Rat Extensor Digitorum Longus Muscles Research Articles

Mechanism for exercise-mediated prevention against muscle wasting on extensor digitorum longus muscle in Spontaneously Diabetic Torii fatty rats

We previously reported the significant increase in limb muscle strength and cross-sectional area of the type IIb muscle fibers in the extensor digitorum longus (EDL) muscle in a type 2 diabetic animal model, with Spontaneously Diabetic Torii (SDT) fatty rats (n = 6) undergoing regular treadmill exercise from 8 to 16 weeks of age compared with sedentary SDT fatty rats (n = 6). This study investigated the mechanism by which exercise training prevented skeletal muscle wasting in the EDL muscle of the SDT fatty rats. The endurance exercise for 8 weeks downregulated the expression of muscle RING-finger protein-1 (an E3 ubiquitin ligase) and upregulated the expression of CD31, insulin receptor substrate-2, and phosphorylated endothelial nitric oxide synthase in the EDL muscle of 16-week-old SDT fatty rats.Endurance exercise training might reduce muscle wasting by preventing muscle degradation and increasing the angiogenic response in the EDL muscle in type 2 diabetes.

Force potentiation during eccentric contractions in rat skeletal muscle.

Postactivation potentiation refers to an acute enhancement of contractile properties following muscle activity. Previously, the effects of prior muscle activation on eccentric force at tetanic activation frequencies have only been sparsely reported. This paper aimed to study acute activity-induced effects on eccentric force of slow and fast-twitch muscles and characterize them in relation to postactivation potentiation. We elicited eccentric contractions in isolated rat extensor digitorum longus and soleus muscles by actively lengthening muscles at a constant velocity. We assessed contractile properties by measuring force over shortly interspaced, identical eccentric, and isometric contractions. We then analyzed stretch force, isometric peak force, rate of force development, and relaxation times. Finally, we compared the time courses for the development and cessation of changes in stretch force to known features of postactivation potentiation. In extensor digitorum longus, muscles stretch force consistently increased in a contraction-to-contraction manner by up to 49% [95% confidence interval (CI): 35-64%] whereas isometric peak force simultaneously showed minor declines (8%, 95% CI: 5-10%). The development and cessation of eccentric force potentiation coincided with the development of twitch potentiation and increases in rate of force development. In soleus muscles we found no consistent eccentric potentiation. Characterization of the increase in eccentric force revealed that force only increased in the very beginning of an active stretch. Eccentric force at tetanic activation frequencies potentiates substantially in extensor digitorum longus muscles over consecutive contractions with a time course coinciding with postactivation potentiation. Such eccentric potentiation may be important in sport performance.NEW & NOTEWORTHY Force during eccentric contractions can increase to a magnitude that may have profound consequences for our understanding of skeletal muscle locomotion. This increase in eccentric force occurs over consecutive, shortly interspaced, tetanic contractions in rat extensor digitorum longus muscles-not in rat soleus muscles-and coincides with well-known traits of postactivation potentiation. Eccentric force potentiation may significantly enhance muscle performance in activities involving stretch-shortening cycles.

Thick filament activation is different in fast- and slow-twitch skeletal muscle.

