In this study, we used single-cell RNA sequencing to delineate the contributions of muscle-resident Schwann cells to neuromuscular junction (NMJ) remodeling by comparing a model of stable innervation with models of reinnervation following partial or complete denervation. We discovered multiple distinct Schwann cell subtypes, including a terminal Schwann cell subtype integral to the denervation-reinnervation cycle, identified by a transcriptomic signature indicative of cell migration and polarization. The data also characterize 3 myelin Schwann cell subtypes, which are distinguished based on enrichment of genes associated with myelin production, mesenchymal differentiation, or collagen synthesis. Importantly, SPP1 signaling emerged as a pivotal regulator of NMJ dynamics, promoting Schwann cell proliferation and muscle reinnervation across nerve injury models. These findings advance our understanding of NMJ maintenance and regeneration and underscore the therapeutic potential of targeting specific molecular pathways to treat neuromuscular and neurodegenerative disorders.

Skeletal muscle is essential for both movement and metabolic processes, characterized by a complex and ordered structure. Despite its importance, a detailed spatial map of gene expression within muscle tissue has been challenging to achieve due to the limitations of existing technologies, which struggle to provide high-resolution views. In this study, we leverage the Seq-Scope technique, an innovative method that allows for the observation of the entire transcriptome at an unprecedented submicron spatial resolution. By applying this technique to the mouse soleus muscle, we analyze and compare the gene expression profiles in both healthy conditions and following denervation, a process that mimics aspects of muscle aging. Our approach reveals detailed characteristics of muscle fibers, other cell types present within the muscle, and specific subcellular structures such as the postsynaptic nuclei at neuromuscular junctions, hybrid muscle fibers, and areas of localized expression of genes responsive to muscle injury, along with their histological context. The findings of this research significantly enhance our understanding of the diversity within the muscle cell transcriptome and its variation in response to denervation, a key factor in the decline of muscle function with age. This breakthrough in spatial transcriptomics not only deepens our knowledge of muscle biology but also sets the stage for the development of new therapeutic strategies aimed at mitigating the effects of aging on muscle health, thereby offering a more comprehensive insight into the mechanisms of muscle maintenance and degeneration in the context of aging and disease.

The maintenance of neuromuscular junctions (NMJs) is crucial for combating degenerative neuromuscular diseases. Maintenance of the NMJ involves turnover and regeneration with these processes dependant on the activation of Schwann cells by specific molecular cues within the muscular environment. Secreted phosphoprotein-1 (SPP1), known as osteopontin, is a chemokine that is rapidly upregulated following nerve injury. Its precise influence on Schwann cell-mediated nerve regeneration, however, remains to be elucidated. Our recent single-cell RNA sequencing data has uncovered a previously unrecognized interaction between myelinating Schwann cells and terminal Schwann cells (tSCs), mediated by SPP1 signaling. We propose that this interaction, orchestrated by SPP1, is crucial for tSC proliferation and ultimately, successful reinnervation of muscle fibers post-injury. To investigate the effect of nerve injury on SPP1 signaling gene expression within skeletal muscle, we conducted sciatic nerve crush experiments in two-month-old C57BL/6 mice. We compared gene expression profiles associated with denervation and SPP1 signaling in the gastrocnemius muscles of uninjured controls to those at 7, 14, and 28 days post-injury (DPI). We found significant transient increases in Spp1 and its receptor genes Cd44 and Itgav, peaking at 7 DPI and returning to baseline by 14 DPI. To further elucidate the role of SPP1 in muscle reinnervation and its impact on tSC responses following nerve injury, we conducted peroneal nerve injuries in S100-GFP transgenic mice and administered either an SPP1 neutralizing antibody (Spp1-nAb) or a saline solution intramuscularly. Direct muscle stimulation force testing revealed comparable evoked forces between both groups, however nerve-evoked muscle forces were significantly diminished by 43% in the Spp1-nAb-treated mice. The Spp1-nAb treated mice also demonstrated a 38% reduction in nerve-to-muscle force ratios compared to the saline group. Histological assessments post-injury showed that mice given Spp1-nAb at 7 DPI had markedly reduced nerve terminal and synaptic areas compared to saline-treated controls. Similarly, a greater percentage of muscle fibers remained denervated with Spp1-nAb treatment (67% vs 33% in saline), accompanied by a reduction in NMJ synaptic area. An analysis of tSCs further indicated a decreased count per NMJ and a reduction in overall tSC area upon SPP1 signaling inhibition. Our comprehensive analysis establishes the essential function of Spp1 in muscle reinnervation, as evidenced by its role in enhancing tSC numbers at the NMJ. These findings provide significant contributions to our understanding of the molecular mechanisms essential for NMJ repair and maintenance, with a particular focus on the role of tSCs in this process. This work was supported by the National institutes of Health (NIH) under the awards R01(AG050676) (SVB), P01 (AG051442) (SVB), P30 (AR069620)(SVB), T32 (AG000114)(SDG), and the American Physiological Society Porter Fellowship (SDG). 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.

