Muscles Of Old Mice Research Articles

Unacylated Ghrelin Protects Against Age-Related Loss of Muscle Mass and Contractile Dysfunction in Skeletal Muscle.

Sarcopenia, the progressive loss of muscle mass and function, universally affects older adults and is closely associated with frailty and reduced quality of life. Despite the inevitable consequences of sarcopenia and its relevance to healthspan, no pharmacological therapies are currently available. Ghrelin is a gut-released hormone that increases appetite and body weight through acylation. Acylated ghrelin activates its receptor, growth hormone secretagogue receptor 1a (GHSR1a), in the brain by binding to it. Studies have demonstrated that acyl and unacylated ghrelin (UnAG) both have protective effects against acute pathological conditions independent of receptor activation. Here, we investigated the long-term effects of UnAG in age-associated muscle atrophy and contractile dysfunction in mice. Four-month-old and 18-month-old mice were subjected to either UnAG or control treatment for 10 months. UnAG did not affect food consumption or body weight. Gastrocnemius and quadriceps muscle weights were reduced by 20%-30% with age, which was partially protected against by UnAG. Specific force, force per cross-sectional area, measured in isolated extensor digitorum longus muscle was diminished by 30% in old mice; however, UnAG prevented the loss of specific force. UnAG also protected from decreases in mitochondrial respiration and increases in hydrogen peroxide generation of skeletal muscle of old mice. Results of bulk mRNA-seq analysis and our contractile function data show that UnAG reversed neuromuscular junction impairment that occurs with age. Collectively, our data revealed the direct role of UnAG in mitigating sarcopenia in mice, independent of food consumption or body weight, implicating UnAG treatment as a potential therapy against sarcopenia.

Chloroquine Affects Frequency-Dependent Endocytosis in Diaphragm Muscle of Old Mice

Chloroquine is an antimalarial agent commonly used as an autophagy inhibitor by preventing autolysosomal fusion. Autophagy is an important cellular process through which defective cytoplasmic structures are recycled and degraded that has been implicated in aging neuromuscular effects. We recently showed that acute chloroquine treatment blunts the maximal forces generated by the diaphragm muscle, likely by impairing neuromuscular transmission generally consistent with aging effects in the diaphragm muscle. In addition, old mice show increased accumulation of autophagy proteins in phrenic motor neurons, consistent with impaired autophagy flux. In the present study, we hypothesized that chloroquine treatment results in accumulation of bulk endosomes pre-synaptically. Each hemidiaphragm with attached phrenic nerve was excised from 24-month-old male and female C57BL/6 mice to evaluate frequency dependent (0 vs 80 Hz stimulation) uptake of the styryl dye FM4-64, given that 80-Hz stimulation frequency induces bulk endocytosis after synaptic vesicle retrieval. Diaphragm-phrenic nerve preparations were incubated in 5 μM FM4-64 for 1 h prior to 6-min nerve stimulation (0.5 ms supramaximal pulses in 80 Hz, 200 ms trains delivered at 40% duty cycle) followed by a 5-min post-stimulation incubation. FM4-64 uptake at diaphragm neuromuscular junctions (NMJs) was measured using confocal microscopy. Preparations were randomly assigned to co-incubation with chloroquine (50 μM) or vehicle (Rees-Simpson solution). There was minimal FM4-64 uptake in the absence of phrenic nerve stimulation, consistent with the activity-dependent uptake of the styryl dye. With 80 Hz stimulation, high levels of FM4-64 uptake were evident in both the chloroquine and vehicle-treated groups without significant differences across groups. FM4-64 fluorescence was evident throughout the presynpatic terminal with few areas of dense accumulation, consistent with retention of endosomes. There was no evidence of greater endosome accumulation with chloroquine treatment. Of note, a slight increase in FM4-64 uptake was evident in the chloroquine treated groups compared to vehicle in the non-stimulated NMJs. These results are consistent with FM4-64 uptake reflecting bulk endocytosis that mainly occurs during high frequency stimulation. The impairment in autophagy flux following chloroquine treatment is expected to result in endosome accumulation but it is possible that the stimulation protocol was insuffcient to elucidate this possibility. Collectively, the findings of this study are consistent with impaired autophagy at diaphragm presynaptic terminals which may underlie neuromuscular deficits observed in old age. Supported by NIH grant R01 AG057052 and the Mayo Clinic. 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.