The contractile properties of fast-twitch and slow-twitch skeletal muscles are primarily determined by the myosin isoform content and modulated by a variety of sarcomere proteins. X-ray diffraction studies of regulatory mechanisms in muscle contraction have focused predominately on fast- or mixed-fibre muscle with slow muscle being much less studied. Here, we used time-resolved X-ray diffraction to investigate the dynamic behaviour of the myofilament proteins in relatively pure slow-twitch-fibre rat soleus (SOL) and pure fast-twitch-fibre rat extensor digitorum longus (EDL) muscle during twitch and tetanic contractions at optimal length. During twitch contractions the diffraction signatures indicating a transition in the myosin heads from ordered OFF states, where heads are held close to the thick filament backbone, to disordered ON states, where heads are free to bind to thin filaments, were found in EDL and not in SOL muscle. During tetanic contraction, changes in the disposition of myosin heads as active tension develops is a quasi-stepwise process in EDL muscle whereas in SOL muscle this relationship appears to be linear. The observed reduced extensibility of the thick filaments in SOL muscle as compared to EDL muscles indicates a molecular basis for this behaviour. These data indicate that for the EDL, thick filament activation is a cooperative strain-induced mechano-sensing mechanism, whereas for the SOL, thick filament activation has a more graded response. These different approaches to thick filament regulation in fast- and slow-twitch muscles may be adaptations for short-duration, strong contractions versus sustained, finely controlled contractions, respectively. KEY POINTS: Fast-twitch muscle and slow-twitch muscle are optimized for strong, short-duration contractions and for tonic postural activity, respectively. Structural events (OFF to ON transitions) in the myosin-containing thick filaments in fast muscle help determine the timing and strength of contractions, but these have not been studied in slow-twitch muscle. The X-ray diffraction signatures of structural OFF to ON transitions are different in fast extensor digitorum longus (EDL) and slow soleus (SOL) muscle, being completely absent during twitches in soleus muscle and blunted during tetanic contractions SOL as compared to EDL Quasi-stepwise thick filament structural OFF to ON transitions in fast twitch muscle may be an adaptation for rapid, ballistic movements, whereas more graded OFF to ON structural transitions in slow-twitch muscle may be an adaptation for slower, finer motions.

Heterogeneity in form and function of the rat extensor digitorum longus motor unit.

The motor unit comprises a variable number of muscle fibres that connect through myelinated nerve fibres to a motoneuron (MN), the central drivers of activity. At the simplest level of organisation there exist phenotypically distinct MNs that activate corresponding muscle fibre types, but within an individual motor pool there typically exists a mixed population of fast and slow firing MNs, innervating groups of Type II and Type I fibres, respectively. Characterising the heterogeneity across multiple levels of motor unit organisation is critical to understanding changes that occur in response to physiological and pathological perturbations. Through a comprehensive assessment of muscle histology and ex vivo function, mathematical modelling and neuronal tracing, we demonstrate regional heterogeneities at the level of the MN, muscle fibre type composition and oxygen delivery kinetics of the rat extensor digitorum longus (EDL) muscle. Specifically, the EDL contains two phenotypically distinct regions: a relatively oxidative medial and a more glycolytic lateral compartment. Smaller muscle fibres in the medial compartment, in combination with a greater local capillary density, preserve tissue O2 partial pressure (PO2) during modelled activity. Conversely, capillary supply to the lateral compartment is calculated to be insufficient to defend active muscle PO2 but is likely optimised to facilitate metabolite removal. Simulation of in vivo muscle length change and phasic activation suggest that both compartments are able to generate similar net power. However, retrograde tracing demonstrates (counter to previous observations) that a negative relationship between soma size and C‐bouton density exists. Finally, we confirm a lack of specificity of SK3 expression to slow MNs. Together, these data provide a reference for heterogeneities across the rat EDL motor unit and re‐emphasise the importance of sampling technique.

Localized Oxygen Exchange Platform for Intravital Video Microscopy Investigations of Microvascular Oxygen Regulation.

Intravital microscopy has proven to be a powerful tool for studying microvascular physiology. In this study, we propose a gas exchange system compatible with intravital microscopy that can be used to impose gas perturbations to small localized regions in skeletal muscles or other tissues that can be imaged using conventional inverted microscopes. We demonstrated the effectiveness of this system by locally manipulating oxygen concentrations in rat extensor digitorum longus muscle and measuring the resulting vascular responses. A computational model of oxygen transport was used to partially validate the localization of oxygen changes in the tissue, and oxygen saturation of red blood cells flowing through capillaries were measured as a surrogate for local tissue oxygenation. Overall, we have demonstrated that this approach can be used to study dynamic and spatial responses to local oxygen challenges to the microenvironment of skeletal muscle.

Evidence for role of capillaries in regulation of skeletal muscle oxygen supply.

We propose individual capillary units can regulate their own O2 supply by direct communication to upstream arterioles via electrically coupled endothelium. To test this hypothesis, we developed a transparent micro-exchange device for localized O2 exchange with surface capillaries of intact tissue. The device was fabricated with an O2 permeable micro-outlet 0.2×1.0mm. Experiments were performed on rat extensor digitorum longus (EDL) muscle using dual wavelength video microscopy to measure capillary hemodynamics and erythrocyte SO2 . Responses to local O2 perturbations were measured with only capillaries positioned over the micro-outlet. Step changes in the gas mixture %O2 caused physiological changes in erythrocyte SO2 , and appropriate changes in flow to offset the O2 challenge if at least 3-4 capillaries were stimulated. These results support our hypothesis that individual capillary units play a role in regulating their erythrocyte supply in response to a changing O2 environment.

