Aging induces sarcopenia by disrupting the crosstalk between the skeletal muscle microenvironment and myofibers

Graphical abstract

Highlights

• •Comprehensively summarize the cellular components and spatial structure of the skeletal muscle microenvironment (SMME).
• •Elucidate the effects of aging on each SMME cellular components.
• •Focus on the crosstalk between myofibers and SMME, as well as its role in sarcopenia during aging.
• •Emphasize the knowledge gap between the pathological changes of SMME components and myofiber atrophy, the endpoint of sarcopenia.
• •Summarize the single-cell and spatial omics in studying the alterations of SMME components and intercellular crosstalk during aging.

Abstract

Background

Sarcopenia is a degenerative muscular disease associated with aging, characterized by a reduction in muscle mass and strength. This disease poses a significant global health challenge, owing to its high prevalence and association with adverse outcomes such as increased frailty, impaired physical function, and elevated mortality risk. A deeper understanding of its underlying mechanisms is urgently warranted for the development of effective therapeutic interventions.

Aim of review

Skeletal muscle is a heterogeneous tissue composed of various cellular components, including myofibers and other muscle-resident cells such as satellite cells, neurons and immune cells. Myofibers serve as the fundamental units determining muscle mass and strength, while muscle-resident cells establish the skeletal muscle microenvironment (SMME), which plays a significant role in maintaining skeletal muscle health. This review aimed to systematically dissect the crosstalk between myofibers and the SMME, and develop potential therapeutic interventions by highlighting novel insights into the pathogenesis of sarcopenia.

Key scientific concepts of review

This review provides a comprehensive overview of the age-related changes in various SMME components, with a specific focus on the disrupted interactions between them and myofibers during aging.

Introduction

Sarcopenia is a degenerative muscular disease associated with aging, characterized by a reduction in muscle mass and strength. This disease poses a significant global health challenge, impacting approximately 10–27 % of older adults globally [1], and is closely linked to numerous adverse outcomes, such as increased frailty, impaired physical function, and elevated mortality risk [2]. The annual cost per patient hospitalized for sarcopenia exceeds $2000 [3]. Despite the huge benefits of targeted interventions, there remains a lack of effective therapeutic options for sarcopenia, and a deeper understanding of its underlying mechanisms is urgently warranted.

Myofibers are the fundamental structures and functional units of skeletal muscle, their quantity, thickness, and contraction properties are decisive factors for skeletal muscle quality (mass and strength). Besides myofibers, skeletal muscle involves multiple other cell types, and they collectively form a complex hierarchical structure that surrounds each myofiber to establish a niche, termed the skeletal muscle microenvironment (SMME), facilitating close interactions and mutual influences. Aging can lead to various pathophysiological changes of the SMME, including dysfunction of satellite cells (SCs), infiltration of fat and fibrotic connective tissue, degeneration of neuromuscular junctions (NMJs), etc. [[4], [5], [6]]. These changes promote the development of sarcopenia via information exchanges with myofibers. For instance, fibro/adipogenic progenitors (FAPs) can specifically secrete growth differentiation factor 10 (GDF10) to maintain myofiber mass; however, the expression of GDF10 is reduced in the aging process, thus exacerbating myofiber atrophy [7]. This underscores the critical role of such crosstalk in elucidating the underlying mechanisms of sarcopenia development. Previous studies have extensively explored how the SMME components influence myofibers. Therefore, it is now necessary to systematically review the current state of research in this field to gain a comprehensive understanding of the pathogenesis of sarcopenia and to guide future research directions.

This review discusses the age-related changes in myofibers and different SMME components. Subsequently, we focus on the interactions between myofibers and their niche, highlighting how aging can reprogram the crosstalk to drive sarcopenia initiation and progression.

Skeletal muscle microenvironment, SMME

In skeletal muscle, a precise and intricate spatial organization exists between myofibers and the cellular components of the SMME. Each myofiber contains multiple myofibrils and is encased by the sarcolemma. SCs are positioned above the sarcolemma, beneath the basal lamina, and adjacent to myonuclei [5]. Bundles of myofibers are encapsulated by the perimysium to form fascicles, and bundles of fascicles are also wrapped by the epimysium. FAPs, fibroblasts, adipocytes, and immune cells such as macrophages are mainly located within the perimysium [[8], [9], [10]]. As a highly innervated and vascularized tissue, skeletal muscle allows nerve fibers and blood vessels to traverse the epimysium and perimysium, ultimately entering the endomysium to establish neuromuscular junctions and local capillaries. Schwann cells envelop nerve fibers, while the vessel walls primarily consist of three cell types: vascular smooth muscle cells, endothelial cells (ECs), and pericytes [11]. Precisely coordinated activities between a myofiber and its niche are essential for the maintenance of muscular health.

Myofiber senescence

Senescence, recognized as the fundamental cellular mechanism underlying aging, was initially defined by Hayflick as the limited replicative capacity of mammalian somatic cells [12]. Cellular senescence can be caused by telomere shortening (replicative senescence) or extrinsic stimuli such as inflammation and radiation (premature senescence). Although myofibers belong to the terminally differentiated cell types, also called post-mitotic cells, with no proliferative potency, they can replicate by endoreplication without myofiber division and duplication [13]. Meanwhile, post-mitotic cells are also vulnerable to external factors that can trigger senescence.

Several studies have detected myofiber senescence from aged mice and humans [4,14]. However, results are inconsistent among these studies, which may have been caused by the heterogeneity of myonuclei. A single myofiber contains hundreds of myonuclei in a shared cytoplasm, each myonucleus can only regulate gene expression in part of the myofiber, so-called myonuclear domain [15,16]. Myonuclei with different senescence levels can co-exist within a single myofiber. Meanwhile, different types of myofibers, distinguished by unique metabolic profiles and protein quality control mechanisms, may vary response to the effects of aging [17]. Hence, the development of interventions targeting senescent myofibers in sarcopenia would be challenging. Application of advanced technologies, such as snRNA-seq and spatial transcriptomics will allow us to better understand the heterogeneity of myofiber and their changes during aging.

Interestingly, myonuclei from obese individuals had elevated levels of DNA damage and senescence-associated secretory phenotypes (SASPs) compared with those from lean individuals [14]. It suggested that adipose tissue may contribute to myofiber senescence via endocrine or paracrine actions. Meanwhile, other SMME cell components can also promote myofiber senescence. For instance, xenotransplantation of senescent human fibroblasts into the skeletal muscle of immunodeficient mice resulted in the production of several senescent markers in adjacent myofibers [18].

Satellite cells

SCs are Paired Box 7 (Pax7)-positive stem cells residing between the myofiber's basal lamina and sarcolemma. Under normal conditions, these cells stay in a quiescent state with low transcriptional and metabolic activity, and only become activated when myofibers encounter damage. SCs can rapidly transit into an active state of proliferation and differentiation in response to autocrine molecules secreted by these damaged myofibers, including mechano-growth factor along with fibroblast growth factor 2 (FGF2) [19,20]. The capacity of SCs for self-renewal and tissue repair plays a vital role in preserving skeletal muscle homeostasis. Notably, SCs are not the sole executors of myofiber repair because recent study has discovered that myonuclei could migrate to the injury sites, facilitating cellular reconstruction and activating self-repair of myofibers independently of SCs [21].

The role of SCs in sarcopenia remains controversial

In general, aging significantly reduces the number and function of SCs, leading to decreased repair and regeneration capacities in aged skeletal muscle. Senescent SCs are only observed at advanced geriatric age and studies have shown that declining basal autophagy and dysfunctional programmed cell death are the decisive regulators [[22], [23], [24]]. Notably, the senescent level of SCs can be greatly influenced by the frequency and extent of SCs’ activation and depletion. SCs undergo telomere shortening and replicative senescence as they pass through multiple rounds of mitosis and approach the Hayflick limit [12]. In addition, the heterochronic parabiosis model can restore the proliferation and regenerative capacities of SCs in aged mice [25], suggesting that exogenous factors such as sex hormones, α-Klotho, and FGF in the blood can reverse SC senescence [[26], [27], [28]].