Skeletal muscle activation using optogenetics has emerged as a promising technique for inducing noninvasive muscle contraction and assessing muscle function both in vivo and in vitro. Transgenic mice overexpressing the optogenetic fusion protein, Channelrhodopsin 2-EYFP (ChR2-EYFP) in skeletal muscle are widely used; however, overexpression of fluorescent proteins can negatively impact the functionality of activable tissues. In this study, we characterized the contractile properties of ChR2-EYFP skeletal muscle and introduced the ChR2-only mouse model that expresses light-responsive ChR2 without the fluorescent EYFP in their skeletal muscles. We found a significant reduction in the contractile ability of ChR2-EYFP muscles compared with ChR2-only and WT mice, observed under both electrical and optogenetic stimulation paradigms. Bulk RNAseq identified the downregulation of genes associated with transmembrane transport and metabolism in ChR2-EYFP muscle, while the ChR2-only muscle did not demonstrate any notable deviations from WT muscle. The RNAseq results were further corroborated by a reduced protein-level expression of ion channel-related HCN2 in ChR2-EYFP muscles and gluconeogenesis-modulating FBP2 in both ChR2-EYFP and ChR2-only muscles. Overall, this study reveals an intrinsic skeletal dysfunction in the widely used ChR2-EYFP mice model and underscores the importance of considering alternative optogenetic models, such as the ChR2-only, for future research in skeletal muscle optogenetics.

Lengthening contractions (i.e., eccentric contractions) are capable of uniquely triggering the nervous system and signaling pathways to promote tissue health/growth. This mode of exercise may be particularly potent for patients suffering from muscle weakness after joint injury. Here we provide a novel framework for eccentric exercise as a safe, effective mode of exercise prescription for muscle recovery.

Whole-body knock-out of Cu,Zn superoxide dismutase (Sod1KO) results in accelerated, age-related loss of muscle mass and function associated with neuromuscular junction (NMJ) breakdown similar to sarcopenia. In order to determine whether altered redox in motor neurons underlies this phenotype, an inducible neuron-specific deletion of Sod1 (i-mnSod1KO) was compared with wild-type (WT) mice of different ages (adult, mid-age, and old) and whole-body Sod1KO mice. Nerve oxidative damage, motor neuron numbers and structural changes to neurons and NMJ were examined. Tamoxifen-induced deletion of neuronal Sod1 from two months of age. No specific effect of a lack of neuronal Sod1 was seen on markers of nerve oxidation (electron paramagnetic resonance of an in vivo spin probe, protein carbonyl, or protein 3-nitrotyrosine contents). i-mnSod1KO mice showed increased denervated NMJ, reduced numbers of large axons and increased number of small axons compared with old WT mice. A large proportion of the innervated NMJs in old i-mnSod1KO mice displayed a simpler structure than that seen in adult or old WT mice. Thus, previous work showed that neuronal deletion of Sod1 induced exaggerated loss of muscle in old mice, and we report that this deletion leads to a specific nerve phenotype including reduced axonal area, increased proportion of denervated NMJ, and reduced acetyl choline receptor complexity. Other changes in nerve and NMJ structure seen in the old i-mnSod1KO mice reflect aging of the mice.

