Rodent Muscle Research Articles

Does Vitamin B6 Act as an Exercise Mimetic in Skeletal Muscle?

Marginal vitamin B6 (B6) deficiency is common in various segments worldwide. In a super-aged society, sarcopenia is a major concern and has gained significant research attention focused on healthy aging. To date, the primary interventions for sarcopenia have been physical exercise therapy. Recent evidence suggests that inadequate B6 status is associated with an increased risk of sarcopenia and mortality among older adults. Our previous study showed that B6 supplementation to a marginal B6-deficient diet up-regulated the expression of various exercise-induced genes in the skeletal muscle of rodents. Notably, a supplemental B6-to-B6-deficient diet stimulates satellite cell-mediated myogenesis in rodents, mirroring the effects of physical exercise. These findings suggest the potential role of B6 as an exercise-mimetic nutrient in skeletal muscle. To test this hypothesis, we reviewed relevant literature and compared the roles of B6 and exercise in muscles. Here, we provide several pieces of evidence supporting this hypothesis and discuss the potential mechanisms behind the similarities between the effects of B6 and exercise on muscle. This research, for the first time, provides insight into the exercise-mimetic roles of B6 in skeletal muscle.

Moderate intensity aerobic exercise prevents myopathic consequences of glucocorticoid administration in aged female mice

Elevated glucocorticoids induce a myopathy characterized by the loss of muscle mass and increased muscle fatiguability. The elderly is highly susceptible to the myopathic effects as they are more likely to experience elevated glucocorticoid levels. Aerobic exercise can prevent glucocorticoid-induced loss of muscle in younger rodents; however, it is unknown whether aerobic exercise can influence glucocorticoid myopathic features in aged muscle. Therefore, the purpose of this study was to define the extent to which moderate intensity aerobic exercise influenced glucocorticoid myopathy in aged muscle. 24-month-old female C57BL/6 mice were randomized into 4 groups of equal bodyweights. Two groups remained sedentary while the other 2 groups began treadmill aerobic exercise performed 5x/week at 10 m/min for 30 min at 5% grade. At the end of the first week of exercise, one group of sedentary mice and one group of exercising mice began receiving once daily subcutaneous injections of dexamethasone (1 mg/kg; DEX). The other group of sedentary mice and group of exercising mice began receiving once daily subcutaneous injections of saline only. All treatments (sedentary/exercise or DEX/saline) continued for an additional 2 weeks. At the end of week 3, the contractile properties of the triceps surae muscle complex were assessed in situ via electrical stimulation of the sciatic nerve. Specific measures included twitch force, sub tetanic force (60 Hz stimulation frequency), tetanic force (125 Hz stimulation frequency), and a fatigue protocol consisting of 60 high force contractions, with each contraction separated by 4 seconds. After testing, muscles of the triceps surae complex were extracted and weighed. Total protein content of the gastrocnemius was determined by the Bradford method. DEX lowered muscle mass, lowered gastrocnemius total protein content, and enhanced fatigue, with aerobic exercise preventing those changes. Interestingly, main effects were noted for DEX to increase twitch and 60 Hz absolute force, normalized force, and RFD in both sedentary and exercise trained mice. Neither DEX nor exercise altered half-relaxation time at any stimulation frequency, nor did they alter tetanic force at the 125 Hz stimulation. These data show that the detrimental effects of elevated glucocorticoids to skeletal muscle morphology and contractile function in aged muscle can be mitigated by moderate intensity aerobic exercise training. They also indicate that there may be some positive benefits to submaximal force generating capacity following a clinically relevant dose of glucocorticoids in aged females. This work was supported by a Doctoral grant from the American College of Sports Medicine (GRL) and NIH R03AG073445 (BSG). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

The influence of new 1,2,3-triazolo-1,4-benzodiazepine derivatives on the muscle tone of rodents