Concomitant excitation and tension development are required for myocellular gene expression and protein synthesis in rat skeletal muscle.

Loading-induced tension development is often assumed to constitute an independent cue to initiate muscle protein synthesis following resistance exercise. However, with traditional physiological models of resistance exercise, changes in loading-induced tension development also reflect changes in neural activation patterns, and direct evidence for a mechanosensitive mechanism is therefore limited. Here, we sought to examine the importance of excitation and tension development per se on initiation of signalling, gene transcription and protein synthesis in rat skeletal muscle. Isolated rat extensor digitorum longus muscles were allocated to the following interventions: (a) Excitation-induced eccentric contractions (ECC); (b) Passive stretching without excitation (PAS); (c) Excitation with inhibition of contractions (STIM+IMA ) and; (d) Excitation in combination with both inhibition of contractions and PAS (STIM+IMA +PAS). Assessment of transcriptional and translational signalling, gene transcription and acute muscle protein synthesis was compared in stimulated vs contra-lateral non-stimulated control muscle. Protein synthesis increased solely in muscles subjected to a combination of excitation and tension development (ECC and STIM+IMA +PAS). The same pattern was true for p38 mitogen-activated protein kinase signalling for gene transcription as well as for gene transcription of immediate early genes FOS and JUN. In contrast, mechanistic target of rapamycin Complex 1 signalling for translation initiation increased in all muscles subjected to increased tension development (ECC and STIM+IMA +PAS as well as PAS). The current study suggests that exercise-induced increases in protein synthesis as well as transcriptional signalling is dependent on the concomitant effect of excitation and tension development, whereas signalling for translation initiation is only dependent of tension development per se.

Principles of the Mechanism for Epimuscular Myofascial Loads Leading to Non-uniform Strain Distributions Along Muscle Fiber Direction: Finite Element Modeling.

Sarcomere lengths and their changes are key determinants of muscle active force production. Recent studies indicate inhomogeneity of sarcomere lengths within the muscle. Studies utilizing magnetic resonance imaging (MRI) analyses for quantifying local muscle tissue strains and diffusion tensor imaging (DTI) analyses allowing for determination of their components along muscle fascicles show that those length changes can be non-uniform. Specifically, two questions arise regarding the muscle’s length change heterogeneities along the muscle fiber direction: (1) How can a passively lengthened muscle show shortened regions? (2) How can an isometric contracting muscle show lengthened parts? Using finite element modeling and studying principles of the mechanism of strain heterogeneity along the muscle fiber direction, the aim was to test the following hypothesis: epimuscular myofascial loads can lead locally to strains opposing those elsewhere within the muscle that are determined by the globally imposed conditions. The geometry of the model was defined by the contour of a longitudinal slice of the rat extensor digitorum longus (EDL) muscle belly. Three models were studied: (1) isolated muscle (muscle modeled fully isolated from its surroundings) and models aiming at representing the principles of a muscle in its in vivo context including (2) extramuscularly connected muscle (muscle’s connections to non-muscular structures are modeled exclusively) and (3) epimuscularly connected muscle (additionally muscle’s connections to neighboring muscle are modeled). Three cases were studied: passive isometric muscle with imposed relative position change (Case I), passive lengthened muscle (Case II), and active isometric muscle with imposed relative position change (Case III). The findings indicated non-uniform strains for all models except for zero strain in model (1) in Case I, but models (2) and (3) also showed strains opposing the imposed effect. Case I: model (3) showed shortened and lengthened sections (up to 35.3%), caused exclusively by imposed relative position change. Case II: models (2) and (3) showed shortened sections (up to 12.7 and 19.5%, respectively) in addition to lengthened sections. Case III: models (2) and (3) showed lengthened sections (up to 5 and 23.4%, respectively) in addition to shortened sections. These effects get more pronounced with stiffer epimuscular connections. Assessments of forces exerted on the muscle by the epimuscular connections showed that such strain heterogeneities are ascribed to epimuscular myofascial loads determined by muscle relative position changes.

