Muscle Of Aged Mice Research Articles

Aging enhances pro-atrogenic gene expression and skeletal muscle loss following respiratory syncytial virus infection.

Aging and many age-related health conditions are associated with skeletal muscle loss. Furthermore, older adults are more susceptible to severe respiratory infections, which can in turn lead to muscle wasting. The mechanisms by which respiratory viral infection can impact skeletal muscle in older adults are not well understood. We determined the effects of acute infection with respiratory syncytial virus (RSV) on the lung and skeletal muscle of aged mice. RSV infection caused more severe disease in aged mice with enhanced weight loss, reduced feeding, higher viral load, and greater airway inflammation. Aged but not young mice showed decreased leg muscle weight at the peak of illness and decreased size of leg muscle fibers. Aged mice increased muscle-specific expression of atrophy-promoting enzymes (Atrogin-1 and MuRF-1) and failed to increase the rate of muscle protein synthesis during RSV infection. In aged mice, the changes in Atrogin-1 and MuRF-1 gene expression in skeletal muscle correlated with IL-6 levels in the lungs. These findings indicate that RSV infection of aged mice provides a model for studying the diverse adverse systemic consequences of respiratory viral infections on health and wellbeing in older adults.

Hypomethylation in promoters of PGC-1α involved in exercise-driven skeletal muscular alterations in old age.

Exercise training can significantly improve skeletal muscle mitochondrial function and has been proven to be highly relevant to alterations in skeletal muscle DNA methylation. However, it remains unclear whether late-in-life exercise has an effect on promoter methylation of PGC-1α, a key regulator of mitochondrial biogenesis. Here we employed two distinct exercise modalities, constant medium intensity exercise training (CMIT) and high-intensity interval exercise training (HIIT), to investigate their impacts on PGC-1α expression and methylation regulation in skeletal muscle of aged mice. The results revealed a notable decrease in PGC-1α expression in skeletal muscle of aged mice, accompanied by elevated methylation levels of the PGC-1α promoter, and increased DNA methyltransferase (DNMT) protein expressions. However, both forms of exercise training significantly corrected PGC-1α epigenetic changes, increased PGC-1α expression, and ameliorated skeletal muscle reduction. Furthermore, exercise training led to elevated expression of proteins related to mitochondrial biogenesis and energy metabolism in skeletal muscle, improving mitochondrial structure and function. In conclusion, late-in-life exercise improved skeletal muscle function, morphology, and mitochondria biogenesis, which may be associated with hypomethylation in promoters of PGC-1α and increased content of skeletal muscle PGC-1α. Notably, there was no clear difference between HIIT and CMIT in PGC-1α expression and skeletal muscle function.

Long-term effects of the chronic administration of doxorubicin on aged skeletal muscle: An exploratory study in mice

The widely used chemotherapeutic drug doxorubicin (DOX) has been associated with adverse effects on the skeletal muscle, which can persist for years after the end of the treatment. These adverse effects may be exacerbated in older patients, whose skeletal muscle might already be impaired by aging. Nonetheless, the mediators responsible for DOX-induced myotoxicity are still largely unidentified, particularly the ones involved in the long-term effects that negatively affect the quality of life of the patients. Therefore, this study aimed to investigate the long-term effects of the chronic administration of DOX on the soleus muscle of aged mice. For that and to mimic the clinical regimen, a dose of 1.5 mg kg−1 of DOX was administered two times per week for three consecutive weeks in a cumulative dose of 9 mg kg−1 to 19-month-old male mice, which were sacrificed two months after the last administration. Body wasting and the atrophy of the soleus muscle, as measured by a decrease in the cross-sectional area of the soleus muscle fibers, were identified as long-term effects of DOX administration. The atrophy observed was correlated with increased reactive oxygen species production and caspase-3 activity. An impaired skeletal muscle regeneration was also suggested due to the correlation between satellite cells activation and the soleus muscle fibers atrophy. Systemic inflammation, skeletal muscle energy metabolism and neuromuscular junction-related markers do not appear to be involved in the long-term DOX-induced skeletal muscle atrophy. The data provided by this study shed light on the mediators involved in the overlooked long-term DOX-induced myotoxicity, paving the way to the improvement of the quality of life and survival rates of older cancer patients.

TRIM16 facilitates SIRT-1-dependent regulation of antioxidant response to alleviate age-related sarcopenia.