Anxiety disorders represent one of the most prevalent categories of psychiatric illnesses, affecting individuals regardless of gender, age, or social standing. They result in substantial personal and societal costs. The pursuit of novel pharmacological approaches for treating these disorders is driven by the increasing medical necessity to enhance the effectiveness and safety profiles of anxiolytic medications. Due to the fact that benzodiazepines and their derivatives have anti-anxiety, hypnotic-sedative, antidepressant, anticonvulsant, and muscle relaxant properties, they occupy a leading place in the treatment of anxiety disorders. An essential aspect of investigating the pharmacological activity of new triazolobenziazepine derivatives is assessing their impact on rodent muscle tone and coordination of movements. The aim of the work is to find out the influence of new 1,2,3-triazolo-1,4-benzodiazepine derivatives on the muscle tone of rodents in the “vertical grid” test and coordination of movements using the rotarod test. Materials and methods. The objects of the study were 5 new derivatives of 1,2,3-triazolo-1,4-benzodiazepines. Before conducting in vivo experiments, these derivatives were mixed with lactose at a ratio of 1:1000. The “vertical grid” test and rotarod test (rotating rod test) were used to reproduce the model of motor behavior of rodents. Results. The presence of a tendency to the manifestation of a myorelaxant effect in the “vertical grid” test was established. The indicator of the total duration of detention at the facilities was similar and did not differ significantly in the control and experimental groups at doses of 0.50 mg/kg and 0.75 mg/kg. Derivatives MA-252, MA-253 and MA-254 at a dose of 1 mg/kg reduced the total duration of retention on the vertical grid, which indicates their mild muscle relaxant effect. In the rotating rod test, MA-253 derivative at a dose of 1 mg/kg increased the retention time on the rotarod, which demonstrates greater physical endurance of the animals of these experimental groups. Conclusions. The study’s findings indicated that the 1,2,3-triazolo-1,4-benzodiazepine derivatives did not exhibit an adverse impact on movement coordination. Some of these derivatives demonstrated a mild muscle relaxant effect. These results support the need for further research into their influence on spontaneous motor activity. Additionally, there’s a necessity to determine the dosage regimen, establish an effective dose, and adapt it for human use.

CDK4-E2F3 signals enhance oxidative skeletal muscle fiber numbers and function to affect myogenesis and metabolism.

Understanding how skeletal muscle fiber proportions are regulated is vital to understanding muscle function. Oxidative and glycolytic skeletal muscle fibers differ in their contractile ability, mitochondrial activity, and metabolic properties. Fiber-type proportions vary in normal physiology and disease states, although the underlying mechanisms are unclear. In human skeletal muscle, we observed that markers of oxidative fibers and mitochondria correlated positively with expression levels of PPARGC1A and CDK4 and negatively with expression levels of CDKN2A, a locus significantly associated with type 2 diabetes. Mice expressing a constitutively active Cdk4 that cannot bind its inhibitor p16INK4a, a product of the CDKN2A locus, were protected from obesity and diabetes. Their muscles exhibited increased oxidative fibers, improved mitochondrial properties, and enhanced glucose uptake. In contrast, loss of Cdk4 or skeletal muscle-specific deletion of Cdk4's target, E2F3, depleted oxidative myofibers, deteriorated mitochondrial function, and reduced exercise capacity, while increasing diabetes susceptibility. E2F3 activated the mitochondrial sensor PPARGC1A in a Cdk4-dependent manner. CDK4, E2F3, and PPARGC1A levels correlated positively with exercise and fitness and negatively with adiposity, insulin resistance, and lipid accumulation in human and rodent muscle. All together, these findings provide mechanistic insight into regulation of skeletal muscle fiber-specification that is of relevance to metabolic and muscular diseases.

Molecular changes in skeletal muscle in chronic kidney disease: A systematic review