Mitochondrial Peroxiredoxin 6 Declines with Aging in Parallel with Increases in Hydrogen Peroxide Generation

Oxidative stress in cells and tissues arise from an imbalance between the generation of free radicals and the ability to detoxify these reactive oxygen species (ROS). Peroxiredoxins (PRDXs) are a family of widely distributed enzymes that act as master antioxidant regulators by scavenging peroxides and peroxynitrites. The accumulation of oxidative damage from chronic exposure to oxidative stress such as on DNA, mitochondrial proteins, and lipid macromolecules, is postulated to result in age-associated pathologies and physiological dysfunctions. Peroxiredoxin-6 (PRDX6) contributes to the maintenance of redox balance in a variety of tissues from lung to skeletal muscle. Here, we hypothesized that PRDX6 localizes to the mitochondria and plays a significant role in maintaining mitochondrial function through targeting mitochondrially generated ROS, and that gradual loss of mitochondrial PRDX6 contributes to increased oxidative stress and accumulation of oxidative damage during aging. In this study, respiratory rates and complex activities in isolated skeletal muscle mitochondria obtained from young (3-6 months) and old (20-24 months) C57/BL6J mice were compared. Amplex UltraRed fluorescence was used to determine production rates of mitochondrial hydrogen peroxide (H2O2). Western blot analysis was used to compare protein abundance and ELISA was used to measure lipid peroxidation in skeletal muscle mitochondrial fractions. Our findings revealed that isolated mitochondria from skeletal muscle in old mice generated higher rates of H2O2 from complex I (CI) during state 3 and state 4 (State 3; State 4) derived respiration (3.93 ± 0.41; 3.56 ± 0.38 pmol H2O2..s−1.mg−1) compared to young mouse mitochondria (1.57 ± 0.48; 1.89 ± 0.59 pmol H2O2.s−1.mg−1, p ≤ 0.05). This was also found when saturating amounts of ADP were used to stimulate maximal rates of oxygen consumption (old: 3.65 ± 0.77; young 1.40 ± 0.21 pmol H2O2−1.mg−1, p ≤ 0.05). Furthermore, CI activity was found to be lower in isolated mitochondria from old mice (0.65 ± 0.006 U.ug−1) compared to young mice (2.11 ± 0.64 U.ug−1, p ≤ 0.05). Similarly, mitochondrial PRDX6 abundance was lower while levels of lipid peroxidation product malondialdehyde were higher in skeletal muscle from old animals (old: 2712 ± 1320 pg.mL−1; young: 489 ± 225 pg.mL−1). These results show higher mitochondrial ROS generation rates and oxidative damage in old animals. These data also suggest that loss of PRDX6 in aging leads to mitochondrial dysfunction mediated by diminished CI activity and loss of membrane integrity. We are currently conducting mechanistic studies in cellular models with disrupted PRDX6 expression to confirm the effect of this protein in maintaining mitochondrial function. This study offers novel observations on the significance of mitochondrially localized PRDX6 in antioxidant defense as well as the sources and roles of ROS generation in contributing to an aging phenotype. National Institute of General Medical Sciences (NIGMS, Award Number R35GM146951). 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 Effects of Aging on Sarcoplasmic Reticulum-Related Factors in the Skeletal Muscle of Mice.

The pathogenesis of sarcopenia includes the dysfunction of calcium homeostasis associated with the sarcoplasmic reticulum; however, the localization in sarcoplasmic reticulum-related factors and differences by myofiber type remain unclear. Here, we investigated the effects of aging on sarcoplasmic reticulum-related factors in the soleus (slow-twitch) and gastrocnemius (fast-twitch) muscles of 3- and 24-month-old male C57BL/6J mice. There were no notable differences in the skeletal muscle weight of these 3- and 24-month-old mice. The expression of Atp2a1, Atp2a2, Sln, and Pln increased with age in the gastrocnemius muscles, but not in the soleus muscles. Subsequently, immunohistochemical analysis revealed ectopic sarcoplasmic reticulum calcium ion ATPase (SERCA) 1 and SERCA2a immunoreactivity only in the gastrocnemius muscles of old mice. Histochemical and transmission electron microscope analysis identified tubular aggregate (TA), an aggregation of the sarcoplasmic reticulum, in the gastrocnemius muscles of old mice. Dihydropyridine receptor α1, ryanodine receptor 1, junctophilin (JPH) 1, and JPH2, which contribute to sarcoplasmic reticulum function, were also localized in or around the TA. Furthermore, JPH1 and JPH2 co-localized with matrix metalloproteinase (MMP) 2 around the TA. These results suggest that sarcoplasmic reticulum-related factors are localized in or around TAs that occur in fast-twitch muscle with aging, but some of them might be degraded by MMP2.

Senescent skeletal muscle fibroadipogenic progenitors recruit and promote M2 polarization of macrophages.