However, it remains controversial whether SCs are involved in the initiation and progression of sarcopenia. Zhao et al. constructed inducible Pax7 gene-knockout mice, and compared their muscle mass at diverse time intervals during aging. They found that a reduction in SCs did not exert any promotive or aggravating effects on sarcopenia [29]. Meanwhile, using the same inducible Pax7 knockout mice, Liu and colleagues found that SC depletion induced NMJ degeneration at a younger age and aggravated myofiber atrophy after neuromuscular deterioration [30,31]. Considering the conflicting results, it can be speculated that the lack of obvious effect of SC depletion on muscle mass may be attributed to compensatory myogenic differentiation by other cells within the SMME. For example, pericytes within the SMME have been identified to possess myogenic differentiation potential​​ [32]. However, the deletion of SC would inevitably disrupt its surrounding environmental structures (e.g., NMJ) and impair skeletal muscle adaptation to environmental challenges like injury. In conclusion, when age-related functional decline of SCs becomes severe enough to exceed the compensatory capacity of other myogenic components in the SMME, this failure ultimately drives the pathogenesis of sarcopenia (Fig. 1).

Fig. 1.

Crosstalk between stem cells and myofibers in aging skeletal muscle. SCs exhibit quantitative depletion, attenuated repair capacity for myofiber regeneration and senescence-associated alterations. Moreover, they secrete S100B, which activates the RAGE on myofibers to induce atrophy, while senescent myofibers reciprocally secrete ANGPTL2, which suppresses SC self-renewal and regenerative activity. This bidirectional crosstalk between senescent SCs and myofibers exacerbates NMJ degeneration, forming a vicious cycle that accelerates skeletal muscle aging. Senescent FAPs undergo enhanced fibrogenic/adipogenic differentiation, driving muscle fibrosis and IMAT. Aging impairs FAP paracrine function, reducing the secretion of GDF10, IGF-1, WISP1, Follistatin, and Periostin, leading to myofiber atrophy, compromised myofiber regeneration, delayed macrophage transition, and Schwann cell deterioration. Concurrently, diminished secretion of SPARC and IL-15 by senescent myofibers also promotes FAP adipogenesis. Abbreviations: ANGPTL2, angiopoietin-like protein 2; FAPs, fibro/adipogenic progenitors; GDF10, growth differentiation factor 10; IGF-1, insulin-like growth factor-1; IL, interleukin; IMAT, intramuscular infiltration of adipose tissue; MuRF1, muscle ring finger 1; NMJ, neuromuscular junction; RAGE, receptor for advanced glycation end-products; SC, satellite cell, SPARC, secreted protein acidic and rich in cysteine; WISP1, WNT1-inducible signaling pathway protein 1.

Crosstalk between SCs and myofibers needs to be further decoded

SCs are located on the myofiber membrane, implying a close information exchange. S100B is a protein capable of binding to Ca²⁺ and it can be expressed and secreted by SCs [33]. It was found to exert an inhibitory effect on the intermediate filament subunit desmin assembly in myofibers [34], and activate receptor for advanced glycation end-products to induce myofiber atrophy in cachexia [35]. Myofibers can reciprocally influence SCs as well. The expression and secretion of angiopoietin-like protein 2 (ANGPTL2) were significantly increased in senescent myofibers. Specific knockout of Angptl2 in skeletal myofibers increased SCs activity and inhibited muscle atrophy [36]. To date, no studies have explored whether quiescent SCs can influence adjacent myofibers, or determined the changes in the secretory profile of senescent SCs and how they affect myofibers. With the application and development of spatial omics, studies will be able to examine the paracrine effects of quiescent SCs in situ, thereby decoding their interactions with myofibers and other SMME components.

Fibro/adipogenic progenitors

FAPs were initially identified in 2010, as a population of mesenchymal cells positive for platelet-derived growth factor receptor α (PDGFRα) and located in the interstitial skeletal muscle with distinct characteristics from SCs [37]. Normally, FAPs provide a supportive environment because they serve as the primary origin of ECM, including collagens, laminin, and fibronectin [38]. FAPs depletion leads to muscle weakness, alterations in myofiber type composition, disruption of vessel organization, impaired revascularization, and denervation at NMJs, ultimately culminating in failure to maintain muscle mass [7]. Besides, FAPs possess the capacity to differentiate into adipocytes, fibroblasts, and osteoblasts, that can cause the intramuscular infiltration of adipose tissue (IMAT), fibrous tissue, or even heterotopic ossification. Accumulation of these non-contractile tissues can significantly impact muscle health [39]. These findings indicate that FAPs represent a key factor in the shift from the state of muscle homeostasis to pathological conditions. Analyses based on single-cell RNA sequencing (scRNA-seq) pinpointed diverse subpopulations of FAPs that exist within skeletal muscle [40]. However, it is not well understood how these FAP subpopulations respond to various extrinsic stimuli. Filling these research gaps can deepen our comprehension of the function that FAPs play in maintaining the health of skeletal muscle.

Aging promotes fibrogenesis and adipogenesis in FAPs

In aged FAPs, a reduction in the truncated PDGFRα variant, which functions as a decoy receptor to suppress PDGF signaling, has been observed, and this reduction is linked to both impaired adipogenesis and an enhanced fibrogenic potential [41,42]. However, a large amount of studies also observed a significant presence of IMAT in the aged skeletal muscle tissue [9]. The presence of fibrous tissue and IMAT in patients with sarcopenia implies that FAPs have a dual-function ability to be activated in two different directions with aging [43,44]. This may be partly attributed to the heterogeneity of FAPs. However, the precise contributions of the various subtypes of FAPs to muscle fibrosis and IMAT during aging remain to be thoroughly explored.

Studies have suggested that the impact of aging on FAPs may be mediated by other components of the SMME. Transplantation of aged bone marrow cells promoted the muscle fibrogenic phenotype in young recipient mice [45], and this effect may be attributed to the mononuclear macrophages [46]. Myeloid cell-specific mutation in Spi1 selectively leads to a reduction in the numbers of M2 macrophages, effectively curbing the muscle fibrosis associated with aging and preventing the occurrence of sarcopenia [47]. Myofibers can also secrete adipogenic inhibitor, the secreted protein acidic and rich in cysteine (SPARC) [48] along with myokines, such as IL-15 [49], to suppress the adipogenic differentiation process of FAPs. However, the regulatory potential of myofibers is diminished with aging, this could potentially account for the enrichment of fatty tissue within degenerative muscle [50] (Fig. 1).

Aging diminishes the paracrine effect of FAPs, leading to myofiber atrophy

GDF10, belonging to the TGF-β superfamily, is uniquely expressed in FAPs. It promotes myofiber hypertrophy through activation of the Smad1/5/8 and Akt signaling pathways, while its deletion leads to muscle atrophy. The level of GDF10 was decreased in mice with age-related sarcopenia, and administration of recombinant GDF10 in aged mice effectively reversed their sarcopenia [7]. IGF-1 is another factor secreted by FAPs that activates the Akt pathway within myofibers, resulting in skeletal muscle hypertrophy [51]. WNT1-Inducible Signaling Pathway Protein 1 and follistatin serve as crucial mediators that facilitate the impact of FAPs on the myogenic potential of SCs. Transwell co-culture experiments showed that neutralizing antibodies against follistatin can significantly diminish the capacity of FAPs to facilitate the generation of multinucleated myotubules from SCs [52]. The expression levels of these soluble factors are involved in muscle health decline during the aging process [42]. Therefore, restoring the healthy paracrine function of aged FAPs may serve as promising therapeutic approaches for treatment of sarcopenia. Transplantation of young FAPs into aged mice skeletal muscle has been demonstrated to alleviate inflammation and increase the cross-sectional area of myofibers. This rejuvenative effect is primarily mediated by Periostin, a FAPs-secreted matricellular protein that stimulates the cell cycle of SCs and activates macrophage anti-inflammatory polarization via paracrine signaling [53]. However, whether prolonged exposure to the aged SMME renders young FAPs dysfunctional, thereby impairing their capacity to restore SMME functionality and rejuvenate myofibers, remains to be further validated.