AbstractBackgroundLoss of skeletal muscle mass is prevalent among patients affected by chronic kidney disease (CKD). It is associated with significant morbidity and mortality. The underlying molecular pathogenesis has yet to be fully understood. The aim of this systematic review is to summarize the current evidence on molecular changes in the skeletal muscle of humans and rodents with CKD and to assess the strength of such evidence.MethodsThe PubMed and EMBASE databases were searched using three main themes: messenger ribonucleic acid/protein/microRNA expression, skeletal muscle and CKD. This study was conducted in accordance with the Preferred Reporting Items for Systematic Reviews and Meta‐Analyses (PRISMA) standards.ResultsA total of 98 studies were included in the systematic review, comprising 26 prospective human clinical studies, four human and rodent studies, and 68 rodent‐only studies (32 mouse and 36 rat models respectively). The sample sizes of human studies were largely small (40% of studies had ≤20 participants). Qualitative polymerase chain reaction (qPCR) was the most commonly used method for gene expression and none of the studies fulfilled the Minimum Information for Publication of qPCR Experiments criteria for quality assessment. Majority of the studies investigated only a few genes or a specific signalling pathway. FBXO32, TRIM63, MSTN, IL6, TNF and IGF1 were the most investigated genes. The identified differentially expressed genes and proteins belonged to eight major pathways, including apoptosis, autophagy, inflammation, insulin/insulin‐like growth factor 1 signalling, lipid metabolism, mitochondrial function, muscle cell growth and differentiation, and protein degradation, similar to other chronic disease states.ConclusionsThe current evidence regarding molecular alterations in the skeletal muscle in CKD is largely derived from small and heterogenous studies. Markedly similar modifications in the major biological pathways between CKD and other chronic diseases supports shared deleterious molecular mechanisms producing muscle atrophy, irrespective of the underlying specific disease.

Protozoan Parasites of Sarcocystis spp. in Rodents from Commercial Orchards

Small mammals are an important group of wildlife that can transmit pathogens to humans and animals. There is a lack of comprehensive studies on the protozoan parasites of the genus Sarcocystis in agricultural areas. The aim of the current research was to evaluate the prevalence of Sarcocystis spp., and to identify the parasite species found in the skeletal muscles of rodents and insectivores from commercial orchards. A total of 679 muscle samples from small mammals, mainly rodents (n = 674), belonging to eight species were examined. Muscle samples were pooled into groups, then digested, and the presence of the Sarcocystis species was confirmed by molecular methods. The examined parasites were determined in five rodent species, Apodemus agrarius, A. flavicollis, Clethrionomys glareolus, Microtus arvalis, and M. oeconomus. The prevalence of Sarcocystis spp. was low: 2.23% in voles and 0.79% in mice. Based on a sequence comparison of cox1 and 28S rDNA, four species were identified: S. myodes, Sarcocystis cf. strixi, Sarcocystis sp. Rod1, and Sarcocystis sp. Rod2. This is the first report of S. myodes in A. agrarius, A. flavicollis, and M. arvalis. The identified species were most closely related to Sarcocystis spp., and were transmitted by predatory mammals and birds. Future studies are needed to describe the species morphologically, as well as to define the host spectrum and to evaluate their possible pathogenicity.

Exercise is associated with younger methylome and transcriptome profiles in human skeletal muscle.

Exercise training prevents age-related decline in muscle function. Targeting epigenetic aging is a promising actionable mechanism and late-life exercise mitigates epigenetic aging in rodent muscle. Whether exercise training can decelerate, or reverse epigenetic aging in humans is unknown. Here, we performed a powerful meta-analysis of the methylome and transcriptome of an unprecedented number of human skeletal muscle samples (n = 3176). We show that: (1) individuals with higher baseline aerobic fitness have younger epigenetic and transcriptomic profiles, (2) exercise training leads to significant shifts of epigenetic and transcriptomic patterns toward a younger profile, and (3) muscle disuse "ages" the transcriptome. Higher fitness levels were associated with attenuated differential methylation and transcription during aging. Furthermore, both epigenetic and transcriptomic profiles shifted toward a younger state after exercise training interventions, while the transcriptome shifted toward an older state after forced muscle disuse. We demonstrate that exercise training targets many of the age-related transcripts and DNA methylation loci to maintain younger methylome and transcriptome profiles, specifically in genes related to muscle structure, metabolism, and mitochondrial function. Our comprehensive analysis will inform future studies aiming to identify the best combination of therapeutics and exercise regimes to optimize longevity.

