Rat Extensor Digitorum Longus Research Articles

Three calpain isoforms are autolyzed in rat fast-twitch muscle after eccentric contractions

The present study investigated changes in autolysis of three calpain isoforms in skeletal muscles undergoing eccentric contractions (ECC), leading to prolonged force deficits. Rat extensor digitorum longus and tibialis anterior muscles were exposed to 200-repeated ECC in situ, excised immediately after or 3 or 6 days after cessation of ECC, and used for measures of force output and for biochemical analyses. Full restoration of tetanic force in ECC-treated muscles was not attained until 6 days of recovery. Maximal calpain activity determined by a fluorogenic substrate was unaltered immediately after ECC, but increased to 313 and 450 % after 3 and 6 days, respectively. Increases in the amount of autolyzed calpain-3 were apparent immediately and developed progressively with recovery time, whereas elevations of autolyzed μ- and m-calpain occurred after 3 and 6 days, respectively. The protein content was augmented only in m-calpain. It is suggested that the three calpain isoforms may be involved in the dismantling, repair, remodeling and/or regeneration processes in ECC-treated muscles.

Proton Fluxes Across the Tubular (T-) System Membrane of Rat Fast-Twitch Fibres

Cytoplasmic pH has major effects on most cellular processes in skeletal muscle, including its ability to develop force. Protons are continuously extruded from the cytoplasm against their electrochemical gradient as shown by the considerably more alkaline pH in the resting muscle than the predicted pH value if protons were distributed passively. We aimed to determine the contribution of the t-system proton extrusion mechanisms to this gradient and the diffusive proton flux of the t-system. To do this we trapped 10 mM of the pH-sensitive dye HPTS inside the tubular (t-) system of mechanically skinned fibres from the rat extensor digitorum longus muscles and continuously imaged dye fluorescence during changes in internal solution pH, [Na+], [K+] and [ATP] by confocal microscopy. Calibrations using monensin showed that in normally polarized fibres with 36 mM [Na+]cyto that pHt-sys was 7.50 ± 0.12 (n=3), 7.91 ± 0.20 (n=3) and 8.31 ± 0.29 (n=4) at pH-cyto of 6.8, 7.2 and 7.5, respectively. In the presence of 162 mM [Na+]cyto, with or without amiloride, the pHt-sys and [Na+]t-sys were similar to cytoplasmic values. The addition of 50 µM amiloride to normally polarized fibres with 36 mM [Na+]cyto increased pHt-sys further indicating that the Na+-H+ exchanger (NHE) was the major protein responsible for extruding protons from the cytoplasm. The pH difference across the t-system membrane at rest is reduced by NHE activity, which moves protons against the inward diffusive proton flux of the resting muscle fibre. We calculated the diffusive proton flux across the t-system to be 2.7 +/- 0.9 e-4 m/s (mean +/- SEM, n=3).

Ca2+ Uptake by the Tubular (T-) System Membrane of Rat Fast-Twitch Fibres

The tubular (t-) system of skeletal muscle is an internalization of the plasma membrane. The t-system forms a junction with the terminal cisternae of the sarcoplasmic reticulum (SR) at every sarcomere of skeletal muscle. At any given moment the [Ca2+] within the small volume bound by the junctional membranes will be critically determined by the leak of Ca2+ through ryanodine receptors and the net Ca2+ handling ability of the t-system. To assess the Ca2+ uptake and handling ability of the t-system we trapped fluo-5N or rhod-5N in the t-system of mechanically skinned fibres of rat extensor digitorum longus muscle and continuously imaged t-system Ca2+-dependent fluorescence on a confocal microscope. In situ calibration determined the half signal of fluo-5N and rhod-5N to be 335 and 872 µM, respectively. Rhod-5N was selected for ongoing experiments. Chronic depletion of [Ca2+]SR with caffeine reduced [Ca2+]t-sys to 0.1 mM via chronic activation of store-operated Ca2+ entry (Launikonis et al 2003, PNAS). We then exposed Ca2+-depleted preparations to 0-800 nM [Ca2+]cyto in 50 mM EGTA. At [Ca2+]cyto > 100 nM the [Ca2+]t-sys reached a plateau at 1.8-1.9 mM after 3-5 s. At [Ca2+]cyto < 100 nM the [Ca2+]t-sys did not always reach this plateau and showed a biphasic uptake of Ca2+. At the plateau [Ca2+]t-sys lowering [Ca2+]cyto to < 1 nM did not cause a significant loss of [Ca2+]t-sys. There was an apparent absence of effect of removing [Na+]cyto on these results. Mathematical modeling of these results suggests that the plasma membrane CaATPase (PMCA) with its low Km for Ca2+ is the major protein responsible for t-system Ca2+ uptake in the resting muscle, despite the higher transport capacity of the Na-Ca exchanger.

