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Abstact: With advancing age, the incidence of sarcopenia increases, eventually leading to a cascade of adverse events. However, there is currently a lack of effective pharmacological treatment for sarcopenia. Sodium-glucose co-transporter 2 inhibitor (SGLT2i) empagliflozin demonstrates anti-fibrotic capabilities in various organs. This study aims to determine whether empagliflozin can improve skeletal muscle fibrosis induced by sarcopenia in naturally aging mice. A natural aging model was established by feeding male mice from 13 months of age to 19 months of age. A fibrosis model was created by stimulating skeletal muscle fibroblasts with TGF-β1. The Forelimb grip strength test assessed skeletal muscle function, and expression levels of COL1A1, COL3A1, and α-SMA were analyzed by western blot, qPCR, and immunohistochemistry. Additionally, levels of AMPKα/MMP9/TGFβ1/Smad signaling pathways were examined. In naturally aging mice, skeletal muscle function declines, expression of muscle fibrosis markers increases, AMPKα expression is downregulated, and MMP9/TGFβ1/Smad signaling pathways are upregulated. However, treatment with empagliflozin reverses this phenomenon. At the cellular level, empagliflozin exhibits similar anti-fibrotic effects, and these effects are attenuated by Compound C and siAMPKα. Empagliflozin exhibits anti-fibrotic effects, possibly associated with the AMPK/MMP9/TGFβ1/Smad signaling pathways., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Sarcopenic obesity (SO) is defined as a combination of obesity and sarcopenia, leading to serious health consequences. However, a lack of suitable animal models has hampered research into this disorder. 12-month-old Sprague-Dawley rats were given a high fat content (HFD, SO group) or standard diet (DC groups) for 28 weeks (until 20 months of age). In addition, 2-month-old rats were fed a standard diet as an age control (YC group) until they reached 10 months of age. At the end of the intervention, quadriceps development in the rats was monitored using magnetic resonance examinations and MR spectroscopy. Age-related changes in muscle mass and strength, histopathology, HFD-induced adiposity, and metabolic disturbances were compared between the three groups. Comparing with DC group, rats of SO (20 months, and fed by high-fat diet) exhibited a more prominent loss of muscle mass and strength, a more pronounced decline in myofibre number, IFM, increase in myocyte apoptosis accompanied with increased visceral fat, remarkable glycolipid metabolic disorders, and insulin resistance. However, DC group rats (20 months with standard diet) only showed a decline in quadriceps cross-sectional area/body weight, forelimb grip strength, myofibre cross-sectional area and number, and intermyofibrillar mitochondria number (IFM), increased myocyte apoptosis, without significant metabolic disorder compared with YC group rats. After verifying, SO animal model was successfully set up by HFD induced obesity concomitant with aging-related sarcopenia., (© 2023. The Author(s), under exclusive licence to Springer Nature B.V.)

The senescence-accelerated mouse (SAM) prone 8 (SAMP8) has been demonstrated for muscular aging research including sarcopenia, but its underlying mechanisms remain scarce. Physiological indices and histology of skeletal muscle were analyzed in SAMP8 mice at different ages. SAMP8 mice exhibited typical features of sarcopenia at 40 weeks of age and were more time-efficient than that at 88 weeks of age in bothSAM resistant 1 (SAMR1) and C57BL/6 mice. Increase in FoxO3a-mediated transcription of Atrogin-1 and MuRF1 and decrease in phosphorylated mTOR/P70 s6k were observed at week 40 in SAMP8 mice. High oxidative stress was observed from week 24 and persisted to week 40 in SAMP8 mice evidenced by overexpression of protein carbonyl groups and reduced activities of CAT, SOD, and GPx. Downregulation of genes involved in mitochondrial biogenesis (PGC-1α, Nrf-1, Tfam, Ndufs8, and Cox5b) and in mitochondrial dynamics fission (Mfn2 and Opa1) from week 24 indicated dysregulation of mitochondrial quality control in SAMP8 mice. Impaired autophagic flux was observed in SAMP8 mice evidenced by elevated Atg13 and LC3-II accompanied with the accumulation of P62 and LAMP1. Increases in inflammatory factors (IL-6 and MCP-1), adipokines (leptin and resistin), and myostatin in serum at week 32 and decline in Pax7+ satellite cell resided next to muscle fibers at week 24 implied that muscle microenvironment contributed to the progression of sarcopenia in SAMP8 mice. Our data suggest that early alterations of mitochondrial quality control and autophagic flux worsen muscle microenvironment prior to the onset of sarcopenia.