Mitochondrial calcium retention capacity correlates with markers of muscle mass, strength, power and functional capacities in young and old adults

RationaleThe aging‐related loss of muscle mass and function is a major risk factor for falls, fractures, frailty, loss of independence, institutionalization and even death. Although the underlying cellular and molecular mechanisms remain partly understood, impaired mitochondrial permeability transition pore (mPTP) function has been proposed as a potential mechanism. Indeed, altered mitochondrial calcium retention capacity (mCRC), a marker of mPTP function, has been documented with aging in both rodent and human skeletal muscles and mPTP opening was shown to cause muscle atrophy. However, studies on the impact of aging on mPTP function in humans remain scarce, and whether impaired mPTP function correlates with markers of muscle mass, performance and functional capacities remain unknown.ObjectiveThe present study aimed at investigating whether mCRC is associated with markers of muscle mass, muscle function and functional capacities in humans.MethodsTwelve young (29.4 ± 4.4y) and fourteen old (80 ± 7.4y) healthy men were enrolled in the present study. Leg lean mass (leg LM) was assessed using DEXA. Maximal isometric knee extension strength (iKES) and power were measured using a strain gauge system attached to a chair and the Nottingham Leg Extensor Power rig, respectively. To assess functional capacities, the performance at the 6‐min walk test and the step test (maximum number of steps in 20s) were evaluated. To assess mPTP function, mCRC was measured in permeabilized myofibers prepared from muscle biopsies obtained in the vastus lateralis.ResultsAs compared to young adults, old individuals displayed significantly lower leg LM (p<0.01), iKES (p<0.01), and power (p<0.01). The performance at the 6‐min walk test (p<0.01) and the step test (p<0.01) were also lower in old individuals. Old individuals also displayed a significantly lower mCRC (p<0.01). mCRC was significantly correlated with leg LM (r=0.34; p=0.045), iKES (r=0.35; p=0.04) and muscle power (r=0.41; p=0.02). In addition, mCRC was significantly correlated with the performance at the 6‐min walking test (r=0.42;p=0.01) and step test (r=0.46; p<0.01).ConclusionsThese results indicate that mPTP function is altered with aging in humans and highlight positive associations between mPTP, muscle mass, strength, power, and functional capacities. These findings therefore strengthen the emerging view that altered mPTP function contributes to the aging‐related loss of muscle mass and function in humans.

Electrostatic–Mechanical Synergistic In Situ Multiscale Tissue Adhesion for Sustainable Residue‐Free Bioelectronics Interfaces (Adv. Mater. 5/2022)

Tissue Adhesives In article number 2105338, Donghee Son, Changhyun Pang, and co-workers report an electrostatic–mechanical synergistic multiscale tissue adhesive for sustainable residue-free bioelectronics interfaces. To deduce the mechanism, a thermodynamic model based on a tailored elastomer–hydrogel combinatory adhesive architecture is proposed. This patch also successfully demonstrates reliable real-time measurements of electrophysiological signals generated by various tissues ranging from rodent sciatic nerve, muscle, brain, and human skin.

P300 or CBP is required for insulin-stimulated glucose uptake in skeletal muscle and adipocytes.

While current thinking posits that insulin signaling to glucose transporter 4 (GLUT4) exocytic translocation and glucose uptake in skeletal muscle and adipocytes is controlled by phosphorylation-based signaling, many proteins in this pathway are acetylated on lysine residues. However, the importance of acetylation and lysine acetyltransferases to insulin-stimulated glucose uptake is incompletely defined. Here, we demonstrate that combined loss of the acetyltransferases E1A binding protein p300 (p300) and cAMP response element binding protein binding protein (CBP) in mouse skeletal muscle caused a complete loss of insulin-stimulated glucose uptake. Similarly, brief (i.e., 1 hour) pharmacological inhibition of p300/CBP acetyltransferase activity recapitulated this phenotype in human and rodent myotubes, 3T3-L1 adipocytes, and mouse muscle. Mechanistically, these effects were due to p300/CBP-mediated regulation of GLUT4 exocytic translocation and occurred downstream of Akt signaling. Taken together, we highlight a fundamental role for acetylation and p300/CBP in the direct regulation of insulin-stimulated glucose transport in skeletal muscle and adipocytes.