Dietary Fat Influences the Expression of Contractile and Metabolic Genes in Rat Skeletal Muscle

Dietary fat plays a major role in obesity, lipid metabolism, and cardiovascular diseases. To determine whether the intake of different types of dietary fats affect the muscle fiber types that govern the metabolic and contractile properties of the skeletal muscle, we fed male Wistar rats with a 15% fat diet derived from different fat sources. Diets composed of soybean oil (n-6 polyunsaturated fatty acids (PUFA)-rich), fish oil (n-3 PUFA-rich), or lard (low in PUFAs) were administered to the rats for 4 weeks. Myosin heavy chain (MyHC) isoforms were used as biomarkers to delineate the skeletal muscle fiber types. Compared with soybean oil intake, fish oil intake showed significantly lower levels of the fast-type MyHC2B and higher levels of the intermediate-type MyHC2X composition in the extensor digitorum longus (EDL) muscle, which is a fast-type dominant muscle. Concomitantly, MyHC2X mRNA levels in fish oil-fed rats were significantly higher than those observed in the soybean oil-fed rats. The MyHC isoform composition in the lard-fed rats was an intermediate between that of the fish oil and soybean oil-fed rats. Mitochondrial uncoupling protein 3, pyruvate dehydrogenase kinase 4, and porin mRNA showed significantly upregulated levels in the EDL of fish oil-fed rats compared to those observed in soybean oil-fed and lard-fed rats, implying an activation of oxidative metabolism. In contrast, no changes in the composition of MyHC isoforms was observed in the soleus muscle, which is a slow-type dominant muscle. Fatty acid composition in the serum and the muscle was significantly influenced by the type of dietary fat consumed. In conclusion, dietary fat affects the expression of genes related to the contractile and metabolic properties in the fast-type dominant skeletal muscle, where the activation of oxidative metabolism is more pronounced after fish oil intake than that after soybean oil intake.

Comparison of Myosin Heavy Chain mRNAs, Protein Isoforms and Fiber Type Proportions in the Rat Slow and Fast Muscles

We studied the expression of myosin heavy chain isoforms at mRNA and protein levels as well as fiber type composition in the fast extensor digitorum longus (EDL) and slow soleus (SOL) twitch muscles of adult inbred Lewis strain rats. Comparison of the results from Real Time RT-PCR, SDS-PAGE and fiber type analysis showed corresponding proportions of MyHC transcripts (MyHC-1, -2a, -2x/d, -2b), protein isoforms (MyHC-1, -2a, -2x/d, -2b) and fiber types (type 1, 2A, 2X/D, 2B) in both muscles. Furthermore, we found that slow MyHC-1 mRNA expression in the SOL was up to three orders higher than that of fast MyHC transcripts. This finding can explain the predominance of MyHC-1 isoform and fiber type 1 and the absence of pure 2X/D and 2B fibers in the SOL muscle. Based on our data presenting quantitative evidence of corresponding proportions between mRNA level, protein content and fiber type composition, we suggest that the Real Time RT-PCR technique can be used as a routine method for analysis of muscle composition changes and could be advantageous for the analysis of scant biological samples such as muscle biopsies in humans.

Different effects of aerobic and anaerobic exercise on the capillary in the extensor digitorum longus: PGC‐1a, VEGF, Flk‐1 and Capillary Architecture.

The rat extensor digitorum longus (EDL) muscles that performed to the different intensities of exercise were used as representatives of fast muscles. Kind of the intensity of exercise to rats was chosen by aerobic low‐intensity (LO) or anaerobic high‐intensity (HI) either. And they were compared with the rat did not do exercise (Cont). Fiber cross‐sectional area and capillary density were not different. Mitochondrial oxidative capacity from SDH of performed LO or HI was higher than that of Cont. And these increased mitochondrial oxidative capacity had different pattern according with muscle fiber type respectively. Moreover level of PGC‐1a was high in order of Cont, LO and HI. Capillary volume and capillary diameter were different, LO was higher than Cont. Moreover HI was higher than Cont and LO. The expression level of VEGF f was not different. However level of vascular endothelial growth factor receptor 2 (Flk‐1) was high in order of Cont, LO and HI. It may be that the effect of the capillary in fast muscle performed exercise is based on the increase in Flk‐1 instead of the increase in VEGF.