Age-related sarcopenia, characterized by reduced skeletal muscle mass and function, significantly affects the health of the elderly individuals. Oxidative stress plays a crucial role in the development of sarcopenia. Tripartite motif containing 16 (TRIM16) is implicated in orchestrating antioxidant responses to mitigate oxidative stress, yet its regulatory role in skeletal muscle remains unclear. This study aims to elucidate the impact of TRIM16 on enhancing antioxidant response through SIRT-1, consequently mitigating age-related oxidative stress, and ameliorating muscle atrophy. Aged mouse models were established utilizing male mice at 18months with D-galactose (D-gal, 200mg/kg) intervention and at 24months with natural aging, while 3-month-old young mice served as controls. Muscle cell senescence was induced in C2C12 myoblasts using 30g/L D-gal. TRIM16 was overexpressed in the skeletal muscle of aged mice and silenced/overexpressed in C2C12 myoblasts. The effects of TRIM16 on skeletal muscle mass, grip strength, morphological changes, myotube formation, myogenic differentiation, and muscle atrophy indicators were evaluated. Reactive oxygen species (ROS) levels and oxidative stress-related parameters were measured. The SIRT-1 inhibitor EX-527 was employed to elucidate the protective role of TRIM16 mediated through SIRT-1. Aged mice displayed significant reductions in lean mass (-11.58%; -14.47% vs. young, P<0.05), hindlimb lean mass (-17.38%; -15.95% vs. young, P<0.05), and grip strength (-22.29%; -31.45% vs. young, P<0.01). Skeletal muscle fibre cross-sectional area (CSA) decreased (-29.30%; -24.12% vs. young, P<0.05). TRIM16 expression significantly decreased in aging skeletal muscle (-56.82%; -66.27% vs. young, P<0.001) and senescent muscle cells (-46.53% vs. control, P<0.001). ROS levels increased (+69.83% vs. control, P<0.001), and myotube formation decreased in senescent muscle cells (-56.68% vs. control, P<0.001). Expression of myogenic differentiation and antioxidant indicators decreased, while muscle atrophy markers increased in vivo and in vitro (all P<0.05). Silencing TRIM16 in myoblasts induced oxidative stress and myotube atrophy, while TRIM16 overexpression partially mitigated aging effects on skeletal muscle. TRIM16 activation enhanced SIRT-1 expression (+75.38% vs. control, P<0.001). SIRT-1 inhibitor EX-527 (100μM) suppressed TRIM16's antioxidant response and mitigating muscle atrophy, offsetting the protective effect of TRIM16 on senescent muscle cells. This study elucidates TRIM16's role in mitigating oxidative stress and ameliorating muscle atrophy through the activation of SIRT-1-dependent antioxidant effects. TRIM16 emerges as a potential therapeutic target for age-related sarcopenia.

Growth Differentiation Factor 11 Evokes Lung Injury, Inflammation, and Fibrosis in Mice through the Activin A Receptor Type II-Like Kinase, 53kDa–Smad2/3 Signaling Pathway

Growth differentiation factor 11 (GDF11) belongs to the transforming growth factor-β superfamily and participates in various pathophysiological processes. Initially, GDF11 was suggested to act as a rejuvenator by improving age-related phenotypes of the heart, brain, and skeletal muscle in aged mice. However, recent studies demonstrate that GDF11 also serves as an adverse risk factor for human frailty and diseases. However, the role of GDF11 in pulmonary fibrosis (PF) remains unclear. In this study, we explored the role and signaling mechanisms of GDF11 in PF. We discovered that GDF11 expression was markedly up-regulated in fibrotic lung tissues of both humans and mice. Intratracheal administration of commercial recombinant GDF11 caused lung injury, inflammation, and fibrogenesis in mice. Furthermore, adenovirus-mediated secretory expression of mature GDF11 was exacerbated, whereas full-length GDF11 or the GDF11 propeptide (GDF111-298) alleviated bleomycin-induced PF in mice. Invitro experiments demonstrated that GDF11 suppressed the growth of alveolar and bronchial epithelial cells (A549 and BEAS-2B) and human pulmonary microvascular endothelial cells, promoted fibroblast activation, and induced epithelial/endothelial-mesenchymal transition. These effects corresponded to the phosphorylation of Smad2/3, and blocking ALK5-Smad2/3 signaling abolished the invivo and invitro effects of GDF11. In conclusion, our findings provide evidence that GDF11 acts as a potent injurious, proinflammatory, and profibrotic factor in the lungs via the ALK5-Smad2/3 pathway.

PARKIN is not required to sustain OXPHOS function in adult mammalian tissues

Loss-of-function variants in the PRKN gene encoding the ubiquitin E3 ligase PARKIN cause autosomal recessive early-onset Parkinson’s disease (PD). Extensive in vitro and in vivo studies have reported that PARKIN is involved in multiple pathways of mitochondrial quality control, including mitochondrial degradation and biogenesis. However, these findings are surrounded by substantial controversy due to conflicting experimental data. In addition, the existing PARKIN-deficient mouse models have failed to faithfully recapitulate PD phenotypes. Therefore, we have investigated the mitochondrial role of PARKIN during ageing and in response to stress by employing a series of conditional Parkin knockout mice. We report that PARKIN loss does not affect oxidative phosphorylation (OXPHOS) capacity and mitochondrial DNA (mtDNA) levels in the brain, heart, and skeletal muscle of aged mice. We also demonstrate that PARKIN deficiency does not exacerbate the brain defects and the pro-inflammatory phenotype observed in mice carrying high levels of mtDNA mutations. To rule out compensatory mechanisms activated during embryonic development of Parkin-deficient mice, we generated a mouse model where loss of PARKIN was induced in adult dopaminergic (DA) neurons. Surprisingly, also these mice did not show motor impairment or neurodegeneration, and no major transcriptional changes were found in isolated midbrain DA neurons. Finally, we report a patient with compound heterozygous PRKN pathogenic variants that lacks PARKIN and has developed PD. The PARKIN deficiency did not impair OXPHOS activities or induce mitochondrial pathology in skeletal muscle from the patient. Altogether, our results argue that PARKIN is dispensable for OXPHOS function in adult mammalian tissues.

