Extensor Digitorum Longus Research Articles

Regulation of injury-induced skeletal myofiber regeneration by glucose transporter 4 (GLUT4)

BackgroundInsulin resistance and type 2 diabetes impair cellular regeneration in multiple tissues including skeletal muscle. The molecular basis for this impairment is largely unknown. Glucose uptake via glucose transporter GLUT4 is impaired in insulin resistance. In healthy muscle, acute injury stimulates glucose uptake. Whether decreased glucose uptake via GLUT4 impairs muscle regeneration is presently unknown. The goal of this study was to determine whether GLUT4 regulates muscle glucose uptake and/or regeneration following acute injury.MethodsTibialis anterior and extensor digitorum longus muscles from wild-type, control, or muscle-specific GLUT4 knockout (mG4KO) mice were injected with the myotoxin barium chloride to induce muscle injury. After 3, 5, 7, 10, 14, or 21 days (in wild-type mice), or after 7 or 14 days (in control & mG4KO) mice, muscles were isolated to examine [3H]-2-deoxyglucose uptake, GLUT4 levels, extracellular fluid space, fibrosis, myofiber cross-sectional area, and myofiber centralized nuclei.ResultsIn wild-type mice, muscle glucose uptake was increased 3, 5, 7, and 10 days post-injury. There was a rapid decrease in GLUT4 protein levels that were restored to baseline at 5–7 days post-injury, followed by a super-compensation at 10–21 days. In mG4KO mice, there were no differences in muscle glucose uptake, extracellular fluid space, muscle fibrosis, myofiber cross-sectional areas, or percentage of centrally nucleated myofibers at 7 days post-injury. In contrast, at 14 days injured muscles from mG4KO mice exhibited decreased glucose uptake, muscle weight, myofiber cross sectional areas, and centrally nucleated myofibers, with no change in extracellular fluid space or fibrosis.ConclusionsCollectively, these findings demonstrate that glucose uptake via GLUT4 regulates skeletal myofiber regeneration following acute injury.

Co-culture of postnatal mouse spinal cord and skeletal muscle explants as an experimental model of neuromuscular interactions.

The intercommunication between nerves and muscles plays an important role in the functioning of our body, and its failure leads to severe neuromuscular disorders such as spinal muscular atrophy and amyotrophic lateral sclerosis. Understanding the cellular and molecular mechanisms underlying nerve-muscle interactions and mediating their mutual influence is an integral part of strategies aimed at curing neuromuscular diseases. Here, we propose a novel ex vivo experimental model for the spinal cord (SC) and skeletal muscle interactions which for the first time utilizes only fully formed (but not yet quite functional) postnatal tissues. The model represents an organotypic co-culture comprising a longitudinal slice of the mouse postnatal SC and an extensor digitorum longus (EDL) muscle explant placed in the "damage zone" of transversally dissected longitudinal slice of the SC. Using this model, we have shown that SC tissue stimulates muscle contractions and reduces the area occupied by acetylcholine receptors on muscle surface. In turn, EDL muscles stimulate the growth of SC-derived neurites. Thus, our organotypic model allows one to assess the mutual influence of neurons and muscles in a nearly natural setting which maintains the architecture and cellular composition of intact tissues. Therefore, this model may provide an effective platform for studying molecular and cellular mechanisms linked to defective neuromuscular interactions in associated pathologies.

Transcriptomic Profiling Reveals Differences in Slow-Twitch and Fast-Twitch Muscles of a Cigarette Smoke-Exposed Rat Model.