Progressive muscle atrophy and loss of muscle strength associated with old age have been well documented. Although impairments in the ability of old mice to regenerate skeletal muscle following injury have been demonstrated, less is known about the way age modulates the regenerative response of motor neurons and the neuromuscular junctions (NMJ) of mice following contraction‐induced injury. The inability for NMJs to effectively regenerate following an injury could lead to the death of the denervated muscle fibers and therefore play a contributing role to age‐related sarcopenia. To investigate the relationship between age and NMJ regeneration following injury, extensor digitorum longus (EDL) muscles of adult (8‐9 months), middle‐aged (18‐19 months), and old mice (27‐28 months) were subjected to a protocol of repeated lengthening‐contractions that resulted in an acute force deficit of ~70%. After 28 days, muscles were evaluated for maximum force generating capacity, muscle mass, NMJ innervation, and endplate fragmentation. Cross sections of muscles taken at three days post injury showed extensive infiltration of mononuclear cells and widespread tissue disruption consistent with the induction of severe injury to the muscles. At 28 days, maximum isometric force recovered completely in adult mice, but was significantly reduced in middle aged and old mice when compared to muscles of adult mice. In each case, approximately 29% of fibers in the cross sections displayed containing central nuclei, whereas controls showed only 2.9%, indicating that the injured muscle fibers had undergone degeneration and regeneration. Analysis of the innervation of the NMJs in the regenerated muscles showed a moderate decrease in the number of fully innervated endplates in old mice as compared to adult and middle‐aged mice. When the architecture of the endplates in regenerated muscles was examined, the relative number of NMJs with intact postsynaptic structure varied little between the age groups, although control muscles of old mice showed an increase in the number of fragmented endplates compared with the adult and middle aged mice. Thus, the diminished ability of the skeletal muscle of old mice to recover following injury may be, in part, due to an age‐related decrease in the ability to adequately regenerate motor neurons and perhaps form NMJs in the injured muscle.

The neuromuscular junction (NMJ) is an excitatory synapse that constitutes the primary site of communication between the nervous system and skeletal muscle and therefore any degenerative changes at the NMJ have the potential to impair muscle function. The primary cellular components of the NMJ are the motor neuron, the muscle fiber and terminal Schwann cells (tSC) that surround the NMJ. The importance of redox homeostasis for the maintenance of NMJ integrity is supported by work from our group showing degenerative changes at NMJs in mice lacking copper zinc superoxide dismutase (CuZnSOD; Sod1 -/- ) as early as 4 months of age. Prior work from others suggests that tSCs contribute trophic support for the NMJ and facilitate motor unit remodeling, but changes in tSC structure and function under conditions of elevated oxidative stress have not been thoroughly explored. Using in vitro assays, we previously showed that primary Schwann cells treated with a sublethal dose of the reactive oxygen species inducing agent Paraquat (PQ) resulted in increased Schwann cell number, ki67+ staining, and gene expression of trophic factors and the critical NMJ organizing protein Agrin. Furthermore, differentiated C2C12 myotubes treated with conditioned media from PQ treated Schwann cell cultures showed increased number of acetylcholine receptor (AChR) plaques. The goal of the current study was to characterize NMJs in young Sod1 -/- mice (2-months) to determine the early effects of elevated oxidative stress on NMJ structure in vivo with a specific focus on tSCs. We hypothesized that loss of Sod1 would lead to aberrant tSC and NMJ structure associated with decreased muscle function. We assessed maximum isometric force, NMJ morphology, and number of Schwann cells in gastrocnemius muscles of WT (n = 3) and Sod1 -/- (n = 3) mice. In Sod1 -/- mice, muscle force was reduced by 26% ( p = 2e-4) with nerve stimulation compared to direct muscle stimulation, and overlap between motor nerve terminals and AChRs was reduced by nearly 60% compared to WT controls. Post-synaptic AChR area was increased by 37% in Sod1 -/- mice, and tSC area and numbers per NMJ were increased by 60% and 155%, respectively in Sod1 -/- mice. Finally, Schwann cells proximal to NMJs showed increased Ki67+ labeling in Sod1 -/- mice compared to WT mice. This analysis demonstrates that early changes in both pre- and post-synaptic NMJ components and reduced neurotransmission due to loss of Sod1 are associated with marked increases in tSC proliferation. Our data implicate a protective role of muscle-resident Schwann cells in preserving NMJ integrity in response to alterations in redox homeostasis that may be mediated in part through activation and proliferation of these cells. These findings indicate that further investigation of the dynamics between Schwann cells and NMJ function is important to increase understanding of neuromuscular degenerativeconditions that involve altered redox homeostasis such as sarcopenia. Supported by the University of Michigan Medical School Office of Research, the American Physiological Society, and NIH AR069620. This is the full abstract presented at the American Physiology Summit 2023 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 development and maintenance of neuromuscular junctions (NMJ) are supported by a specialized population of myonuclei that are referred to as the subsynaptic myonuclei (SSM). The relationship between the number of SSM and the integrity of the NMJ as well as the impact of a loss of innervation on SSM remain unclear. This study aimed to clarify these associations by simultaneously analyzing SSM counts and NMJ innervation status in three distinct mouse models of acute and chronic NMJ disruption. SSM were identified using fluorescent immunohistochemistry for Nesprin1 expression, which is highly enriched in SSM, along with anatomical location beneath the muscle fiber motor endplate. Acute denervation, induced by surgical nerve transection, did not affect SSM number after 7 days. Additionally, no significant changes in SSM number were observed during normal aging or in mice with chronic oxidative stress (Sod1−/−). Both aging WT mice and Sod1−/− mice accumulated degenerating and denervated NMJ in skeletal muscle, but there was no correlation between innervation status of a given NMJ and SSM number in aged or Sod1−/− mice. These findings challenge the notion that a loss of SSM is a primary driver of NMJ degradation and leave open questions of the mechanisms that regulate SSM number as well as the physiological significance of the precise SSM number. Further investigations are required to define other properties of the SSM, such as transcriptional profiles and structural integrity, to better understand their role in NMJ maintenance.