Increased Serum Soluble (Pro)renin Receptor Levels in Patients with Sarcopenia

Purpose. Sarcopenia is an advancing and widespread skeletal muscle condition characterized by the depletion of skeletal muscle mass. The (pro)renin receptor ((P)RR) is a multifaceted protein with both pathophysiological and physiological functions. (P)RR is cleaved to generate soluble (P)RR, whose blood concentration may reflect the expression level of tissue (P)RR. We have discovered that the expression of (P)RR was elevated in the atrophied skeletal muscles of aged mice and humans. Methods. In this study, we examined the correlation between muscle atrophy and serum soluble (P)RR levels in bedridden patients with sarcopenia. Results. In total, 45 subjects were enrolled. Their ages were 81 (59–89) y.o., and serum soluble (P)RR levels were 30.5 ± 7.3 ng/ml. Lean body mass ratio (lean body mass/body weight), an index of muscle mass, was independently significantly and negatively correlated with serum soluble (P)RR concentrations in all patients. In addition, the lean body mass ratio was independently significantly and negatively correlated with serum soluble (P)RR concentrations in older patients (≥82 y.o.) but not in younger patients (&lt;82 y.o.). Conclusion. These data suggested that patients with sarcopenia, especially in elderly patients, may be associated with increased (P)RR expression. The serum soluble (P)RR level could be a biomarker of sarcopenia although this issue should be investigated by future studies.

Handelin alleviates cachexia- and aging-induced skeletal muscle atrophy by improving protein homeostasis and inhibiting inflammation.

Handelin is a bioactive compound from Chrysanthemum indicum L. that improves motor function and muscle integrity during aging in Caenorhabditis elegans. This study aimed to further evaluate the protective effects and molecular mechanisms of handelin in a mouse muscle atrophy model induced by cachexia and aging. A tumour necrosis factor (TNF)-α-induced atrophy model was used to examine handelin activity in cultured C2C12 myotubes in vitro. Lipopolysaccharide (LPS)-treated 8-week-old model mice and 23-month-old (aged) mice were used to examine the therapeutic effects of handelin on cachexia- and aging-induced muscle atrophy, respectively, in vivo. Protein and mRNA expressions were analysed by Western blotting, ELISA and quantitative PCR, respectively. Skeletal muscle mass was measured by histological analysis. Handelin treatment resulted in an upregulation of protein levels of early (MyoD and myogenin) and late (myosin heavy chain, MyHC) differentiation markers in C2C12 myotubes (P<0.05), and enhanced mitochondrial respiratory (P<0.05). In TNF-α-induced myotube atrophy model, handelin maintained MyHC protein levels, increased insulin-like growth factor (Igf1) mRNA expression and phosphorylated protein kinase B protein levels (P<0.05). Handelin also reduced atrogin-1 expression, inhibited nuclear factor-κB activation and reduced mRNA levels of interleukin (Il)6, Il1b and chemokine ligand 1 (Cxcl1) (P<0.05). In LPS-treated mice, handelin increased body weight (P<0.05), the weight (P<0.01) and cross-sectional area (CSA) of the soleus muscle (P<0.0001) and improved motor function (P<0.05). In aged mice, handelin slightly increased the weight of the tibialis anterior muscle (P=0.06) and CSA of the tibialis anterior and gastrocnemius muscles (P<0.0001). In the tibialis anterior muscle of aged mice, handelin upregulated mRNA levels of Igf1 (P<0.01), anti-inflammatory cytokine Il10 (P<0.01), mitochondrial biogenesis genes (P<0.05) and antioxidant-related enzymes (P<0.05) and strengthened Sod and Cat enzyme activity (P<0.05). Handelin also reduced lipid peroxidation and protein carbonylation, downregulated mRNA levels of Fbxo32, Mstn, Cxcl1, Il1b and Tnf (P<0.05), and decreased IL-1β levels in serum (P<0.05). Knockdown of Hsp70 or using an Hsp70 inhibitor abolished the ameliorating effects of handelin on myotube atrophy. Handelin ameliorated cachexia- and aging-induced skeletal muscle atrophy in vitro and in vivo, by maintaining homeostasis of protein synthesis and degradation, possibly by inhibiting inflammation. Handelin is a potentially promising drug candidate for the treatment of muscle wasting.

Farnesol prevents aging-related muscle weakness in mice through enhanced farnesylation of Parkin-interacting substrate.

Peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) is a master regulator of mitochondrial biogenesis. Reduced PGC-1α abundance is linked to skeletal muscle weakness in aging or pathological conditions, such as neurodegenerative diseases and diabetes; thus, elevating PGC-1α abundance might be a promising strategy to treat muscle aging. Here, we performed high-throughput screening and identified a natural compound, farnesol, as a potent inducer of PGC-1α. Farnesol administration enhanced oxidative muscle capacity and muscle strength, leading to metabolic rejuvenation in aged mice. Moreover, farnesol treatment accelerated the recovery of muscle injury associated with enhanced muscle stem cell function. The protein expression of Parkin-interacting substrate (PARIS/Zfp746), a transcriptional repressor of PGC-1α, was elevated in aged muscles, likely contributing to PGC-1α reduction. The beneficial effect of farnesol on aged muscle was mediated through enhanced PARIS farnesylation, thereby relieving PARIS-mediated PGC-1α suppression. Furthermore, short-term exercise increased PARIS farnesylation in the muscles of young and aged mice, whereas long-term exercise decreased PARIS expression in the muscles of aged mice, leading to the elevation of PGC-1α. Collectively, the current study demonstrated that the PARIS-PGC-1α pathway is linked to muscle aging and that farnesol treatment can restore muscle functionality in aged mice through increased farnesylation of PARIS.

Physical training promotes remodeling of the skeletal muscle extracellular matrix: An ultrastructural study in a murine model of Down syndrome.

Down syndrome (DS) is a genetically based disease caused by triplication of chromosome 21. DS is characterized by multi-systemic premature aging associated with deficit in motor coordination, balance, and postural control. Using a morphological, morphometrical, and immunocytochemical ultrastructural approach, this study investigated in vastus lateralis muscle of Ts65Dn mouse, a murine model of DS, the effect of an adapted physical training on the extracellular matrix (ECM) characteristics and whether the forecasted exercise-induced ECM remodeling impacts on sarcomere organization. Morphometry demonstrated thicker basement membrane and larger collagen bundles with larger interfibrillar spacing as well as irregularly arrayed myofibrils and lower telethonin density on Z-lines in trisomic versus euploid sedentary mice. In agreement with the multi-systemic premature aging described in DS, these ECM alterations were similar to those previously observed in skeletal muscle of aged mice. Adapted physical training induced remodeling of ECM in both trisomic and euploid mice, that is, enlargement of the collagen bundles associated with hypertrophy of collagen fibrils and reduction of the interfibrillar spacing. A re-alignment of the myofibrils and a higher telethonin density on Z-line was found in trisomic mice. Altogether, our findings suggest that physical training is an effective tool in limiting/counteracting the trisomy-associated musculoskeletal structural anomalies. The current findings constitute a solid experimental background for further study investigating the possible positive effect of physical training on skeletal muscle performance. RESEARCH HIGHLIGHTS: Vastus lateralis muscle of trisomic mice shows aging-like alterations of extracellular matrix. Training promotes extracellular matrix remodeling. Training may be an effective tool to counteract trisomy-associated alterations of skeletal muscle.

Physical exercise attenuates age-related muscle atrophy and exhibits anti-ageing effects via the adiponectin receptor 1 signalling.

Although the adiponectin signalling exerts exercise-mimicking effects, whether this pathway contributes to the anti-ageing benefits of physical exercise has not been established yet. Swim exercise training and wheel running were used to measure lifespan in the nematode Caenorhabditis elegans and skeletal muscle quality in mice, respectively. Muscle weight, muscle fibre cross-sectional area (CSA) and myonuclei number were used to evaluate muscle mass. RNA sequencing (RNA-Seq) analysis of skeletal muscle in exercised mice was used to study the underlying mechanisms. Western blot and immunofluorescence were performed to explore autophagy- and senescence-related markers. The C.elegans adiponectin receptor PAQR-1/AdipoR1, but not PAQR-2/AdipoR2, was activated (3.55-fold and 3.48-fold increases in p-AMPK on Days 1 and 6, respectively, P<0.001), which was involved in lifespan extension in exercised worms. Exercise training increased skeletal muscle mass index (1.29-fold, P<0.01), muscle weight (1.75-fold, P<0.001), myonuclei number (1.33-fold, P<0.05), muscle fibre CSA (1.39-fold, P<0.05) and capillary abundance (2.19-fold, P<0.001 for capillary density; 1.58-fold, P<0.01 for capillary number) in aged mice. Physical exercise reduced protein (2.94-fold, P<0.001) and mRNA levels (1.70-fold, P<0.001) of p16INK4a , a marker for cellular senescence, in skeletal muscle of aged mice. These beneficial effects of exercise on skeletal muscle of mice were dependent on AdipoR1. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis for differentially expressed genes in skeletal muscle between exercised mice with and without AdipoR1 knockdown by RNA-Seq analysis revealed that several KEGG pathways, such as 'AMPK signalling pathway' (P<0.001), 'FOXO signalling pathway' (P<0.001) and 'autophagy' (P<0.001) were overrepresented. Knockdown of FoxO3a inhibited exercise-mediated beneficial effects on skeletal muscle quality of mice by inhibiting autophagy/mitophagy (3.81-fold reduction in LC3-II protein, P<0.001; 1.53-fold reduction in BNIP3 protein, P<0.05). Knockdown of daf-16, the FoxO homologue in C.elegans, reduced autophagy (2.77-fold and 2.06-fold reduction in GFP::LGG-1 puncta in seam cells and the intestine, respectively, P<0.05) and blocked lifespan extension by exercise in worms. Our findings provide insights into how the AdipoR1 pathway has an impact on the anti-ageing benefits of exercise and implicate that activation of the AdipoR1 signalling may represent a potential therapeutic strategy for reducing age-related loss of skeletal muscle.