Age-related changes in ventral lumbar spinal cord (L3-L5) were compared in young [3 month, (M)] and old (27 M) C57BL/6J male mice. The aged mice had previously been shown to exhibit sarcopenia and changes to peripheral nerve morphology. The putative connectivity of β-III tubulin positive α-motor neurons was compared in immunostained transverse sections using excitatory and inhibitory terminal markers vesicular glutamate transporter-1 (VGLUT1) and vesicular GABA transporter (VGAT). Glial fibrillary acidic protein (GFAP) and ionized calcium binding adaptor molecule 1 (Iba1) immunostaining was used to monitor changes in astrocyte and microglial phenotype respectively. For a given motor neuron, the neuronal perimeter was outlined and terminals immunoreactive for VGLUT1 or VGAT in close apposition to the soma were identified. By 27 M, the percentage coverage and total number of VGLUT1 immunoreactive terminals immediately adjacent to the soma of α-motor neurons was significantly decreased compared with young mice. However, percentage coverage of immunoreactive VGAT inhibitory terminals did not change significantly with age. The gray matter of 27 M spinal cords showed increased astrocytic and microglial activity. The loss of VGLUT1 terminals on α-motor neurons, terminals known to be derived from proprioceptive muscle afferents, may further impair sensorimotor control of hind limb skeletal muscle function in old mice.

Sarcopenia is a condition of the loss of muscle mass that is associated with aging and that increases the risk for bedridden state, thereby warranting studies of interventions that attenuate sarcopenia. Here the effects of 2-month dietary L-lysine (Lys) supplementation (1.5-3.0 %) on myofibrillar protein degradation and major proteolytic systems were investigated in senescence-accelerated mouse prone 8 (SAMP8). At 36 weeks of age, skeletal muscle and lean body mass was reduced in SAMP8 when compared with control senescence-accelerated mouse resistant 1 (SAMR1). The myofibrillar protein degradation, which was evaluated by the release of 3-methylhistidine, was stimulated in SAMP8, and the autophagy activity, which was evaluated by light chain 3-II, was stimulated in the skeletal muscle of SAMP8. The activation of ubiquitin-proteasome system was not observed in the muscles of SAMP8. However, myofibrillar protein degradation and autophagic activity in skeletal muscles of SAMP8 were suppressed by dietary intake of 3.0 % Lys. The present data indicate that myofibrillar protein degradation by bulk autophagy is stimulated in the skeletal muscles of SAMP8 and that dietary Lys supplementation attenuates sarcopenia in SAMP8 by suppressing autophagic proteolysis.

A common characteristic of ageing is disrupted homeostasis between growth and atrophy of skeletal muscle resulting in loss of muscle mass and function, which is associated with sarcopenia. Sarcopenia is related to impaired balance, increased falls and decline in quality of life of older people. Ageing-related transcriptome and proteome changes in skeletal muscle have been characterised, however the molecular mechanisms underlying sarcopenia are still not fully understood. microRNAs are novel regulators of gene expression known to modulate skeletal muscle development and homeostasis. Expression of numerous microRNAs is disrupted in skeletal muscle with age however, the functional consequences of this are not yet understood. Given that a single microRNA can simultaneously affect multiple signalling pathways, microRNAs are potent modulators of pathophysiological changes occurring during ageing. Here we use microRNA and transcript expression profiling together with microRNA functional assays to show that disrupted microRNA:target interactions play an important role in maintaining muscle homeostasis. We identified miR-181a as a regulator of the sirtuin1 (Sirt1) gene expression in skeletal muscle and show that the expression of miR-181a and its target gene is disrupted in skeletal muscle from old mice. Moreover, we show that miR-181a:Sirt1 interactions regulate myotube size. Our results demonstrate that disrupted microRNA:target interactions are likely related to the pathophysiological changes occurring in skeletal muscle during ageing.