Inhibition of glycogenolysis prolongs action potential repriming period and impairs muscle function in rat skeletal muscle.

Muscle glycogen content is associated with muscle function, but the physiological link between the two is poorly understood. This study investigated the effects of inhibiting glycogenolysis, while maintaining high overall energy status, on different aspects of muscle function. We demonstrate here that Na+ ,K+ -ATPase activity depends on glycogenolytically derived ATP regardless of high global ATP, with a decrease in activity leading to reduced force production and accelerated fatigue development. The results support the concept of compartmentalized energy transfer with glycogen metabolism playing a crucial role in intramuscular ATP resynthesis and ion regulation. This study gives specific insights into muscular function and may help towards a better understanding of glycogen storage diseases and muscle fatigue. Skeletal muscle glycogen content is associated with muscle function and fatigability. However, little is known about the physiological link between glycogen content and muscle function. Here we aimed to investigate the importance of glycogenolytically derived ATP per se on muscle force and action potential (AP) repriming period, i.e. the time before a second AP can be produced (indicative of Na+ ,K+ -ATPase activity). Single fibres from rat extensor digitorum longus muscles were isolated and mechanically skinned in order to investigate force production and the AP repriming period while global ATP and PCr concentrations were kept high. The importance of glycogenolytically derived ATP was studied by inhibition of glycogen phosphorylase (1,4-dideoxy-1,4-imino-d-arabinitol (DAB; 2mm) or CP-316,819 (CP; 10µm)) or glycogen removal (amyloglucosidase, 20Uml-1 ). Tetanic force decreased by (mean (SD)) 21(15)% (P<0.001) and 76(28)% (DAB) or 94(6)% (CP, P<0.001) in well-polarized and partially depolarized fibres, respectively. In depolarized fibres, twitch force decreased by 16(10)% and 55(26)% with DAB and CP, respectively, with no effect in well-polarized fibres (84(10)%, P=0.14). There was no effect of glycogen phosphorylase inhibition on repriming period in well-polarized fibres (median (25th, 75th percentile): 5(4,5) vs. 4(4,5)ms, P=0.26), while the repriming period was prolonged from 6(5,7) to 8(7,10)ms (P=0.01) in partially depolarized fibres. In line with this, glycogen removal increased repriming period from 5(5,6) to 6(5,7)ms (P=0.003) in depolarized fibres. Together, these data strongly indicate that blocking glycogenolysis attenuates Na+ ,K+ -ATPase activity, which in turn increases the repriming period and reduces force, demonstrating a functional link between glycogenolytically derived ATP and force production.

Shark liver oil consumption decreases contractility in EDL muscle of trained rats

Abstract Introduction: Professional and recreational athletes make daily use of nutritional supplements to improve physical performance. Polyunsaturated fatty acids (PUFAs) have been used in this sense. N-3 PUFA, particularly eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids are involved in important physiological functions and the benefits of supplementation are demonstrated in several types of users. Shark liver oil (SLO) is a natural source of n-3 PUFA. Objective: To evaluate the effect of supplementation with SLO on contractility of skeletal muscles with different metabolic characteristics, soleus and extensor digitorum longus (EDL) from rats submitted to eight weeks of interval training of progressive intensity on a motorized treadmill. In the supplemented group, animals were supplemented with SLO (1 g/kg) five times a week for eight weeks. Method: Contractile parameters as maximum isometric twitch force (Tmax), maximum speed of force development (+dF/dt), maximum speed of force decrease (-dF/dt), maximum tetanic force (Fmax) and resistance to fatigue were analyzed in isolated muscle. Results: Compared to the control group, EDL muscles from the supplemented group reduced Tmax at the first (10.82 ± 0.89 vs 14.30 ± 0.67 mN/mm2. p &lt; 0.01) and second minutes of experimentation (9.85 ± 0.63 vs 13.12 ± 0.70 mN/mm2. p &lt; 0.01). However, it increased resistance to fatigue (22.80 ± 0.97 vs 18.60 ± 0.51 seconds. p = 0.005). Conclusion: No difference was observed in the soleus muscle.

Hyperinsulinemia does not cause de novo capillary recruitment in rat skeletal muscle.