Tissue-engineered skeletal muscle is a promising novel therapy for the treatment of volumetric muscle loss (VML). Our laboratory has developed tissue-engineered skeletal muscle units (SMUs) and engineered neural conduits (ENCs), and modularly scaled them to clinically relevant sizes for the treatment of VML in a large animal (sheep) model. In a previous study, we evaluated the effects of the SMUs and ENCs in treating a 30% VML injury in the ovine peroneus tertius muscle after a 3-month recovery period. The goal of the current study was to expand on our 3-month study and evaluate the SMUs and ENCs in restoring muscle function after a 6-month recovery period. Six months after implantation, we found that the repair groups with the SMU (VML+SMU and VML+SMU+ENC) restored muscle mass to a level that was statistically indistinguishable from the uninjured contralateral muscle. In contrast, the muscle mass in the VML-Only group was significantly less than groups repaired with an SMU. Following the 6-month recovery from VML, the maximum tetanic force was significantly lower for all VML injured groups compared with the uninjured contralateral muscle. However, we did demonstrate the ability of our ENCs to effectively regenerate nerve between the distal stump of the native nerve and the repair site in 14 of the 15 animals studied. Impact Statement Volumetric muscle loss (VML) is a clinically relevant problem for which current treatment options are lacking and for which tissue-engineered skeletal muscle presents a promising novel therapeutic option. However, the fabrication of tissues of clinically relevant sizes is necessary for advancement of the technology to the clinic. This study aimed to evaluate the efficacy of our scaled-up tissue-engineered skeletal muscle to treat VML in a large animal (sheep) model after a 6-month recovery.