Egg White Protein Promotes Developmental Growth in Rodent Muscle Independently of Leucine Content

BackgroundLeucine has unique anabolic properties, serving as a nutrient signal that stimulates muscle protein synthesis. ObjectiveWe tested whether the leucine concentration is the only factor determining protein quality for muscle development. MethodsWe selected 3 dietary proteins: casein (CAS), egg white protein (EWP), and albumin (ALB), representing the leucine concentrations of ∼8.3%, 7.7%, and 6.7% of the total protein (wt:wt), respectively. In the chronic feeding experiment, these proteins were pair-fed to growing male Wistar rats [110–135 g body weight (BW)] for 14 d as a protein source, providing 10% of total energy intake, after which soleus and extensor digitorum longus (EDL) muscles were used to estimate muscle growth. In the acute administration experiment, we injected CAS, ALB, and EWP to rats by oral gavage (0.3 g protein/100 g BW), and after 1 or 3 h EDL muscle was excised for capillary electrophoresis-MS–based metabolomics. In another chronic feeding experiment, rats were pair-fed either CAS or a CAS diet supplemented with arginine to the same level as in the EWP diet for 14 d. ResultsAt the end of the 14-d feeding, soleus and EDL muscle weight was 20% and 17% higher, respectively, when rats were fed EWP as compared with CAS (P < 0.05). In addition, the 14-d EWP diet increased the expression of p70S6K by 117% compared with CAS (P < 0.05). These results suggest the possibility that some amino acids (excluding leucine), derived from EWP, promote muscle growth. Metabolomics analysis showed that muscle arginine concentration, following acute protein administration, appeared to match muscle growth over the 14-d feeding period. In addition, 14-d arginine supplementation to a CAS diet increased EDL muscle weight by 15% when compared with the plain CAS diet (P < 0.05). ConclusionsEWP promotes rat developmental muscle growth compared with CAS, which can be partly explained by the arginine-rich EWP.

Changes in Myonuclear Number During Postnatal Growth - Implications for AAV Gene Therapy for Muscular Dystrophy.

Adult skeletal muscle is a relatively stable tissue, as the multinucleated muscle fibres contain post-mitotic myonuclei. During early postnatal life, muscle growth occurs by the addition of skeletal muscle stem cells (satellite cells) or their progeny to growing muscle fibres. In Duchenne muscular dystrophy, which we shall use as an example of muscular dystrophies, the muscle fibres lack dystrophin and undergo necrosis. Satellite-cell mediated regeneration occurs, to repair and replace the necrotic muscle fibres, but as the regenerated muscle fibres still lack dystrophin, they undergo further cycles of degeneration and regeneration.AAV gene therapy is a promising approach for treating Duchenne muscular dystrophy. But for a single dose of, for example, AAV coding for microdystrophin, to be effective, the treated myonuclei must persist, produce sufficient dystrophin and a sufficient number of nuclei must be targeted. This latter point is crucial as AAV vector remains episomal and does not replicate in dividing cells. Here, we describe and compare the growth of skeletal muscle in rodents and in humans and discuss the evidence that myofibre necrosis and regeneration leads to the loss of viral genomes within skeletal muscle. In addition, muscle growth is expected to lead to the dilution of the transduced nuclei especially in case of very early intervention, but it is not clear if growth could result in insufficient dystrophin to prevent muscle fibre breakdown. This should be the focus of future studies.

Effect of methanol extract of Anacardium occidentale stem bark on agonist-induced contraction in rat uterine muscle

Anacardium occidentale (Ao) is widely used in ethnomedicine and the effect on contractility of the gut and heart muscles of laboratory rodents have been established however, its effect on the contractility of uterine muscle has been rarely researched. The present study investigated the effect of stem bark methanolic extract of Anacardium occidentale (AoME) on Isolated Rat Uterus Strips (IRUS). The Anacardium occidentale methanol extract (AoME) was obtained by solvent extraction. The IRUS were suspended in a bath containing 15 ml of De Jalon's solution through a silk connected to a force transducer. A tension of 1.0 g was applied. The strips were allowed to equilibrate for a period of 60–90 min before the commencement of the experiment. The extract (0.025- 0.3mg/ml) added cumulatively did not modify the normal tonus in most of the IRUS used except in one where it feebly contracted it. However, the contractile responses induced by acetylcholine (10-9 -10-5 M) and oxytocin (10-9 -10-5 M) on the IRUS were almost completely blocked by pre-incubated AoME at a concentration of 0.2 mg/ml. Stem bark methanol extract of Anacardium occidentale shifted the log concentration curve of both agonists to the right. The present results report the antagonistic effects of stem bark methanol extract of Anacardium occidentale on oxytocin and acetylcholine-induced contraction in rat uterus strips.&#x0D; Keywords: Anacardium occidentale, Oxytocin, Acetylcholine, Uterus, Smooth muscle