Cigarette smoking is known to affect muscle function and exercise capacity, including muscle fatigue resistance. Most studies showed diminished cross-sectional area and fibre type shifting in slow-twitch muscles such as the soleus, while effects on fast-twitch muscles were seldom reported and the differential responses between muscle types in response to exposureto cigarette smoke (CS) were largely unknown. This study aimed to elucidate the histomorphological, biochemical and transcriptomic changes induced by CS on both slow-twitch and fast-twitch muscles. Male Sprague-Dawley rats were randomly divided into two groups: sham air (SA) and CS. The rats were exposed to CS for 8 weeks using an exposure chamber system to mimic smoking conditions. Histomorphological analyses on muscle fibre type and cross-sectional area were determined in soleus and extensor digitorum longus (EDL). Transcriptomic profiles were investigated for identifying differentially expressed genes (DEGs) and potential mechanistic pathways involved. Inflammatory responses in terms of the macrophage population and the level of inflammatory cytokines were measured. Markers for muscle-specific proteolysis were also examined. Soleus muscle, but not in EDL, exhibited a significant increase in Type IIa fibres (SA: 9.0 ± 3.3%; CS: 19.8 ± 2.4%, p = 0.002) and decrease in Type I fibres (SA: 90.1 ± 3.6%; CS: 77.9 ± 3.3%, p = 0.003) after CS exposure. RNA sequencing revealed 165 identified DEGs in soleus including upregulation of 'Cd68', 'Ccl2' and 'Ucp2' as well as downregulation of 'Ucp3', etc. Pathways enrichment analysis revealed that the upregulated pathways in soleus were related to immune system and cellular response, while the downregulated pathways were related to oxidative metabolism. Only 10 DEGs were identified in EDL with less enriched pathways. The soleus also showed elevated pro-inflammatory cytokines, and the total macrophage marker CD68 was significantly higher in soleus of CS compared to the SA group (CD68+/no. of fibre: SA = 60.3 ± 39.3%; CS = 106.5 ± 27.2%, p = 0.0039), while the two groups in EDL muscle showed no significant difference. The expression of E3 ubiquitin ligase atrogin-1 associated with muscle degradation pathways was 1.63-fold higher in the soleus after CS, while no significant differences were observed in the EDL. The CS-induced inflammatory responses on soleus muscle are likely mediated via targeting mitochondrial-related signalling, resulting in mitochondrial dysfunction and impaired oxidative capacity. The presumably less active mitochondrial-related signalling in EDL renders it less susceptible to changes towards CS, accounting for differential impacts between muscle types.

Perfusion staining methods for visualization of the intact microvascular networks in whole mount skeletal muscle preparations.

Visualization of the intact microvascular network in skeletal muscle requires labeling the entire network in whole mount preparations where muscle fibre length can be set to near optimal but the tools to do this are not clear. We intravascularly injected CD-1 mice with different fluorescently labelled lectins (fluorescent isolectin GS-IB4 (ISO), wheat germ agglutinin (WGA), lycopersicon esculentum (tomato) (LYCO) or FITC-labelled gel. Soleus, extensor digitorum longus, diaphragm, gluteus maximus and cremaster muscles were excised, pinned at optimal sarcomere length and viewed using fluorescence microscopy. WGA and LYCO were the effective at labeling the entire vascular network with WGA labeling capillaries more bright. ISO labelled the arteriolar vasculature and early segments of the capillaries but not the full length of the capillaries or the venular network. FITC-labelled gel was effective at labelling the microvascular network but not all small vessels were consistently labelled. The pattern of staining for each labelling method was similar across all muscle fibre-types tested. WGA was optimal for perfusion labeling and visualization of the intact microvascular network in whole mount skeletal muscle preparations and can be used in combination with ISO to distinguish the arteriolar and venous sides of the network.

Micro‐Doses of DNP Preserve Motor and Muscle Function with a Period of Functional Recovery in Amyotrophic Lateral Sclerosis Mice

ObjectiveMitochondrial dysfunction is one of the earliest pathological events observed in amyotrophic lateral sclerosis (ALS). The aim of this study is to evaluate the therapeutic efficacy of 2,4‐dinitrophenol (DNP), a mild mitochondrial uncoupler, in an ALS mouse model to provide preclinical proof‐of‐concept evidence of using DNP as a potential therapeutic drug for ALS.MethodshSOD1G93A mice were treated with 0.5–1.0 mg/kg DNP through daily oral gavage from presymptomatic stage or disease onset until 18 weeks old. Longitudinal behavioral studies were performed weekly or biweekly from 6 to 18 weeks old. In situ muscle contraction measurements in extensor digitorum longus muscles were conducted to evaluate the preservation of contractile force and motor unit numbers in hSOD1G93A mice following DNP treatment. Muscle innervation and inflammatory markers were assessed using immunostaining. Extent of protein oxidation and activation of Akt pathway were also examined.ResultsDNP delayed disease onset; improved motor coordination and muscle performance in vivo; preserved muscle contractile function, neuromuscular junction morphology, and muscle innervation; and reduced inflammation and protein oxidation at 18 weeks old in hSOD1G93A mice. Strikingly, symptomatic hSOD1G93A mice exhibited a period of recovery in running ability at 20 cm/s several weeks after 2,4‐dinitrophenol treatment started at disease onset, offering the first observation in disease phenotype reversal using a small molecule.InterpretationOur results strongly support that micro‐dose DNP may be used as a potential novel treatment for ALS patients, with a possibility for recovery, when used at optimal doses and time of intervention. ANN NEUROL 2024

Unilateral two-headed extensor digitorum longus muscle: atypical configuration and clinical implications.