Phosphorylation of rat skeletal muscle pyruvate dehydrogenase phosphatase in response to insulin stimulation

Pyruvate dehydrogenase phosphatase (PDP) regulates carbohydrate oxidation through the mitochondrial pyruvate dehydrogenase (PDH) complex. PDP dephosphorylates and activates PDH, enabling increased carbohydrate flux towards oxidative energy production. In cell culture, both PDP1 and 2 appear to undergo covalent activation in direct response to insulin–stimulation by PKCδ. Our objective was to examine the effect of insulin on PDP phosphorylation and PDH activation. Intact rat extensor digitorum longus muscles were incubated (oxygenated at 25°C, fixed to 1g of resting tension) tendon–to–tendon for 30min in basal or insulin–stimulated (10mU/mL) media. PDH activity increased 58% following insulin stimulation, (p=0.057, n=11). Serine phosphorylation of PDP1 (p=0.037) and PDP2 (p=0.027) increased by 44% and 54% respectively following stimulation (n=4), and PKCδ protein content was enriched in the mitochondrial fraction by 45% in response to insulin stimulation (p=0.0009, n=8). These data suggest that the insulin–stimulated increase in PDH activity in whole tissue is mediated through mitochondrial migration of PKCδ and subsequent PDP phosphorylation.Research supported by NSERC, Canada.

Efeito de diferentes doses de nandrolona associado ao treinamento de força sobre o perfil fenotípico e área de secção transversa do músculo de ratos

O presente estudo avaliou a influência de diferentes doses de decanoato de nandrolona (DN) associado ao Treinamento de Força (TF) sobre o fenótipo de fibras e área de secção transversa (AST) do músculo extensor longo dos dedos (EDL) em ratos "Wistar". Os animais foram divididos em sete grupos: controle (GC) e grupos de acordo com a concentração de DN (0,1, 1, 2, 5, 10 e 20 mg/kg) administrada intramuscular 3 vezes/semana. O TF consistiu de saltos em meio líquido (carga 50-70% do peso corporal) 3x/semana, durante cinco semanas. A associação do TF e DN promoveu ação modulatória sobre os tipos de fibras. Houve hipertrofia das fibras de contração rápida (tipo II) em comparação com as fibras de contração lenta (tipo I). Em conclusão, apesar da associação do TF com DN aumentar a AST muscular e alterar o fenótipo das fibras, não houve efeito gradual das doses mais altas.

Isolation of Sarcolemmal Plasma Membranes by Mechanically Skinning Rat Skeletal Muscle Fibers for Phospholipid Analysis

Membrane phospholipid (PL) composition has been shown to affect cellular function by altering membrane physical structure. The sarcolemma plasma membrane (SLpm) is integral to skeletal muscle function and health. Previous studies assessing SLpm PL composition have demonstrated contamination from transverse (t)-tubule, sarcoplasmic reticulum, and nuclear membranes. This study assessed the possibility of isolating SL by mechanically skinning skeletal muscle fiber segments for the analysis of SLpm PL composition. Mechanically skinned SLpm from rat extensor digitorum longus (EDL) muscle fibers underwent Western blot analysis to assess contamination from t-tubule, sarcoplasmic reticulum, nuclear and mitochondrial membranes. The results indicate that isolated SLpm had minimal nuclear and mitochondrial membrane contamination and was void of contamination from sarcoplasmic reticulum and t-tubule membranes. After performing both high-performance thin layer chromatography and gas chromatography, we found that the SLpm obtained by mechanical skinning had higher sphingomyelin and total fatty acid saturation and lower phosphatidylcholine when compared to previous literature. Thus, by avoiding the use of various chemical treatments and membrane fractionation, we present data that may truly represent the SLpm and future studies can use this technique to assess potential changes under various perturbations and disease conditions such as insulin resistance and muscular dystrophy.

Excitation-induced exchange of Na+, K+, and Cl- in rat EDL muscle in vitro and in vivo: physiology and pathophysiology.