The neuromuscular junction (NMJ) is a tripartite structure made up of the pre-synaptic motor neuron terminal, the post-synaptic muscle end plate, and the perisynaptic Schwann cells (SC). Degeneration of the NMJ is strongly associated with age-associated muscle mass and strength declines as well as other neurodegenerative diseases; however, the cellular interactions and signals that initiate the degeneration are poorly understood. In particular, understanding of the role of perisynaptic SCs in NMJ maintenance is scant, whereas the signaling between motor neurons and muscle fibers has been extensively studied. For example, agrin is a motor neuron derived secreted factor that is the primary signal for the induction of acetylcholine receptor (AChR) clustering in myofibers, and treatment of myotubes in culture with agrin has been shown to similarly promote AChR clustering in the absence of neurons. The presence of SCs at motor nerve terminals implicates signaling from these cells in both NMJ formation and maintenance. Thus, the goal was to determine if SC derived signals impact AChR clustering. We hypothesized that SC derived factors would potentiate agrin-induced aneural AChR clustering in C2C12 myotubes. Differentiated C2C12 myotubes were treated with agrin for either 10 minutes or 4 hours to minimally or maximally induce AChR clustering, respectively. Additional myotubes were treated with agrin for 10 minutes followed by treatment with conditioned media (CM) derived from SW10 Schwann cell line cultures for the remaining 4 hours. Finally, additional myotube cultures were treated for 4 hours with CM alone or normal complete media as controls. The number of clusters and mean cluster area were increased in myotube cultures treated with the combination of a 10-minute agrin pulse followed by and CM from SW10 cells compared to cultures treated for either 10 minutes or 4 hours with agrin. Neither cluster number nor area were elevated compared with controls in cultures treated with CM alone. These data suggest that SC-derived signals can potentiate agrin-induced post-synaptic AChR clustering in vitro. We are currently investigating the critical SC-derived signals that potentiate AChR clustering to determine factors that may provide therapeutic benefit in neurodegenerative phenotypes including aging.

Aging results in the progressive accumulation of senescent cells in tissues that display loss of proliferative capacity and acquire a senescence-associated secretory phenotype (SASP). The tumor suppressor, p16 INK4A , which slows the progression of the cell cycle, is highly expressed in most senescent cells and the removal of p16-expressing cells has been shown to be beneficial to tissue health. Although much work has been done to assess the effects of cellular senescence on a variety of different organs, little is known about the effects on skeletal muscle and whether reducing cellular senescent load would provide a therapeutic benefit against age-related muscle functional decline. We hypothesized that whole-body ablation of p16-expressing cells in the advanced stages of life in mice would provide a therapeutic benefit to skeletal muscle structure and function. Treatment of transgenic p16-3MR mice with ganciclovir (GCV) from 20 to 26 months of age resulted in reduced p16 mRNA levels in muscle. At 26 months of age, the masses of tibialis anterior, extensor digitorum longus, gastrocnemius and quadriceps muscles were significantly larger in GCV-treated compared with vehicle-treated mice, but this effect was limited to male mice. Maximum isometric force for gastrocnemius muscles was also greater in GCV-treated male mice compared to controls. Further examination of muscles of GCV- and vehicle-treated mice showed fewer CD68-positive macrophages present in the tissue following GCV treatment. Plasma cytokine levels were also measured with only one, granulocyte colony stimulating factor (G-CSF), out of 22 chemokines analyzed was reduced in GCV-treated mice. These findings show that genetic ablation of p16+ senescent cells provides moderate and sex specific therapeutic benefits to muscle mass and function.