This report describes a new configuration of the extensor digitorum longus (EDL) with two heads and two main tendons divided into four slips. During routine cadaver dissection, unilateral (right) EDL muscle belly and tendon variations were identified. The morphometric measurements of the EDL were conducted using Image J software. Two bellies and two tendons of the EDL muscle were observed in the right leg of an 87-year-old male cadaver. The second belly of the EDL muscle originates 2cm below the first belly. The tendons originate at the ends of the muscle bellies. First and second tendons split into two more slips after passing through the same tunnel below the extensor retinaculum. The first tendon was divided into two tendon slips attached to the extensor expansion of the second and third toes, whereas the second tendon was divided into two tendon slips attached to the extensor expansion of the fourth and fifth toes on the dorsum of the foot. The width of the muscle belly for the tendons of 2nd and 3rd toes was 1.10 ± 0.06cm, while it was 1.39 ± 0.04cm for that of the 4th and 5th toes. The developmental variations in the EDL muscles can be asymptomatic. This variant of the EDL muscle may cause entrapment under the extensor retinaculum, potentially restricting ankle dorsiflexion. This limitation can also affect walking. Consequently, paying attention to this variance is crucial for surgical planning and interpretation of radiological scans.

Investigating in vivo force and work production of rat medial gastrocnemius at varying locomotor speeds using a muscle avatar.

Traditional work loop studies, that use sinusoidal length trajectories with constant frequencies, lack the complexities of in vivo muscle mechanics observed in modern studies. This study refines methodology of the 'avatar' method (a modified work loop) to infer in vivo muscle mechanics using ex vivo experiments with mouse extensor digitorum longus (EDL) muscles. The 'avatar' method involves using EDL muscles to replicate in vivo time-varying force, as demonstrated by previous studies focusing on guinea fowl lateral gastrocnemius (LG). The present study extends this method by using in vivo length trajectories and electromyographic activity from rat medial gastrocnemius (MG) during various gaits on a treadmill. Methodological enhancements from previous work, including adjusted stimulation protocols and systematic variation of starting length, improved predictions of in vivo time-varying force production (R2=0.80-0.96). The study confirms there is a significant influence of length, stimulation and their interaction on work loop variables (peak force, length at peak force, highest and average shortening velocity, and maximum and minimum active velocity), highlighting the importance of these interactions when muscles produce in vivo forces. We also investigated the limitations of traditional work loops in capturing muscle dynamics in legged locomotion (R2=0.01-0.71). While in vivo length trajectories enhanced force prediction, accurately predicting work per cycle remained challenging. Overall, the study emphasizes the utility of the 'avatar' method in elucidating dynamic muscle mechanics and highlights areas for further investigation to refine its application in understanding in vivo muscle function.

Repeatability of diffusion kurtosis tensor parameters in muscles of the lower legs.

The aim of this study was to provide measurements from and investigate the repeatability of diffusion kurtosis tensor parameters in the muscles of the lower legs. Test-retest acquisition of a kurtosis tensor sequence was performed in 13 healthy volunteers. Quantitative kurtosis tensor parameters were derived, and repeatability of each parameter was evaluated by muscle group and over the whole muscle through intraclass correlation coefficient (ICC) and within-subject coefficient of variation (wsCV). Bland-Altman analysis was also conducted. Differences in parameter values by muscle group were investigated through an analysis of variance. Axial kurtosis and radial kurtosis values from the test data were 0.63 ± 0.04 and 0.70 ± 0.05, respectively. Kurtosis tensor parameters from all muscle groups and over the whole muscle had wsCV below 15%. ICC for the parameters from most muscle groups was above 85%, with the lowest ICC over the whole muscle being 88.39%. The medial gastrocnemius and extensor digitorum longus showed highest repeatability. Mean, axial, and radial diffusivity had higher wsCV despite being lower-order terms than kurtosis. This study sought to examine the repeatability of diffusion kurtosis tensor-derived parameters in the legs and verify that they could potentially be used as longitudinal imaging metrics. wsCV values from test-retest data indicated high repeatability throughout all examined muscle groups. There were minimal differences in kurtosis and diffusivity parameters between muscle groups in this healthy volunteer cohort.

The importance of muscle activation on the interpretation of muscle mechanical performance.