Sarcopenia is a major contributor to the loss of independence and deteriorating quality of life in elderly individuals, it manifests as a decline in skeletal muscle mass and strength beyond the age of 65. Muscle fibre atrophy is a major contributor to sarcopenia and the most severely atrophic fibres are commonly found in elderly muscles to have permanently lost their motor nerve input. By contrast with elderly fibres, when fibres in young animals lose their motor input they normally mount a response to induce restoration of nerve contact, and this is mediated in part by upregulated expression of the nerve cell adhesion molecule (NCAM). Therefore, skeletal muscles appear to progressively lose their ability to become reinnervated, and here we have investigated whether this decline occurs via loss of the muscle's ability to upregulate NCAM in response to denervation. We performed partial denervation (by peripheral nerve crush) of the extensor digitorum longus muscle of the lower limb in both young and elderly mice. We used immunohistochemistry to compare relative NCAM levels at denervated and control innervated muscle fibres, focused on measurements at neuromuscular junctional, extra-junctional and cytoplasmic locations. Muscle fibres in young animals responded to denervation with significant (32.9%) increases in unpolysialylated NCAM at extra-junctional locations, but with no change in polysialylated NCAM. The same partial denervation protocol applied to elderly animals resulted in no significant change in either polysialylated or unpolysialylated NCAM at junctional, extra-junctional or cytoplasmic locations, therefore muscle fibres in young mice upregulated NCAM in response to denervation but fibres in elderly mice failed to do so. Elevation of NCAM levels is likely to be an important component of the muscle fibre's ability to attract or reattract a neural input, so we conclude that the presence of increasing numbers of long-term denervated fibres in elderly muscles is due, at least in part, to the fibre's declining ability to mount a normal response to loss of motor input.

The age-related loss of skeletal muscle mass and strength (sarcopenia) is predominantly attributed to myofiber atrophy, however the role or existence of myofiber death is currently unclear. We recently discovered dysmorphic myofibers in normal elderly mice resembling those that characterize the Autophagic Vacuolar Myopathies, and speculated that they may be myofibers caught in the act of dying. Since these myofibers were identifiable by Dystrophin Encircled Vacuoles and invaginations with Intracellular Localization we coined the acronym DEVILs and aimed to determine their frequency, pathogenesis and correlation with myofiber loss. In whole transverse sections of young (1-6 month) and elderly (22-26 month) C57Bl/6j mouse muscles, DEVILated myofiber number correlated with myofiber loss, being increasingly prevalent in aged extensor digitorum longus (R = 0.7, p < 0.001) and soleus (R = 0.6, p = 0.004) muscles, whilst rare in myofiber loss resistant muscles (cleido- and sternomastoid). In a cell viability dye-exclusion test, 17 ± 14% of DEVILated myofibers stained positive and were accompanied by immunoglobulin infiltration compared to 1 ± 1% of normal myofibers (p = 0.029). Virtually all DEVILs were acid-phosphatase reactive but contained p62 immunoreactivity and periodic acid-Schiff stained plaques. Compared to normal myofibers, BNIP3 immunostaining in DEVILated myofibers was reduced, whilst MAP-LC3b was indifferent. Cleaved-caspase 3 immunoreactivity was marginally elevated in DEVILated myofibers, but unaccompanied by nuclear DNA fragmentation. DEVILated myofibers were also identified in elderly rat (24 month) and cadaveric human (78 years) muscles. We argue that DEVIL formation reflects a previously undescribed fibre death process via a mechanism involving autophagic dysfunction and that the process may represent our first direct insight into the mechanism by which myofibers are lost in old age.

