Integration of single-cell datasets depicts profiles of macrophages and fibro/adipogenic progenitors in dystrophic muscle.

Single-cell technologies have recently expanded the possibilities for researchers to gain, at an unprecedented resolution level, knowledge about tissue composition, cell complexity, and heterogeneity. Moreover, the integration of data coming from different technologies and sources also offers, for the first time, the possibility to draw a holistic portrait of how cells behave to sustain tissue physiology during the human lifespan and disease. Here, we interrogated and integrated publicly available single-cell RNAseq data to advance the understanding of how macrophages, fibro/adipogenic progenitors, and other cell types establish gene regulatory networks and communicate with each other in the muscle tissue. We identified altered gene signatures and signaling pathways associated with the dystrophic condition, including an enhanced Spp1-Cd44 signaling in dystrophic macrophages. We shed light on the differences among dystrophic muscle aging, considering wild type, mdx, and more severe conditions as in the case of the mdx-2d model. Contextually, we provided details on existing communication relations between muscle niche cell populations, highlighting increased interactions and distinct signaling events that these cells stablish in the dystrophic microenvironment. We believe our findings can help scientists to formulate and test new hypotheses by moving towards a more complete understanding of muscle regeneration and immune system biology.

IL-33 Facilitates Fibro-Adipogenic Progenitors to Establish the Pro-Regenerative Niche after Muscle Injury.

During the process of muscle regeneration post-injury in adults, muscle stem cells (MuSCs) function is facilitated by neighboring cells within the pro-regenerative niche. However, the precise mechanism triggering the initiation of signaling in the pro-regenerative niche remains unknown. Using single-cell RNA sequencing, 14 different muscle cells are comprehensively mapped during the initial stage following injury. Among these, macrophages and fibro-adipogenic progenitor cells (FAPs) exhibit the most pronounced intercellular communication with other cells. In the FAP subclusters, the study identifies an activated FAP phenotype that secretes chemokines, such as CXCL1, CXCL5, CCL2, and CCL7, to recruit macrophages after injury. Il1rl1, encoding the protein of the interleukin-33 (IL-33) receptor, is identified as a highly expressed signature surface marker of the FAP phenotype. Following muscle injury, autocrine IL-33, an alarmin, has been observed to activate quiescent FAPs toward this inflammatory phenotype through the IL1RL1-MAPK/NF-κB signaling pathway. Il1rl1 deficiency results in decreased chemokine expression and recruitment of macrophages, accompanied by impaired muscle regeneration. These findings elucidate a novel mechanism involving the IL-33/IL1RL1 signaling pathway in promoting the activation of FAPs and facilitating muscle regeneration, which can aid the development of therapeutic strategies for muscle-related disorders and injuries.

Multiparametric identification of putative senescent cells in skeletal muscle via mass cytometry.

Senescence is an irreversible arrest of the cell cycle that can be characterized by markers of senescence such as p16, p21, and KI-67. The characterization of different senescence-associated phenotypes requires selection of the most relevant senescence markers to define reliable cytometric methodologies. Mass cytometry (a.k.a. Cytometry by time of flight, CyTOF) can monitor up to 40 different cell markers at the single-cell level and has the potential to integrate multiple senescence and other phenotypic markers to identify senescent cells within a complex tissue such as skeletal muscle, with greater accuracy and scalability than traditional bulk measurements and flow cytometry-based measurements. This article introduces an analysis framework for detecting putative senescent cells based on clustering, outlier detection, and Boolean logic for outliers. Results show that the pipeline can identify putative senescent cells in skeletal muscle with well-established markers such as p21 and potential markers such as GAPDH. It was also found that heterogeneity of putative senescent cells in skeletal muscle can partly be explained by their cell type. Additionally, autophagy-related proteins ATG4A, LRRK2, and GLB1 were identified as important proteins in predicting the putative senescent population, providing insights into the association between autophagy and senescence. It was observed that sex did not affect the proportion of putative senescent cells among total cells. However, age did have an effect, with a higher proportion observed in fibro/adipogenic progenitors (FAPs), satellite cells, M1 and M2 macrophages from old mice. Moreover, putative senescent cells from muscle of old and young mice show different expression levels of senescence-related proteins, with putative senescent cells of old mice having higher levels of p21 and GAPDH, whereas putative senescent cells of young mice had higher levels of IL-6. Overall, the analysis framework prioritizes multiple senescence-associated proteins to characterize putative senescent cells sourced from tissue made of different cell types.