The work loop technique was developed to assess muscle performance during cyclical length changes with phasic activation, simulating the in vivo conditions of many muscles, particularly during locomotion. To estimate muscle function in vivo, the standard approach involves subjecting a muscle to length trajectories and activation timings derived from in vivo measurements, whilst simultaneously measuring force. However, the stimulation paradigm typically used, supramaximal, 'square-wave' stimulation, does not accurately reflect the graded intensity of activation observed in vivo. While the importance of the timing and duration of stimulation within the cycle on estimates of muscle performance has long been established, the importance of graded muscle activation has not been investigated. In this study, we investigated how the activation pattern affects muscle performance by comparing square-wave, supramaximal activation with a graded in vivo activation pattern. First, we used in vivo electromyography-derived activation patterns and fibre strains from the rabbit digastric muscle during mastication and replayed them in situ. Second, we used Hill-type musculoskeletal model-derived activation patterns and fibre strains in a trotting mouse, replayed ex vivo in the soleus (SOL) and extensor digitorum longus (EDL) muscles. In the rabbit digastric muscle, square-wave activation led to an 8-fold higher estimate of net power, compared with the in vivo graded activation pattern. Similarly, in the mouse SOL and EDL, supramaximal, square-wave activation resulted in significantly greater positive and negative muscle work. These findings highlight that realistic interpretations of in vivo muscle function rely upon more accurate representations of muscle activation intensity.

Sucla2 Knock-Out in Skeletal Muscle Yields Mouse Model of Mitochondrial Myopathy With Muscle Type-Specific Phenotypes.

Pathogenic variants in subunits of succinyl-CoA synthetase (SCS) are associated with mitochondrial encephalomyopathy in humans. SCS catalyses the conversion of succinyl-CoA to succinate coupled with substrate-level phosphorylation of either ADP or GDP in the TCA cycle. This report presents a muscle-specific conditional knock-out (KO) mouse model of Sucla2, the ADP-specific beta subunit of SCS, generating a novel invivo model of mitochondrial myopathy. The mouse model was generated using the Cre-Lox system, with the human skeletal actin (HSA) promoter driving Cre-recombination of a CRISPR-Cas9-generated Sucla2 floxed allele within skeletal muscle. Inactivation of Sucla2 was validated using RT-qPCR and western blot, and both enzyme activity and serum metabolites were quantified by mass spectrometry. To characterize the model invivo, whole-body phenotyping was conducted, with mice undergoing a panel of strength and locomotor behavioural assays. Additionally, exvivo contractility experiments were performed on the soleus (SOL) and extensor digitorum longus (EDL) muscles. SOL and EDL cryosections were also subject to imaging analyses to assess muscle fibre-specific phenotypes. Molecular validation confirmed 68% reduction of Sucla2 transcript within the mutant skeletal muscle (p < 0.001) and 95% functionally reduced SUCLA2 protein (p < 0.0001). By 3 weeks of age, Sucla2 KO mice were 44% the size of controls by body weight (p < 0.0001). Mutant mice also exhibited 34%-40% reduced grip strength (p < 0.01) and reduced spontaneous exercise, spending about 88% less cumulative time on a running wheel (p < 0.0001). Contractile function was also perturbed in a muscle-specific manner; although no genotype-specific deficiencies were seen in EDL function, SUCLA2-deficient SOL muscles generated 40% less specific tetanic force (p < 0.0001), alongside slower contraction and relaxation rates (p < 0.001). Similarly, a SOL-specific threefold increase in mitochondria (p < 0.0001) was observed, with qualitatively increased staining for both COX and SDH, and the proportion of Type 1 myosin heavy chain expressing fibres within the SOL was nearly doubled (95% increase, p < 0.0001) in the Sucla2 KO mice compared with that in controls. SUCLA2 loss within murine skeletal muscle yields a model of SCS-deficient mitochondrial myopathy with reduced body weight, muscle weakness and exercise intolerance. Physiological and morphological analyses of hindlimb muscles showed remarkable differences in exvivo function and cellular consequences between the EDL and SOL muscles, with SOL muscles significantly more impacted by Sucla2 inactivation. This novel model will provide an invaluable tool for investigations of muscle-specific and fibre type-specific pathogenic mechanisms to better understand SCS-deficient myopathy.

Lower limb interosseous membrane in foetuses.

The leg interosseous membrane (LIM) stabilises the tibia and the fibula. These two bones articulate at the proximal and distal tibiofibular joints. In addition, the LIM is the place of attachment of tibialis anterior muscle, extensor digitorum longus muscle, fibularis tertius muscle (anatomical variant), tibialis posterior muscle and flexor hallucis longus muscle. The specific structure of the collagen fibre network of the LIM provides durability comprising collagenous fibres that are predominately projected longitudinally, obliquely, and often transversely. 222 human foetuses (Male: 120, Female: 102) between 117 and 197 (median 177) days of foetal life were available for the study. The material derived from the foetal collection is stored in the Department of Human Morphology and Embryology, Division of Anatomy of the Medical University of Wroclaw. In this study, we assessed the variability of the foetal LIM using a novel dyeing technique to identify the LIM syndesmotic structure. Overall, the study of the three types of interosseous fibres (transverse, oblique, longitudinal) of the right/left leg revealed that the fibres run in all three directions with frequencies approximating 60-70%. However, there were differences in the frequency of fibre directions and in the size of LIM between sexes. After consideration of the directions and size of fibres of LIM, parts of it can be used for reconstruction of the upper limb interosseous membrane. Sexually dimorphic features of the LIM in the studied material confirm the different dynamics of lower limb growth in each sex.