Whole body knock out of Cu, Zn superoxide dismutase1 (Sod1KO) results in accelerated, age-related loss of muscle mass and function associated with a breakdown of neuromuscular junctions (NMJ) similar to sarcopenia. In order to determine whether altered redox in motor neurons is integral to this phenotype, an inducible neuron specific deletion of Sod1 (i-mnSod1KO) was compared with wild type (WT) mice of different ages (adult, mid-age and old) and whole body Sod1KO mice. Nerve oxidative damage, motor neuron numbers and structural changes to neurons and NMJ were examined. Deletion of neuronal Sod1 (induced by tamoxifen injection at 6 months of age) caused the exaggerated, age-associated loss of muscle mass and force generation previously reported. No effect of age or lack of neuronal Sod1 was seen on oxidation in the sciatic nerve assessed by electron paramagnetic resonance of the in vivo spin probe 1-hydroxy-3-carboxy-2,2,5,5 tetramethylpyrrolidine (CPH), analysis of protein 3-nitrotyrosines or carbonyl content. i-mnSod1KO mice showed increased numbers of denervated NMJs, a reduced number of large axons and increased number of small axons compared with age-matched old WT mice. A large proportion of the remaining innervated NMJs in i-mnSod1KO mice also displayed a much simpler structure than that seen in WT mice. Thus, while Sod1KO mice recapitulate substantially the neuromuscular phenotypes of old WT mice, deletion of Sod1 specifically in neurons induces exaggerated loss of muscle mass and force only in old (24-29 month) mice indicating that significant muscle declines require the accumulation of age-related changes such that a threshold is reached past which maintenance of structure and function is not possible. Significance statement Sarcopenia is the age-related loss of muscle mass and function. It is a significant contributor to frailty and to increased falls in the elderly. While multifactorial, changes in redox status have been shown to have significant influence over neuromuscular aging, recent work suggests that changes in motor neurons may be the driving factor in muscle atrophy. The current study confirmed that a specific lack of Sod1 in the motor neuron causes significant alteration in axonal architecture and the neuromuscular junctions which can drive reduced muscle mass and function. Pinpointing early changes in motor neurons may provide therapeutic targets critical for maintaining muscle in the elderly.

Aging is accompanied by loss of muscle mass and force, known as sarcopenia. Muscle atrophy, weakness, and neuromuscular junction (NMJ) degeneration reminiscent of normal muscle aging are observed early in adulthood for mice deficient in Cu, Zn-superoxide dismutase (SOD, Sod1(-/-)). Muscles of Sod1(-/-) mice also display impaired mitochondrial ATP production and increased mitochondrial reactive oxygen species (ROS) generation implicating oxidative stress in sarcopenia. Restoration of CuZnSOD specifically in neurons of Sod1(-/-) mice (SynTgSod1(-/-)) prevents muscle atrophy and loss of force, but whether muscle mitochondrial function is preserved is not known. To establish links among CuZnSOD expression, mitochondrial function, and sarcopenia, we examined contractile properties, mitochondrial function and ROS production, intracellular calcium transients (ICT), and NMJ morphology in lumbrical muscles of 7-9 month wild type (WT), Sod1(-/-), and SynTgSod1(-/-) mice. Compared with WT values, mitochondrial ROS production was increased 2.9-fold under basal conditions and 2.2-fold with addition of glutamate and malate in Sod1(-/-) muscle fibers while oxygen consumption was not significantly altered. In addition, NADH recovery was blunted following contraction and the peak of the ICT was decreased by 25%. Mitochondrial function, ROS generation and calcium handling were restored to WT values in SynTgSod1(-/-) mice, despite continued lack of CuZnSOD in muscle. NMJ denervation and fragmentation were also fully rescued in SynTgSod1(-/-) mice suggesting that muscle mitochondrial and calcium handling defects in Sod1(-/-) mice are secondary to neuronal oxidative stress and its effects on the NMJ rather than the lack of muscle CuZnSOD. We conclude that intact neuronal function and innervation are key to maintaining excitationcontraction coupling and muscle mitochondrial function.