Senescent cells compromise tissue structure and function in older organisms. We recently identified senescent fibroadipogenic progenitors (FAPs) with activated chemokine signaling pathways in the skeletal muscle of old mice, and hypothesized these cells may contribute to the age-associated accumulation of immune cells in skeletal muscle. In this study, through cell-cell communication analysis of skeletal muscle single-cell RNA-sequencing data, we identified unique interactions between senescent FAPs and macrophages, including those mediated by Ccl2 and Spp1. Using mouse primary FAPs invitro, we verified increased expression of Ccl2 and Spp1 and secretion of their respective proteins in the context of both irradiation- and etoposide-induced senescence. Compared to non-senescent FAPs, the medium of senescent FAPs markedly increased the recruitment of macrophages in an invitro migration assay, an effect that was mitigated by preincubation with antibodies to either CCL2 or osteopontin (encoded by Spp1). Further studies demonstrated that the secretome of senescent FAPs promotes polarization of macrophages toward an M2 subtype. These data suggest the unique secretome of senescent FAPs may compromise skeletal muscle homeostasis by recruiting and directing the behavior of macrophages.

Older mice show decreased regeneration of neuromuscular junctions following lengthening contraction-induced injury.

Progressive muscle atrophy and loss of muscle strength associated with old age have been well documented. Although age-associated impairments in skeletal muscle regeneration following injury have been demonstrated, less is known about whether aging impacts the regenerative response of neuromuscular junctions (NMJ) following contraction-induced injury. Reduced ability of NMJs to regenerate could lead to increased numbers of 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 middle-aged (18-19 months) and old mice (27-28 months) were subjected to a protocol of lengthening contractions (LC) that resulted in an acute force deficit of ~55% as well as functional and histological evidence of a similar magnitude of injury 3 days post LCs that was not different between age groups. After 28 days, the architecture and innervation of the NMJs were evaluated. The numbers of fragmented endplates increased and of fully innervated NMJs decreased post-injury for the muscle of both middle-aged and old mice and for contralateral uninjured muscles of old compared with uninjured muscles of middle-aged controls. Thus, the diminished ability of the skeletal muscle of old mice to recover following injury may be due in part to an age-related decrease in the ability to regenerate NMJs in injured muscles. The impaired ability to regenerate NMJs may be a triggering factor for degenerative changes at the NMJ contributing to muscle fiber weakness and loss in old age.

Deletion of Sod1 in Motor Neurons Exacerbates Age-Related Changes in Axons and Neuromuscular Junctions in Mice.

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.

The role of the aging microenvironment on the fate of PDGFRβ lineage cells in skeletal muscle repair

BackgroundDuring aging, perturbation of muscle progenitor cell (MPC) constituents leads to progressive loss of muscle mass and accumulation of adipose and fibrotic tissue. Mesenchymal stem cells (MSCs) give rise to adipocytes and fibroblasts that accumulate in injured and pathological skeletal muscle through constitutive activation of platelet-derived growth factor receptors (PDGFRs). Although the role of the PDGFRα has been widely explored, there is a paucity of evidence demonstrating the role of PDGFRβ in aged skeletal muscle.MethodsIn this study, we investigated the role of PDGFRβ lineage cells in skeletal muscle during aging by using Cre/loxP lineage tracing technology. The PDGFR-Cre mice were crossed with global double-fluorescent Cre reporter mice (mTmG) that indelibly marks PDGFRβ lineage cells. Those cells were analyzed and compared at different ages in the skeletal muscle of the mice.ResultsOur results demonstrated that PDGFRβ lineage cells isolated from the muscles of young mice are MPC-like cells that exhibited satellite cell morphology, expressed Pax7, and undergo myogenic differentiation producing myosin heavy chain expressing myotubes. Conversely, the PDGFRβ lineage cells isolated from muscles of old mice displayed MSC morphology with a reduced myogenic differentiation potential while expressing adipogenic and fibrotic differentiation markers. PDGFRβ lineage cells also gave rise to newly regenerated muscle fibers in young mice after muscle injury, but their muscle regenerative process is reduced in old mice.ConclusionsOur data suggest that PDGFRβ lineage cells function as MPCs in young mice, while the same PDGFRβ lineage cells from old mice undergo a fate switch participating in adipose and fibrotic tissue infiltration in aged muscle. The inhibition of fate-switching in PDGFRβ lineage cells may represent a potential approach to prevent fibrosis and fatty infiltration in skeletal muscle during the aging process.

Older Mice Show Decreased Regeneration of Neuromuscular Junctions Following Lengthening‐Contraction Injury

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.