Cellular interactions and microenvironment dynamics in skeletal muscle regeneration and disease.

Skeletal muscle regeneration relies on the intricate interplay of various cell populations within the muscle niche-an environment crucial for regulating the behavior of muscle stem cells (MuSCs) and ensuring postnatal tissue maintenance and regeneration. This review delves into the dynamic interactions among key players of this process, including MuSCs, macrophages (MPs), fibro-adipogenic progenitors (FAPs), endothelial cells (ECs), and pericytes (PCs), each assuming pivotal roles in orchestrating homeostasis and regeneration. Dysfunctions in these interactions can lead not only to pathological conditions but also exacerbate muscular dystrophies. The exploration of cellular and molecular crosstalk among these populations in both physiological and dystrophic conditions provides insights into the multifaceted communication networks governing muscle regeneration. Furthermore, this review discusses emerging strategies to modulate the muscle-regenerating niche, presenting a comprehensive overview of current understanding and innovative approaches.

miR-27b-3p reduces muscle fibrosis during chronic skeletal muscle injury by targeting TGF-ßR1/Smad pathway.

BACKGROUND: Fibrosis is a significant pathological feature of chronic skeletal muscle injury, profoundly affecting muscle regeneration. Fibro-adipogenic progenitors (FAPs) have the ability to differentiate into myofibroblasts, acting as a primary source of extracellular matrix (ECM). the process by which FAPs differentiate into myofibroblasts during chronic skeletal muscle injury remains inadequately explored. METHOD: mouse model with sciatic nerve denervated was constructed and miRNA expression profiles between the mouse model and uninjured mouse were analyzed. qRT/PCR and immunofluorescence elucidated the effect of miR-27b-3p on fibrosis in vivo and in vitro. Dual-luciferase reporter identified the target gene of miR-27b-3p, and finally knocked down or overexpressed the target gene and phosphorylation inhibition of Smad verified the influence of downstream molecules on the abundance of miR-27b-3p and fibrogenic differentiation of FAPs. RESULT: FAPs derived from a mouse model with sciatic nerves denervated exhibited a progressively worsening fibrotic phenotype over time. Introducing agomiR-27b-3p effectively suppressed fibrosis both in vitro and in vivo. MiR-27b-3p targeted Transforming Growth Factor Beta Receptor 1 (TGF-ßR1) and the abundance of miR-27b-3p was negatively regulated by TGF-ßR1/Smad. CONCLUSION: miR-27b-3p targeting the TGF-ßR1/Smad pathway is a novel mechanism for regulating fibrogenic differentiation of FAPs. Increasing abundance of miR-27b-3p, suppressing expression of TGF-ßR1 and inhibiting phosphorylation of smad3 presented potential strategies for treating fibrosis in chronic skeletal muscle injury.

Parathyroid hormone promotes induction of beige adipocytes and reversibly ameliorates muscle quality and atrophy following chronic rotator cuff tear in a rat model.