ObjectiveThe effect of insulin on blood flow distribution within muscle microvasculature has been suggested to be important for glucose metabolism. However, the “capillary recruitment” hypothesis is still controversial and relies on studies using indirect contrast‐enhanced ultrasound (CEU) methods.MethodsWe studied how hyperinsulinemia effects capillary blood flow in rat extensor digitorum longus (EDL) muscle during euglycemic hyperinsulinemic clamp using intravital video microscopy (IVVM). Additionally, we modeled blood flow and microbubble distribution within the vascular tree under conditions observed during euglycemic hyperinsulinemic clamp experiments.ResultsEuglycemic hyperinsulinemia caused an increase in erythrocyte (80 ± 25%, P < .01) and plasma (53 ± 12%, P < .01) flow in rat EDL microvasculature. We found no evidence of de novo capillary recruitment within, or among, capillary networks supplied by different terminal arterioles; however, erythrocyte flow became slightly more homogenous. Our computational model predicts that a decrease in asymmetry at arteriolar bifurcations causes redistribution of microbubble flow among capillaries already perfused with erythrocytes and plasma, resulting in 25% more microbubbles flowing through capillaries.ConclusionsOur model suggests increase in CEU signal during hyperinsulinemia reflects a redistribution of arteriolar flow and not de novo capillary recruitment. IVVM experiments support this prediction showing increases in erythrocyte and plasma flow and not capillary recruitment.

Changes in local capillarity of pure and hybrid MyHC muscle fiber types after nerve injury in rat extensor digitorum longus muscle (EDL).

Recently, we evaluated capillary indices without discrimination by fiber type in rat extensor digitorum longus muscle (EDL) 4weeks after nerve cut (NC), after double nerve crush (double NCR) and in two controls, from the start (CON-1) and the end (CON-2) of the experiment. In the present study, we determined the capillary indices related to specific myosin heavy chain (MyHC) fiber types. Fiber-type composition and local capillarity were assessed from a single, composite, multicolor image, where different MyHC-fiber types and capillaries were shown simultaneously. Applying local capillary indices [the number of capillaries around fiber (CAF) and the CAF scaled to fiber perimeter (CAF/FP)], to specific MyHC-fiber types, we found changes relevant to neuro-muscular studies. In the NC group, only type-2x fibers had a significantly lower CAF, and in the double NCR group, only type-2a fibers had a higher CAF in comparison with both controls. Both types of nerve injury elicited two responses: a coupled regulation of fiber size and capillarity in the oxidative, type 2a fibers and a capillarity independent regulation of fiber size in the glycolytic type-2b fibers. All subtypes of type-2 fibers had a better capillary supply (higher CAF/FP) in the NC and double NCR than in CON-2. The highest improvement was observed in type-2b fibers; this change was mirrored in an oxidative shift only in the double NCR group. Adopting fiber-type-specific capillary indices improves data analysis of rat EDL muscle samples.

Increased Expression of miR-1 in Fast-Twitch Skeletal Muscle in Response to Resistance Exercise

Background: The response of microRNA (miR-1), upstream regulators (Myogenic Differentiation 1 (MyoD1) and Myocyte Enhancer Factor 2C (Mef2C)), and a downstream target (Histone Deacetylase 4 (Hdac4)) to an acute bout of Resistance Exercise (RE) in Extensor Digitorum Longus (EDL) and soleus muscles has remained elusive. Objectives: In this experimental study, we investigated the effect of an acute bout of RE on the expression of miR-1, MyoD1, Mef2C, and Hdac4 genes in the slow and fast-twitch muscle of rats. Methods: The current study was conducted at Tarbiat Modares University, Tehran, Iran, in 2017. Fifteen male Wistar rats were randomly divided into two groups, control (n = 5) and RE (n = 10). The RE protocol consisted of four sets of five repetitions of climbing a ladder with weights attached to the tails of rats. The soleus and EDL muscles of rats were collected at 3 h (n = 5) and 6 h (n = 5) post-RE. The real-time polymerase chain reaction was used to measure the levels of miR-1, MyoD1, Mef2C, and Hdac4 mRNA expression. Results: The miR-1 expression in EDL muscle was significantly lower at 3 h and 6 h post-exercise in the RE group than in the control group (P < 0.01). The miR-1 expression in soleus muscle was significantly lower at 3 h post-exercise in the RE group than in the control group (P < 0.007) but it was significantly higher at 6 h post-exercise (P < 0.036). The expression of MyoD1, Mef2C, and Hdac4 genes in the EDL muscle was higher in the RE group than in the control group (P < 0.01). The expression of these genes in response to RE had more fluctuations in soleus muscle. Conclusions: It can be concluded that miR-1 expression in extensor digitorum longus and soleus muscles of rats responds to resistance exercise in different manners and this coincides with a change in upstream regulators and downstream target.