Montelukast, a LRRC8A inhibitor, decreases the contractile response to Endothelin-1 in the vaginal smooth muscle of aged mice

Introduction: Vaginal smooth muscle (VaSM) is an important contributor to female sexual function, desire, and arousal. Increased vaginal blood flow via vasodilation of the vaginal blood vessels is key to the of lubrication and creating an enjoyable sexual experience. Female sexual dysfunction (FSD) is common in hypertensive and diabetic women, as well as many who suffer from hormonal imbalances, including menopause. Despite the substantial population that suffers from FSD, research into the mechanisms and non-hormonal treatments are limited. Leucine rich repeat containing channel 8A (LRRC8A) is a volume regulated anion channel (VRAC) that regulates Nox1 and can modulate the extracellular production of superoxide (O2∙-). In aged tissues, there are increased in extracellular ROS production as well as vascular and smooth muscle dysfunction. The objective of these experiments is to elucidate the mechanisms behind female sexual dysfunction that present with vascular and smooth muscle dysfunction and determine if the LRRC8A inhibitor, Montelukast, can alleviate some of the symptoms of FSD and improve quality of life. In this study, we hypothesize that the inhibition of LRRC8A in will decrease the contraction to Endothelin-1 in the vaginal smooth muscle of the aged mice. Methods: Aged (16 mo and 20 mo old) female lean mice underwent distal vaginal dissection. Vaginal tissue was cleaned and mounted on a strip myograph in Krebs solution and the contraction elicited by Endothelin-1 was performed in the absence and repeated in the presence of the LRRC8A inhibitor, Montelukast (1uM) after a 30-minute incubation. Data was analyzed using Prism. The maximal contraction in the absence of the inhibitor was taken as 100%, and the contraction in the presence of the inhibitor was calculated as a percentage of the maximal contraction attained in the absence of the inhibitor. Results: Contraction induced by Endothelin-1 (10-12 M to 3✕10 -8 M) alone compared to in the presence of Montelukast (1 uM) showed that Montelukast decreased both the potency and the maximum force of contraction in the aged VaSM. After incubation with 1 mM Montelukast, the EC50 and Emax were significantly different (0.0430 and &lt;0.0001, respectively). Conclusion: This is the first study investigating the vaginal smooth muscle from the perspective of ROS production and the influence of LRRC8A in aged animals. The change in the contractile response in the treated animals may be due to the modulation of the production of O2∙-. Patients that suffer from FSD are a large and overlooked community in need of new therapeutic approaches. Treatment with LRRC8A offers a potential novel approach to treat FSD and should be further researched to observe its overall impact in FSD. 1R01DK132948-01, SMSNA Student Grant 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.

A chronic high-fat diet does not exacerbate muscle atrophy in fast-twitch skeletal muscle of aged mice.

What is the central question of this study? Ageing leads to a loss of mass in skeletal muscle, but the effect of obesity on ageing-related muscle wasting is unclear. In this study, we aimed to demonstrate the specific effect of obesity on fast-twitch skeletal muscle in ageing. What is the main finding and its importance? Our findings show that the obesity induced by long-term ingestion of a high-fat diet does not aggravate muscle wasting in fast-twitch skeletal muscle of aged mice, indicating that the present study provides morphological characteristics for skeletal muscle of sarcopenic obesity. Obesity and ageing reduce muscle mass and lead to deficits in muscle maintenance, but it is not known whether obesity accelerates muscle wasting additively in the setting of ageing. We investigated morphological characteristics in fast-twitch extensor digitorum longus (EDL) muscle of mice fed a low-fat diet (LFD) or a high-fat diet (HFD) for 4 or 20 months. The fast-twitch EDL muscle was harvested, and the muscle fibre-type composition, individual muscle cross-sectional area and myotube diameter were measured. We found an increase in the percentage of type IIa and IIx myosin heavy chain fibres in the whole EDL muscle, but a decrease in type IIB myosin heavy chain in both HFD protocols. The cross-sectional area and myofibre diameter were lower in both groups of aged mice (after 20 months of LFD or HFD) compared with young mice (after 4 months of the diets), but there were no differences between mice fed LFD or HFD for 20 months. These data suggest that long-term feeding of HFD does not aggravate muscle wasting in fast-twitch EDL muscle of male mice.

Accumulation of polyunsaturated fatty acid-derived metabolites in the sarcopenic muscle of aging mice.