BACKGROUND: Fatty infiltration (FI) and muscle atrophy (MA) in the rotator cuff muscles following rotator cuff tears (RCT) persist post repair, increasing the risk of re-tears. Brown adipocyte-like "beige adipocytes" are expected to have a therapeutic effect on intramuscular FI and MA due to their lipolytic activity and the muscle regenerative effects of their secreted factors. However, whether parathyroid hormone (PTH) ameliorates the already advanced FI and MA remains unknown. Therefore, this study aimed to clarify whether PTH promotes the expression of beige adipocytes and ameliorates advanced FI and MA following chronic RCT in rats. METHODS: Supraspinatus muscles were harvested from rats with chronic RCT after 4 or 8 weeks of PTH treatment and compared to those in the control group or to those at the start of treatment. FI was assessed by Oil Red O staining, and the staining area was evaluated as a percentage of the muscle cross-sectional area. MA was evaluated by measuring muscle wet weight and cross-sectional area of muscle fiber. Beige adipocyte expression was evaluated by immunostaining for uncoupling protein 1 (UCP1). Fibro-adipogenic progenitors (FAPs) were separated from muscle-injured mice. We assessed whether PTH could diminish fat droplet accumulation by promoting the differentiation of FAPs into beige adipocytes. RESULTS: After four weeks, PTH reduced the area fraction of FI in the rat supraspinatus muscle following chronic RCT compared with that at the beginning of treatment (P = .028). In addition, PTH increased wet muscle mass (P < .001) and muscle fiber cross-sectional area (P < .018) compared with measurements at the start of treatment. PTH administration promoted the expression of UCP1, a beige adipocyte marker, in the supraspinatus muscle (P = .019). PTH increased gene expression of beige adipocyte-related markers and suppressed fat droplet accumulation even after adipogenic differentiation of FAPs (P = .004) but did not reduce fat droplets that had already accumulated in in vitro experiments. CONCLUSIONS: PTH facilitated beige adipocyte expression and reversibly ameliorated muscle quality and atrophy following chronic RCT by hindering fat droplet accumulation and facilitating muscle regeneration. Therefore, PTH may be a medicament for FI and MA following RCT, leading to expanded rotator cuff repair indications.

IL-33-ST2 signaling in fibro-adipogenic progenitors alleviates immobilization-induced muscle atrophy in mice.

BACKGROUND: The regenerative and adaptive capacity of skeletal muscles reduces with age, leading to severe disability and frailty in the elderly. Therefore, development of effective therapeutic interventions for muscle wasting is important both medically and socioeconomically. In the present study, we aimed to elucidate the potential contribution of fibro-adipogenic progenitors (FAPs), which are mesenchymal stem cells in skeletal muscles, to immobilization-induced muscle atrophy. METHODS: Young (2-3 months), adult (12-14 months), and aged (20-22 months) mice were used for analysis. Muscle atrophy was induced by immobilizing the hind limbs with a steel wire. FAPs were isolated from the hind limbs on days 0, 3, and 14 after immobilization for transcriptome analysis. The expression of ST2 and IL-33 in FAPs was evaluated by flow cytometry and immunostaining, respectively. To examine the role of IL-33-ST2 signaling in vivo, we intraperitoneally administered recombinant IL-33 or soluble ST2 (sST2) twice a week throughout the 2-week immobilization period. After 2-week immobilization, the tibialis anterior muscles were harvested and the cross-sectional area of muscle fibers was evaluated. RESULTS: The number of FAPs increased with the progression of muscle atrophy after immobilization in all age-groups. Transcriptome analysis of FAPs collected before and after immobilization revealed that Il33 and Il1rl1 transcripts, which encode the IL-33 receptor ST2, were transiently induced in young mice and, to a lesser extent, in aged mice. The number of FAPs positive for ST2 increased after immobilization in young mice. The number of ST2-positive FAPs also increased after immobilization in aged mice, but the difference from the baseline was not statistically significant. Immunostaining for IL-33 in the muscle sections revealed a significant increase in the number of FAPs expressing IL-33 after immobilization. Administration of recombinant IL-33 suppressed immobilization-induced muscle atrophy in aged mice but not in young mice. CONCLUSIONS: Our data reveal a previously unknown protective role of IL-33-ST2 signaling against immobilization-induced muscle atrophy in FAPs and suggest that IL-33-ST2 signaling is a potential new therapeutic target for alleviating disuse muscle atrophy, particularly in older adults.

Cellular and Molecular Mechanisms of Heterotopic Ossification in Fibrodysplasia Ossificans Progressiva.