Sex differences in metabolic pathways are regulated by Pfkfb3 and Pdk4 expression in rodent muscle

Skeletal muscles display sexually dimorphic features. Biochemically, glycolysis and fatty acid β-oxidation occur preferentially in the muscles of males and females, respectively. However, the mechanisms of the selective utilization of these fuels remains elusive. Here, we obtain transcriptomes from quadriceps type IIB fibers of untreated, gonadectomized, and sex steroid-treated mice of both sexes. Analyses of the transcriptomes unveil two genes, Pfkfb3 (phosphofructokinase-2) and Pdk4 (pyruvate dehydrogenase kinase 4), that may function as switches between the two sexually dimorphic metabolic pathways. Interestingly, Pfkfb3 and Pdk4 show male-enriched and estradiol-enhanced expression, respectively. Moreover, the contribution of these genes to sexually dimorphic metabolism is demonstrated by knockdown studies with cultured type IIB muscle fibers. Considering that skeletal muscles as a whole are the largest energy-consuming organs, our results provide insights into energy metabolism in the two sexes, during the estrus cycle in women, and under pathological conditions involving skeletal muscles.

Confocal Endomicroscopy of Neuromuscular Junctions Stained with Physiologically Inert Protein Fragments of Tetanus Toxin.

Live imaging of neuromuscular junctions (NMJs) in situ has been constrained by the suitability of ligands for inert vital staining of motor nerve terminals. Here, we constructed several truncated derivatives of the tetanus toxin C-fragment (TetC) fused with Emerald Fluorescent Protein (emGFP). Four constructs, namely full length emGFP-TetC (emGFP-865:TetC) or truncations comprising amino acids 1066–1315 (emGFP-1066:TetC), 1093–1315 (emGFP-1093:TetC) and 1109–1315 (emGFP-1109:TetC), produced selective, high-contrast staining of motor nerve terminals in rodent or human muscle explants. Isometric tension and intracellular recordings of endplate potentials from mouse muscles indicated that neither full-length nor truncated emGFP-TetC constructs significantly impaired NMJ function or transmission. Motor nerve terminals stained with emGFP-TetC constructs were readily visualised in situ or in isolated preparations using fibre-optic confocal endomicroscopy (CEM). emGFP-TetC derivatives and CEM also visualised regenerated NMJs. Dual-waveband CEM imaging of preparations co-stained with fluorescent emGFP-TetC constructs and Alexa647-α-bungarotoxin resolved innervated from denervated NMJs in axotomized WldS mouse muscle and degenerating NMJs in transgenic SOD1G93A mouse muscle. Our findings highlight the region of the TetC fragment required for selective binding and visualisation of motor nerve terminals and show that fluorescent derivatives of TetC are suitable for in situ morphological and physiological characterisation of healthy, injured and diseased NMJs.

In vivo human gracilis whole-muscle passive stress-sarcomere strain relationship.

We measured the passive mechanical properties of intact, living human gracilis muscles (n=11 individuals, 10 male and 1 female, age: 33±12 years, mass: 89±23 kg, height: 177±8 cm). Measurements were performed in patients undergoing surgery for free-functioning myocutaneous tissue transfer of the gracilis muscle to restore elbow flexion after brachial plexus injury. Whole-muscle force of the gracilis tendon was measured in four joint configurations (JC1-JC4) with a buckle force transducer placed at the distal tendon. Sarcomere length was also measured by biopsy from the proximal gracilis muscle. After the muscle was removed, a three-dimensional volumetric reconstruction of the muscle was created via photogrammetry. Muscle length from JC1 to JC4 increased by 3.3±1.0, 7.7±1.2, 10.5±1.3 and 13.4±1.2 cm, respectively, corresponding to 15%, 34%, 46% and 59% muscle fiber strain, respectively. Muscle volume and an average optimal fiber length of 23.1±0.7 cm yielded an average muscle physiological cross-sectional area of 6.8±0.7cm2 which is approximately 3 times that measured previously from cadaveric specimens. Absolute passive tension increased from 0.90±0.21N in JC1 to 16.50±2.64N in JC4. As expected, sarcomere length also increased from 3.24±0.08 µm at JC1 to 3.63±0.07 µm at JC4, which are on the descending limb of the human sarcomere length-tension curve. Peak passive muscle stress was 27.8±5.5 kPa in JC4 and muscle modulus ranged from 44.8 MPa in JC1 to 125.7 MPa in JC4. Comparison with other mammalian species indicates that human muscle passive mechanical properties are more similar to rodent muscle than to rabbit muscle. These data provide direct measurements of whole-human muscle passive mechanical properties that can be used in modeling studies and for understanding comparative passive mechanical properties among mammalian muscles.