In skeletal muscle, excitation leads to increased [Na+]i, loss of K+, increased [K+]o, depolarization, and Cl− influx. This study quantifies these changes in rat extensor digitorum longus (EDL) muscles in vitro and in vivo using flame photometric determination of Na+ and K+ and 36Cl as a tracer for Cl−. In vitro, 5-Hz stimulation for 300 s increased intracellular Na+ content by 4.6 ± 1.2 µmol/g wet wt (P < 0.002) and decreased intracellular K+ content by 5.5 ± 2.3 µmol/g wet wt (P < 0.03). This would increase [K+]o by 28 ± 12 mM, sufficient to cause severe loss of excitability as the result of inactivation of Na+ channels. In rat EDL, in vivo stimulation at 5 Hz for 300 s or 60 Hz for 60 s induced significant loss of K+ (P < 0.01), sufficient to increase [K+]o by 71 ± 22 mM and 73 ± 15 mM, respectively. In spite of this, excitability may be maintained by the rapid and marked stimulation of the electrogenic Na+,K+ pumps already documented. This may require full utilization of the transport capacity of Na+,K+ pumps, which then becomes a limiting factor for physical performance. In buffer containing 36Cl, depolarization induced by increasing [K+]o to 40–80 mM augmented intracellular 36Cl by 120–399% (P < 0.001). Stimulation for 120–300 s at 5–20 Hz increased intracellular 36Cl by 100–188% (P < 0.001). In rats, Cl− transport in vivo was examined by injecting 36Cl, where electrical stimulation at 5 Hz for 300 s or 60 Hz for 60 s increased 36Cl uptake by 81% (P < 0.001) and 84% (P < 0.001), respectively, indicating excitation-induced depolarization. Cl− influx favors repolarization, improving K+ clearance and maintenance of excitability. In conclusion, excitation-induced fluxes of Na+, K+, and Cl− can be quantified in vivo, providing new evidence that in working muscles, extracellular accumulation of K+ is considerably higher than previously observed and the resulting depression of membrane excitability may be a major cause of muscle fatigue.

Simultaneous Visualization of Myosin Heavy Chain Isoforms in Single Muscle Sections

We developed a staining protocol that enables simultaneous visualization of myosin heavy chain (MHC) pure and hybrid muscle fiber types in rat skeletal muscle. Up to eight different muscle fiber types can be visualized in a single section of the rat extensor digitorum longus muscle, which contains all four adult MHC isoforms and shows plasticity during the denervation-reinnervation process. Triple immunofluorescent staining of MHC-1, MHC-2a and MHC-2b with primary antibodies BA-D5 (isotype IgG2b), SC-71 (isotype IgG1) and BF-F3 (isotype IgM) and with three fluorophore-labeled isotype-specific secondary antibodies displays different muscle fiber types in a merged image of red, green and blue channels, each in its own color. Immunoperoxidase staining with primary antibody 6H1 directed against MHC-2x can be additionally applied on the same tissue section to facilitate the identification of muscle fibers containing MHC-2x. Triple staining can also be used in combination with other staining procedures to derive more information about the number of capillaries or the oxidative potential of muscle fiber types. Simultaneous visualization of multiple fiber types in a single merged image enables economical use of muscle samples and provides simple and rapid identification of all fiber types that are present in rat limb muscles.

The Effect of Intra-Muscular Injections of Alpha-tocopherol on the Activity of Phospho- Fructokinase in the Slow- and Fast-Twitch Skeletal Muscles of Metabolic Stress-Induced and Malnourished Rats

It was been hypothesized that critical illness will change the activity of phospho-fructokinase (PFK) and that intramuscular (i.m) injections of the antioxidant α-tocopherol would protect this rate- limiting enzyme of glycolysis in the slow-twitch soleus and in the fast-twitch extensor digitorum longus (EDL) muscles of metabolically-stressed and malnourished rats. Twenty eight male Wistar rats were divided into four groups: controls (CNs), zymosan-injected (ZY), α- tocopherol-injected (ZYE), and pair-fed (PF). Rats were fed Osmolite-HN, and were sacrificed on day three. PFK activity was measured in EDL and soleus muscles using a fluorometric assay, and was found to be significantly lower in both the soleus and EDL of ZY rats. The PFK activity in the EDL of ZYE rats was comparable to the PFK of PF rats and was significantly higher than ZY rats on day three. Glycolysis in the slow- and fast-twitch muscle of ZY rats is adversely affected, and three days of i.m injections of α-tocopherol significantly (p<0.05) protected PFK activity, in fast-twitch but not in slow- twitch muscles.