Fibrodysplasia ossificans progressiva (FOP) is a debilitating genetic disorder characterized by recurrent episodes of heterotopic ossification (HO) formation in muscles, tendons, and ligaments. FOP is caused by a missense mutation in the ACVR1 gene (activin A receptor type I), an important signaling receptor involved in endochondral ossification. The ACVR1R206H mutation induces increased downstream canonical SMAD-signaling and drives tissue-resident progenitor cells with osteogenic potential to participate in endochondral HO formation. In this article, we review aberrant ACVR1R206H signaling and the cells that give rise to HO in FOP. FOP mouse models and lineage tracing analyses have been used to provide strong evidence for tissue-resident mesenchymal cells as cellular contributors to HO. We assess how the underlying mutation in FOP disrupts muscle-specific dynamics during homeostasis and repair, with a focus on muscle-resident mesenchymal cells known as fibro-adipogenic progenitors (FAPs). Accumulating research points to FAPs as a prominent HO progenitor population, with ACVR1R206H FAPs not only aberrantly differentiating into chondro-osteogenic lineages but creating a permissive environment for bone formation at the expense of muscle regeneration. We will further discuss the emerging role of ACVR1R206H FAPs in muscle regeneration and therapeutic targeting of these cells to reduce HO formation in FOP.

Metformin mitigates adipogenesis of fibro-adipogenic progenitors after rotator cuff tears via activating mTOR/ULK1-mediated autophagy.

Muscular fatty infiltration is a common issue after rotator cuff tears (RCTs), which impair shoulder function. Females suffer a higher prevalence and a more severe degree of muscular fatty infiltration after RCT when compared with males, with the underlying mechanisms remaining unclear. Fibro-adipogenic progenitors (FAPs) are the primary source of muscular fatty infiltration following RCT. Our findings disclose that gender-specific disparities in muscular fatty infiltration are linked to mTOR/ULK1-mediated autophagy of FAPs. Decreased autophagic activity contributes to adipogenic differentiation in female FAPs after RCT. Furthermore, metformin could enhance mTOR/ULK1-mediated autophagic processes of FAPs, thereby alleviating fatty infiltration and improving shoulder functionality after RCT. Together, our study reveals that gender differences in muscular fatty infiltration arise from distinct autophagic activities. Metformin could be a promising noninvasive intervention to ameliorate muscular fatty infiltration of RCT.NEW & NOTEWORTHY The current study demonstrated that gender-specific disparities in muscular fatty infiltration are attributed to mTOR/ULK1-mediated autophagy of FAPs. Decreased autophagic activity contributes to adipogenic differentiation in female FAPs after RCT. Moreover, metformin could enhance mTOR/ULK1-mediated autophagic processes of FAPs, thereby alleviating fatty infiltration and improving shoulder functionality after RCT. Therefore, metformin could be a promising noninvasive intervention to ameliorate muscular fatty infiltration of RCT.

The reciprocity of skeletal muscle and bone: an evolving view from mechanical coupling, secretory crosstalk to stem cell exchange.

Introduction: Muscle and bone constitute the two main parts of the musculoskeletal system and generate an intricately coordinated motion system. The crosstalk between muscle and bone has been under investigation, leading to revolutionary perspectives in recent years. Method and results: In this review, the evolving concept of muscle-bone interaction from mechanical coupling, secretory crosstalk to stem cell exchange was explained in sequence. The theory of mechanical coupling stems from the observation that the development and maintenance of bone mass are largely dependent on muscle-derived mechanical loads, which was later proved by Wolff's law, Utah paradigm and Mechanostat hypothesis. Then bone and muscle are gradually recognized as endocrine organs, which can secrete various cytokines to modulate the tissue homeostasis and remodeling to each other. The latest view presented muscle-bone interaction in a more direct way: the resident mesenchymal stromal cell in the skeletal muscle, i.e., fibro-adipogenic progenitors (FAPs), could migrate to the bone injury site and contribute to bone regeneration. Emerging evidence even reveals the ectopic source of FAPs from tissue outside the musculoskeletal system, highlighting its dynamic property. Conclusion: FAPs have been established as the critical cell connecting muscle and bone, which provides a new modality to study inter-tissue communication. A comprehensive and integrated perspective of muscle and bone will facilitate in-depth research in the musculoskeletal system and promote novel therapeutic avenues in treating musculoskeletal disorders.

