Fibroadipogenic Progenitor Cell Research Articles

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.

210-OR: ADA Presidents' Select Abstract: Discovering Stimulatory State-Specific Type 2 Diabetes GWAS Mechanisms with Single-Cell Multiomics on iPSC-Derived Fibro-adipogenic Progenitor Cell Villages

210-OR: ADA Presidents' Select Abstract: Discovering Stimulatory State-Specific Type 2 Diabetes GWAS Mechanisms with Single-Cell Multiomics on iPSC-Derived Fibro-adipogenic Progenitor Cell Villages

Abstract 1601: The role of complement in pancreatic cancer cachexia

Abstract Cancer cachexia is a multifactorial condition characterized by skeletal muscle atrophy and dysfunction that impairs longevity and quality of life for the vast majority of cancer patients. Recently, we discovered through unbiased proteomic analyses that the complement system is activated in the skeletal muscle of cachectic pancreatic cancer patients. Therefore, the purpose of this study was to investigate the role of complement in pancreatic cancer cachexia. To accomplish this, we obtained genetically modified, C57Bl6/J congenic mice lacking the central regulator of complement, C3 (C3−/− mice). Wild-type (C57Bl6/J, WT, n=12) and C3−/− mice (n=11) (male, 12 weeks old) received orthotopic injections of mouse pancreatic cancer cells (KPC) into the pancreas, and as non-cancer controls, WT (n=8) and C3−/− (n=4) mice received injections of PBS into the pancreas. Mice were monitored daily and euthanized at IACUC-mandated tumor endpoint (body condition score ≤ 2; range: 14-15 days). In-vivo diaphragm function was assessed immediately prior to euthanasia via M-mode ultrasound. Skeletal muscles (soleus, SOL; tibialis anterior, TA; diaphragm, DIA) were harvested and ex-vivo contractile function was assessed on strips of costal diaphragm. Diaphragmatic excursion and respiratory rate were reduced with KPC tumor burden in WT mice, but preserved in C3−/− mice. Deletion of C3 did not influence the KPC-induced reduction of body mass or KPC tumor growth. Atrophy of limb muscle mass (SOL, TA) and fiber size (TA) during KPC tumor burden was attenuated in C3−/− mice. Maximal specific force of DIA was greater in C3−/− versus WT KPC-bearing mice. KPC tumor burden increased collagen content in the DIA of WT mice, but not C3−/− mice. Diaphragm fiber size was reduced in WT KPC-bearing mice, but preserved in C3−/− mice. KPC tumor burden induced significant increases in immune cell (CD45+) infiltration and fibroadipogenic progenitor cell (PDGFRa+) abundance in the DIA of WT tumor-bearing mice, but to a lesser extent in C3−/− mice. These findings suggest complement activation is causative in pancreatic cancer-induced skeletal muscle wasting and dysfunction. Inhibition of complement during cancer cachexia may possess therapeutic potential for the preservation of skeletal muscle mass and function. Citation Format: Andrew C. D'Lugos, Chandler Callaway, Sarah M. Judge, Andrew R. Judge. The role of complement in pancreatic cancer cachexia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1601.

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.

Fibroblast growth factor 8b (FGF-8b) enhances myogenesis and inhibits adipogenesis in rotator cuff muscle cell populations in vitro.

Fatty expansion is one of the features of muscle degeneration due to muscle injuries, and its presence interferes with muscle regeneration. Specifically, poor clinical outcomes have been linked to fatty expansion in rotator cuff tears and repairs. Our group recently found that fibroblast growth factor 8b (FGF-8b) inhibits adipogenic differentiation and promotes myofiber formation of mesenchymal stem cells in vitro. This led us to hypothesize that FGF-8b could similarly control the fate of muscle-specific cell populations derived from rotator cuff muscle involved in muscle repair following rotator cuff injury. In this study, we isolate fibro-adipogenic progenitor cells (FAPs) and satellite stem cells (SCs) from rat rotator cuff muscle tissue and analyzed the effects of FGF-8b supplementation. Utilizing a cell plating protocol, we successfully isolate FAPs-rich fibroblasts (FIBs) and SCs-rich muscle progenitor cells (MPCs). Subsequently, we demonstrate that FIB adipogenic differentiation can be inhibited by FGF-8b, while MPC myogenic differentiation can be enhanced by FGF-8b. We further demonstrate that phosphorylated ERK due to FGF-8b leads to the inhibition of adipogenesis in FIBs and SCs maintenance and myofiber formation in MPCs. Together, these findings demonstrate the powerful potential of FGF-8b for rotator cuff repair by altering the fate of muscle undergoing degeneration.