Combined transcriptomic, proteomic and synthesis profiling reveal distinct proteostatic signatures for skeletal muscle of adult and old mice

Muscle mass is reduced with age and numerous studies have examined the potential role of global changes in proteostasis, through protein synthesis and/or degradation in the overall loss of muscle mass with age, although no consensus has been reached.We hypothesise that changes in expression, synthesis and degradation may be very different for individual proteins and such findings may be overshadowed by examining global changes in protein turnover. In addition, we hypothesize that alterations in the synthesis rates of specific individual proteins in the muscles of old mice when compared with that of adult are likely to impact on the age‐related loss of muscle mass. We used deuterated water labelling to determine the synthesis rates of individual muscle proteins and have integrated these data with RNA Sequencing data and protein abundances to provide a powerful analysis of individual proteins in muscles of adult (6‐8months) and old (26months) C57BL/6J mice by which time ~15% of gastrocnemius muscle had been lost.For protein synthesis analysis, all animals received an initial loading dose of 2H2O via two IP injections of 99% 2H2O in PBS, 4hrs apart. Mice were then given 8% 2H2O in normal drinking water to maintain plasma enrichment of 2H2O at around 4.5% and gastrocnemius muscles were harvested for up to 60 days when mice were culled. Muscle RNA was isolated, RNA libraries generated using poly‐A selection, sequenced using Illumina HiSeq 4000 platform and analysed with DESEQ2 and gene ontology and pathway tools. To determine the effect of age on differentially expressed genes, protein abundance and turnover, we analysed the common list of 185 proteins and cognate transcripts for which we had data on transcript and turnover parameters.Functional annotation of proteins in the unlabelled dataset demonstrated an upregulation of protein degradation pathways with aging, including ubiquitin protein ligase binding and the unfolded protein response suggesting a clear role of protein degradation as a major contributor to the loss of muscle mass with age in mice. The top four KEGG disease pathways enriched when comparing protein abundance in the muscles of adult and old mice were all degenerative diseases caused or exacerbated by protein misfolding and accumulation. For most proteins, synthesis rates were unchanged, and thus, any change in abundance has to be reconciled with a commensurate change in degradation rate. These global data demonstrate that the net negative protein balance within the muscles of old mice is driven by elevated protein degradation rather than reduced translation. However, there were some notable outliers from the analysis of protein synthesis data showing a decrease in the synthesis of several structural proteins and an increase in the synthesis of metabolic or cytoprotective proteins in muscles of old compared with adult mice. The rate of synthesis of a protein was not always associated with transcript level suggesting caution in extrapolating the suggestion that static mRNA levels reflect the rate of protein synthesis or the protein levels in skeletal muscle.

Altered oxidative DNA damage repair systems in muscles of old mice: role in the age‐related increase in muscle production of cytokines/chemokines

An increased presence of the oxidised DNA base lesion 8‐oxo‐7,8‐dihydroguanine (8‐oxoG) is seen in muscles of old mice. 8‐oxoG is repaired by the 8‐oxoguanine DNA glycosylase‐1 (OGG1)‐initiated DNA base excision repair pathway, a process proposed to lead to activation of nuclear factor kappa‐light‐chain‐enhancer of activated B cells (NF‐kB). We hypothesised that chronic oxidative DNA damage in muscles of old mice leads to the chronic increase in activation of NF‐kB by OGG1 and the subsequent production of inflammatory cytokines by skeletal muscle, contributing to age‐related deterioration of muscle function.An in vitro model of oxidative DNA damage was established by treating C2C12 myoblasts with H2O2. DNA damage repair responses including 8‐oxoG, OGG1, PARP‐1, NF‐kB activation and cytokine/chemokine gene expression and release by muscle cells were determined using Luminex multiplex analysis. The effect of TH5487 (5µM), a selective OGG1 inhibitor on NF‐kB activation and cytokine/chemokine production was determined. The role of OGG1 in the chronic production of cytokines/chemokines by isolated muscle fibres of adult mice treated with H2O2 and of old mice was examined. Cell viability was maintained in myoblasts treated with 50µM H2O2 but Ogg1 mRNA levels were significantly increased as was acetylation of OGG1, PARP‐1 and cleaved PARP‐1 protein levels. Activation of NF‐κB was evident and accompanied by an increased expression and release of a number of cytokines/chemokines (3.7 fold, 4.2 fold, 3 fold increases in IL‐6 TNF‐a and CXCL1 mRNA respectively). The H2O2‐mediated increase in NF‐kB activation and increase in cytokine/chemokine mRNA levels were normalized when cells were pre‐treated with TH5487 prior to treatment with H2O2. Experiments using isolated muscle fibers from adult mice demonstrated an increase in NF‐κB activation following treatment with H2O2. This H2O2‐mediated increase in NF‐kB DNA binding activity was significantly decreased in muscle fibers pre‐treated with TH5487. Isolated muscle fibers from old compared with adult mice showed an increase in cytokine/chemokine release which was also reduced by treatment with TH5487. Increases in IL‐6, CCL2,CXCL1,CCL7,CCL27,CXCL5,CCL11,CXCL16 and CXCL‐12 were seen in the media of fibers from adult mice following treatment with H2O2 and most of these were then decreased to normal values by pre‐treatment with TH5487. Isolated muscle fibers from old mice showed a significant increase in IL‐6, CXCL1,CCL2,CCL7, of which all were reduced following treatment with TH5487, suggesting that activation of the OGG1 pathway may be responsible, at least in part for the increased production of cytokines/chemokines by muscle of old mice. Data on proteomic and mRNA analyses of muscles from adult and old mice for evidence of changes in DNA repair pathways with age will also be presented. A computationalmodel of DNA damage repair is being designed to identify DNA damage repair process therapeutic targets to modify activation of NF‐κB in muscles of old mice.