Utilization of the Rat Tibial Nerve Transection Model to Evaluate Cellular and Molecular Mechanisms Underpinning Denervation-Mediated Muscle Injury.

Peripheral nerve injury denervates muscle, resulting in muscle paralysis and atrophy. This is reversible if timely muscle reinnervation occurs. With delayed reinnervation, the muscle's reparative ability declines, and muscle-resident fibro-adipogenic progenitor cells (FAPs) proliferate and differentiate, inducing fibro-fatty muscle degradation and thereby physical disability. The mechanisms by which the peripheral nerve regulates FAPs expansion and differentiation are incompletely understood. Using the rat tibial neve transection model, we demonstrated an increased FAPs content and a changing FAPs phenotype, with an increased capacity for adipocyte and fibroblast differentiation, in gastrocnemius muscle post-denervation. The FAPs response was inhibited by immediate tibial nerve repair with muscle reinnervation via neuromuscular junctions (NMJs) and sensory organs (e.g., muscle spindles) or the sensory protection of muscle (where a pure sensory nerve is sutured to the distal tibial nerve stump) with reinnervation by muscle spindles alone. We found that both procedures reduced denervation-mediated increases in glial-cell-line-derived neurotrophic factor (GDNF) in muscle and that GDNF promoted FAPs adipogenic and fibrogenic differentiation in vitro. These results suggest that the peripheral nerve controls FAPs recruitment and differentiation via the modulation of muscle GDNF expression through NMJs and muscle spindles. GDNF can serve as a therapeutic target in the management of denervation-induced muscle injury.

Sex as a Critical Variable in Basic and Pre-Clinical Studies of Fibrodysplasia Ossificans Progressiva.

Heterotopic ossification (HO) is most dramatically manifested in the rare and severely debilitating disease, fibrodysplasia ossificans progressiva (FOP), in which heterotopic bone progressively accumulates in skeletal muscles and associated soft tissues. The great majority of FOP cases are caused by a single amino acid substitution in the type 1 bone morphogenetic protein (BMP) receptor ACVR1, a mutation that imparts responsiveness to activin A. Although it is well-established that biological sex is a critical variable in a range of physiological and disease processes, the impact of sex on HO in animal models of FOP has not been explored. We show that female FOP mice exhibit both significantly greater and more variable HO responses after muscle injury. Additionally, the incidence of spontaneous HO was significantly greater in female mice. This sex dimorphism is not dependent on gonadally derived sex hormones, and reciprocal cell transplantations indicate that apparent differences in osteogenic activity are intrinsic to the sex of the transplanted cells. By circumventing the absolute requirement for activin A using an agonist of mutant ACVR1, we show that the female-specific response to muscle injury or BMP2 implantation is dependent on activin A. These data identify sex as a critical variable in basic and pre-clinical studies of FOP.

Exercise-induced Musclin determines the fate of fibro-adipogenic progenitors to control muscle homeostasis.

The effects of exercise on fibro-adipogenic progenitors (FAPs) are unclear, and the direct molecular link is still unknown. In this study, we reveal that exercise reduces the frequency of FAPs and attenuates collagen deposition and adipose formation in injured or disused muscles through Musclin. Mechanistically, Musclin inhibits FAP proliferation and promotes apoptosis in FAPs by upregulating FILIP1L. Chromatin immunoprecipitation (ChIP)-qPCR confirms that FoxO3a is the transcription factor of FILIP1L. In addition, the Musclin/FILIP1L pathway facilitates the phagocytosis of apoptotic FAPs by macrophages through downregulating the expression of CD47. Genetic ablation of FILIP1L in FAPs abolishes the effects of exercise or Musclin on FAPs and the benefits on the reduction of fibrosis and fatty infiltration. Overall, exercise forms a microenvironment of myokines in muscle and prevents the abnormal accumulation of FAPs in a Musclin/FILIP1L-dependent manner. The administration of exogenous Musclin exerts a therapeutic effect, demonstrating a potential therapeutic approach for muscle atrophy or acute muscle injury.