MERG1A Protein Abundance Increases in the Atrophied Skeletal Muscle of Denervated Mice, But Does Not Affect NFκB Activity.

Skeletal muscle atrophy may occur with disease, injury, decreased muscle use, starvation, and normal aging. No reliably effective treatments for atrophy are available, thus research into the mechanisms contributing to muscle loss is essential. The ERG1A K+ channel contributes to muscle loss by increasing ubiquitin proteasome proteolysis (UPP) in the skeletal muscle of both unweighted and cachectic mice. Because the mechanisms which produce atrophy vary based upon the initiating factor, here we investigate atrophy produced by denervation. Using immunohistochemistry and immunoblots, we demonstrate that ERG1A protein abundance increases significantly in the Gastrocnemius muscle of rodents 7 days after both sciatic nerve transection and hind limb unweighting. Further, we reveal that ectopic expression of a Merg1a encoded plasmid in normal mouse Gastrocnemius muscle has no effect on activity of the NFκB transcription factor family, a group of proteins which contribute to muscle atrophy by modulation of the UPP. Further, although NFκB activity increases significantly after denervation, we show that expression of a plasmid encoding a dominant negative Merg1a mutant in Gastrocnemius muscle prior to denervation, has no effect on NFκB activity. Thus, although the ERG1A K+ channel increases UPP, it does not do so through modulation of NFκB transcription factors.

3: IGF-1 Hydrogel-based Nanofiber Drug Delivery System to Improve Nerve Regeneration and Functional Recovery After Peripheral Nerve Repair

Purpose:Insulin-like growth factor 1 (IGF-1) is a potent mitogen with the potential to enhance axonal regeneration and minimize muscle atrophy and Schwann cell senescence following prolonged denervation after peripheral nerve injury. IGF-1 is a small protein with a half-life of 5 min, making local delivery a challenge. Our group has demonstrated over 6 weeks of sustained release of bioactive IGF-1 encapsulated within biodegradable nanoparticles (NP) and subsequently developed a nanofiber fiber hydrogel composite (NHC) carrier to retain IGF-1 NPs at target tissue locally for the duration of drug release. The aim of this study was to further characterize and refine the IGF-1 NP-NHC drug delivery and then investigate its efficacy in both rodent and non-human primate (NHP) median nerve injury models.Methods:IGF-1 was encapsulated in biodegradable PCL NPs and then embedded within the NHC composed of hyaluronic acid and PCL nanofibers. Release kinetics and biocompatibility were evaluated and optimized both in vitro and in vivo. The drug delivery system was assessed using a chronic denervation median nerve injury rat model and an acute median nerve repair NHP model. IGF-1NP/NHC was injected along the median nerve and within denervated muscle. In rodents, a range of IGF-1 doses (300, 900 and 1500 μg/mL) were investigated to evaluate dose-response relationships. Axonal regeneration, muscle atrophy, neuromuscular junction reinnervation and recovery of grip strength were assessed.Results:The refined NP-NHC delivery system provided sustained release of bioactive IGF-1, in vivo, for at least 42 days by serial ELISA. IGF-1 treated rodents demonstrated a 35% increase in functional recovery (stimulated grip strength) compared to untreated rodents, with no differences observed between the different concentrations of IGF-1 that were evaluated. Median nerve histomorphometry demonstrated a significantly greater total number of axons at each concentration of IGF-1 compared to untreated rodents (p<0.0001). IGF-1 treated rodents also demonstrated a greater percentage of reinnervation of neuromuscular junctions by 17% (from 14% to 31%). In addition, the IGF-1 treated non-human primate demonstrated a 31% increase in functional recovery compared to the untreated animal (N=1 per group).Conclusion:The IGF-1 NP/NHC delivery system provided sustained delivery for over 42 days in rodents and NHP. IGF-1 improves motor functional recovery by enhancing axonal regeneration and neuromuscular junction reinnervation while limiting denervation-induced muscle and Schwann cell atrophy in rodents. Our NHP pilot study has established a used pre-clinical model with robust functional analysis that will serve as a platform for a formal NHP study prior to clinical testing. The components of the NP-NHC delivery system are already used in FDA approved formulations, which will facilitate clinical translation.