Specialized pro-resolving mediators actively limit inflammation and support tissue regeneration, but their role in age-related muscle dysfunction has not been explored. We profiled the mediator lipidome of aging muscle via liquid chromatography-tandem mass spectrometry and tested whether treatment with the pro-resolving mediator resolvin D1 (RvD1) could rejuvenate the regenerative ability of aged muscle. Aged mice displayed chronic muscle inflammation and this was associated with a basal deficiency of pro-resolving mediators 8-oxo-RvD1, resolvin E3, and maresin 1, as well as many anti-inflammatory cytochrome P450-derived lipid epoxides. Following muscle injury, young and aged mice produced similar amounts of most pro-inflammatory eicosanoid metabolites of cyclooxygenase (e.g., prostaglandin E 2 ) and 12-lipoxygenase (e.g., 12-hydroxy-eicosatetraenoic acid), but aged mice produced fewer markers of pro-resolving mediators including the lipoxins (15-hydroxy-eicosatetraenoic acid), D-resolvins/protectins (17-hydroxy-docosahexaenoic acid), E-resolvins (18-hydroxy-eicosapentaenoic acid), and maresins (14-hydroxy-docosahexaenoic acid). Similar absences of downstream pro-resolving mediators including lipoxin A 4 , resolvin D6, protectin D1/DX, and maresin 1 in aged muscle were associated with greater inflammation, impaired myofiber regeneration, and delayed recovery of strength. Daily intraperitoneal injection of RvD1 had minimal impact on intramuscular leukocyte infiltration and myofiber regeneration but suppressed inflammatory cytokine expression, limited fibrosis, and improved recovery of muscle function. We conclude that aging results in deficient local biosynthesis of specialized pro-resolving mediators in muscle and that immunoresolvents may be attractive novel therapeutics for the treatment of muscular injuries and associated pain in the elderly, due to positive effects on recovery of muscle function without the negative side effects on tissue regeneration of non-steroidal anti-inflammatory drugs.

Skeletal muscle suffers atrophy and weakness with aging. Denervation, oxidative stress, and mitochondrial dysfunction are all proposed as contributors to age-associated muscle loss, but connections between these factors have not been established. We examined contractility, mitochondrial function, and intracellular calcium transients (ICTs) in muscles of mice throughout the life span to define their sequential relationships. We performed these same measures and analyzed neuromuscular junction (NMJ) morphology in mice with postnatal deletion of neuronal Sod1 (i-mn-Sod1-/- mice), previously shown to display accelerated age-associated muscle loss and exacerbation of denervation in old age, to test relationships between neuronal redox homeostasis, NMJ degeneration and mitochondrial function. In control mice, the amount and rate of the decrease in mitochondrial NADH during contraction was greater in middle than young age although force was not reduced, suggesting decreased efficiency of NADH utilization prior to the onset of weakness. Declines in both the peak of the ICT and force were observed in old age. Muscles of i-mn-Sod1-/- mice showed degeneration of mitochondrial and calcium handling functions in middle-age and a decline in force generation to a level not different from the old control mice, with maintenance of NMJ morphology. Together, the findings support the conclusion that muscle mitochondrial function decreases during aging and in response to altered neuronal redox status prior to NMJ deterioration or loss of mass and force suggesting mitochondrial defects contribute to sarcopenia independent of denervation.

Specialized proresolving mediators (SPMs) actively limit inflammation and expedite its resolution by modulating leukocyte recruitment and function. Here we profiled intramuscular lipid mediators via liquid chromatography-tandem mass spectrometry–based metabolipidomics following myofiber injury and investigated the potential role of SPMs in skeletal muscle inflammation and repair. Both proinflammatory eicosanoids and SPMs increased following myofiber damage induced by either intramuscular injection of barium chloride or synergist ablation–induced functional muscle overload. Daily systemic administration of the SPM resolvin D1 (RvD1) as an immunoresolvent limited the degree and duration of inflammation, enhanced regenerating myofiber growth, and improved recovery of muscle strength. RvD1 suppressed inflammatory cytokine expression, enhanced polymorphonuclear cell clearance, modulated the local muscle stem cell response, and polarized intramuscular macrophages to a more proregenerative subset. RvD1 had minimal direct impact on in vitro myogenesis but directly suppressed myokine production and stimulated macrophage phagocytosis, showing that SPMs can modulate both infiltrating myeloid and resident muscle cell populations. These data reveal the efficacy of immunoresolvents as a novel alternative to classical antiinflammatory interventions in the management of muscle injuries to modulate inflammation while stimulating tissue repair.