Contractile benefits of doublet-initiated low-frequency stimulation in rat extensor digitorum longus muscle exposed to high extracellular [K+] or fatiguing contractions.

During dynamic contractions, high-frequency muscle activation is needed to achieve optimal power. This must be balanced against an increased excitation-induced accumulation of extracellular K+, which can reduce excitability and ultimately may prevent adequate responses to high-frequency activation. Mean activation frequencies in vivo are often low (subtetanic), but activation patterns contain bursts of high (supratetanic) frequencies known as doublets, which enhance dynamic contraction in rested muscles at normal extracellular K+ concentration ([K+]o). Here, we examine how dynamic contractions in fast-twitch fibers stimulated by high frequency/doublets are affected during exposure to 11 mM [K+]o and during fatigue. Dynamic contractions were elicited by electrical stimulation in isolated rat extensor digitorum longus muscles incubated at 4 or 11 mM K+. When stimulation frequency was maintained constant, an increase from 150 to 300 Hz enhanced maximal power (Pmax), maximal velocity (Vmax), and rate of force development (RFD) at 4 mM K+ but only Vmax at 11 mM K+. With the use of subtetanic frequency trains (50 Hz) with or without an initiating doublet (300 Hz), the addition of a doublet increased maximal force, Pmax, Vmax, and RFD at both 4 and 11 mM K+. Furthermore, a work-matched fatiguing protocol was performed comparing a doublet-initiated subtetanic train (DT) of 60 Hz with a constant-frequency train (CFT) of 71 Hz during 100 dynamic contractions. We found that DT produced higher power, velocity, and RFD than CFT throughout the fatiguing protocol. The results indicate that doublets enhance dynamic contraction in fast-twitch muscles stimulated at subtetanic frequency during both normal and fatiguing conditions.

Phosphodiesterase 4B knockout prevents skeletal muscle atrophy in rats with burn injury.

The phosphodiesterase 4 (PDE4)-cAMP pathway plays a predominant role in mediating skeletal muscle proteolysis in burn injury. The present investigations to determine the PDE4 isoform(s) involved in this action revealed that burn injury increased the expression of rat skeletal muscle PDE4B mRNA by sixfold but had little or no effect on expression of other PDE4 isoforms. These observations led us to study the effects of burn in PDE4B knockout (KO) rats. As reported by us previously, burn injury significantly increased extensor digitorum longus (EDL) muscle total and myofibrillar proteolysis in wild-type (WT) rats, but there were no significant effects on either total or myofibrillar protein breakdown in EDL muscle of PDE4B KO rats with burn injury. Moreover, burn injury increased PDE4 activity in the skeletal muscle of WT rats, but this was reduced by >80% in PDE4B KO rats. Also, burn injury decreased skeletal muscle cAMP concentration in WT rats but had no significant effects in the muscles of PDE4B KO rats. Incubation of the EDL muscle of burn-PDE4B KO rats with an inhibitor of the exchange factor directly activated by cAMP, but not with a protein kinase A inhibitor, eliminated the protective effects of PDE4B KO on EDL muscle proteolysis and increased muscle proteolysis to the same extent as in the EDL of burn-WT rats. These novel findings confirm a major role for PDE4B in skeletal muscle proteolysis in burn injury and suggest that an innovative therapy based on PDE4B-selective inhibitors could be developed to treat skeletal muscle cachexia in burn injury without the fear of causing emesis, which is associated with PDE4D inhibition.

Sporadic amyotrophic lateral sclerosis (SALS) - skeletal muscle response to cerebrospinal fluid from SALS patients in a rat model.

