Aim Interventions that decrease atrophy during disuse are desperately needed to maintain muscle mass. We recently found that massage as a mechanotherapy can improve muscle regrowth following disuse atrophy. Therefore, we aimed to determine if massage has similar anabolic effects when applied during normal weight bearing conditions (WB) or during atrophy induced by hindlimb suspension (HS) in adult rats. Methods Adult (10 months) male Fischer344‐Brown Norway rats underwent either hindlimb suspension (HS, n = 8) or normal WB (WB, n = 8) for 7 days. Massage was applied using cyclic compressive loading (CCL) in WB (WBM, n = 9) or HS rats (HSM, n = 9) and included four 30‐minute bouts of CCL applied to gastrocnemius muscle every other day. Results Massage had no effect on any anabolic parameter measured under WB conditions (WBM). In contrast, massage during HS (HSM) stimulated protein turnover, but did not mitigate muscle atrophy. Atrophy from HS was caused by both lowered protein synthesis and higher degradation. HS and HSM had lowered total RNA compared with WB and this was the result of significantly higher ribosome degradation in HS that was attenuated in HSM, without differences in ribosomal biogenesis. Also, massage increased protein turnover in the non‐massaged contralateral limb during HS. Finally, we determined that total RNA degradation primarily dictates loss of muscle ribosomal content during disuse atrophy. Conclusion We conclude that massage is an effective mechanotherapy to impact protein turnover during muscle disuse in both the massaged and non‐massaged contralateral muscle, but it does not attenuate the loss of muscle mass.

Systemic deletion of senescent cells leads to robust improvements in cognitive, cardiovascular, and whole‐body metabolism, but their role in tissue reparative processes is incompletely understood. We hypothesized that senolytic drugs would enhance regeneration in aged skeletal muscle. Young (3 months) and old (20 months) male C57Bl/6J mice were administered the senolytics dasatinib (5 mg/kg) and quercetin (50 mg/kg) or vehicle bi‐weekly for 4 months. Tibialis anterior (TA) was then injected with 1.2% BaCl2 or PBS 7‐ or 28 days prior to euthanization. Senescence‐associated β‐Galactosidase positive (SA β‐Gal+) cell abundance was low in muscle from both young and old mice and increased similarly 7 days following injury in both age groups, with no effect of D+Q. Most SA β‐Gal+ cells were also CD11b+ in young and old mice 7‐ and 14 days following injury, suggesting they are infiltrating immune cells. By 14 days, SA β‐Gal+/CD11b+ cells from old mice expressed senescence genes, whereas those from young mice expressed higher levels of genes characteristic of anti‐inflammatory macrophages. SA β‐Gal+ cells remained elevated in old compared to young mice 28 days following injury, which were reduced by D+Q only in the old mice. In D+Q‐treated old mice, muscle regenerated following injury to a greater extent compared to vehicle‐treated old mice, having larger fiber cross‐sectional area after 28 days. Conversely, D+Q blunted regeneration in young mice. In vitro experiments suggested D+Q directly improve myogenic progenitor cell proliferation. Enhanced physical function and improved muscle regeneration demonstrate that senolytics have beneficial effects only in old mice.

New Findings What is the central question of this study?What is the impact of stress‐induced premature senescence on skeletal muscle myoblast‐derived extracellular vesicles (EVs) and myoblast–endothelial cell crosstalk? What is the main finding and its importance?Hydrogen peroxide treatment of human myoblasts induced stress‐induced premature senescence (SIPS) and increased the release of exosome‐sized EVs (30–150 nm in size) five‐fold compared to untreated controls. Treatment of SIPS myoblast‐derived EVs on endothelial cells increased senescence markers and decreased proliferation. Gene expression analysis of SIPS myoblast‐derived EVs revealed a four‐fold increase in senescence factor transforming growth factor‐β. These results highlight potential mechanisms by which senescence imparts deleterious effects on the cellular microenvironment. Abstract Cellular senescence contributes to numerous diseases through the release of pro‐inflammatory factors as part of the senescence‐associated secretory phenotype (SASP). In skeletal muscle, resident muscle progenitor cells (satellite cells) express markers of senescence with advancing age and in response to various pathologies, which contributes to reduced regenerative capacities in vitro. Satellite cells regulate their microenvironment in part through the release of extracellular vesicles (EVs), but the effect of senescence on EV signaling is unknown. Primary human myoblasts were isolated following biopsies of the vastus lateralis from young healthy subjects. Hydrogen peroxide (H2O2) treatment was used to achieve stress‐induced premature senescence (SIPS) of myoblasts. EVs secreted by myoblasts with and without H2O2 treatment were isolated, analysed and used to treat human umbilical vein endothelial cells (HUVECs) to assess senescence and angiogenic impact. H2O2 treatment of primary human myoblasts in vitro increased markers of senescence (β‐galactosidase and p21Cip1), decreased proliferation and increased exosome‐like EV (30–150 nm) release approximately five‐fold. In HUVECs, EV treatment from H2O2‐treated myoblasts increased markers of senescence (β‐galactosidase and transforming growth factor β), decreased proliferation and impaired HUVEC tube formation. Analysis of H2O2‐treated myoblast‐derived EV mRNA revealed a nearly four‐fold increase in transforming growth factor β expression. Our novel results highlight the impact of SIPS on myoblast communication and identify a VasoMyo Crosstalk by which SIPS myoblast‐derived EVs impair endothelial cell function in vitro.