Self-transducible LRS-UNE-L peptide enhances muscle regeneration.

BackgroundMuscle regeneration includes proliferation and differentiation of muscle satellite cells, which involves the mammalian target of rapamycin (mTOR). We identified the C‐terminal unique attached sequence motif (UNE) domain of leucyl‐tRNA synthetase (LRS‐UNE‐L) as an mTORC1 (mTOR complex1)‐activating domain that acts through Vps34 and phospholipase D1 (PLD1) when introduced in the form of a muscle‐enhancing peptide.Methods In vitro Vps34 lipid kinase assay, phosphatidylinositol 3‐phosphate (PI(3)P) measurement, in vivo PLD1 assay, and western blot assay were performed in HEK293 cells to test the effect of the LRS‐UNE‐L on the Vps34‐PLD1‐mTOR pathway. Adeno‐associated virus (AAV)‐LRS‐UNE‐L was transduced in C2C12 cells in vitro, in BaCl2‐injured tibialis anterior (TA) muscles, and in 18‐month‐old TA muscles to analyse its effect on myogenesis, muscle regeneration, and aged muscle, respectively. The muscle‐specific cell‐permeable peptide M12 was fused with LRS‐UNE‐L and tested for cell integration in C2C12 and HEK293 cells using FACS analysis and immunocytochemistry. Finally, M12‐LRS‐UNE‐L was introduced into BaCl2‐injured TA muscles of 15‐week‐old Pld1 +/+ or Pld1 −/− mice, and its effect was analysed by measurement of cross‐sectional area of regenerating muscle fibres.ResultsThe LRS‐UNE‐L expression restored amino acid‐induced S6K1 phosphorylation in LRS knockdown cells in a RagD GTPases‐independent manner (421%, P = 0.007 vs. LRS knockdown control cells). The LRS‐UNE‐L domain was directly bound to Vps34; this interaction was accompanied by increases in Vps34 activity (166%, P = 0.0352), PI(3)P levels (146%, P = 0.0039), and PLD1 activity (228%, P = 0.0294) compared with amino acid‐treated control cells, but it did not affect autophagic flux. AAV‐delivered LRS‐UNE‐L domain augmented S6K1 phosphorylation (174%, P = 0.0013), mRNA levels of myosin heavy chain (MHC) (122%, P = 0.0282) and insulin‐like growth factor 2 (IGF2) (146%, P = 0.008), and myogenic fusion (133%, P = 0.0479) in C2C12 myotubes. AAV‐LRS‐UNE‐L increased the size of regenerating muscle fibres in BaCl2‐injured TA muscles (124%, P = 0.0279) (n = 9–10), but it did not change the muscle fibre size of TA muscles in old mice. M12‐LRS‐UNE‐L was preferentially delivered into C2C12 cells compared with HEK293 cells and augmented regeneration of BaCl2‐injured TA muscles in a PLD1‐dependent manner (116%, P = 0.0022) (n = 6).ConclusionsOur results provide compelling evidence that M12‐LRS‐UNE‐L could be a muscle‐enhancing protein targeting mTOR.