Although it is known that advanced age alters skeletal muscle lipid metabolism, the role(s) of polyunsaturated fatty acid-derived metabolites (mostly eicosanoids and docosanoids) in sarcopenia are not clear. We therefore examined the changes in the metabolites of arachidonic acid, eicosapentaenoic acid and docosahexaenoic acid in the sarcopenic muscle of aged mice. We used 6- and 24-month-old male C57BL/6J mice as healthy and sarcopenic muscle models, respectively. Skeletal muscles were removed from the lower limb and subjected to a liquid chromatography-tandem mass spectrometry analysis. The liquid chromatography-tandem mass spectrometry analysis detected distinct changes of metabolites in the muscles of the aged mice. Of the 63 metabolites identified, nine were significantly higher in the sarcopenic muscle of aged mice compared with the healthy muscle of young mice. In particular, prostaglandin E2 , prostaglandin F2a , thromboxane B2 , 5-hydroxyeicosatetraenoic acid, and 15-oxo-eicosatetraenoic acid (arachidonic acid-derived metabolites), 12-hydroxy-eicosapentaenoic acid and 14,15-epoxy-eicosatetraenoic acid (eicosapentaenoic acid-derived metabolites) and 10-hydroxydocosa-hexaenoic acid and 14-hydroxyoctadeca-pentaenoic acid (docosahexaenoic acid-derived metabolites) were significantly higher in aged tissue compared with young tissue (all P< 0.05). We observed the accumulation of metabolites in the sarcopenic muscle of aged mice. Our results may provide new insights into the pathogenesis and progression of aging- or disease-related sarcopenia. Geriatr Gerontol Int 2023; 23: 297-303.

Pyruvate dehydrogenase B regulates myogenic differentiation via the FoxP1-Arih2 axis.

Sarcopenia, the age-related decline in skeletal muscle mass and function, diminishes life quality in elderly people. Improving the capacity of skeletal muscle differentiation is expected to counteract sarcopenia. However, the mechanisms underlying skeletal muscle differentiation are complex, and effective therapeutic targets are largely unknown. The human Gene Expression Omnibus database, aged mice and primary skeletal muscle cells were used to assess the expression level of pyruvate dehydrogenase B (PDHB) in human and mouse aged state. d-Galactose (d-gal)-induced sarcopenia mouse model and two classic cell models (C2C12 and HSkMC) were used to assess the myogenic effect of PDHB and the underlying mechanisms via immunocytochemistry, western blotting, quantitative real-time polymerase chain reaction, RNA interference or overexpression, dual-luciferase reporter assay, RNA sequencing and untargeted metabolomics. We identified that a novel target PDHB promoted myogenic differentiation. PDHB expression decreased in aged mouse muscle relative to the young state (-50% of mRNA level, P<0.01) and increased during mouse and primary human muscle cell differentiation (+3.97-fold, P<0.001 and +3.79-fold, P<0.001). Knockdown or overexpression of PDHB modulated the expression of genes related to muscle differentiation, namely, myogenic factor 5 (Myf5) (-46%, P<0.01 and -27%, P<0.05; +1.8-fold, P<0.01), myogenic differentiation (MyoD) (-55%, P<0.001 and -34%, P<0.01; +2.27-fold, P<0.001), myogenin (MyoG) (-60%, P<0.001 and -70%, P<0.001; +5.46-fold, P<0.001) and myosin heavy chain (MyHC) (-70%, P<0.001 and -69%, P<0.001; +3.44-fold, P<0.001) in both C2C12 cells and HSkMC. Metabolomic and transcriptomic analyses revealed that PDHB knockdown suppressed pyruvate metabolism (P<0.001) and up-regulated ariadne RBR E3 ubiquitin protein ligase 2 (Arih2) (+7.23-fold, P<0.001) in cellular catabolic pathways. The role of forkhead box P1 (FoxP1) (+4.18-fold, P<0.001)-mediated Arih2 transcription was the key downstream regulator of PDHB in muscle differentiation. PDHB overexpression improved d-gal-induced muscle atrophy in mice, which was characterized by significant increases in grip strength, muscle mass and mean muscle cross-sectional area (1.19-fold to 1.5-fold, P<0.01, P<0.05 and P<0.001). The comprehensive results show that PDHB plays a sarcoprotective role by suppressing the FoxP1-Arih2 axis and may serve as a therapeutic target in sarcopenia.

Activation of eIF4E-binding-protein-1 rescues mTORC1-induced sarcopenia by expanding lysosomal degradation capacity.