ABSTRACTSkeletal muscle atrophy is the most prominent feature of amyotrophic lateral sclerosis (ALS), an adult-onset neurodegenerative disease of motor neurons. However, the contribution of skeletal muscle to disease progression remains elusive. Our previous studies have shown that intrathecal injection of cerebrospinal fluid from sporadic ALS patients (ALS-CSF) induces several degenerative changes in motor neurons and glia of neonatal rats. Here, we describe various pathologic events in the rat extensor digitorum longus muscle following intrathecal injection of ALS-CSF. Adenosine triphosphatase staining and electron microscopic (EM) analysis revealed significant atrophy and grouping of type 2 fibres in ALS-CSF-injected rats. Profound neuromuscular junction (NMJ) damage, such as fragmentation accompanied by denervation, were revealed by α-bungarotoxin immunostaining. Altered expression of key NMJ proteins, rapsyn and calpain, was also observed by immunoblotting. In addition, EM analysis showed sarcolemmal folding, Z-line streaming, structural alterations of mitochondria and dilated sarcoplasmic reticulum. The expression of trophic factors was affected, with significant downregulation of vascular endothelial growth factor (VEGF), marginal reduction in insulin-like growth factor-1 (IGF-1), and upregulation of brain-derived neurotrophic factor (BDNF) and glial-derived neurotrophic factor (GDNF). However, motor neurons might be unable to harness the enhanced levels of BDNF and GDNF, owing to impaired NMJs. We propose that ALS-CSF triggers motor neuronal degeneration, resulting in pathological changes in the skeletal muscle. Muscle damage further aggravates the motor neuronal pathology, because of the interdependency between them. This sets in a vicious cycle, leading to rapid and progressive loss of motor neurons, which could explain the relentless course of ALS.This article has an associated First Person interview with the first author of the paper.

Regional variation in the mechanical properties and fibre-type composition of the rat extensor digitorum longus muscle.

What is the central question of this study? Mammalian muscle is typically heterogeneous in fibre-type distribution, with distinct regional variation in composition. The effects this might have on mechanical performance are largely unknown. What is the main finding and its importance? Contractile properties vary regionally within a heterogeneous muscle. The mixed extensor digitorum longus muscle has phenotypically distinct compartments that differ in their isometric twitch kinetics, the optimal cycle frequency for maximal power generation and fatigue resistance. The mechanisms underpinning the decline in performance during fatigue differ between compartments. Regional variation in mechanical performance suggests that regions of the extensor digitorum longus muscle might be differentially recruited during locomotion, depending upon functional demand. Fibre-type composition is heterogeneous, and distribution varies spatially in many muscles, indicating that there might be regional variation in recruitment and mechanical output. The rat extensor digitorum longus muscle is composed of predominantly fast-twitch fibres and exhibits a gradient in phenotype, resulting in oxidative medial (areal composition 24.3% type I/IIa) and glycolytic lateral (92.4% type IIx/IIb) compartments. Here, we investigated the variation in mechanical performance between the medial and lateral compartments during isometric, isotonic and cyclical contractions. Isometric tetanic stress and force-velocity relationships were similar in both compartments, but isometric twitch kinetics were slower in the medial compared with the lateral compartment. The medial compartment also had a lower optimal cycle frequency for maximal net power generation (11 versus 15Hz; P<0.05) attributable to slower isometric kinetics, resulting in a lower level of activation and reduced net work generation at higher cycle frequencies, compared with the lateral compartment. The more oxidative, medial compartment had higher fatigue resistance, maintaining net power 26% longer than the lateral compartment. The predominant mechanisms underpinning the decrease in net power varied between the compartments, resulting from an increase in the work to extend the muscle and from a reduction in work during shortening in the medial and lateral compartments, respectively. Regional variation in mechanical performance and resistance to fatigue within a mixed muscle suggests that a differential recruitment pattern is likely during locomotion, with the medial compartment being used during slow-speed locomotion and the lateral compartment during burst activities.

Segmental Distribution of Myosin Heavy Chain Isoforms Within Single Muscle Fibers.