Regulation of acetylcholinesterase activity by nitric oxide in rat neuromuscular junction via N‐methyl‐d‐aspartate receptor activation

Acetylcholinesterase (AChE) is an enzyme that hydrolyses the neurotransmitter acetylcholine, thereby limiting spillover and duration of action. This study demonstrates the existence of an endogenous mechanism for the regulation of synaptic AChE activity. At the rat extensor digitorum longus neuromuscular junction, activation of N-methyl-d-aspartate (NMDA) receptors by combined application of glutamate and glycine led to enhancement of nitric oxide (NO) production, resulting in partial AChE inhibition. Partial AChE inhibition was measured using increases in miniature endplate current amplitude. AChE inhibition by paraoxon, inactivation of NO synthase by N(x)-nitro-L-arginine methyl ester, and NMDA receptor blockade by DL-2-amino-5-phosphopentanoic acid prevented the increase in miniature endplate current amplitude caused by amino acids. High-frequency (10 Hz) motor nerve stimulation in a glycine-containing bathing solution also resulted in an increase in the amplitude of miniature endplate currents recorded during the interstimulus intervals. Pretreatment with an NO synthase inhibitor and NMDA receptor blockade fully eliminated this effect. This suggests that endogenous glutamate, released into the synaptic cleft as a co-mediator of acetylcholine, is capable of triggering the NMDA receptor/NO synthase-mediated pathway that modulates synaptic AChE activity. Therefore, in addition to well-established modes of synaptic plasticity (e.g. changes in the effectiveness of neurotransmitter release and/or the sensitivity of the postsynaptic membrane), another mechanism exists based on the prompt regulation of AChE activity.

Both short intense and prolonged moderate in vitro stimulation reduce the mRNA expression of calcium-regulatory proteins in rat skeletal muscle

Sarcoplasmic and t-tubule membrane proteins regulating sarcoplasmic Ca(2+) concentration exhibit fibre-type-dependent isoform expression, and play central roles in muscle contraction and relaxation. The purpose of this study was to evaluate the effects of in vitro electrical stimulation on the mRNA expression of components involved in Ca(2+) regulation in oxidative and glycolytic skeletal muscle. The mRNA level of Ca(2+)-ATPase (SERCA1, 2), calsequestrin (CASQ1, 2), ryanodine receptor (RyR1), and dihydropyridine receptor (Cacna1) was assessed in rat extensor digitorum longus (EDL) and soleus (SOL) muscles at 4 h of recovery following in vitro stimulations (either short intensive (SHO) 60 Hz, 5 min, or prolonged moderate (PRO) 20 Hz, 40 min). Stimulation induced acute regulation of the mRNA level of Ca(2+)-regulating proteins in a manner that does not follow typical fibre-type-specific transitions. In general, stimulation decreased mRNA content of all proteins studied. Most prominent down-regulation was observed for Cacna1 (26 and 32 % after SHO and PRO, respectively, in SOL; 19 % after SHO in EDL). SERCA1, SERCA2, CASQ1, CASQ2, and RyR1 mRNA content also decreased significantly in both muscles relative to resting control. Of notice is that hexokinase II mRNA content was increased in EDL and unchanged in SOL underlining the specificity of the down-regulation of mRNA of Ca(2+) regulatory proteins. The results demonstrate contraction-induced down-regulation of mRNAs for the main components of Ca(2+)-regulating system in skeletal muscle. The down-regulation of both isoforms of SERCA and CASQ after a single electrical stimulation session suggests that adaptations to repeated stimulation involve further regulatory mechanisms in addition to acute mRNA responses.