Single-Cell RNA Sequencing Reveals the Cellular Landscape of Longissimus Dorsi in a Newborn Suhuai Pig.

The introduction of single-cell RNA sequencing (scRNA-seq) technology has spurred additional advancements in analyzing the cellular composition of tissues. The longissimus dorsi (LD) in pigs serves as the primary skeletal muscle for studying meat quality in the pig industry. However, the single-cell profile of porcine LD is still in its infancy stage. In this study, we profiled the transcriptomes of 16,018 cells in the LD of a newborn Suhuai pig at single-cell resolution. Subsequently, we constructed a cellular atlas of the LD, identifying 11 distinct cell populations, including endothelial cells (24.39%), myotubes (18.82%), fibro-adipogenic progenitors (FAPs, 18.11%), satellite cells (16.74%), myoblasts (3.99%), myocytes (5.74%), Schwann cells (3.81%), smooth muscle cells (3.22%), dendritic cells (2.99%), pericytes (1.86%), and neutrophils (0.33%). CellChat was employed to deduce the cell-cell interactions by evaluating the gene expression of receptor-ligand pairs across different cell types. The results show that FAPs and pericytes are the primary signal contributors in LD. In addition, we delineated the developmental trajectory of myogenic cells and examined alterations in the expression of various marker genes and molecular events throughout various stages of differentiation. Moreover, we found that FAPs can be divided into three subclusters (NR2F2-FAPs, LPL-FAPs, and TNMD-FAPs) according to their biological functions, suggesting that the FAPs could be associated with the differentiation of tendon cell. Taken together, we constructed the cellular atlas and cell communication network in LD of a newborn Suhuai pig, and analyzed the developmental trajectory of myogenic cells and the heterogeneity of FAPs subpopulation cells. This enhances our comprehension of the molecular features involved in skeletal muscle development and the meat quality control in pigs.

MuSCs and IPCs: roles in skeletal muscle homeostasis, aging and injury.

Skeletal muscle is a highly specialized tissue composed of myofibres that performs crucial functions in movement and metabolism. In response to external stimuli and injuries, a range of stem/progenitor cells, with muscle stem cells or satellite cells (MuSCs) being the predominant cell type, are rapidly activated to repair and regenerate skeletal muscle within weeks. Under normal conditions, MuSCs remain in a quiescent state, but become proliferative and differentiate into new myofibres in response to injury. In addition to MuSCs, some interstitial progenitor cells (IPCs) such as fibro-adipogenic progenitors (FAPs), pericytes, interstitial stem cells expressing PW1 and negative for Pax7 (PICs), muscle side population cells (SPCs), CD133-positive cells and Twist2-positive cells have been identified as playing direct or indirect roles in regenerating muscle tissue. Here, we highlight the heterogeneity, molecular markers, and functional properties of these interstitial progenitor cells, and explore the role of muscle stem/progenitor cells in skeletal muscle homeostasis, aging, and muscle-related diseases. This review provides critical insights for future stem cell therapies aimed at treating muscle-related diseases.

Combinatorial activation of the WNT-dependent fibrogenic program by distinct complement subunits in dystrophic muscle.

Fibrosis is associated with compromised muscle functionality in Duchenne muscular dystrophy (DMD). We report observations with tissues from dystrophic patients and mice supporting a model to explain fibrosis in DMD, which relies on the crosstalk between the complement and the WNT signaling pathways and the functional interactions of two cellular types. Fibro-adipogenic progenitors and macrophages, which populate the inflamed dystrophic muscles, act as a combinatorial source of WNT activity by secreting distinct subunits of the C1 complement complex. The resulting aberrant activation of the WNT signaling in responsive cells, such as fibro-adipogenic progenitors, contributes to fibrosis. Indeed, pharmacological inhibition of the C1r/s subunits in a murine model of DMD mitigated the activation of the WNT signaling pathway, reduced the fibrogenic characteristics of the fibro-adipogenic progenitors, and ameliorated the dystrophic phenotype. These studies shed new light on the molecular and cellular mechanisms responsible for fibrosis in muscular dystrophy and open to new therapeutic strategies.