Immune compartment

Skeletal muscle harbors a significant population of immune cells, comprising neutrophils, monocyte-lineage cells, and lymphocytes. Under conditions of skeletal muscle injury, repair, or aging, myofibers and various SMME components, including SCs and FAPs, secrete chemokines such as C–C-chemokine ligand 2 (CCL2) and C-X-C motif ligand 8 (CXCL8). These ligands bind to G protein-coupled receptors (GPCRs) on immune cell surfaces, thereby directing immune cell migration toward skeletal muscle [[54], [55], [56]]. Immune cells exhibit heterogeneity and remarkable plasticity, allowing them to assume diverse roles based on the specific microenvironment in which they reside. This adaptability enables immune cells to respond effectively and function in a wide range of situations. In general, neutrophils and monocyte-lineage cells are important in the innate immune response by performing essential functions such as phagocytosis and pathogen clearance. Meanwhile, lymphocytes actively contribute to the adaptive immune response through tasks such as antigen presentation and immune regulation. These distinct types of immune cells synergize seamlessly to carry out crucial functions in pathophysiological processes such as tissue damage, infection, and aging (Fig. 2).

Fig. 2.

Crosstalk between immune cells and myofibers in aging skeletal muscle. Senescent neutrophils exhibit impaired phagocytosis, while excessive production of ROS and NETs damages myofibers. It demonstrates hyperactivation of the PI3K pathway, secreting TNF-α to activate TLR7/8/9-mediated ERK1/2 phosphorylation in FAPs, thereby promoting fibrogenic differentiation and compromising muscle force generation. M1 macrophages in the aged SMME exacerbate inflammation and oxidative stress, leading to myofiber atrophy. And M2 macrophages promote IMAT to influence muscle force generation. Senescent NK cells show diminished immune surveillance capacity and senescent B cells exhibit reduced antibody secretion, but their crosstalk with myofibers remains elusive. Critically, senescent T cells secrete elevated levels of IFN-γ, inducing mitochondrial dysfunction in myofibers and accelerating muscle degeneration. Abbreviations: ERK, extracellular regulated protein kinase; FAPs, fibro/adipogenic progenitors; IFN-γ, interferon-gamma; IMAT, intramuscular infiltration of adipose tissue; NET, neutrophil extracellular trap; PI3K, phosphoinositide 3-kinase; ROS, reactive oxygen species; TLR, toll-like receptor; TNF, tumor necrosis factor.

Neutrophils

Neutrophils, characterized by positivity for CD11b and CD16, function as the first line of immune defense by killing/eliminating pathogens via mechanisms such as degranulation, phagocytosis, the generation of reactive oxygen species (ROS), and the formation of neutrophil extracellular trap (NET). Neutrophils stay in the bloodstream for a brief period (6–8 h) and then migrate through the walls of blood vessels to enter the surrounding body tissues, including skeletal muscle [57]. In the injured skeletal muscle, platelets localize and promote neutrophil recruitment by releasing CXCL7 as a neutrophil chemokine. Then neutrophil infiltration facilitates muscle repair through clearing cellular debris [58]​. The continuous maintenance of resident neutrophil turnover and replenishment from the bloodstream is achieved through a fine-tuned balance between host-mediated responses and microbial forces. Any disruption to this equilibrium can have an immediate impact on the host's well-being.

Aging impairs the quality and quantity of neutrophils

Elevated blood neutrophil levels in elderly people are positively correlated with frailty [59], and there was an increase in the neutrophil-to-lymphocyte ratio in middle-aged and older sarcopenia patients [60]. In the skeletal muscle of mice, aging also causes an increase in the infiltration of neutrophils [61]. The possible explanation is that IMAT associated with aging releases a series of chemokines into skeletal muscle [62]. Although the number of neutrophils increases, delayed recruitment and impaired phagocytosis have been observed in neutrophils during aging, which could arise through a weakened chemotactic response and over-activation of the phosphoinositide 3-kinase pathway [63]. Furthermore, neutrophils derived from elderly individuals exhibit a pre-activated basal condition accompanied by augmented ROS production [64]. Therefore, the quality and quantity of neutrophils are both affected by aging, but findings for the role of neutrophils in the context of sarcopenia are scarce.

Crosstalk between neutrophils and myofibers

Contraction exercise leads to neutrophil recruitment through increased expression of neutrophil chemotactic factors such as CXCL1 and ICAM-1 from myofibers and the endothelium. Subsequently, moderate IL-1β released by neutrophils can enhance glucose transporter type 4 (GLUT4) translocation of myofibers and endurance capability [65]. However, extensive neutrophil infiltration with robust NET formation in the skeletal muscle can generate large amounts of IL-10 and TNF-α, which activate Toll-like receptor (TLR) 7/8/9-ERK 1/2 phosphorylation signaling of FAPs to promote their fibrosis [66]. In dystrophic muscle, Cathelicidin-related antimicrobial peptide (Cramp) became activated within muscle-infiltrating neutrophils and was then internalized into myofibers. Genetic ablation of Cramp ameliorated muscle pathology, such as muscle force and fiber size, fibrofatty infiltration, fibrosis, and inflammation. Mechanistically, Cramp binds and inactivates sarcoplasmic/endoplasmic reticulum Ca²⁺-ATPase1 within myofibers, resulting in the activation of Ca²⁺-dependent calpain proteases that aggravate the development of muscle dystrophy [67]. These findings establish neutrophils as vital immune regulators in muscle atrophy.

Eosinophils

Eosinophils, alongside neutrophils, belong to the granulocyte lineage and have been implicated in maintaining skeletal muscle homeostasis. Following skeletal muscle injury, eosinophils are recruited to damaged sites by chemotactic signals including IL-5​​ and ​​CCL11 [68]. At injury sites, eosinophils promote muscle regeneration through ​​IL-4 secretion​​, which activates the proliferation of ​FAPs​​ while simultaneously suppressing their adipogenic differentiation, thereby preventing IMAT [69]. However, the functional role of eosinophils in ​​aging SMME​​ remains poorly defined. Notably, a prospective cohort study revealed a ​​positive correlation​​ between circulating eosinophil counts and reduced muscle mass in elderly patients with type 2 diabetes, suggesting potential pathological involvement in sarcopenia [70]. Further investigation is warranted to elucidate eosinophil-mediated mechanisms in ​​sarcopenia pathogenesis and their therapeutic relevance.

Monocyte-macrophages

Monocytes have a relatively short lifespan within the vascular system. Once these cells enter the tissues, they undergo differentiation into macrophages and dendritic cells. The current understanding of dendritic cells in muscle is very limited. In the SMME, macrophages, a primary population of immune cells, are located in the region within the endomysium and the perimysium. Embryo-derived self-renewing macrophages and adult bone marrow-derived non-renewing macrophages are the two types of muscle-resident macrophages [71]. Skeletal muscle's resident macrophages exhibit significant heterogeneity, and their phenotypes alter during aging progresses, which might intensify the degradation of skeletal muscle and impede effective regeneration [72].