Effects of Fiber Type and Size on the Heterogeneity of Oxygen Distribution in Exercising Skeletal Muscle

The process of oxygen delivery from capillary to muscle fiber is essential for a tissue with variable oxygen demand, such as skeletal muscle. Oxygen distribution in exercising skeletal muscle is regulated by convective oxygen transport in the blood vessels, oxygen diffusion and consumption in the tissue. Spatial heterogeneities in oxygen supply, such as microvascular architecture and hemodynamic variables, had been observed experimentally and their marked effects on oxygen exchange had been confirmed using mathematical models. In this study, we investigate the effects of heterogeneities in oxygen demand on tissue oxygenation distribution using a multiscale oxygen transport model. Muscles are composed of different ratios of the various fiber types. Each fiber type has characteristic values of several parameters, including fiber size, oxygen consumption, myoglobin concentration, and oxygen diffusivity. Using experimentally measured parameters for different fiber types and applying them to the rat extensor digitorum longus muscle, we evaluated the effects of heterogeneous fiber size and fiber type properties on the oxygen distribution profile. Our simulation results suggest a marked increase in spatial heterogeneity of oxygen due to fiber size distribution in a mixed muscle. Our simulations also suggest that the combined effects of fiber type properties, except size, do not contribute significantly to the tissue oxygen spatial heterogeneity. However, the incorporation of the difference in oxygen consumption rates of different fiber types alone causes higher oxygen heterogeneity compared to control cases with uniform fiber properties. In contrast, incorporating variation in other fiber type-specific properties, such as myoglobin concentration, causes little change in spatial tissue oxygenation profiles.

How physical exercise changes rat myotendinous junctions: an ultrastructural study

Myotendinous junctions can be easily injured by overloading or trauma, and exercise training may be a way of increasing their resistance to mechanical stress. To this end, we examined herein the morphological changes induced by moderate exercise training in the myotendinous junctions of extensor digitorum longus and gastrocnemius muscles in rats. Twelve Sprague-Dawley rats were used in this investigation. Six of them were trained to run on a treadmill for 1 h/day, 3 days/week over 10 weeks in order for them to achieve a running rate of 25 m/min at the end of the training period. Six age-matched sedentary rats were used as controls. The rats were sacrificed 24 h after the final training session, and the extensor digitorum longum (EDL) and the gastrocnemium were excised; the myotendinous junctions (MTJ) were then prepared and observed with electron microscopy. Digitation branching was evaluated by counting the bifurcations in the MTJ protrusions. Our observations indicate that exercise does indeed induce changes in MTJ morphology. In both muscles the number of bifurcated interdigitations increased significantly, as well as, in gastrocnemius, the branching of the finger-like processes. It was demonstrated that the MTJ is able to adapt to an increase in tensile force by enlarging the muscle-tendon contact area and, consequently, mechanical resistance.

Effects of β2-agonists on force during and following anoxia in rat extensor digitorum longus muscle

Electrical stimulation of isolated muscles may lead to membrane depolarization, gain of Na(+), loss of K(+) and fatigue. These effects can be counteracted with β(2)-agonists possibly via activation of the Na(+)-K(+) pumps. Anoxia induces loss of force; however, it is not known whether β(2)-agonists affect force and ion homeostasis in anoxic muscles. In the present study isolated rat extensor digitorum longus (EDL) muscles exposed to anoxia showed a considerable loss of force, which was markedly reduced by the β(2)-agonists salbutamol (10(-6) M) and terbutaline (10(-6) M). Intermittent stimulation (15-30 min) clearly increased loss of force during anoxia and reduced force recovery during reoxygenation. The β(2)-agonists salbutamol (10(-7)-10(-5) M) and salmeterol (10(-6) M) improved force development during anoxia (25%) and force recovery during reoxygenation (55-262%). The effects of salbutamol on force recovery were prevented by blocking the Na(+)-K(+) pumps with ouabain or by blocking glycolysis with 2-deoxyglucose. Dibutyryl cAMP (1 mM) or theophylline (1 mM) also improved force recovery remarkably. In anoxic muscles, salbutamol decreased intracellular Na(+) and increased (86)Rb uptake and K(+) content, indicating stimulation of the Na(+)-K(+) pumps. In fatigued muscles salbutamol induced recovery of excitability. Thus β(2)-agonists reduce the anoxia-induced loss of force, leading to partial force recovery. These data strongly suggest that this effect is mediated by cAMP stimulation of the Na(+)-K(+) pumps and that it is not related to recovery of energy status (PCr, ATP, lactate).