Single-nucleus RNA sequencing reveals heterogeneity among multiple white adipose tissue depots

Abstract Regardless of its anatomical site, adipose tissue shares a common energy-storage role but exhibits distinctive properties. Exploring the cellular and molecular heterogeneity of white adipose tissue (WAT) is crucial for comprehending its function and properties. However, existing single-nucleus RNA sequencing (snRNA-seq) studies of adipose tissue heterogeneity have examined only one or two depots. In this study, we employed snRNA-seq to test five representative depots including inguinal, epididymal, mesenteric, perirenal, and pericardial adipose tissues in mice under physiological conditions. By analyzing the contents of main cell categories and gene profiles of various depots, we identified their distinctive physiological properties. Immune cells and fibro-adipogenic progenitor cells (FAPs) showed dramatic differences among WAT depots, while adipocytes seemed to be conserved. The heightened presence of regulatory macrophages and B cells in pericardial adipose tissues implied their potential contribution to the preservation of coronary vascular function. Moreover, the selective aggregation of pericytes within mesenteric adipose tissue was likely associated with the maintenance of intestinal barrier homeostasis. Using a combination of RNA sequencing and snRNA-seq analysis, the major subpopulations of FAPs derived from these depots determined the site characteristics of FAPs to a certain extent. Our work establishes a systematic and reliable foundation for investigating the heterogeneity of WAT depots and elucidating the unique roles these depots play in coordinating the function of adjacent organs.

PSVII-18 Applying the Marbling Window Concept to Enhance Intramuscular Fat Development in Beef Cattle

Abstract The increase in marbling deposition without increasing the overall fatness of the carcass is one of the most challenge goals in meat industry. Previous studies have suggested the nutritional interventions at the neonatal stages as a strategy to enhance the commitment of muscular mesenchymal stem cells to adipogenesis, which may contribute to enhance marbling deposition at the finishing phase. As such the objective of this study was designed the evaluate the impact nutrient supplementation during the pre-weaning stage of beef calves has on intramuscular adipogenic determination. Thirty-four female calves were assigned to two experimental treatments: Control (CON, n = 17), where calves received only a mineral mixture as a supplement; Supplemented (SUP, n = 17), where calves received an energy-protein supplement (0.5 kg·100 kg of BW-1·day-1; the same mineral mixture was used in the CON supplement). Calves were supplemented from 100 to 250 days of age. Once animals reached 250 days of age, skeletal muscle tissue samples were biopsied from the 10th and 11th rib and used for identification of mesenchymal stem cells and preadipocytes through immunofluorescence, mRNA and protein abundance of adipo-fibrogenic markers, and abundance of candidate miRNA controlling expression of adipogenic commitment. Data were analyzed considering the completely randomized design of the experiment. Heifers were randomized across paddocks, and paddocks were randomized across treatments. The PROC MIXED of SAS 9.2 was used for all analyses. The average daily gain (P = 0.08) and final body weight (P = 0.07) tended to be greater in SUP than CON calves. The number of fibro-adipogenic progenitor cells (P = 0.69) did not differ between treatments, while a greater number of intramuscular pre-adipocytes were observed in SUP than CON calves (P = 0.01). The expression of miRNA-4429 (P = 0.20) did not differ between treatments, while the expression of miRNA-129-5p (P = 0.09) and miRNA-129-2-3p (P = 0.05) tended to be greater in CON than SUP calves. Our findings demonstrate that energy-protein supplementation from 100 to 250 days of age is capable to enhance intramuscular adipogenesis in beef cattle. Moreover, our results suggest that regulation of adipogenic commitment occurs by post-transcriptional regulation through miR-129 family.

Single-Cell RNA Sequencing of Coronary Perivascular Adipose Tissue From End-Stage Heart Failure Patients Identifies SPP1+ Macrophage Subpopulation as a Target for Alleviating Fibrosis.

Perivascular adipose tissue (PVAT) is vital for vascular homeostasis, and PVAT dysfunction is associated with increased atherosclerotic plaque burden. But the mechanisms underlining coronary PVAT dysfunction in coronary atherosclerosis remain elusive. We performed single-cell RNA sequencing of the stromal vascular fraction of coronary PVAT from 3 groups of heart transplant recipients with end-stage heart failure, including 3 patients with nonobstructive coronary atherosclerosis, 3 patients with obstructive coronary artery atherosclerosis, and 4 nonatherosclerosis control subjects. Bioinformatics was used to annotate the cellular populations, depict the cellular developmental trajectories and interactions, and explore the differences among 3 groups of coronary PVAT at the cellular and molecular levels. Pathological staining, quantitative real-time polymerase chain reaction, and in vitro studies were performed to validate the key findings. Ten cell types were identified among 67 936 cells from human coronary PVAT. Several cellular subpopulations, including SPP1+ (secreted phosphoprotein 1) macrophages and profibrotic fibroadipogenic progenitor cells, were accumulated in PVAT surrounding atherosclerotic coronary arteries compared with nonatherosclerosis coronary arteries. The fibrosis percentage was increased in PVAT surrounding atherosclerotic coronary arteries, and it was positively associated with the grade of coronary artery stenosis. Cellular interaction analysis suggested OPN (osteopontin) secreted by SPP1+ macrophages interacted with CD44 (cluster of differentiation 44)/integrin on fibroadipogenic progenitor cells. Strikingly, correlation analyses uncovered that higher level of SPP1 in PVAT correlates with a more severe fibrosis degree and a higher coronary stenosis grade. In vitro studies showed that conditioned medium from atherosclerotic coronary PVAT promoted the migration and proliferation of fibroadipogenic progenitor cells, while such effect was prevented by blocking CD44 or integrin. SPP1+ macrophages accumulated in the PVAT surrounding atherosclerotic coronary arteries, and they promoted the migration and proliferation of fibroadipogenic progenitor cells via OPN-CD44/integrin interaction and thus aggravated the fibrosis of coronary PVAT, which was positively correlated to the coronary stenosis burden. Therefore, SPP1+ macrophages in coronary PVAT may participate in the progression of coronary atherosclerosis.