Polarized macrophages regulate fibro/adipogenic progenitor (FAP) adipogenesis through exosomes.

BACKGROUND: Macrophage polarization has been observed in the process of muscle injuries including rotator cuff (RC) muscle atrophy and fatty infiltration after large tendon tears. In our previous study, we showed that fibrogenesis and white adipogenesis of muscle residential fibro/adipogenic progenitors (FAPs) cause fibrosis and fatty infiltration and that brown/beige adipogenesis of FAPs promotes rotator cuff muscle regeneration. However, how polarized macrophages and their exosomes regulate FAP differentiation remains unknown. METHODS: We cultured FAPs with M0, M1, and M2 macrophages or 2 × 109 exosomes derived from M0, M1 and M2 with and without GW4869, an exosome inhibitor. In vivo, M0, M1, and M2 macrophages were transplanted or purified macrophage exosomes (M0, M1, M2) were injected into supraspinatus muscle (SS) after massive tendon tears in mice (n = 6). SS were harvested at six weeks after surgery to evaluate the level of muscle atrophy and fatty infiltration. RESULTS: Our results showed that M2 rather than M0 or M1 macrophages stimulates brown/beige fat differentiation of FAPs. However, the effect of GW4869, the exosome inhibitor, diminished this effect. M2 exosomes also promoted FAP Beige differentiation in vitro. The transplantation of M2 macrophages reduced supraspinatus muscle atrophy and fatty infiltration. In vivo injections of M2 exosomes significantly reduced muscle atrophy and fatty infiltration in supraspinatus muscle. CONCLUSION: Results from our study demonstrated that polarized macrophages directly regulated FAP differentiation through their exosomes and M2 macrophage-derived exosomes may serve as a novel treatment option for RC muscle atrophy and fatty infiltration.

Interleukin 4 improved adipose-derived stem cells engraftment via interacting with fibro/adipogenic progenitors in dystrophic mice.

Adipose-derived stem cells (ADSC) therapy shows promise as an effective treatment for dystrophinopathy. Fibro-/adipogenic progenitors (FAPs) play an essential role in the myogenesis of muscle satellite cells and contribute to muscle fibrosis and adipocyte infiltration. The interleukin 4 (IL-4) pathway acts as a switch that regulates the functions of FAPs. The interaction between FAPs and engrafted cells remains unclear. In this study, we used a co-culture system to investigate possible crosstalk between the FAPs of dystrophic mice and ADSC overexpressing IL4 (IL4-ADSC) and control ADSC. Systemic transplantation of IL4-ADSC and control ADSC in dystrophic mice was conducted for 16 weeks, after which motor function and molecular improvements were evaluated. Overexpression of IL4 in ADSC significantly promoted myogenesis in vitro, increasing the expression of Pax7, Myogenin, and MyHC. Co-culture indicated that although myoblasts derived from control ADSC promoted adipogenic and fibrogenic differentiation of FAPs, FAPs did not significantly affect myogenesis of ADSC-derived myoblasts. However, overexpression of IL4 in ADSC inhibited their myotube-dependent promotion of FAPs differentiation on the one hand and promoted FAPs to enhance myogenesis on the other. Dystrophic mice administered with IL4-ADSC-derived myoblasts displayed significantly better motor ability, more engrafted cells showing dystrophin expression, and less muscle fibrosis, intramuscular adipocytes, and macrophage infiltration than mice administered control-ADSC-derived myoblasts. In conclusion, IL4 activation enhanced the therapeutic potential of ADSC transplantation in dystrophic mice, possibly by improving the myogenesis of IL4-ADSC and altering the crosstalk between engrafted stem cells and resident FAPs.

Gastric inhibitory polypeptide receptor antagonism suppresses intramuscular adipose tissue accumulation and ameliorates sarcopenia.