Aging leads to dynamic changes in M1 and M2 macrophages

Macrophages are known to polarize to pro-inflammatory M1 state or anti-inflammatory M2 state [73]. The number of M2 macrophages in the skeletal muscle of the elderly was markedly higher than that in the skeletal muscle of the young [74]. This might be because FAPs in aged skeletal muscle highly express CCL2 to recruit macrophages, and their secretome promotes macrophage polarization toward the M2 subtype [56].​ Moreover, M2 macrophages were often co-localized with IMAT in aging skeletal muscle [75]. In contrast, pro-inflammatory M1 macrophages were decreased in the muscles of elderly people [10]. However, comparisons in mouse skeletal muscle yielded different results. Flow cytometry analyses showed that ly6c-positive M1 macrophages in skeletal muscle were significantly increased in mice aged 20 to 27 months, accompanied by elevated levels of CCL2, increased inflammation and oxidative stress [61]. Two comparative studies involving scRNA-seq analyses demonstrated that LYVE1-negative macrophages were predominant in aging mouse skeletal muscles, and associated with glycolysis, pro-inflammatory pathways, and activation of pathways related to ECM synthesis and angiogenesis [76,77]. Although no increase in M1 macrophages was observed in human aging skeletal muscle, the pro-inflammatory levels were still elevated. This may be mechanistically linked to the following potential mechanisms. In aging skeletal muscle, the increased M2 macrophages may replace M1 macrophages to exert pro-inflammatory effects. When exposed to complexed IgG and TLR ligands, M2 macrophage undergo “non canonical” differentiation, producing the pro-inflammatory cytokines TNF-α, IL-1β, and IL-6 [78]. Furthermore, other aging tissues are contributing to the increase in circulating pro-inflammatory cytokines, resulting in aging skeletal muscle pro-inflammatory levels elevated. For example, aging adipose tissue macrophages are activated by SPARC, which induces the interferon-response through the TLR4 signaling pathway, increasing the concentrations of pro-inflammatory cytokines such as TNF-α and IL-1β in serum, thereby exacerbating chronic inflammation related to aging [79]. In conclusion, macrophages in aging skeletal muscle undergo dynamic changes. M1 macrophages potentially lead to elevated pro-inflammatory levels and chronic cellular damage, while M2 macrophages may contribute to the development of IMAT.

Crosstalk between macrophages and myofibers

A prior study discovered a substantial rise in macrophage infiltration after 1-week hindlimb immobilization-induced sarcopenia, suggesting a mutual interaction between macrophages and myofiber atrophy [80]. Conditioned medium from M1 macrophages decreased IkappaB-alpha and phosphorylated Akt, increased phosphorylated c-Jun N-terminal kinase, and eventually led to an elevation in atrophy markers within myofibers [81]. M1 macrophages can also cause a significant decrease in insulin-mediated glucose uptake that is linked with myofiber atrophy [82]. CD206-positive macrophages residing within skeletal muscle express Klotho [83], a well-known anti-aging protein with crucial roles in myofiber hypertrophy, SC mobilization, and myogenic differentiation [84]. Transplanting bone marrow cells derived from Klotho transgenic mice into mdx recipient mice revealed that macrophage-derived Klotho promotes myofiber anabolism and growth [83]. In addition, macrophages may also influence muscle mass and strength by interacting with other SMME components, including SCs [85,86], FAPs [87] and etc. In the future, single-cell and spatial transcriptomics technologies will help to achieve a more comprehensive understanding of macrophage heterogeneity and how these cells interact with myofibers and the SMME components.

Lymphocytes

Adult lymphocytes are characterized as CD3^-CD56⁺ natural killer (NK) cells, CD3⁺ T lymphocytes (T cells), and CD19⁺ B lymphocytes (B cells) based on the functions and origins. NK cells are integral components of the natural immune system. In contrast, B and T cells primarily mediate adaptive immunity. Lymphocytes consist of a wide variety of different subsets, and each subset is crucial for the proper functioning of the immune system.

Aging induces alterations in lymphocyte numbers and functional impairments

Alterations in the immune system are the most pronounced changes that occur during aging, including an augmented infiltration of T cells and B cells detected in the aging quadriceps. Subtype analyses showed that the quantity of CD4⁺ and CD8⁺ T cells in skeletal muscle increased and the CD4 to CD8 proportion decreased with advancing age [88]. ScRNA-seq data reveal that several SMME components, including fibroblasts, SCs, arterial ECs, and pericytes, express pro-inflammatory chemokines, which may mediate lymphocytes recruitment to skeletal muscle [89]. Although the proportion of regulatory T (Treg) cells within CD4⁺ T cell populations in the uninjured skeletal muscle of mice does not change with age, the accumulation of Tregs in skeletal muscle during recovery from injury is significantly diminished in aged mice, attributed to impaired recruitment, proliferation, and retention of Tregs [90,91]. A relatively overlooked issue of immunosenescence pertains to the age-associated malfunction of NK cells, including diminished cytokine secretion, a decline in cytotoxicity to target cell, and even an increase in the quantity of NK cells with aging [92]. In addition, emerging research has demonstrated that NK cells play a pivotal role in the immunosurveillance of senescent cells, and act as the primary lymphocytes that play a crucial role in eliminating these cells [93]. Thus, more attention should be paid to understanding the factors contributing to the age-related decline in NK cell function. Such insights could pave the way for therapeutic strategies aimed at enhancing NK cell-mediated immunosurveillance of senescent cells or even treat age-associated diseases such as sarcopenia. Nevertheless, the roles of NK cells and B cells in sarcopenia remain poorly defined and necessitate further investigations. Consequently, this part will concentrate on the established roles of T cells.

Crosstalk between T cells and myofibers

On the one hand, within the SMME, T cells can influence myofibers through modulation of inflammatory microenvironment. Mechanistically, muscle-resident Tregs can mitigate inflammation and prevent myofiber damage by suppressing interferon-γ (IFN-γ) production, which is a key driver of chronic inflammation and aging [94]. On the other hand, T cells engage in intricate crosstalk with key components of the SMME, including SCs and FAPs, thereby modulating myofiber dynamics. FAPs regulate Treg homeostasis within skeletal muscle by secreting IL-33, while the expression level of IL-33 decreases in the skeletal muscle of aging mice [90]. In T cell-specific IL-6Rα deficient (TKO) mice, the functionality of muscle-resident Tregs is significantly impaired. Of note, after establishing model of colitis-induced sarcopenia, IL-6Rα TKO mice exhibited a reduction in their maximal muscle grip strength compared with control group [95]. In the denervation-induced sarcopenia model mice, CD4⁺IL17⁺ and CD4⁺IFN-γ⁺ T cells were significantly increased in the spleen [96]. Through applying conditioned medium from activated T cells to cultured mouse myoblasts derived from young mice, researchers found that ERK1/2 phosphorylation and the proliferation rate of myoblasts were enhanced, while the myogenic differentiation potential was decreased [97]. Another study using SCs from aged mouse muscle revealed that T cell cytokines had no effects on proliferation and migration, but similarly impaired myogenesis [98]. These findings highlight age-dependent differences in the response of muscle cells to T cell signaling. Notably, these in vitro experiments were all conducted on spleen-derived T cells. However, skeletal muscle-resident T cells display unique transcriptomic profiles, T-cell receptor repertoires, and effector capabilities compared to those of classical, lymphoid-organ T cells. Due to the rarity of muscle T cells and their fragility upon isolation, studying the provenance and functions of these unique features has proven to be challenging [99].

Motor neurons

Muscle strength is largely determined by the recruitment of myofibers. Each myofiber is innervated and regulated by a single α motor neuron, collectively referred to as a motor unit. The nerve-related components in skeletal muscle include neuronal axons, Schwann cells, and specialized structures called NMJs. NMJs are synapses that bridge the neuron and the myofibers. The pre-synaptic boutons of NMJs are positioned directly above the post-synaptic indentations of the sarcolemma, known as junctional folds [100]. They are the locations of high-density aggregations of acetylcholine receptors (AChRs) (∼10,000 AChRs/μm²) [101]. NMJs are crucial structures for molecular information exchange between the neuron and the myofibers. Chief among the crosstalk molecules is the neurotransmitter acetylcholine (ACh). ACh released from the pre-synapse binds to AChRs, thereby triggering the generation of an endplate potential, which is a local depolarization. Subsequently, this depolarization spreads across the entirety of the myofibers in the form of a propagated action potential, ultimately leading to muscle contraction. In addition, Schwann cells influence skeletal muscle homeostasis through paracrine effects. Thus, molecular communication between nerves and myofibers is essential for maintaining the normal function and healthy status of skeletal muscle.