Fibrillation potentials of denervated rat skeletal muscle are associated with expression of cardiac-type voltage-gated sodium channel isoform Nav1.5

ObjectiveThe molecular mechanisms underlying fibrillation potentials are still unclear. We hypothesised that expression of the cardiac-type voltage-gated sodium channel isoform Nav1.5 in denervated rat skeletal muscle is associated with the generation of such potentials. MethodsMuscle samples were extracted and analysed biologically from surgically denervated rat extensor digitorum longus muscle after concentric needle electromyographic recording at various time points after denervation (4h to 6days). ResultsBoth nav1.5 messenger RNA (mRNA) signal on northern blotting and Nav1.5 protein expression on immunohistochemistry appeared on the second day after denervation, exactly when fibrillation potentials appeared. Administration of lidocaine, which has much stronger affinity for sodium channels in cardiac muscle than for those in skeletal muscle, dramatically decreased fibrillation potentials, but had no effect on contralateral compound muscle action potentials. ConclusionsExpression of Nav1.5 participates in the generation of fibrillation potentials in denervated rat skeletal muscle. SignificanceWe proposed an altered expression of voltage-gated sodium channel isoforms as a novel mechanism to explain the occurrence of fibrillation potentials following skeletal muscle denervation.

Cholesterol Depletion Uncouples β-dystroglycans from Discrete Sarcolemmal Domains, Reducing the Mechanical Activity of Skeletal Muscle

Background/Aims: β-Dystroglycan (β-DG) is a transmembrane glycoprotein that links the intracellular cytoskeleton to the extracellular matrix and is crucial for the molecular pathway of lateral force transmission in muscle. We aimed to investigate the effect of decreasing sarcolemmal cholesterol on the distribution of β-DG, its interaction with dystrophin and the impact on the contraction efficiency of muscle. Methods: Isolated rat extensor digitorum longus muscles were incubated with methyl β-cyclodextrin (MβCD) to deplete cholesterol and with MβCD-cholesterol to restore cholesterol. Electric stimulation protocols were used to determine muscle force and fatigue. Detergent-resistant membranes (lipid rafts) were separated from isolated skeletal muscle sarcolemma. The distribution and interactions of β-DG, caveolin-3 and dystrophin were determined by an immunoreactivity analysis. Results: Cholesterol depletion in muscle results in a weakened force of contraction, which recovers after cholesterol restoration. The rate of fatigue is unaffected, but fatigue recovery is dependent upon cholesterol restoration. MβCD modifies the structures of lipid rafts obtained from MβCD-treated muscles by, displacing the membrane proteins β-DG and caveolin-3 f from the lipid raft, thus reducing the interaction of β-DG with dystrophin. Conclusion: Cholesterol depletion weakens the muscle contractile force by disturbing the sarcolemmal distribution of β-dystroglycan and its interaction with dystrophin, two key proteins in the alignment of lateral force transmission pathway.

Absolute amounts and diffusibility of HSP72, HSP25, and αB-crystallin in fast- and slow-twitch skeletal muscle fibers of rat

Heat shock proteins (HSPs) are essential for normal cellular stress responses. Absolute amounts of HSP72, HSP25, and αB-crystallin in rat extensor digitorum longus (EDL) and soleus (SOL) muscle were ascertained by quantitative Western blotting to better understand their respective capabilities and limitations. HSP72 content of EDL and SOL muscle was only ∼1.1 and 4.6 μmol/kg wet wt, respectively, and HSP25 content approximately twofold greater (∼3.4 and ∼8.9 μmol/kg, respectively). αB-crystallin content of EDL muscle was ∼4.9 μmol/kg but in SOL muscle was ∼30-fold higher (∼140 μmol/kg). To examine fiber heterogeneity, HSP content was also assessed in individual fiber segments; every EDL type II fiber had less of each HSP than any SOL type I fiber, whereas the two SOL type II fibers examined were indistinguishable from the EDL type II fibers. Sarcolemma removal (fiber skinning) demonstrated that 10-20% of HSP25 and αB-crystallin was sarcolemma-associated in SOL fibers. HSP diffusibility was assessed from the extent and rate of diffusion out of skinned fiber segments. In unstressed SOL fibers, 70-90% of each HSP was readily diffusible, whereas ∼95% remained tightly bound in fibers from SOL muscles heated to 45°C. Membrane disruption with Triton X-100 allowed dispersion of HSP72 and sarco(endo)plasmic reticulum Ca(2+)-ATPase pumps but did not alter binding of HSP25 or αB-crystallin. The amount of HSP72 in unstressed EDL muscle is much less than the number of its putative binding sites, whereas SOL type I fibers contain large amounts of αB-crystallin, suggesting its importance in normal cellular function without upregulation.