Effects of Glycine Supplementation on Mitochondrial Function and Protein Degradation in Skeletal Muscle of Old Mice

Glycine is the simplest amino acid and it has a pivotal role in different metabolic processes, such as being a building block of glutathione, collagen and purine bases, or taking part in methylation reactions, detoxication and ammonia metabolism. Although considered for many years a non-essential amino acid, glycine levels are decreased in certain conditions, as the endogenous synthesis cannot fulfill the needs required to sustain all the cellular processes in which glycine is involved. Here we describe that glycine levels are significantly lower in skeletal muscle of aged zebrafish and mice and in plasma of humans compared to young subjects. We therefore fed healthy old mice for 6 weeks with a glycine-supplemented diet and observed a significant restoration of glycine levels in skeletal muscle and liver towards young mouse levels. Moreover, old mice showed decreased mitochondrial function in glycolytic and oxidative fibers, and a significant increase in oxygen consumption was observed in glycolytic fibers after glycine supplementation. The improvement of mitochondrial function is not associated to an increased mitochondrial biogenesis or an increased antioxidant capacity, but glycine supplementation increases both total GSH and GSSG levels, suggestive of a pro-oxidant environment. Overall, glycine supplementation induced an increase in the cross-sectional area of fibers. Finally, we carried out RNA-Seq study to decipher the impact of higher glycine intake. Our results suggest that age-associated glycine deficiency plays an important role in atrophy of muscle, especially in glycolytic fibers, and is reversible with a dietary supplementation.

Age-related structural changes show that loss of fibers is not a significant contributor to muscle atrophy in old mice.

Age-related loss of skeletal muscle mass is widely considered a consequence of both fiber atrophy and fiber death. Evidence for fiber death derives largely from an age-related reduction in fiber numbers in muscle cross-sections, however it is unclear how age-related alterations in muscle morphology affect accuracy of such counts. To explore this we performed an examination of muscle and tendon length, muscle mass and girth, and pennation angle, in addition to histological section fiber counts of parallel-fibered (sternomastoid), fusiform (biceps brachii), and pennate (tibialis anterior, extensor digitorum longus, soleus) muscles from 31 mice aged 6-32months. Age-related decline in mass and girth occurred in soleus (p=0.026; p=0.040), tibialis anterior (p=0.004; p=0.039), and extensor digitorum longus (p=0.040; p=0.022) muscles, for which location of maximal girth also changed. Tendon length and pennation angle remained consistent across the lifespan in all except soleus which showed elongation of both proximal and distal tendons coupled with alterations in pennation angle. Age-related decreases in fiber number were observed in transversely sectioned soleus and extensor digitorum longus muscles however when age-related changes in morphology were accounted for via oblique sectioning the age-related decrease in fiber number was eliminated. Findings show loss of fibers is not a significant contributor to age-related muscle wasting in mice, and that age-related changes in connective tissue selectively impact muscle structure. Fiber shortening is a likely contributor to loss of mass and change in function in muscles of old mice.

MiR-24 and its target gene Prdx6 regulate viability and senescence of myogenic progenitors during aging.

Satellite cell‐dependent skeletal muscle regeneration declines during aging. Disruptions within the satellite cells and their niche, together with alterations in the myofibrillar environment, contribute to age‐related dysfunction and defective muscle regeneration. In this study, we demonstrated an age‐related decline in satellite cell viability and myogenic potential and an increase in ROS and cellular senescence. We detected a transient upregulation of miR‐24 in regenerating muscle from adult mice and downregulation of miR‐24 during muscle regeneration in old mice. FACS‐sorted satellite cells were characterized by decreased levels of miR‐24 and a concomitant increase in expression of its target: Prdx6. Using GFP reporter constructs, we demonstrated that miR‐24 directly binds to its predicted site within Prdx6 mRNA. Subtle changes in Prdx6 levels following changes in miR‐24 expression indicate miR‐24 plays a role in fine‐tuning Prdx6 expression. Changes in miR‐24 and Prdx6 levels were associated with altered mitochondrial ROS generation, increase in the DNA damage marker: phosphorylated‐H2Ax and changes in viability, senescence, and myogenic potential of myogenic progenitors from mice and humans. The effects of miR‐24 were more pronounced in myogenic progenitors from old mice, suggesting a context‐dependent role of miR‐24 in these cells, with miR‐24 downregulation likely a part of a compensatory response to declining satellite cell function during aging. We propose that downregulation of miR‐24 and subsequent upregulation of Prdx6 in muscle of old mice following injury are an adaptive response to aging, to maintain satellite cell viability and myogenic potential through regulation of mitochondrial ROS and DNA damage pathways.