Aging deteriorates the structure and function of motor neurons

Studies conducted on both animals and humans have revealed that aging causes degenerative changes in the motor neuron structures, typified by axonal denervation and reinnervation [102]. These changes happen before the onset of sarcopenia and are slightly dependent on the muscle types and their activity levels [103]. For example, fast fibers and fibers lacking exercise stimulation are more prone to age-related deterioration [104,105]. Interestingly, some denervated fast fibers become reinnervated through axonal sprouting from slow motor neurons. This process leads to a transformation of these fast-twitch fibers into slow-twitch fibers, and representing one of the typical pathological changes in sarcopenia [106]. Meanwhile, in aged mice, the NMJs exhibit a growing tendency to fragment, and there is a concomitant loss of their junctional folds [107]. They also display a diminished quantity of pre-synaptic vesicles and nerve terminals [108], along with a spreading out in the region of motor endplates [104]. Moreover, there are declines in the quantity of AChRs [109] and their binding affinity to ACh [110]. It is noteworthy that human motor neuron axon diameters and NMJs are significantly smaller than those in rodents [111]. Additionally, in human NMJs, there are extensive post-synaptic junctional folds. These folds serve to augment the local membrane area [112,113]. Due to the significant disparities in motor neurons between mice and humans, evident age-related changes in mice, such as NMJ fragmentation, may not be observed in humans [111]. Other changes, such as decreased postsynaptic fold number and regularity, are more obvious in humans than in rodents [114].

In aging skeletal muscle, disrupted mitochondrial homeostasis within myofibers surrounding the NMJ precipitates NMJ dysfunction and neurotransmission impairment [115]. Evidence from human and animal studies demonstrated mitochondrial dysfunction in aging skeletal muscle, manifesting as reduced electron transport chain complex activity, impaired oxidative phosphorylation, and diminished mitochondrial content [116,117]. These culminate in attenuated ATP synthesis that directly constrains synaptic vesicle release efficiency [118]. Concurrently, compromised mitochondrial Ca²⁺ buffering capacity disrupts intracellular Ca²⁺ homeostasis, activating calcium-dependent proteases (e.g., calpains) which dissociate rapsyn from AChRs, thereby inducing AChR dispersion alongside a decline of postsynaptic fold length and density [106,119]. The pathological cascade of mitochondrial degeneration and NMJ destabilization that synergistically undermines neurotransmission and exacerbates age-related myofiber atrophy.

Crosstalk between motor neurons and myofibers

Neurotransmitters are important molecules that facilitate information exchange between motor neurons and myofibers. They can influence the intracellular levels of metal ions, especially calcium ions, thereby regulating the myofiber function. For example, action potentials can cause an increase in calcium ions release from the sarcoplasmic reticulum, leading to elevated expression of IGF-1 and decreased atrogin-1 and muscle ring finger 1 (MuRF1) levels in the ubiquitin–proteasome pathway, ultimately resulting in myofiber hypertrophy [120,121]. Aging causes a significant reduction in the myoplasmic calcium concentration through a decline in L-type calcium channel (dihydropyridine receptor) charge movement [122].

Besides neurotransmitters, motor neurons also communicate with myofibers via other types of molecules. Agrin, which is secreted from presynaptic terminals, binds with low-density lipoprotein receptor-related protein 4 (Lrp4) in myofibers. This crosstalk activates muscle-specific kinase (MuSK), contributing to the anchoring of AChRs at the sarcolemma [123]. With a conditional knockout of agrin in motor neurons, 5–6 months mice exhibited almost complete disappearance of presynaptic nerve terminals and a reduction in the size of motor units, further demonstrating the importance of agrin in maintaining the structure and function of NMJ [124]. Meanwhile, 4-month-old transgenic mice with overexpression of neurotrypsin, which is a neuronal protease capable of cleaving agrin within motor neurons, exhibited numerous traits associated with sarcopenia, with a decrease in fiber number, fiber atrophy, augmented proportion of slow-twitch fibers, along with fragmented NMJs [125]. In addition, emerging research has demonstrated that the Agrin level in mouse skeletal muscle decreases with age, contributing to the pathogenesis of age-related sarcopenia [126]. Similar to agrin, connective tissue growth factor, neuregulin-1, and R-spondin 2 derived from motor neurons separately bind to Lrp4, epidermal growth factor receptors, and leucine-rich repeat-containing G-protein coupled receptor 5 on myofibers to enhance MuSK phosphorylation, leading to focused clustering of AChRs and increased expression of NMJ-specific genes in the postsynaptic region [127]. Moreover, α-Calcitonin-gene-related peptide secreted from the presynaptic membrane has been proposed to stabilize AChRs at the NMJ by suppressing the transcriptional activity of forkhead box O (FoxO), thereby inhibiting autophagy in denervated skeletal muscles [128]. Schwann cells secrete extracellular vesicles (EVs) enriched with antioxidants and anti-inflammatory factors, which suppress oxidative stress and inflammatory responses in skeletal muscle. These EVs promote the proliferation and differentiation of SCs and enhance vascular ECs proliferation, consequently improving the muscle perfusion and preventing myofiber atrophy [129]. However, the age-related changes in these molecules and their roles in sarcopenia warrant further investigation (Fig. 3).

Fig. 3.

Crosstalk between motor neurons and myofibers in aging skeletal muscle. In aging skeletal muscle, myofiber denervation occurs, followed by reinnervation through axonal sprouting from slow motor neurons. This drives fast-twitch fiber transformation into slow-twitch fibers. Concurrently, motor neurons exhibit slowed action potential propagation velocity. The NMJ undergoes significant remodeling characterized by structural fragmentation and functional decline: (1) presynaptic deficits: reduced ACh vesicles and diminished Agrin secretion; (2) postsynaptic impairments: decreased AChR density, attenuated ACh-AChR binding affinity, and loss of their junctional folds. Notably, mitochondrial dysfunction and elevated intracellular Ca²⁺ concentrations in myofibers surrounding the NMJ promote AChR dispersion. Additionally, senescent Schwann cells show reduced secretion of EVs containing antioxidant and anti-inflammatory components, further compromising neuromuscular integrity. Abbreviations: ACh, acetylcholine; AChR, acetylcholine receptor; AP, action potential; EVs, extracellular vesicles; NMJ, neuromuscular junction.

Vascular cells

Skeletal muscle features a high degree of vascularization, where individual myofibers are surrounded and nourished by multiple capillaries. Their metabolic and endocrine functions are closely associated with the density of blood vessels [130]. ECs, pericytes, and vascular smooth muscle cells constitute the vascular compartment. In general, various cell populations arranged in concentric layers make up the cellular wall of blood vessels, namely ECs, which constitute the tunica intima, and mural cells situated within the tunica media (pericytes in capillaries or vascular smooth muscle in larger vessels) [131]. ECs form a lining along the vessel lumen. They possess thromboresistant characteristics, exhibit selective permeability towards substances, and create a dynamic barrier for the cells that are circulating within the blood. ECs also function as detectors of the metabolic requirements of myofibers and trigger the dilation of capillaries by regulating the balance between vasoconstrictors and vasodilators, thereby guaranteeing a blood flow that is precisely tailored to the oxygen and nutrient demands [132]. Pericytes are perivascular cells wrapped by the capillary basal lamina that preserve the integrity of muscular capillaries through encircling ECs [133]. They are considered the precursor cells of mesenchymal stem cells possessing the potential for multi-lineage differentiation. Currently, pericytes still lack a distinctive cell surface marker, the accepted markers include platelet-derived growth factor receptor-β, CD146, neuron-glial antigen (NG2), and alkaline phosphatase [[134], [135], [136], [137]]. ECs and pericytes display significant heterogeneity, with diverse cell subtypes contributing to the intricate regulation of skeletal muscle homeostasis. By performing scRNA‐seq on skeletal muscle, Peng et al. identified four distinct EC subtypes, among which the EC subtype with high expression of guanylate-binding protein 2 exhibited compromised endothelial tube formation ability that was linked with sarcopenia [138]. Three pericyte subpopulations are present in skeletal muscle: type 1 (Nestin⁻/NG2⁺) pericytes possess fibrogenic/adipogenic potential [139]; type 2 (Nestin⁺/NG2⁺) pericytes are myogenic [32,139]; type 3 (PECAM1⁺/VWF⁺) pericytes are endothelial-like [140]. Currently, few data are available concerning the exact functions of these blood vessel cell subpopulations in skeletal muscle homoeostasis.