Compound heterozygous RYR1-RM mouse model reveals disease pathomechanisms and muscle adaptations to promote postnatal survival.

Pathogenic variants in the type I ryanodine receptor (RYR1) result in a wide range of muscle disorders referred to as RYR1-related myopathies (RYR1-RM). We developed the first RYR1-RM mouse model resulting from co-inheritance of two different RYR1 missense alleles (Ryr1TM/SC-ΔL mice). Ryr1TM/SC-ΔL mice exhibit a severe, early onset myopathy characterized by decreased body/muscle mass, muscle weakness, hypotrophy, reduced RYR1 expression, and unexpectedly, incomplete postnatal lethality with a plateau survival of ~50% at 12 weeks of age. Ryr1TM/SC-ΔL mice display reduced respiratory function, locomotor activity, and invivo muscle strength. Extensor digitorum longus muscles from Ryr1TM/SC-ΔL mice exhibit decreased cross-sectional area of type IIb and type IIx fibers, as well as a reduction in number of type IIb fibers. Exvivo functional analyses revealed reduced Ca2+ release and specific force production during electrically-evoked twitch stimulation. In spite of a ~threefold reduction in RYR1 expression in single muscle fibers from Ryr1TM/SC-ΔL mice at 4 weeks and 12 weeks of age, RYR1 Ca2+ leak was not different from that of fibers from control mice at either age. Proteomic analyses revealed alterations in protein synthesis, folding, and degradation pathways in the muscle of 4- and 12-week-old Ryr1TM/SC-ΔL mice, while proteins involved in the extracellular matrix, dystrophin-associated glycoprotein complex, and fatty acid metabolism were upregulated in Ryr1TM/SC-ΔL mice that survive to 12 weeks of age. These findings suggest that adaptations that optimize RYR1 expression/Ca2+ leak balance, sarcolemmal stability, and fatty acid biosynthesis provide Ryr1TM/SC-ΔL mice with an increased survival advantage during postnatal development.

Moderate intensity exercise training ameliorates the heart failure with preserved ejection fraction phenotype in a preclinical model by improving cardiometabolic fitness

Abstract Introduction and Aim Exercise intolerance is a hallmark feature of patients with heart failure with preserved ejection fraction (HFpEF).While clinical evidence suggests potential benefits of exercise training in HFpEF, its mechanisms remain largely unknown.In this study, we used a translationally relevant murine model of cardiometabolic HFpEF(1) to assess whether moderate intensity continuous training(MICT) can ameliorate the HFpEF phenotype in mice and promote cardiometabolic health. Methods Twenty-five adult, male C57BL/6N were fed with the combination of high fat diet(HFD;D12492,Research diet)and L-NAME(0.5 g/L,in drinking water) for 15 weeks to elicit HFpEF as previously described(1).After the establishment of the HFpEF phenotype, mice were randomized in two groups:sedentary(SED,n= 10) and exercised(MICT,n=15)mice.MICT was carried out according to the protocol:running on 10-degree positive inclination treadmill for 8 consecutive weeks,5 times/week,60 minutes/day,at a speed gradually increasing from 0.1 to 0.2 m/sec on a weekly basis.Morphometry, echocardiography(TTE),isoproterenol-induced stress echocardiography(SE),time domain nuclear magnetic resonance, and treadmill exhaustion tests were performed at the beginning and end of MICT.Additionally, at the end of MICT blood and tissues were collected for proteomics analysis(Fig1).Non-trained male C57BL/6N mice(37w) were used as controls for the HFpEF mice. Results Before randomization, all HFpEF mice were similar in terms of phenotypic characteristics. Compared to SED mice, MICT induced a significantly decrease in body weight driven by a reduction of total fat mass with an increased in lean mass(Fig2A+B).Interestingly, MICT had no impact on skeletal muscle mass for all different muscle groups evaluated (extensor digitorum longus, soleus and tibialis anterior).Importantly, MICT led to a significant improvement in exercise capacity in HFpEF mice, as shown by increased running distance and time, surpassing even the running distance seen in non-HFpEF mice (SED:62.8±18.7,MICT:305.7±13.9,non-HFpEF mice: 129.72±15.2meters,p&amp;lt;0.001).Preclinical surrogate of HFpEF such as pulmonary congestion, evaluated by index lung weights, improved after aerobic training.Following MICT there was a marked improvement in diastolic dysfunction(measured by E/E’ratio) both at rest(SED:36.5±3.3,MICT:23.3±5.09,p&amp;lt;0.001) and after isoproterenol challenge(SED:42.6±15.1,MICT:26.09±3.75,p&amp;lt;0.001)(Fig2C).Moreover, the wet cardiac mass was lower in the MICT group(Fig2A), suggesting potential amelioration in cardiac hypertrophy following aerobic training. Conclusion Eight weeks of supervised MICT on treadmill improves functional status, exercise capacity, and diastolic function in a cardiometabolic HFpEF murine model.The most significant effects were seen on weight loss and fat mass reduction.Our findings suggest a beneficial impact of aerobic exercise in mitigating HFpEF by improving metabolic fitness in the preclinical HFpEF model.Fig1-Study DesignFig2-Characteristics SED vs MICT groups