BACKGROUND: Intramuscular adipose tissue (IMAT) formation derived from muscle fibro-adipogenic progenitors (FAPs) has been recognized as a pathological feature of sarcopenia. This study aimed to explore whether genetic and pharmacological gastric inhibitory polypeptide (GIP) receptor antagonism suppresses IMAT accumulation and ameliorates sarcopenia in mice. METHODS: Whole body composition, grip strength, skeletal muscle weight, tibialis anterior (TA) muscle fibre cross-sectional area (CSA) and TA muscle IMAT area were measured in young and aged male C57BL/6 strain GIP receptor (Gipr)-knockout (Gipr-/- ) and wild-type (Gipr+/+ ) mice. FAPs isolated from lower limb muscles of 12-week-old Gipr+/+ mice were cultured with GIP, and their differentiation into mature adipocytes was examined. Furthermore, TA muscle IMAT area and fibre CSA were measured in untreated Gipr-/- mice and GIP receptor antagonist-treated Gipr+/+ mice after glycerol injection into the TA muscles. RESULTS: Body composition analysis revealed that 104-week-old Gipr-/- mice had a greater proportion of lean tissue mass (73.7 ± 1.2% vs. 66.5 ± 2.7%, P < 0.05 vs. 104-week-old Gipr+/+ mice) and less adipose tissue mass (13.1 ± 1.3% vs. 19.4 ± 2.6%, P < 0.05 vs. 104-week-old Gipr+/+ mice). Eighty-four-week-old Gipr-/- mice exhibited increases in grip strength (P < 0.05), weights of TA (P < 0.05), soleus (P < 0.01), gastrocnemius (P < 0.05) and quadriceps femoris (P < 0.01) muscles, and average TA muscle fibre CSA (P < 0.05) along with a reduction in TA muscle IMAT area assessed by the number of perilipin-positive cells (P < 0.0001) compared with 84-week-old Gipr+/+ mice. Oil Red O staining analysis revealed 1.6- and 1.7-fold increased adipogenesis in muscle FAPs cultured with 10 and 100 nM of GIP (P < 0.01 and P < 0.001 vs. 0 nM of GIP, respectively). Furthermore, both untreated Gipr-/- mice and GIP receptor antagonist-treated Gipr+/+ mice for 14 days after glycerol injection into the TA muscles at 12 weeks of age showed reduced TA muscle IMAT area (1.39 ± 0.38% and 2.65 ± 0.36% vs. 6.54 ± 1.30%, P < 0.001 and P < 0.01 vs. untreated Gipr+/+ mice, respectively) and increased average TA muscle fibre CSA (P < 0.01 and P < 0.05 vs. untreated Gipr+/+ mice, respectively). CONCLUSIONS: GIP promotes the differentiation of muscle FAPs into adipocytes and its receptor antagonism suppresses IMAT accumulation and promotes muscle regeneration. Pharmacological GIP receptor antagonism may serve as a novel therapeutic approach for sarcopenia.

Single-Cell RNA-Sequencing Provides Insight into Skeletal Muscle Evolution during the Selection of Muscle Characteristics.

Skeletal muscle comprises a large, heterogeneous assortment of cell populations that interact to maintain muscle homeostasis, but little is known about the mechanism that controls myogenic development in response to artificial selection. Different pig (Sus scrofa) breeds exhibit distinct muscle phenotypes resulting from domestication and selective breeding. Using unbiased single-cell transcriptomic sequencing analysis (scRNA-seq), the impact of artificial selection on cell profiles is investigated in neonatal skeletal muscle of pigs. This work provides panoramic muscle-resident cell profiles and identifies novel and breed-specific cells, mapping them on pseudotime trajectories. Artificial selection has elicited significant changes in muscle-resident cell profiles, while conserving signs of generational environmental challenges. These results suggest that fibro-adipogenic progenitors serve as a cellular interaction hub and that specific transcription factors identified here may serve as candidate target regulons for the pursuit of a specific muscle phenotype. Furthermore, a cross-species comparison of humans, mice, and pigs illustrates the conservation and divergence of mammalian muscle ontology. The findings of this study reveal shifts in cellular heterogeneity, novel cell subpopulations, and their interactions that may greatly facilitate the understanding of the mechanism underlying divergent muscle phenotypes arising from artificial selection.