compared to fast MyHC isoforms.     

Modeling of skeletal muscle: the influence of tendon and aponeuroses compliance on the force–length relationship

The aim of this study was to investigate the influence of changing elastic properties of tendon and aponeuroses on force production and muscle geometry. A three-dimensional, structural, continuum mechanics model of the cat medial gastrocnemius was used for this purpose. Increasing compliance in tendon and aponeuroses caused a decrease in the peak isometric force and a shift of the force–length relationship to the right of the length axis (i.e. toward greater muscle lengths). This result can be explained with the stability condition of the force–length relationship which produced a history dependence of force production that is conceptually in agreement with experimental observations.     

A study of the mechanisms of excitation–contraction coupling in frog skeletal muscle based on measurements of [Ca<Superscript>2+</Superscript>] transients inside the sarcoplasmic reticulum

[Ca²⁺] transients inside the sarcoplasmic reticulum (SR) were recorded in frog skeletal muscle twitch fibers under voltage clamp using the low affinity indicator Mag Fluo 4 (loaded in its AM form) with the purpose of studying the effect on Ca²⁺ release of extrinsic Ca²⁺ buffers (i.e. BAPTA) added at high concentration to the myoplasm. When the extrinsic Ca²⁺ buffer is added to the myoplasm, part of the released Ca²⁺ binds to it, reducing the Ca²⁺ signal reported by a myoplasmic indicator. This, in turn, hinders the quantification of the amount of Ca²⁺ released. Monitoring release by measuring [Ca²⁺] inside the SR avoids this problem. The application of extrinsic buffers at high concentration reduced the resting [Ca²⁺] in the SR ([Ca²⁺]_SR) continuously from a starting value close to 400 μM reaching the range of 100 μM in about half an hour. The effect of reducing resting [Ca²⁺]_SR on the Ca²⁺ permeability of the SR activated by voltage clamp depolarization to 0 mV was studied in cells where the myoplasmic [Ca²⁺] ([Ca²⁺]_myo) transients were simultaneously recorded with Rhod2. The Ca²⁺ release flux was calculated from [Ca²⁺]_myo and divided by [Ca²⁺]_SR to obtain the permeability. Peak permeability was significantly reduced, from 0.026?±?0.005 ms^?1 at resting [Ca²⁺]_SR?=?372?±?5 μM to 0.021?±?0.004 ms^?1 at resting [Ca²⁺]_SR?=?120?±?16 μM (n?=?4, p?=?0.03). The time averaged permeability was not significantly changed (0.009?±?0.003 and 0.010?±?0.003 ms^?1, at the higher and lower [Ca²⁺]_SR respectively). Once the cells were equilibrated with the high buffer intracellular solution, the change in [Ca²⁺]_SR (Δ[Ca²⁺]_SR) in response to voltage clamp depolarization (0 mV, 200 ms) in 20 mM BAPTA was significantly lower (Δ[Ca²⁺]_SR?=?30.2?±?3.5 μM from resting [Ca²⁺]_SR?=?88.8?±?13.6 μM, n?=?5) than in 40 mM EGTA (Δ[Ca²⁺]_SR?=?72.2?±?10.4 μM from resting [Ca²⁺]_SR?=?98.2?±?15.6 μM, n?=?4) suggesting that a Ca²⁺ activated component of release was suppressed by BAPTA.     

Effects of sphingosine 1-phosphate on excitation–contraction coupling in mammalian skeletal muscle

Sphingosine 1-phosphate (S1P) activates a subset of plasma membrane receptors of the endothelial differentiation gene family (EdgRs) in many cell types. In C2C12 myoblasts, exogenous S1P elicits Ca2+ transients by activating voltage-independent plasma membrane Ca2+ channels and intracellular Ca2+-release channels. In this study, we investigated the effects of exogenous S1P on voltage-dependent L-type Ca2+ channels in skeletal muscle fibers from adult mice. To this end, intramembrane charge movements (ICM) and L-type Ca2+ current (I(Ca)) were measured in single cut fibers using the double Vaseline-gap technique. Our data showed that submicromolar concentrations of S1P (100 nM) caused a approximately 10-mV negative shift of the voltage threshold and transition voltages of q(gamma) and q(h) components of ICM, and of I(Ca) activation and inactivation. Biochemical studies showed that EdgRs are expressed in skeletal muscles. The involvement of EdgRs in the above S1P effects was tested with suramin, a specific inhibitor of Edg-3Rs. Suramin (200 microM) significantly reduced, by approximately 90%, the effects of S1P on ICM and I(Ca), suggesting that most of S1P action occurred via Edg-3Rs. Moreover, SIP at concentration above 10 microM elicited intracellular Ca2+ transients in muscle fibers loaded with the fluorescent Ca2+ dye Fluo-3, as detected by confocal laser scanning microscopy.     