Exercise-induced gene expression changes in skeletal muscle of old mice

Exercise is believed to be beneficial for skeletal muscle functions across all ages. Regimented exercise is often prescribed as an effective treatment/prophylaxis for age-related loss of muscle mass and function, known as sarcopenia, and plays an important role in the maintenance of mobility and functional independence in the elderly. However, response to exercise declines with aging, resulting in limited gain of muscle strength and endurance. These changes likely reflect age-dependent alterations in transcriptional response underlying the muscular adaptation to exercise. The exact changes in gene expression accompanying exercise, however, are largely unknown, and elucidating them is of a great clinical interest for understanding and optimizing the exercise-based therapies for sarcopenia.In order to characterize the exercise-induced transcriptomic changes in aged muscle, a paired-end RNA sequencing was performed on rRNA-depleted total RNA extracted from the gastrocnemius muscles of 24 months-old mice after 8 weeks of regimented exercise (exercise group) or no formal exercise program (sedentary group).Differential gene expression analysis of aged skeletal muscle revealed upregulations in the group of genes involved in neurotransmission and neuroexcitation, as well as equally notable absence of anabolic gene upregulations in the exercise group. In particular, genes encoding the transporters and receptor components of glutaminergic transmission were significantly upregulated in exercised muscles, as exemplified by Gria 1, Gria 2 and Grin2c encoding glutamate receptor 1, 2 and 2C respectively, Grin1 and Grin2b encoding N-methyl-d-aspartate receptors (NMDARs), Nptx1 responsible for glutaminergic receptor clustering, and Slc1a2 and Slc17a7 regulating synaptic uptake of glutamate. These changes were accompanied by an increase in the post-synaptic density of NMDARs and acetylcholine receptors (AChRs), as well as their innervation at neuromuscular junctions (NMJs).These results suggest that neural responses predominate the adaptive response of aged skeletal muscle to exercise, and indicate a possibility that glutaminergic transmission at NMJs may be present and responsible for synaptic protection and neural remodeling accompanying the exercise-induced functional enhancement in aged skeletal muscle. In addition, the absence of upregulations in the anabolic pathways highlights them as the area of potential pharmacological targeting for optimizing exercise-led sarcopenia therapy.

TMPRSS11a is a novel age-altered, tissue specific regulator of migration and wound healing.

Aging is a gradual biological process characterized by a decrease in cellular and organism functions. Aging-related processes involve changes in the expression and activity of several proteins. Here, we identified the transmembrane protease serine 11a (TMPRSS11a) as a new age-specific protein that plays an important role in skin wound healing. TMPRSS11a levels increased with age in rodent and human skin and gingival samples. Strikingly, overexpression of TMPRSS11a decreased cell migration and spreading, and inducing cellular senescence. Mass spectrometry, bioinformatics, and functional analyses revealed that TMPRSS11a interacts with integrin β1 through an RGD sequence contained within the C-terminal domain and that this motif was relevant for cell migration. Moreover, TMPRSS11a was associated with cellular senescence, as shown by overexpression and downregulation experiments. In agreement with tissue-specific expression of TMPRSS11a, shRNA-mediated downregulation of this protein improved wound healing in the skin, but not in the skeletal muscle of old mice, where TMPRSS11a is undetectable. Collectively, these findings indicate that TMPRSS11a is a tissue-specific factor relevant for wound healing, which becomes elevated with aging, promoting cellular senescence and inhibiting cell migration and skin repair.

Iron accumulation in skeletal muscles of old mice is associated with impaired regeneration after ischaemia-reperfusion damage.

BackgroundOxidative stress is implicated in the insidious loss of muscle mass and strength that occurs with age. However, few studies have investigated the role of iron, which is elevated during ageing, in age‐related muscle wasting and blunted repair after injury. We hypothesized that iron accumulation leads to membrane lipid peroxidation, muscle wasting, increased susceptibility to injury, and impaired muscle regeneration.MethodsTo examine the role of iron in age‐related muscle atrophy, we compared the skeletal muscles of 3‐month‐old with 22‐ to 24‐month‐old 129SvEv FVBM mice. We assessed iron distribution and total elemental iron using laser ablation inductively coupled plasma mass spectrometry and Perls' stain on skeletal muscle cross‐sections. In addition, old mice underwent ischaemia–reperfusion (IR) injury (90 min ischaemia), and muscle regeneration was assessed 14 days after injury. Immunoblotting was used to determine lipid peroxidation (4HNE) and iron‐related proteins. To determine whether muscle iron content can be altered, old mice were treated with deferiprone (DFP) in the drinking water, and we assessed its effects on muscle regeneration after injury.ResultsWe observed a significant increase in total elemental iron (+43%, P < 0.05) and lipid peroxidation (4HNE: +76%, P < 0.05) in tibialis anterior muscles of old mice. Iron was further increased after injury (adult: +81%, old: +135%, P < 0.05) and associated with increased lipid peroxidation (+41%, P < 0.05). Administration of DFP did not impact iron or measures of lipid peroxidation in skeletal muscle or modulate muscle mass. Increased muscle iron concentration and lipid peroxidation were associated with less efficient regeneration, evident from the smaller fibres in cross‐sections of tibialis anterior muscles (−24%, P < 0.05) and an increased percentage of fibres with centralized nuclei (+4124%, P < 0.05) in muscles of old compared with adult mice. Administration of DFP lowered iron after IR injury (PRE: −32%, P < 0.05 and POST: −41%, P < 0.05), but did not translate to structural improvements.ConclusionsMuscles from old mice have increased iron levels, which are associated with increased lipid peroxidation, increased susceptibility to IR injury, and impaired muscle regeneration. Our results suggest that iron is involved in effective muscle regeneration, highlighting the importance of iron homeostasis in muscle atrophy and muscle repair.