Aging causes vascular rarefaction

Muscle biopsy and immunostaining analyses have revealed decreased angiogenic potential, impaired vascular constrictor response, microvascular senescence, and decreased capillary-to-fiber ratio in aged muscle, reflecting a condition known as vascular rarefaction [141]. Vascular rarefaction leads to reduced perfusion and impairment of tissue function, and it occurs in skeletal muscle as well as in various other organs during aging. Mice treated with vascular endothelial growth factor (VEGF) exhibited both a prolonged lifespan and an extended period of health, reflected by a decrease in muscle loss (sarcopenia) and enhanced maintenance of the ability to generate muscle force. Moreover, in VEGF-treated mice, hallmarks of aging like mitochondrial malfunction, impaired metabolic adaptability, cell senescence, and inflammation were also mitigated [142]. These findings indicate that vascular aging plays a preeminent role in propelling the overall aging process of the organism, unraveling the cause of this phenomenon is crucial for developing therapeutic interventions.

Cellular senescence and EC apoptosis are the main contributing factors to vascular rarefaction. ECs are continuously exposed to circulating pathogenic stimuli, such as ROS and inflammatory cytokines, as well as the hemodynamic forces of blood flow, that can cause EC damage and a high turnover rate, leading to senescence and apoptosis [143,144]. Senescent ECs exhibit a flat and enlarged morphological phenotype, which can increase their adhesion and denudation resistance, allowing them to persist for long periods of time [145]. Studies have revealed that EC senescence leads to reduction and disorganization of junctional proteins, such as cadherin-5, facilitating the exposure of myofibers to harmful substances in the blood [146]. Aging also decreases the pericyte-covered vessel ratio, leading to skeletal muscle capillary destabilization and eventually contributing to vascular rarefaction [147]. Furthermore, there are notable changes in the pericyte subpopulations that contribute to age-related muscular pathological processes. In aged skeletal muscle, the myogenic potential of a specific pericyte subpopulation is significantly reduced, limiting their capacity for muscle regeneration [148]. Conversely, another pericyte subpopulation promotes increased fibrous and adipose tissue deposition, further exacerbating age-related muscle degeneration [139].

Crosstalk between microvascular components and myofibers

Nitric oxide (NO), a vasodilator, regarded as one of the most significant molecules secreted by ECs [149]. The decrease in NO combines with the increase in vasoconstrictors during aging, leading to reduced muscle perfusion [150]. Furthermore, NO and its derivatives also can enhance GLUT4 translocation via the cGMP-PKG (cGMP-dependent protein kinase) dependent pathway, thereby stimulating glucose uptake in myofibers [151]. Angiopoietin 1, another molecule secreted by ECs, can facilitate the proliferation and myogenic differentiation of SCs by up-regulating myogenic regulatory factors MyoD and Myogenin [152]. Senescent ECs also exhibit SASPs and produce numerous inflammatory cytokines [153,154], as well as a variety of cellular adhesive molecules and chemokines, such as C-X-C motif chemokines [155], that induce immune cell infiltration into skeletal muscle. Meanwhile, myofibers can reciprocally influence the composition of microvascular cells. Apelin, a myokine that can strongly promote muscular capillarization through enhancing ECs proliferation, was revealed by scRNA-seq analysis to exhibit the most abundant apelin receptor levels in ECs among all cell populations within the SMME [156,157]. The knockout of apelin receptors specifically in ECs resulted in vascular defects, impaired SC function, muscle fibrosis, myofiber necrosis, and reduced force generation [158]. Decoding the crosstalk between ECs and myofibers is vital to identify the crucial elements participating in the maintenance of skeletal muscle health.

CD146⁺Lin⁻ pericyte subgroup can respond to muscle contraction and upregulate gene expression such as Igf2 and Fgf2. The intramuscular injection of CD146⁺Lin⁻ pericytes failed to remarkably augment the size of myofibers. However, it promoted the remodeling of the ECM and angiogenesis when subjected to repeated sessions of electrical stimulation over a period of 4 weeks [159]. Wu et al. demonstrated that healthy pericytes can produce small EVs containing large amounts of ECM remodeling proteins, anti-inflammatory cytokines, and antioxidant cytokines. Data from scRNA-seq analyses revealed that pericytes in disused and aging skeletal muscle lose the ability to synthesize these small EVs. Intramuscular injection of these small EVs efficiently restored the size of myofibers in disused and aged skeletal muscle [160]. Currently, information remains minimal in relation to how pericytes respond to muscle disuse and aging, and how it affects sarcopenia (Fig. 4).

Fig. 4.

Crosstalk between microvascular components and myofibers in aging skeletal muscle. Vascular rarefaction and decreased capillary-to-fiber ratio are core pathological alterations in aging skeletal muscle, caused by reduced angiogenic potential, impaired vascular constrictor response, ECs senescence. Together, these drive diminished blood perfusion and metabolic dysfunction in myofibers. Concomitantly, ECs exhibit reduced expression of junctional proteins and decreased pericyte-covered vessel ratio, compromising vascular integrity. Senescent ECs further demonstrate attenuated tube formation ability and impaired molecular crosstalk with myofibers, alongside reduced secretion of NO, impairing myofiber glucose uptake and diminished Angiopoietin 1 production, which suppresses SC proliferation and differentiation, thereby compromising myofiber regeneration. Additionally, senescent ECs produce SASPs to influence myofiber, and paracrine signaling from senescent myofibers is also disrupted, evidenced by reduced Apelin secretion that inhibits EC activity. Meanwhile, senescent type 1 pericytes (Nestin^–/NG2⁺) exhibit enhanced fibrogenic/adipogenic differentiation, promoting muscle fibrosis and IMAT, while senescent type 2 pericytes (Nestin⁺/NG2⁺) display declined myogenic potential, contributing to myofiber atrophy. Abbreviations: EC, endothelial cell; IMAT, intramuscular infiltration of adipose tissue; NO, nitric oxide; SASPs, senescence-associated secretory phenotypes; SC, satellite cell.

Conclusion

Skeletal muscle is of vital significance in maintenance of body health and preservation of functional mobility. It is a diverse tissue containing myofibers and various other cell components, and influenced by their complex intercellular communication. A comprehensive overview of the SMME and the age-related changes in its components is presented by this review. It also highlights the intricate crosstalk between the SMME components and myofibers, which will help to advance the understanding of sarcopenia pathophysiology.