Sarcopenia, the progressive loss of muscle mass and strength, is a phenomenon characterizing human aging whose etiology is still not clear. While there is increasing evidence for the influence of inter-muscular adipose tissue infiltration in the development of sarcopenia, much less is known about a possible role for intra-muscular triglycerides (IMTG). IMTG accumulate in form of lipid droplets decorated by proteins such as Perilipins (Plins). In skeletal muscle the most abundant are Plin2 and Plin5. In this study we compared the expression of these two Plins in Vastus lateralis muscle samples of subjects of different age, both healthy donors (HD) and patients with limited lower limb mobility (LLMI). These latter are characterized by a condition of chronic physical inactivity. Plin2 expression resulted higher in old age for both HD and LLMI patients, while Plin5 slightly decreased only in LLMI patients. Moreover, in these patients, only Plin2 was associated with the decrease of muscle strength and the expression of factors related to muscle atrophy (MuRF1, Atrogin and p53). An increase in Plin2 and a concomitant decrease of Plin5 was also observed when we considered animal model of disuse-induced muscle atrophy. As a whole, these data indicate that Plin2 and Plin5 have a different expression pattern during muscle aging and inactivity, and only Plin2 appears to be associated with functional alterations of the muscle.

Mammalian skeletal muscles exhibit age-related adaptive and pathological remodeling. Several muscles in particular undergo progressive atrophy and degeneration beyond median lifespan. To better understand myocellular responses to aging, we used semi-quantitative global metabolomic profiling to characterize trends in metabolic changes between 15-month-old adult and 32-month-old aged Fischer 344 × Brown Norway (FBN) male rats. The FBN rat gastrocnemius muscle exhibits age-dependent atrophy, whereas the soleus muscle, up until 32 months, exhibits markedly fewer signs of atrophy. Both gastrocnemius and soleus muscles were analyzed, as well as plasma and urine. Compared to adult gastrocnemius, aged gastrocnemius showed evidence of reduced glycolytic metabolism, including accumulation of glycolytic, glycogenolytic, and pentose phosphate pathway intermediates. Pyruvate was elevated with age, yet levels of citrate and nicotinamide adenine dinucleotide were reduced, consistent with mitochondrial abnormalities. Indicative of muscle atrophy, 3-methylhistidine and free amino acids were elevated in aged gastrocnemius. The monounsaturated fatty acids oleate, cis-vaccenate, and palmitoleate also increased in aged gastrocnemius, suggesting altered lipid metabolism. Compared to gastrocnemius, aged soleus exhibited far fewer changes in carbohydrate metabolism, but did show reductions in several glycolytic intermediates, fumarate, malate, and flavin adenine dinucleotide. Plasma biochemicals showing the largest age-related increases included glycocholate, heme, 1,5-anhydroglucitol, 1-palmitoleoyl-glycerophosphocholine, palmitoleate, and creatine. These changes suggest reduced insulin sensitivity in aged FBN rats. Altogether, these data highlight skeletal muscle group-specific perturbations of glucose and lipid metabolism consistent with mitochondrial dysfunction in aged FBN rats.

Oxidative stress caused by reactive oxygen species is proposed to cause age related muscle wasting (sarcopenia). Reversible oxidation of protein thiols by reactive oxygen species can affect protein function, so we evaluated whether muscle wasting in normal aging was associated with a pervasive increase in reversible oxidation of protein thiols or with an increase in irreversible oxidative damage to macromolecules. In gastrocnemius muscles of C57BL/6J female mice aged 3, 15, 24, 27, and 29 months there was no age related increase in protein thiol oxidation. In contrast, there was a significant correlation (R (2) = 0.698) between increasing protein carbonylation, a measure of irreversible oxidative damage to proteins, and loss of mass of gastrocnemius muscles in aging female mice. In addition, there was an age-related increase in lipofuscin content, an aggregate of oxidised proteins and lipids, in quadriceps limb muscles in aging female mice. However, there was no evidence of an age-related increase in malondialdehyde or F2-isoprostanes levels, which are measures of oxidative damage to lipids, in gastrocnemius muscles. In summary, this study does not support the hypothesis that a pervasive increase in protein thiol oxidation is a contributing factor to sarcopenia. Instead, the data are consistent with an aging theory which proposes that molecular damage to macromolecules leads to the structural and functional disorders associated with aging.