Chronic mTORC1 activation in skeletal muscle is linked with age-associated loss of muscle mass and strength, known as sarcopenia. Genetic activation of mTORC1 by conditionally ablating mTORC1 upstream inhibitor TSC1 in skeletal muscle accelerates sarcopenia development in adult mice. Conversely, genetic suppression of mTORC1 downstream effectors of protein synthesis delays sarcopenia in natural aging mice. mTORC1 promotes protein synthesis by activating ribosomal protein S6 kinases (S6Ks) and inhibiting eIF4E-binding proteins (4EBPs). Whole-body knockout of S6K1 or muscle-specific over-expression of a 4EBP1 mutant transgene (4EBP1mt), which is resistant to mTORC1-mediated inhibition, ameliorates muscle loss with age and preserves muscle function by enhancing mitochondria activities, despite both transgenic mice showing retarded muscle growth at a young age. Why repression of mTORC1-mediated protein synthesis can mitigate progressive muscle atrophy and dysfunction with age remains unclear. Mice with myofiber-specific knockout of TSC1 (TSC1mKO), in which mTORC1 is hyperactivated in fully differentiated myofibers, were used as a mouse model of sarcopenia. To elucidate the role of mTORC1-mediated protein synthesis in regulating muscle mass and physiology, we bred the 4EBP1mt transgene or S6k1 floxed mice into the TSC1mKO mouse background to generate 4EBP1mt-TSC1mKO or S6K1-TSC1mKO mice, respectively. Functional and molecular analyses were performed to assess their role in sarcopenia development. Here, we show that 4EBP1mt-TSC1mKO, but not S6K1-TSC1mKO, preserved muscle mass (36.7% increase compared with TSC1mKO, P<0.001) and strength (36.8% increase compared with TSC1mKO, P<0.01) at the level of control mice. Mechanistically, 4EBP1 activation suppressed aberrant protein synthesis (two-fold reduction compared with TSC1mKO, P<0.05) and restored autophagy flux without relieving mTORC1-mediated inhibition of ULK1, an upstream activator of autophagosome initiation. We discovered a previously unidentified phenotype of lysosomal failure in TSC1mKO mouse muscle, in which the lysosomal defect was also conserved in the naturally aged mouse muscle, whereas 4EBP1 activation enhanced lysosomal protease activities to compensate for impaired autophagy induced by mTORC1 hyperactivity. Consequently, 4EBP1 activation relieved oxidative stress to prevent toxic aggregate accumulation (0.5-fold reduction compared with TSC1mKO, P<0.05) in muscle and restored mitochondrial homeostasis and function. We identify 4EBP1 as a communication hub coordinating protein synthesis and degradation to protect proteostasis, revealing therapeutic potential for activating lysosomal degradation to mitigate sarcopenia.

Effects of an exogenous ketone ester using multi-omics in skeletal muscle of aging C57BL/6J male mice.

Exogenous ketone ester supplementation provides a means to increase circulating ketone concentrations without the dietary challenges imposed by ketogenic diets. Our group has shown that oral R,S-1,3, butanediol diacetoacetate (BD-AcAc2) consumption results in body weight loss or maintenance with moderate increases in circulating ketones. We have previously shown a diet consisting of 25% BD-AcAc2 can maintain lean body mass (LBM) and induce fat mass (FM) loss in young, healthy male mice, but the underlying mechanisms are still unknown. Therefore, the purpose of this study was to determine if a diet consisting of 25% BD-AcAc2 (ketone ester, KE) would alter body composition, transcriptional regulation, the proteome, and the lipidome of skeletal muscle in aged mice. We hypothesized that the KE group would remain weight stable with improvements in body composition compared to controls, resulting in a healthy aging phenotype. Male C57BL/6J mice (n = 16) were purchased from Jackson Laboratories at 72 weeks of age. After 1 week of acclimation, mice were weighed and randomly assigned to one of two groups (n = 8 per group): control (CON) or KE. A significant group by time interaction was observed for body weight (P < 0.001), with KE fed mice weighing significantly less than CON. FM increased over time in the control group but was unchanged in the KE group. Furthermore, LBM was not different between CON and KE mice despite KE mice weighing less than CON mice. Transcriptional analysis of skeletal muscle identified 6 genes that were significantly higher and 21 genes that were significantly lower in the KE group compared to CON. Lipidomic analysis of skeletal muscle identified no differences between groups for any lipid species, except for fatty acyl chains in triacylglycerol which was 46% lower in the KE group. Proteomics analysis identified 44 proteins that were different between groups, of which 11 were lower and 33 were higher in the KE group compared to CON. In conclusion, 72-week-old male mice consuming the exogenous KE, BD-AcAc2, had lower age-related gains in body weight and FM compared to CON mice. Furthermore, transcriptional and proteomics data suggest a signature in skeletal muscle of KE-treated mice consistent with markers of improved skeletal muscle regeneration, improved electron transport chain utilization, and increased insulin sensitivity.

Atrophic skeletal muscle fibre-derived small extracellular vesicle miR-690 inhibits satellite cell differentiation during ageing.