Plasticity of seven-transmembrane-helix receptor heteromers in human vascular smooth muscle cells.

Recently, we reported that the chemokine (C-X-C motif) receptor 4 (CXCR4) and atypical chemokine receptor 3 (ACKR3) heteromerize with α1A/B/D-adrenoceptors (ARs) and arginine vasopressin receptor 1A (AVPR1A) in recombinant systems and in rodent and human vascular smooth muscle cells (hVSMCs). In these studies, we observed that heteromerization between two receptor partners may depend on the presence and the expression levels of other partnering receptors. To test this hypothesis and to gain initial insight into the formation of these receptor heteromers in native cells, we utilized proximity ligation assays in hVSMCs to visualize receptor-receptor proximity and systematically studied how manipulation of the expression levels of individual protomers affect heteromerization patterns among other interacting receptor partners. We confirmed subtype-specific heteromerization between endogenously expressed α1A/B/D-ARs and detected that AVPR1A also heteromerizes with α1A/B/D-ARs. siRNA knockdown of CXCR4 and of ACKR3 resulted in a significant re-arrangement of the heteromerization patterns among α1-AR subtypes. Similarly, siRNA knockdown of AVPR1A significantly increased heteromerization signals for seven of the ten receptor pairs between CXCR4, ACKR3, and α1A/B/D-ARs. Our findings suggest plasticity of seven transmembrane helix (7TM) receptor heteromerization in native cells and could be explained by a supramolecular organization of these receptors within dynamic clusters in the plasma membrane. Because we previously observed that recombinant CXCR4, ACKR3, α1a-AR and AVPR1A form hetero-oligomeric complexes composed of 2–4 different protomers, which show signaling properties distinct from individual protomers, re-arrangements of receptor heteromerization patterns in native cells may contribute to the phenomenon of context-dependent GPCR signaling. Furthermore, these findings advise caution in the interpretation of functional consequences after 7TM receptor knockdown in experimental models. Alterations of the heteromerization patterns among other receptor partners may alter physiological and pathological responses, in particular in more complex systems, such as studies on the function of isolated organs or in in vivo experiments.

Dissection, in vivo imaging and analysis of the mouse epitrochleoanconeus muscle.

Analysis of rodent muscles affords an opportunity to glean key insights into neuromuscular development and the detrimental impact of disease‐causing genetic mutations. Muscles of the distal leg, for instance the gastrocnemius and tibialis anterior, are commonly used in such studies with mice and rats. However, thin and flat muscles, which can be dissected, processed and imaged without major disruption to muscle fibres and nerve‐muscle contacts, are more suitable for accurate and detailed analyses of the peripheral motor nervous system. One such wholemount muscle is the predominantly fast twitch epitrochleoanconeus (ETA), which is located in the upper forelimb, innervated by the radial nerve, and contains relatively large and uniformly flat neuromuscular junctions (NMJs). To facilitate incorporation of the ETA into the experimental toolkit of the neuromuscular disease field, here, we describe a simple method for its rapid isolation (<5 min), supported by high‐resolution videos and step‐by‐step images. Furthermore, we outline how the ETA can be imaged in live, anaesthetised mice, to enable examination of dynamic cellular processes occurring at the NMJ and within intramuscular axons, including transport of organelles, such as mitochondria and signalling endosomes. Finally, we present reference data on wild‐type ETA fibre‐type composition in young adult, male C57BL6/J mice. Comparative neuroanatomical studies of different muscles in rodent models of disease can generate critical insights into pathogenesis and pathology; dissection of the wholemount ETA provides the possibility to diversify the repertoire of muscles analysed for this endeavour.