Our previous work has demonstrated that loss of superoxide dismutase1 (Sod1) in whole body KO mice (Sod1KO) results in an accelerated loss of muscle mass and function that is associated with a breakdown of neuromuscular junctions (NMJ). Deletion of Sod1 specifically in muscle or nerve caused a milder phenotype. These observations led us to hypothesise that altered redox signalling in muscle or nerve alone is not sufficient to induce sarcopenic changes but requires defects in both tissues. Inducible motor neuron Sod1 KO (i‐mnSod1KO) mice were created in order to examine whether loss of Sod1 in nerves in adult‐life affected age‐associated muscle wasting and weakness.This current work has examined changes in the neuromuscular system of Sod1KO, nerve rescue (SynTgSod1KO), i‐mnSod1KO mice and WT mice with advancing age.Hind limb skeletal muscles and sciatic nerves from adult (8–10 months), mid age (16–18 months) and old WT (24–27 months) mice, mid‐age and old i‐mnSod1KO mice, adult Sod1KO and adult SynTgSod1KO were used. Fixed and stained EDL muscles were used for NMJ analysis. Alterations in NMJ structure were quantified including pre‐and post‐synaptic overlap, fragmentation, complexity and area. Schwann cells were stained with fluorescently tagged s100 antibody. Western blot analysis of the sciatic nerves was performed to compare levels of oxidative damage assessed by the contents of 3‐NT and protein carbonylation. Electron paramagnetic resonance (EPR) analysis was carried out by infusing mice with a CPH spin probe and excising the gastrocnemius (GTN) muscle and sciatic nerve to examine the level of oxidised adducts. Samples were placed in Krebs buffer, frozen in liquid nitrogen and scanned on Bruker e‐scan EPR.A decline in NMJ structure was seen with advancing age. Despite some structural alterations, mid age mice showed no significant changes in NMJ structure in either WT or i‐mnSod1KO mice. There were significant alterations in all mice at 24 months and older. NMJs were substantially more disrupted in old i‐mnSod1Ko mice compared with old WT mice. Overlap and complexity of NMJs were both significantly reduced, but fragmentation was similar. The pattern of s100 staining was also altered with advancing age.The concentration of the 3‐carboxy‐proxyl radical (CP) measured by EPR in the sciatic nerve or GTN of adult WT, i‐mnSod1KO and SynTgSod1KO mice was not significantly different, although Sod1KO mice demonstrated a significant increase in CP concentration compared with adult WT.Data suggest that an accumulation of age‐related changes in the muscle alongside altered redox signalling in the nerve due to lack of Sod1 is needed to bring about a significant breakdown of the NMJ.Support or Funding InformationWith thanks to National Institute of Ageing (AG051442) and University of Liverpool for their support.

Much effort has been made to fabricate engineered tissues on a scale that is clinically relevant to humans; however, scale-up remains one of the most significant technological challenges of tissue engineering to date. To address this limitation, our laboratory has developed tissue-engineered skeletal muscle units (SMUs) and engineered neural conduits (ENCs), and modularly scaled them to clinically relevant sizes for the treatment of volumetric muscle loss (VML). The goal of this study was to evaluate the SMUs and ENCs in vitro, and to test the efficacy of our SMUs and ENCs in restoring muscle function in a clinically relevant large animal (sheep) model. The animals received a 30% VML injury to the peroneus tertius muscle and were allowed to recover for 3 months. The animals were divided into three experimental groups: VML injury without a repair (VML only), repair with an SMU (VML+SMU), or repair with an SMU and ENC (VML+SMU+ENC). We evaluated the SMUs before implantation and found that our single scaled-up SMUs were characterized by the presence of contracting myotubes, linearly aligned extracellular matrix proteins, and Pax7+ satellite cells. Three months after implantation, we found that the repair groups (VML+SMU and VML+SMU+ENC) had restored muscle mass and tetanic force production to a level that was statistically indistinguishable from the uninjured contralateral muscle after 3 months in vivo. Furthermore, we demonstrated the ability of our ENCs to effectively bridge the gap between native nerve and the repair site by eliciting a muscle contraction through direct electrical stimulation of the re-routed nerve. Impact statement The fabrication of tissues of clinically relevant sizes is one of the largest obstacles preventing engineered tissues from achieving widespread use in the clinic. This study aimed to combat this limitation by developing a fabrication method to scale-up tissue-engineered skeletal muscle for the treatment of volumetric muscle loss in a large animal (sheep) model and evaluating the efficacy of the tissue-engineered constructs after a 3-month recovery.