Despite substantial amounts of research and accumulation of experimental data on the roles of aging SMME in the onset of sarcopenia, numerous questions remain unanswered. First, several SMME components, such as pericytes, Schwann cells, B cells, and NK cells, are known to undergo substantial alterations in the aging process [161]. However, their effects on myofibers are less well studied and warrant further investigation. Second, single-cell and spatial omics has made it possible to comprehensively characterize the cellular heterogeneity and achieve in situ detection of SMME (Table 1, Table 2). It revealed that there are multiple subpopulations of each cell type in the SMME, and the characteristics of these subpopulations and their responses to the external microenvironment are markedly different. However, the alterations within these subpopulations over the course of aging, along with their functions in the progression of sarcopenia, have been barely investigated. For example, FAPs can be classified into 11 transcriptomically unique subpopulations with completely different behavioral traits [162], but their pathological changes in aging and sarcopenia have not been clearly elucidated. Last but not the least, due to the holistic nature of the SMME, it is essential to go beyond the present concentration on single cell types of concern and instead adopt a more integrated system-level methodology where all components of the SMME are incorporated to pinpoint the crucial factors contributing to the development of sarcopenia.

Table 1.

Summary of single-cell omics analyses decoding the aging skeletal muscle microenvironment.

Omics type	Method	Species	Sample	Major findings	Reference
ScRNA‐seq	10 × Genomics	Mouse	Tibialis anterior muscle from 1-, 3-, 18-, 21-, 24-, and 30-month-old mice	Constructed a comprehensive atlas of single-cell transcriptomics of Mus musculus, covering the lifespan and integrating data from 23 tissues and organs, including skeletal muscle.	[163]
	10 × Genomics	Mouse	Quadriceps, gastrocnemius, tibialis anterior, and extensor digitorum longus muscles from 3 young (6-month-old) and 3 aged (24-month-old) mice	Discovered a subpopulation of aged FAPs characterized by elevated p16Ink4a expression, co-expressed senescence-associated genes, and displayed features of DNA damage and chromatin remodeling.	[4]
	10 × Genomics	Human	Muscle samples from surgically discarded tissue from 10 donors (age: 41–81  years)	Utilized unsupervised clustering on a comprehensive dataset to identify 16 unique populations of cells residing within skeletal muscle.	[164]
	10 × Genomics	Human	Gluteus maximus muscle from 2 younger (age: 29 and 34 years) and 3 older (age: >70 years) adults	Identified a unique subpopulation of macrophages within aged skeletal muscle, while CD4+ T cells exhibited Th1-like differentiation during aging.	[165]
SnRNA‐seq	10 × Genomics	Mouse	Tibialis anterior muscle from 5-, 24-, and 30-month-old mice	Observed the appearance of unique myonuclear populations exclusively in skeletal muscle from 30-month-old mice, characterized by the expression of Nr4a3, Ampd3, and Enah.	[166]
	10 × Genomics	Monkey	Quadriceps muscle from 8 younger (age: 4–6 years) and 8 older (age: 18–21 years) cynomolgus monkeys	Constructed a comprehensive single-nucleus transcriptomic landscape of primate skeletal muscle aging, and discovered the decreased expression of FOXO3 in the skeletal muscle of aged primates, which serves as a pivotal transcription factor in sustaining the homeostasis of skeletal muscle.	[167]
	10 × Genomics	Human	Vastus lateralis muscle from 6 younger (mean age: 20 years) and 11 older (mean age: 75 years) adults	Observed a rare subpopulation of myonuclei that exhibit the expression of CDKN1A, which is exclusively present in aged skeletal muscle.	[168]
	DNBelab C	Mouse	Paravertebral muscle from 1-, 6-, 12-, and 24-month-old mice	Discovered the enrichment of an energy metabolism-related gene set named TCA CYCLE IN SENESCENCE within SCs.	[169]
Multi-omics	ScRNA-seq, SnRNA-seq (10 × Genomics)	Human	Intercostal muscle biopsies from 8 young (age: approximately 20–40  years) and 9 older (age: approximately 60–75  years) donors	Conducted profiling on 90,902 single cells and 92,259 single nuclei obtained from 17 donors with the aim of mapping out the aging process within the adult human intercostal muscle, discovering the cellular alterations occurring in each muscle compartment.	[89]
	ScRNA-seq, SnRNA-seq, SnATAC-seq (DNBelab C)	Human	Hindlimb muscle from 12 younger (age: 15–46 years) and 19 older (age: 74–99 years) adults	Constructed a single-cell/single-nucleus atlas of human limb skeletal muscles, covering over 387,000 cells/nuclei from individuals aged 15–99, characterizing age-related cell population changes, and cell-specific and multicellular network features at transcriptomic and epigenetic levels.	[170]

FAPs, fibro/adipogenic progenitors; ScRNA‐seq, single-cell RNA sequencing; SCs, satellite cells; SnATAC-seq, single-nucleus assay for transposase‐accessible chromatin using sequencing; SnRNA-seq, single-nucleus RNA sequencing.

Table 2.

Summary of spatial omics analyses applied to skeletal muscle.

Omics type	Method	Species	Sample	Major findings	Reference
Spatial transcriptomics	10 × Genomics	Mouse	Tibialis anterior muscles of 5-month-old mice injected with notexin at 2, 5, and 7 days prior to collection	Observed the spatiotemporal patterns of gene expression related to Midkine during the regeneration of skeletal muscle, and discovered that Mdk is co-expressed spatially with Ncl and Lrp1, which implied a locally coordinated paracrine signaling system.	[171]
	10 × Genomics	Mouse	Gastrocnemius muscle of 8-week-old D2-mdx mice	Detected SA-β-gal and the activity of senescence genes in dystrophic skeletal muscle, which are specifically localized in the areas of active muscle repair and inflammation.	[172]
	10 × Genomics	Mouse	Tibialis anterior and associated extensor digitorum longus muscles before and after reversible nerve injury at 3 and 30 days after sciatic nerve compression	Determined the spatial arrangement and nerve dependence of the atrophic signaling pathway and polyamine metabolism within glycolytic fibers; discovered that disruptions in the polyamine pathway could impact muscle function.	[173]
	10 × Genomics	Mouse	Tibialis anterior muscles from young adult mice at 7 days post-injury	Found that volumetric muscle loss resulted a distinct spatial profibrotic pattern, which was driven by the interaction between fibrotic and inflammatory macrophages and mesenchymal-derived cells.	[174]
	10 × Genomics	Mouse	Gastrocnemius/plantaris muscle complex from 6-week-old wild-type (DBA2/J; n = 3) and severely dystrophic (D2-mdx; n = 5) mice	Profiled a high-resolution cellular and molecular spatial atlas of dystrophic skeletal muscle by integrating spatial transcriptomics and scRNA‐seq datasets.	[175]
	10 × Genomics	Mouse	Gastrocnemius/plantaris muscle complex from 6-week-old mdx mice in the DBA2/J background (D2-mdx)	Found that galectin-3+ macrophages establish interactions with stromal cells, such as FAPs, through Spp1 within degenerative lesions, thereby facilitating the process of fibrosis.	[176]
	10 × Genomics	Mouse	Quadriceps muscle of healthy (C57BL10 and DBA/2J) and dystrophic (mdx and D2-mdx) mice	Applied spatial transcriptomics to two DMD mouse models with distinct disease severities; demonstrated elevated expression levels of specific genes in regions associated with muscle regeneration, including Myl4, Sparc, and Hspg2; fibrosis, including Vim, Fn1, and Thbs4; and calcification, including Bgn, Ctsk, and Spp1.	[177]
	10 × Genomics	Mouse	Diaphragms at embryonic days E14.5 and E18.5 for CaV1.1 KO, β-cat KO, and wild-type mice	Discovered gene characteristic of specific muscle regions that vary in maturity and fiber type composition, as well as for central NMJ and peripheral MTJ regions.	[178]
	10 × Genomics	Rabbit	Posterior distal region of the supraspinatus muscle belly, free from fascia and tendon/junction	Detected heterogeneous fiber degeneration-regeneration process that occurs subsequent to tenotomy.	[179]
	10 × Genomics	Chicken	Muscle samples (1 cm3) from the cranial part of the right pectoralis major muscle from 3 randomly sampled broiler chickens at 23 days post-hatching	Characterized the unique histological features by analysing gene expression profiles, including myositis, myofibers, lipid-laden macrophages, connective tissue, and the vasculature system.	[180]
	10 × Genomics	Human	Whole embryonic limb samples at PCW6–PCW8	Conducted spatial and temporal mapping of cells within a whole fetal hindlimb, providing a comprehensive analysis of human embryonic limb development.	[181]
	10 × Genomics	Human	Quadriceps muscle biopsies from 2 patients affected by muscular sarcoidosis	Elucidated pathophysiological mechanisms by demonstrating that immune cells within granulomas directly influence adjacent skeletal muscle, driving its fibrotic transformation through the expression of pro-inflammatory and profibrotic factors.	[182]
	10 × Genomics	Human	Muscle biopsies from 3 control subjects, 2 immune-mediated necrotizing myopathy patients, and 3 inclusion body myositis patients	Discovered distinct spatially restricted niches in both control and idiopathic inflammatory myopathy samples, revealing that gene expression patterns associated with muscle damage and immune cell subtypes were spatially correlated.	[183]
	Seq-Scope	Mouse	Soleus muscle from 11 mice: untreated (3 mice; 12 sections), control at 3 days (1 mouse; 2 sections), control at 7 days (1 mouse; 2 sections), 3 days after denervation (2 mice; 6 sections); 7 days after denervation (4 mice, 9 sections)	Generated a comprehensive transcriptomic atlas of the mouse soleus muscle, achieving microscopic spatial resolution, and depicted the dynamic alterations taking place subsequent to denervation.	[184]
Spatial metabolomics	MALDI-MSI; AP-MALDI-MSI	Mouse	Gastrocnemius-soleus muscle cryosections	Conducted metabolomic profiling with high spatial resolution to analyze slow- and fast-twitch myofibers; identified a novel subtype of superfast type 2B myofibers characterized by enhanced fatty acid oxidative metabolism.	[185]
	MALDI‐MSI	Rat	Soleus, extensor digitorum longus, and gastrocnemius muscles from 6-month-old rats	Indicated that biomolecules like the antioxidant anserine and acylcarnitines were present in higher abundances within glycolytic fibers, while taurine and certain nucleotides were detected to be localized specifically in oxidative fibers.	[186]
	AP-MALDI‐MSI	Rat	Gastrocnemius muscle cryosections	Found that [M + K]+ of phosphatidylcholine (36:4), [M + Na]+ of phosphatidylcholine (38:4), along with [M + K]+ of phosphatidylcholine (40:6) exhibited elevated intensity in areas where type I and IIA fibers were clustered.	[187]
	MALDI‐MSI	Human	Paired biopsies of two rotator cuff muscles, torn infraspinatus and intact teres minor, together with an intact shoulder muscle (deltoid)	Differentiate degenerated areas from intact areas by their metabolome profiles and find that in degenerated areas, heme is the most ample metabolite while in intact areas, Heme oxygenase-1, which catabolizes heme, exists.	[188]