Effect of foot type on electromyography characteristics and synergy of lower limb muscles during running

The foot structure is associated with different running mechanics. The central nervous system is responsible for using the muscles through synergies during locomotion. The purpose of the present study was to examine the effect of foot structure on the electromyography factors and the synergy of the selected muscles of the lower extremity. Tibialis anterior, extensor digitorum longus, peroneus longus, soleus, biceps femoris, vastus lateralis, gastrocnemius lateralis and medialis muscles activity of 60 barefoot recreational runners with different foot structures was recorded while running at a speed of 3.3 m/s. Muscle activity was measured in the running cycle. Besides, muscle synergies were extracted using non-negative matrix factorization algorithm. The results showed that there were differences between groups with different foot type in muscle activity under different phases of running in some muscles. Furthermore, the findings indicated that the number of synergies was similar in different groups and the relative weight of muscles was not different across groups. In conclusion, despite the difference in muscle activity under different phases of the running cycle, muscle synergies are similar among the groups. This can indicate similar control by the central nervous system in runners with different arch structures while running and the observed changes in muscle activity can be attributed to the type of forces exerted on the body, the length-tension relationship, and changes in the direction of the lower limbs in people with different arch structures.

Nrf2 deficiency in muscle attenuates experimental autoimmune myositis-induced muscle weakness.

Idiopathic inflammatory myopathies (IIMs) are systemic autoimmune diseases characterised by muscle weakness. Although multiple physiological and pathological processes are associated with IIMs, T-lymphocyte infiltration into muscle plays a key role in the development and exacerbation of IIMs. Nuclear factor erythroid 2-related factor 2 (Nrf2) is a key transcription factor that regulates inflammatory responses; therefore, muscle Nrf2 may serve an important role in the development of IIMs. In this study, we demonstrated that experimental autoimmune myositis (EAM) causes loss of muscle mass and function in oxidative and glycolytic muscles in C57BL/6 mice. EAM increased CD4+ and CD8+ T-lymphocyte infiltration, as well as interferon-gamma (IFN-γ) and tumour necrosis factor-alpha (TNF-α) mRNA expression in oxidative soleus and glycolytic extensor digitorum longus muscles, along with elevated chemokine mRNA levels (i.e. CCL3, CCL5, CXCL9, CXCL10 and CXCL16). IFN-γ and TNF-α treatments increased the mRNA expression levels of these chemokines in C2C12 myotubes. EAM also increased phosphorylated Nrf2 at Ser40 in soleus and glycolytic white vastus lateralis muscle. Although the expression of several chemokines was affected by Nrf2 activation following tert-butylhydroquinone treatment or Keap1 knockdown, CCL5 mRNA expression significantly increased in C2C12 myotubes and mouse skeletal muscle. Moreover, muscle-specific Nrf2 knockout in mice attenuates EAM-induced loss of muscle mass and function, which was associated with the inhibition of CCL5 mRNA expression, CD8+ T-lymphocyte infiltration and IFN-γ mRNA expression. Collectively, these findings reveal that regulating Nrf2 activity is a promising therapeutic approach for treating IIM-mediated muscle weakness. KEY POINTS: Experimental autoimmune myositis (EAM) causes loss of muscle mass and function. Loss of muscle mass and function in EAM were associated with increased chemokine mRNA expression (i.e. CCL3, CCL5, CXCL9, CXCL10 and CXCL16), T-lymphocyte infiltration and inflammatory cytokine mRNA expression (i.e. IFN-γ and TNF-α) in the skeletal muscle. EAM activated Nrf2 in muscle and increased Nrf2 activity in vivo and in vitro increased CCL5 mRNA expression. Muscle-specific Nrf2 knockout in mice attenuated EAM-induced muscle weakness by inhibiting CCL5 mRNA expression, CD8+ T-lymphocyte migration and IFN-γ mRNA expression in muscles. These results provide further evidence for the potential therapeutic targeting of Nrf2 to mitigate EAM-induced muscle weakness.