Although adult skeletal muscle is composed of fully differentiated fibers, it retains the capacity to regenerate in response to injury and to modify its contractile and metabolic properties in response to changing demands. The major role in the growth, remodeling and regeneration is played by satellite cells, a quiescent population of myogenic precursor cells that reside between the basal lamina and plasmalemma and that are rapidly activated in response to appropriate stimuli. However, in pathologic conditions or during aging, the complete regenerative program can be precluded by fibrotic tissue formation and resulting in functional impairment of the skeletal muscle. Our study, along with other studies, demonstrated that although the regenerative program can also be impaired by the limited proliferative capacity of satellite cells, this limit is not reached during normal aging, and it is more likely that the restricted muscle repair program in aging is presumably due to missing signals that usually render the damaged muscle a permissive environment for regenerative activity.

Obesity has been suggested as a risk factor for sarcopenia. However, the underlying pathogenic concept of sarcopenic obesity is mainly based on phenotypical data from clinical observation. The present pilot study describes a rodent animal model which opens up prospects to carry out translational research of sarcopenic obesity in an experimental setting. Starting with 2 months, male Wistar rats were fed with a diet containing either 25 en % (control diet, CD) versus 45 en % (high fat diet, HFD) of neutral fat. At the age of 20 and 23 months quadriceps muscles were examined in vivo by magnetic resonance techniques which revealed a positive correlation between muscular fat and body weight (r = 0.639) and a negative correlation between muscular fat content and muscle volume (r = -0.742). Expression and phosphorylation status of proteins within the PKB/Akt and AMPK-dependent signaling pathway were examined in muscles of the 24 month-old animals which significantly showed a 50 percent upregulation of Ser(473)P-PKB/Akt and a 90 % constitutive downregulation of S6K1 in the HFD rats. Notably, S6K1 is a key mediator for muscular protein biosynthesis with additional negative feedback on PKB/Akt. Furthermore, muscular expression of the mitochondrial key regulator PGC-1α in the aged HFD rats was only 25 % of that concurrent controls (p = 0.029). These explorative findings in the aging high-fat fed rat might serve as a firm starting point for controlled longitudinal observations in a larger animal cohort of both sexes studying the natural history of sarcopenic obesity.

Sarcopenia is the age-related loss of skeletal muscle mass and strength, attributable in part to muscle fibre loss. We are currently unable to prevent fibre loss because we do not know what causes it. To provide a platform from which to better understand the causes of muscle fibre death we have quantified fibre loss in several muscles of aged C57Bl/6J mice. Comparison of muscle fibre numbers on dystrophin-immunostained transverse tissue sections at 6 months of age with those at 24 months shows a significant fibre loss in extensor digitorum longus and soleus, but not in sternomastoid or cleidomastoid muscles. The muscles of the elderly mice were mostly lighter than their younger counterparts, but fibres in the elderly muscles were of about the same cross-sectional area. This study shows that the contribution of fibre death to sarcopenia is highly variable and that there is no consistent pattern of age-related fibre loss between skeletal muscles.

The frailty syndrome is increasingly recognized by geriatricians to identify elders at an extreme risk of adverse health outcomes. The physiological changes that result in frailty are complex and up to now have been extremely difficult to characterize due to the frequent coexistence of acute and chronic illness. Frailty is characterized by an decline in the functional reserve with several alterations in diverse physiological systems, including lower energy metabolism, decreased skeletal muscle mass and quality, altered hormonal and inflammatory functions. This altered network leads to an extreme vulnerability for disease, functional dependency, hospitalization and death. One of the most important core components of the frailty syndrome is a decreased reserve in skeletal muscle functioning which is clinically characterized by a loss in muscle mass and strength (sarcopenia), in walking performance and in endurance associated with a perception of exhaustion and fatigue. There are a number of physiological changes that occur in senescent muscle tissues that have a critical effect on body metabolism. The causes of sarcopenia are multi-factorial and can include disuse, changing hormonal function, chronic diseases, inflammation, insulin resistance, and nutritional deficiencies. In this review, we will explore the dysregulation of some biological mechanisms that may contribute to the pathophysiology of the frailty syndrome through age-related changes in skeletal muscle mass and function.