AP, atmospheric pressure; DMD, Duchenne muscular dystrophy; FAPs, fibro/adipogenic progenitors; MALDI‐MSI, matrix-assisted laser desorption/ionization-based mass spectrometry imaging; MTJ, myotendinous junction; NMJ, neuromuscular junction; PCW, post-conception week; SA-β-gal, senescence-associated beta-galactosidase; ScRNA‐seq, single-cell RNA sequencing.

Compliance with ethics requirements

This article does not contain any studies with human or animal subjects.

CRediT authorship contribution statement

Ning Wang: Conceptualization, Funding acquisition, Project administration, Writing – original draft. Aojie Zheng: Conceptualization, Visualization, Writing – original draft. Youzhen Yan: Formal analysis, Writing – review & editing. Tailai He: Supervision, Writing – review & editing. Xuan Wu: Investigation, Writing – review & editing. Menglin Xian: Writing – review & editing, Resources. Jingyuan Luo: Writing – review & editing, Formal analysis. Changjun Li: Writing – review & editing, Methodology. Jie Wei: Writing – review & editing, Methodology. Yilun Wang: Funding acquisition, Writing – review & editing. Chao Zeng: Funding acquisition, Supervision, Writing – review & editing. Guanghua Lei: Funding acquisition, Project administration, Writing – review & editing.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Biographies

Ning Wang MD & PhD, serves as an associate professor and master's supervisor within the Department of Orthopaedics at Xiangya Hospital, Central South University. Having completed a 28-month research scholar program at the School of Medicine, University of Pittsburgh, he has since honed his focus on degenerative diseases of the musculoskeletal system. This area of interest encompasses conditions such as sarcopenia and osteoarthritis. In the past five years, he has published seven papers as the first or corresponding author in journals such as Osteoarthritis and Cartilage and Bone Research.

Aojie Zheng is a PhD student in the Department of Orthopaedics at Xiangya Hospital of Central South University, China. His main research interests include skeletal muscle microenvironment and intercellular crosstalk.

YouzhenYan is a Master student in the Department of Orthopaedics at Xiangya Hospital of Central South University, China. His current research focuses on fibro/adipogenic progenitors in skeletal muscle.

TailaiHe is a PhD student in the Department of Orthopaedics at Xiangya Hospital of Central South University, China. His current research focuses on cell death in skeletal muscle.

Xuan Wu has received her Master’s degree from Xiangya School of Basic Medical Sciences, Central South University, China. Her research area is metabolism of skeletal muscle.

Menglin Xian has received her Master’s degree from School of Basic Medical Sciences, Chongqing Medical University, China. Her main research interest is skeletal muscle homeostasis.

JingyuanLuo is a PhD student in Department of Orthopaedics at Xiangya Hospital of Central South University, China. His current research focuses on skeletal muscle single cell atlas.

ChangjunLi is a professor, doctoral supervisor, and vice director of the Key Laboratory of Aging-Related Bone and Joint Diseases Prevention and Treatment, Ministry of Education, Xiangya Hospital, Central South University, China. In recent years, he has published a series of papers, including Cell Metabolism, Science Bulletin, and Bone Research.

JieWei is a professor, doctoral supervisor, and vice director of the Key Laboratory of Aging-Related Bone and Joint Diseases Prevention and Treatment, Ministry of Education, Xiangya Hospital, Central South University, China. In recent years, she has published 52 SCI papers as the first/corresponding author in journals, including Ann Intern Med, JAMA Intern Med, and Ann Rheum Dis.

YilunWang is an associate professor and master's supervisor within the Department of Orthopaedics at Xiangya Hospital, Central South University, China. In recent 5 years, he has published 21 SCI papers as the first/corresponding author in journals, including JAMA Internal Med, Arthritis Rheumatol, JAMA Network Open, EBioMedicine, and J Cachexia Sarcopenia Muscle.

Chao Zeng is a professor, doctoral supervisor, and vice director of the Key Laboratory of Aging-Related Bone and Joint Diseases Prevention and Treatment, Ministry of Education, Xiangya Hospital, Central South University, China. He has long been committed to the research of pathogenesis, clinical prevention, treatment, and translational medicine of degenerative diseases of the musculoskeletal system. In the past five years, he has published 40 SCI papers as the first/corresponding author in journals, including JAMA, JAMA Intern Med, Ann Rheum Dis, Eur Heart J, and Nat Aging.

GuanghuaLei is a professor, doctoral supervisor, the director of the National Clinical Research Center for Geriatric Disorders and the director of Key Laboratory of Aging-related Bone and Joint Diseases Prevention and Treatment, Ministry of Education, Xiangya Hospital, Central South University, China, with a long-standing commitment to both the clinical and basic research of degenerative diseases of the musculoskeletal system. He has obtained nine grants from the National Key Research and Development Project and the National Natural Science Foundation of China. To date, he has published 191 manuscripts as corresponding author in peer-reviewed SCI journals, including JAMA and BMJ.