Skeletal muscle fibre type-dependent effects of atorvastatin on the PI3K/Akt/mTOR signalling pathway and atrophy-related genes in rats

BackgroundOne of the probable causes of statin myotoxicity is an imbalance between protein synthesis and degradation. These processes are regulated by the PI3K/Akt/mTOR pathway and the ubiquitin‒proteasome system (UPS). The aim of this study was to assess whether the effects of atorvastatin on PI3K/Akt/mTOR pathway downstream proteins, the FoxO3a transcription factor and the UPS genes, i.e., MuRF-1 and MAFbx, depend on muscle fibre type.Methods and resultsAtorvastatin (50 mg/kg) was administered to Wistar rats. The levels of selected PI3K/Akt/mTOR pathway proteins were assayed via Western blotting, whereas MuRF-1, MAFbx and FoxO3a mRNA levels were measured using reverse transcription quantitative polymerase chain reaction (RT‒qPCR). Gomöri trichrome staining was performed to assess skeletal muscle pathology. A decrease in the P-Akt/Akt ratio was observed in the gastrocnemius muscle (MG), whereas an increase in the P-Akt/Akt ratio was observed in the soleus muscle (SOL). FoxO3a gene expression increased in the SOL and extensor digitorum longus (EDL) muscles. MuRF-1 gene expression increased in the MG, and MAFbx expression increased in the EDL. No histopathological changes were observed in any of the tested muscles.ConclusionsIn the absence of overt muscle damage, atorvastatin decreased the P-Akt/Akt ratio in the MG, indicating an increase in inactive Akt. Consistent with the decrease in Akt activation, rpS6 phosphorylation decreased. In SOL, atorvastatin increased the P-Akt/Akt ratio, indicating Akt activation. P-FoxO3a and the P-FoxO3a/FoxO3a ratio increased, suggesting that FoxO3a inactivation occurred. Moreover, in the SOL, atorvastatin did not affect the expression of atrophy-related genes. These findings indicate that atorvastatin has no adverse effect on the Akt pathway in the SOL. Our results showed that the effects of atorvastatin on the Akt signalling pathway and atrophy-related gene expression depend on muscle type.

Reduced K+ build-up in t-tubules contributes to resistance of the diaphragm to myotonia.

Patients with myotonia congenita suffer from slowed muscle relaxation caused by hyperexcitability. The diaphragm is only mildly affected in myotonia congenita; discovery of the mechanism underlying its resistance to myotonia could identify novel therapeutic targets. Intracellular recordings from two mouse models of myotonia congenita revealed the diaphragm had less myotonia than either the extensor digitorum longus (EDL) or the soleus muscles. A mechanism contributing to resistance of the diaphragm to myotonia was reduced depolarization of the interspike membrane potential during repetitive firing of action potentials, a process driven by build-up of K+ in small invaginations of muscle membrane known as t-tubules. We explored differences between diaphragm and EDL that might underlie reduction of K+ build-up in diaphragm t-tubules. Smaller size of diaphragm fibres, which promotes diffusion of K+ out of t-tubules, was identified as a contributor. Intracellular recording revealed slower repolarization of action potentials in diaphragm suggesting reduced Kv conductance. Higher resting membrane conductance was identified suggesting increased Kir conductance. Computer simulation found that a reduction of Kv conductance had little effect on K+ build-up whereas increased Kir conductance lessened build-up, although the effect was modest. Our data and computer simulation suggest opening of K+ channels during action potentials has little effect on K+ build-up whereas opening of K+ channels during the interspike interval slightly lessens K+ build-up. We conclude that activation of K+ channels may lessen myotonia by opposing depolarization to action potential threshold without worsening K+ build-up in t-tubules. KEY POINTS: In mouse models of the muscle disease myotonia congenita, the diaphragm has much less myotonia (muscle stiffness) than the extensor digitorum longus or soleus muscles. Identifying why the diaphragm is resistant to myotonia may help in developing novel therapy. We found the reason the diaphragm has less myotonia is that it is less prone to depolarization caused by K+ build-up in t-tubules during repetitive firing of action potentials. Smaller fibre size contributes to resistance to K+ build-up with differences in K+ currents playing little role. Our data suggest drugs that open K+ channels may be effective in treating myotonia as they may lessen excitability without worsening K+ build-up in t-tubules.