Sarcopenia is a common and progressive skeletal muscle disorder characterized by atrophic muscle fibres and contractile dysfunction. Accumulating evidence shows that the number and function of satellite cells (SCs) decline and become impaired during ageing, which may contribute to impaired regenerative capacity. A series of myokines/small extracellular vesicles (sEVs) released from muscle fibres regulate metabolism in muscle and extramuscular tissues in an autocrine/paracrine/endocrine manner during muscle atrophy. It is still unclear whether myokines/sEVs derived from muscle fibres can affect satellite cell function during ageing. Aged mice were used to investigate changes in the myogenic capacity of SCs during ageing-induced muscle atrophy. The effects of atrophic myotube-derived sEVs on satellite cell differentiation were investigated by biochemical methods and immunofluorescence staining. Small RNA sequencing was performed to identify differentially expressed sEV microRNAs (miRNAs) between the control myotubes and atrophic myotubes. The target genes of the miRNA were predicted by bioinformatics analysis and verified by luciferase activity assays. The effects of identified miRNA on the myogenic capacity of SCs in vivo were investigated by intramuscular injection of adeno-associated virus (AAV) to overexpress or silence miRNA in skeletal muscle. Our study showed that the myogenic capacity of SCs was significantly decreased (50%, n=6, P<0.001) in the tibialis anterior muscle of aged mice. We showed that atrophic myotube-derived sEVs inhibited satellite cell differentiation in vitro (n=3, P<0.001) and in vivo (35%, n=6, P<0.05). We also found that miR-690 was the most highly enriched miRNA among all the screened sEV miRNAs in atrophic myotubes [Log2 (Fold Change)=7, P<0.001], which was verified in the atrophic muscle of aged mice (threefold, n=6, P<0.001) and aged men with mean age of 71±5.27years (2.8-fold, n=10, P<0.001). MiR-690 can inhibit myogenic capacity of SCs by targeting myocyte enhancer factor 2, including Mef2a, Mef2c and Mef2d, in vitro (n=3, P<0.05) and in vivo (n=6, P<0.05). Specific silencing of miR-690 in the muscle can promote satellite cell differentiation (n=6, P<0.001) and alleviate muscle atrophy in aged mice (n=6, P<0.001). Our study demonstrated that atrophic muscle fibre-derived sEV miR-690 may inhibit satellite cell differentiation by targeting myocyte enhancer factor 2 during ageing.

Macrophage immunomodulation accelerates skeletal muscle functional recovery in aged mice following disuse atrophy.

Poor recovery of muscle size and strength with aging coincides with a dysregulated macrophage response during the early stages of regrowth. Immunomodulation in the form of ex vivo cytokine (macrophage-colony stimulating factor) or polarized macrophage delivery has been demonstrated to improve skeletal muscle regeneration. However, it is unclear if these macrophage-promoting approaches would be effective to improve skeletal muscle recovery following disuse in aged animals. Here, we isolated bone marrow-derived macrophages from donor mice of different ages under various experimental conditions and polarized them into proinflammatory macrophages. Macrophages were delivered intramuscularly into young adult or aged recipient mice during the early recovery period following a period of hindlimb unloading (HU). Delivery of proinflammatory macrophages from donor young adults or aged mice was sufficient to increase muscle function of aged mice during the recovery period. Moreover, proinflammatory macrophages derived from aged donor mice collected during recovery were similarly able to increase muscle function of aged mice following disuse. In addition to the delivery of macrophages, we showed that the intramuscular injection of the cytokine, macrophage-colony stimulating factor, to the muscle of aged mice following HU was able to increase muscle macrophage content and muscle force production during recovery. Together, these results suggest that macrophage immunomodulation approaches in the form of ex vivo proinflammatory macrophage or macrophage-colony stimulating factor delivery during the early recovery phase following disuse atrophy were sufficient to restore the loss of aged skeletal muscle function.NEW & NOTEWORTHY A single intramuscular administration of polarized macrophages into muscles of aged mice following a bout of disuse atrophy was sufficient to improve functional recover similarly to young adults after disuse atrophy regardless of the age or experimental condition of the donor mice. Additionally, intramuscular delivery of macrophage-colony stimulating factor into aged mice was similarly effective. Targeting macrophage function early during the regrowth phase may be a novel tool to bolster muscle recovery in aging.

운동 형태에 따른 노화 쥐 골격근 내 Apoptosis 작용기전에 미치는 영향

The purpose of this study is to investigate the effect of exercise type on the mechanism of apoptosis in the skeletal muscle of aging mice.BR For the experiment, it was classified into a young group (n=10), an aging group (n=10), an uphill exercise(n=10), and a downhill exercise(n=10). The exercise group performed treadmill exercises for 60 minutes, five days a week and eight weeks at exercise intensity 13 m/min, slope 10° (uphill excise) and eccentric excise 23 m/min, -16° (downhill excise).BR The results of this study are as follows.BR 1. Two forms of endurance training in skeletal muscle building in aging mice and changes in expression of oxidative stress-related factors have had a positive effect on aging mice.BR 2. In changes in expression of factors related to apoptosis in skeletal muscle and interaction of IGFBP-3 in aging mice, two forms of endurance exercise training had a positive effect on aging mice.BR 3. In changes in expression of factors related to cell growth and differentiation in skeletal muscle in aging mice and the interaction-related factors of IGFBP-3, two forms of endurance exercise training had a positive effect on aging mice.BR Therefore, the expression mechanism of IGFBP-3, a key factor of stress caused by exercise, not only presented academically meaningful results but also presented important data to suggest safer exercise methods.