TrkB signaling contributes to transdiaphragmatic pressure generation in aged mice.

Ventilatory deficits are common in old age and may result from neuromuscular dysfunction. Signaling via the tropomyosin-related kinase receptor B (TrkB) regulates neuromuscular transmission and, in young mice, is important for the generation of transdiaphragmatic pressure (Pdi). Loss of TrkB signaling worsened neuromuscular transmission failure and reduced maximal Pdi, and these effects are similar to those observed in old age. Administration of TrkB agonists such as 7,8-dihydroxyflavone (7,8-DHF) improves neuromuscular transmission in young and old mice (18 mo; 75% survival). We hypothesized that TrkB signaling contributes to Pdi generation in old mice, particularly during maximal force behaviors. Old male and female TrkBF616A mice, with a mutation that induces 1NMPP1-mediated TrkB kinase inhibition, were randomly assigned to systemic treatment with vehicle, 7,8-DHF, or 1NMPP1 1 h before experiments. Pdi was measured during eupneic breathing (room air), hypoxia-hypercapnia (10% O2/5% CO2), tracheal occlusion, spontaneous deep breaths ("sighs"), and bilateral phrenic nerve stimulation (Pdimax). There were no differences in the Pdi amplitude across treatments during ventilatory behaviors (eupnea, hypoxia-hypercapnia, occlusion, or sigh). As expected, Pdi increased from eupnea and hypoxia-hypercapnia (∼7 cm H2O) to occlusion and sighs (∼25 cm H2O), with no differences across treatments. Pdimax was ∼50 cm H2O in the vehicle and 7,8-DHF groups and ∼40 cm H2O in the 1NMPP1 group (F8,74 = 2; P = 0.02). Our results indicate that TrkB signaling is necessary for generating maximal forces by the diaphragm muscle in old mice and are consistent with aging effects of TrkB signaling on neuromuscular transmission.NEW & NOTEWORTHY TrkB signaling is necessary for generating maximal forces by the diaphragm muscle. In 19- to 21-mo-old TrkBF616A mice susceptible to 1NMPP1-induced inhibition of TrkB kinase activity, maximal Pdi generated by bilateral phrenic nerve stimulation was ∼20% lower after 1NMPP1 compared with vehicle-treated mice. Treatment with the TrkB agonist 7,8-dihydroxyflavone did not affect Pdi generation when compared with age-matched mice. Inhibition of TrkB kinase activity did not affect the forces generated during lower force behaviors in old age.

Alterations of macrophage and neutrophil content in skeletal muscle of aged versus young mice.

BackgroundSkeletal muscle inflammation and oxidative stress are associated with aging‐related loss of muscle mass and may be attributable to alterations in the number and types of leukocytes in skeletal muscle. Here, we tested the hypothesis that aging changes the number and composition of leukocyte subsets in skeletal muscle tissue.MethodsSkeletal muscle was sampled from 4‐mo‐old (young) and 27‐mo‐old (old) C57BL/6J mice. Mononuclear cells of the gastrocnemius muscle were isolated, and flow cytometry was used to characterize the number and types of immune cells.ResultsThe number of neutrophils and Ly‐6C+ inflammatory macrophages in the skeletal muscle was significantly higher in old mice than in young mice. Inflammation and oxidative stress (measured using the markers phosphorylated JNK and nitrotyrosine) were also higher in the skeletal muscle of old mice than in that of young mice.ConclusionsIncreasing age promotes skeletal muscle inflammation and oxidative stress, as well as infiltration of inflammatory macrophages and neutrophils.