Spatial multi-omics in whole skeletal muscle reveals complex tissue architecture

Myofibers are large multinucleated cells that have long thought to have a rather simple organization. Single-nucleus transcriptomics, spatial transcriptomics and spatial metabolomics analysis have revealed distinct transcription profiles in myonuclei related to myofiber type. However, the use of local tissue collection or dissociation methods have obscured the spatial organization. To elucidate the full tissue architecture, we combine two spatial omics, RNA tomography and mass spectrometry imaging. This enables us to map the spatial transcriptomic, metabolomic and lipidomic organization of the whole murine tibialis anterior muscle. Our findings on heterogeneity in fiber type proportions are validated with multiplexed immunofluorescence staining in tibialis anterior, extensor digitorum longus and soleus. Our results demonstrate unexpectedly strong regionalization of gene expression, metabolic differences and variable myofiber type proportion along the proximal-distal axis. These new insights in whole-tissue level organization reconcile sometimes conflicting results coming from previous studies relying on local sampling methods.

7284 Ablation of hexose-6-phosphate dehydrogenase in mice causes skeletal muscle atrophy and dramatically impairs exercise capacity

Abstract Disclosure: M. Zogg: None. A.D. Grötsch: None. J. Birk: None. P. Pelczar: None. J. Bouitbir: None. A. Odermatt: None. Hexose-6-phosphate dehydrogenase (H6PD) catalyzes the first two steps of the pentose-phosphate pathway (PPP) within the endoplasmic reticulum (ER), generating the redox equivalent NADPH. It has been shown that mice lacking H6PD develop defects mainly in skeletal muscles [1]. Nevertheless, the mechanisms underlying the myopathy in H6PD KO mice is unknown. Here, we further assessed muscle responses to H6PD deficiency and aimed to uncover the mechanisms that may cause myopathy in mice. We conducted a detailed characterization of the muscle structure and function as well as energy metabolism in oxidative and glycolytic muscles of H6PD KO mice. At the age of 15 weeks, H6PD KO mice displayed lower body weight than WT mice, despite similar food and water intake. Grip strength was significantly lower in H6PD KO than in WT mice. This was associated with decreased weight of the mixed (gastrocnemius, quadriceps) and fast fiber type rich muscles of the hind leg (extensor digitorum longus, tibialis anterior) in H6PD KO animals. Atrogin1 and Murf1 mRNA expression, markers of atrophy, increased only in the white (glycolytic) quadriceps. In this context, fast-twitch fibers in WT mice displayed higher H6pd mRNA expression than slow-twitch fibers. Electron microscopy analysis of red and white gastrocnemius muscles displayed the presence of large intramyofibrillar vacuoles, and morphologic changes of the sarcoplasmic reticulum and mitochondria in H6PD KO muscle. Metabolically, H6PD KO muscles presented an accumulation of glycogen granules and significantly decreased mRNA expression of glucose transporter Glut4, glucose-6-phosphate transporter G6pt and glycogen phosphorylase Pygm. The exercise capacity, measured by maximal running distance, decreased by more than 50% in H6PD KO mice. Upon exercise, both WT and H6PD KO animals were able to mobilize the majority of glycogen stored in their muscles. However, immediately after exercise, there was a significant increase in plasma lactate and glucose levels in H6PD KO animals. Disturbances in glucose metabolism were further supported by in vitro experiments using stable C2C12 H6PD KO myoblasts, revealing a shift in basal state mitochondrial fuel oxidation usage from glucose to fatty acid and glutamine in metabolic dependency assays. In conclusion, our findings revealed that H6PD seems to play a key role in proper function and energy metabolism of skeletal muscles.

Intramuscular ganglionic cyst in the extensor digitorum longus muscle of the leg: A case report and review of literature

Ganglionic cysts are benign soft-tissue lesions with unclear etiology and pathogenesis commonly occurring on the wrists and hands. Although their diagnosis and management in these characteristic locations are easy, rare occurrences in atypical locations are difficult to diagnose. Ultrasonography and magnetic resonance imaging (MRI) are helpful in diagnosing and differentiating a ganglionic cyst from other soft-tissue lesions. Here, we report a case of 60-year-old male patient with intramuscular ganglionic cyst, measuring 68×41×48 mm, arising in the extensor digitorum muscle of the left leg, which was diagnosed using MRI and treated using excision and biopsy.