The role of the acetylcholine — Cholinesterase system in the origin of bioelectrical phenomena in skeletal muscle

Summary Poisoning of the isolated sartorius muscle in frog with eserine and strychnine in the 110⁵ and 110⁴ concentrations causes the change of the value and the form of the excitation biocurrent. Strychnine and eserine in the 110⁵, 110⁴ and 110³ concentrations have no effect on the electromotive force of the sartorius muscle injury. These data lead us to presume certain differences in the mechanism of the origin of the excitation and injury biocurrents in the skeletal muscle, at least with relation to the participation of the acetylcholine-cho-linesterase system in this process.Presented by Academician the late L. A. Orbeli     

Effect of chronic γ-irradiation and β-carotene on lipid metabolism in presynaptic membranes in rat brain

Lysophosphatidylcholine is involved in radiation-induced modulation of presynaptic membranes in rat brain. High sensitivity of the cortical integrative functions to chronic low-dose γ-irradiation is demonstrated. β-Carotene produced a protective effect during chronic irradiation. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 129, No. 6, pp. 629–632, June, 2000     

Effects of 14 days of microgravity on fast hindlimb and diaphragm muscles of the rat

The purpose of the present study was to determine the effects of 14 days of microgravity on specific rat fast-twitch muscles, and to compare these data with previous data from rat fast-twitch muscles exposed to microgravity for 10 days (Kraemer et al. 2000). Hindlimb muscles containing predominately fast fibers [extensor digitorum longus (EDL), superficial “white” (GSW) and deep “red” (GDR) gastrocnemius] and the diaphragm (DIA) were removed from flight and ground-based control animals and analyzed for: muscle mass, fiber type distribution, cross-sectional area, and myosin heavy chain (MHC) isoform content. Gravitational unloading for 14 days caused significant decreases in muscle mass (8–9%) and cross-sectional area of almost all fiber types (10–35%) from both EDL and gastrocnemius muscles. However, microgravity had little effect on fiber type composition in these muscles with significant changes occurring only in the EDL type IID fiber population (9.5% decrease). Similarly, relative MHC isoform content was only slightly altered by exposure to microgravity (increased content of MHCIIa in flight EDL). No changes in area, fiber type percentages, or MHC isoform content were detected in the DIA following the 14-day spaceflight. Similar to data gathered following a 10-day spaceflight (Kraemer et al. 2000), the 14-day flight did not appear to cause significant slow-to-fast (I → IIA) or fast-to-faster (IIA → IID → IIB) transformations in hindlimb muscles containing predominantly fast-twitch fibers. However, the longer period of gravitational unloading did result in additional loss in muscle fiber cross-sectional area with involvement of more major fiber types.     

Isoproterenol and GTPγS inhibit L-type calcium channels of differentiating rat skeletal muscle cells

Summary In adult skeletal muscle, G-proteins have been shown to modulate the calcium channels both directly and through a cAMP-dependent phosphorylating mechanism. We have investigated the action of G-proteins on the L-type calcium current in cultured rat muscle cells (myoballs) under voltage clamp in whole cell or perforated patch modes. Intracellular photolytic release of 200 M GTPS inhibited the L-type calcium current. Inclusion of 500 M uncaged GTPS in the patch pipette in the whole cell configuration reduced the calcium current by a similar amount. Under perforated patch conditions external application of 10 M of the -adrenergic agonist isoproterenol also reduced the calcium current. Pretreatment of the cells with pertussis toxin reversed the effect of GTPS and removed that of isoproterenol. We conclude that rat myoballs contain -adrenergic receptors that inhibit the L-type calcium current, and that this inhibition is mediated by a pertussis toxinsensitive G-protein.