Despite many studies looking at the distribution of myosin heavy chain (MHC) isoforms across a transverse section of muscle, knowledge of MHC distribution along the longitudinal axis of a single skeletal muscle fiber has been relatively overlooked. Immunocytochemistry was performed on serial sections of rat extensor digitorum longus (EDL) muscle to identify MHC types I, IIA, IIX, IIY, and IIB. Sixteen fascicles which contained a total of 362 fibers were randomly and systematically sampled from the three EDL muscles. All MHC type I and type II isoforms were expressed. Segmental expression occurred within a very limited segment. MHC isoform expression followed the accepted traditional order from I⇔IIA⇔IIX⇔IIB, however, in some samples expression of an isoform was circumvented from IIB to I or from I to IIB directly. Segmental distribution of MHC isoforms along a single muscle fiber may be because of the myonuclear domain. Anat Rec, 300:1636-1642, 2017. © 2017 Wiley Periodicals, Inc.

Role of calpain in eccentric contraction-induced proteolysis of Ca2+-regulatory proteins and force depression in rat fast-twitch skeletal muscle.

The aim of this study was to examine the in vivo effects of eccentric contraction (ECC) on calpain-dependent proteolysis of Ca2+-regulatory proteins and force production in fast-twitch skeletal muscles. Rat extensor digitorum longus muscles were exposed to 200 repeated ECC in situ and excised immediately [recovery 0 (REC0)] or 3 days [recovery 3 (REC3)] after cessation of ECC. Calpain inhibitor (CI)-treated rats were intraperitoneally injected with MDL-28170 before ECC and during REC3. Tetanic force was markedly reduced at REC0 and remained reduced at REC3. CI treatment ameliorated the ECC-induced force decline but only at REC3. No evidence was found for proteolysis of dihydropyridine receptor (DHPR), junctophilin (JP)1, JP2, ryanodine receptor (RyR), sarcoplasmic reticulum Ca2+-ATPase (SERCA)1a, or junctional face protein-45 at REC0. At REC3, ECC resulted in decreases in DHPR, JP1, JP2, RyR, and SERCA1a. CI treatment prevented the decreases in DHPR, JP1, and JP2, whereas it had little effect on RyR and SERCA1a. These findings suggest that DHPR, JP1, and JP2, but not RyR and SERCA1a, undergo calpain-dependent proteolysis in in vivo muscles subjected to ECC and that impaired function of DHPR and/or JP might cause prolonged force deficits with ECC.NEW & NOTEWORTHY Calpain-dependent proteolysis is one of the contributing factors to muscle damage that occurs with eccentric contraction (ECC). It is unclear, however, whether calpains account for proteolysis of Ca2+-regulatory proteins in in vivo muscles subjected to ECC. Here, we provide evidence that dihydropyridine receptor and junctophilin, but not ryanodine receptor and sarcoplasmic reticulum Ca2+-ATPase, undergo calpain-dependent proteolysis.

Higher insulin sensitivity in EDL muscle of rats fed a low-protein, high-carbohydrate diet inhibits the caspase-3 and ubiquitin-proteasome proteolytic systems but does not increase protein synthesis

Compared with the extensor digitorum longus (EDL) muscle of control rats (C), the EDL muscle of rats fed a low-protein, high-carbohydrate diet (LPHC) showed a 36% reduction in mass. Muscle mass is determined by the balance between protein synthesis and proteolysis; thus, the aim of this work was to evaluate the components involved in these processes. Compared with the muscle from C rats, the EDL muscle from LPHC diet-fed rats showed a reduction (34%) in the in vitro basal protein synthesis and a 22% reduction in the in vitro basal proteolysis suggesting that the reduction in the mass can be associated with a change in the rate of the two processes. Soon after euthanasia, in the EDL muscles of the rats fed the LPHC diet for 15days, the activity of caspase-3 and that of components of the ubiquitin-proteasome system (atrogin-1 content and chymotrypsin-like activity) were decreased. The phosphorylation of p70S6K and 4E-BP1, proteins involved in protein synthesis, was also decreased. We observed an increase in the insulin-stimulated protein content of p-Akt. Thus, the higher insulin sensitivity in the EDL muscle of LPHC rats seemed to contribute to the lower proteolysis in LPHC rats. However, even with the higher insulin sensitivity, the reduction in p-E4-BP1 and p70S6K indicates a reduction in protein synthesis, showing that factors other than insulin can have a greater effect on the control of protein synthesis.