The decline of skeletal muscle mass during illness, injury, disuse, and aging is associated with poor health outcomes. Therefore, it is important to pursue a greater understanding of the mechanisms that dictate skeletal muscle adaptation. In this review, we propose that RNA-binding proteins (RBPs) comprise a critical regulatory node in the orchestration of adaptive responses in skeletal muscle. While RBPs have broadly pleiotropic molecular functions, our discussion is constrained at the outset by observations from hibernating animals, which suggest that RBP regulation of RNA stability and its impact on translational reprogramming is a key component of skeletal muscle response to anabolic and catabolic stimuli. We discuss the limited data available on the expression and functions of RBPs in adult skeletal muscle in response to disuse, aging, and exercise. A model is proposed in which dynamic changes in RBPs play a central role in muscle adaptive processes through their differential effects on mRNA stability. While limited, the currently available data suggest that understanding how adaptive (and maladaptive) changes in the expression of RBPs regulate mRNA stability in skeletal muscle could be an informative and productive research area for finding new strategies to limit atrophy and promote hypertrophy.

A mechanistic understanding of cell-autonomous skeletal muscle changes after injury can lead to novel interventions to improve functional recovery in an aged population. However, major knowledge gaps persist owing to limitations of traditional biological aging models. 2D cell culture represents an artificial environment, while aging mammalian models are contaminated by influences from non-muscle cells and other organs. Here, a 3D muscle aging system is created to overcome the limitations of these traditional platforms. It is shown that old muscle constructs (OMC) manifest a sarcopenic phenotype, as evidenced by hypotrophic myotubes, reduced contractile function, and decreased regenerative capacity compared to young muscle constructs. OMC also phenocopy the regenerative responses of aged muscle to two interventions, pharmacological and biological. Interrogation of muscle cell-specific mechanisms that contribute to impaired regeneration over time further reveals that an aging-induced increase of complement component 4b (C4b) delays muscle progenitor cell amplification and impairs functional recovery. However, administration of complement factor I, a C4b inactivator, improves muscle regeneration in vitro and in vivo, indicating that C4b inhibition may be a novel approach to enhance aged muscle repair. Collectively, the model herein exhibits capabilities to study cell-autonomous changes in skeletal muscle during aging, regeneration, and intervention.

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Aging is accompanied by reduced remodeling of skeletal muscle extracellular matrix (ECM), which is exacerbated during recovery following periods of disuse atrophy. Mechanotherapy has been shown to promote ECM remodeling through immunomodulation in adult muscle recovery, but not during the aged recovery from disuse. In order to determine if mechanotherapy promotes ECM remodeling in aged muscle, we performed single cell RNA sequencing (scRNA-seq) of all mononucleated cells in adult and aged rat gastrocnemius muscle recovering from disuse, with (REM) and without mechanotherapy (RE). We show that fibroadipogenic progenitor cells (FAPs) in aged RE muscle are highly enriched in chemotaxis genes (Csf1), but absent in ECM remodeling genes compared to adult RE muscle (Col1a1). Receptor-ligand (RL) network analysis of all mononucleated cell populations in aged RE muscle identified chemotaxis-enriched gene expression in numerous stromal cell populations (FAPs, endothelial cells, pericytes), despite reduced enrichment of genes related to phagocytic activity in myeloid cell populations (macrophages, monocytes, antigen presenting cells). Following mechanotherapy, aged REM mononuclear cell gene expression resembled adult RE muscle as evidenced by RL network analyses and KEGG pathway activity scoring. To validate our transcriptional findings, ECM turnover was measured in an independent cohort of animals using in vivo isotope tracing of intramuscular collagen and histological scoring of the ECM, which confirmed mechanotherapy-mediated ECM remodeling in aged RE muscle. Our results highlight age-related cellular mechanisms underpinning the impairment to complete recovery from disuse, and also promote mechanotherapy as an intervention to enhance ECM turnover in aged muscle recovering from disuse.

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