FSHD muscle shows perturbation in fibroadipogenic progenitor cells, mitochondrial function and alternative splicing independently of inflammation.

Facioscapulohumeral muscular dystrophy (FSHD) is a prevalent, incurable myopathy. FSHD is highly heterogeneous, with patients following a variety of clinical trajectories, complicating clinical trials. Skeletal muscle in FSHD undergoes fibrosis and fatty replacement that can be accelerated by inflammation, adding to heterogeneity. Well controlled molecular studies are thus essential to both categorize FSHD patients into distinct subtypes and understand pathomechanisms. Here, we further analyzed RNA-sequencing data from 24 FSHD patients, each of whom donated a biopsy from both a non-inflamed (TIRM-) and inflamed (TIRM+) muscle, and 15 FSHD patients who donated peripheral blood mononucleated cells (PBMCs), alongside non-affected control individuals. Differential gene expression analysis identified suppression of mitochondrial biogenesis and up-regulation of fibroadipogenic progenitor (FAP) gene expression in FSHD muscle, which was particularly marked on inflamed samples. PBMCs demonstrated suppression of antigen presentation in FSHD. Gene expression deconvolution revealed FAP expansion as a consistent feature of FSHD muscle, via meta-analysis of 7 independent transcriptomic datasets. Clustering of muscle biopsies separated patients in an unbiased manner into clinically mild and severe subtypes, independently of known disease modifiers (age, sex, D4Z4 repeat length). Lastly, the first genome-wide analysis of alternative splicing in FSHD muscle revealed perturbation of autophagy, BMP2 and HMGB1 signalling. Overall, our findings reveal molecular subtypes of FSHD with clinical relevance and identify novel pathomechanisms for this highly heterogeneous condition.

Single-Nuclei RNA-Sequencing of the Gastrocnemius Muscle in Peripheral Artery Disease.

Lower extremity peripheral artery disease (PAD) is a growing epidemic with limited effective treatment options. Here, we provide a single-nuclei atlas of PAD limb muscle to facilitate a better understanding of the composition of cells and transcriptional differences that comprise the diseased limb muscle. We obtained gastrocnemius muscle specimens from 20 patients with PAD and 12 non-PAD controls. Nuclei were isolated and single-nuclei RNA-sequencing was performed. The composition of nuclei was characterized by iterative clustering via principal component analysis, differential expression analysis, and the use of known marker genes. Bioinformatics analysis was performed to determine differences in gene expression between PAD and non-PAD nuclei, as well as subsequent analysis of intercellular signaling networks. Additional histological analyses of muscle specimens accompany the single-nuclei RNA-sequencing atlas. Single-nuclei RNA-sequencing analysis indicated a fiber type shift with patients with PAD having fewer type I (slow/oxidative) and more type II (fast/glycolytic) myonuclei compared with non-PAD, which was confirmed using immunostaining of muscle specimens. Myonuclei from PAD displayed global upregulation of genes involved in stress response, autophagy, hypoxia, and atrophy. Subclustering of myonuclei also identified populations that were unique to PAD muscle characterized by metabolic dysregulation. PAD muscles also displayed unique transcriptional profiles and increased diversity of transcriptomes in muscle stem cells, regenerating myonuclei, and fibro-adipogenic progenitor cells. Analysis of intercellular communication networks revealed fibro-adipogenic progenitors as a major signaling hub in PAD muscle, as well as deficiencies in angiogenic and bone morphogenetic protein signaling which may contribute to poor limb function in PAD. This reference single-nuclei RNA-sequencing atlas provides a comprehensive analysis of the cell composition, transcriptional signature, and intercellular communication pathways that are altered in the PAD condition.

Regulatory T cells require IL6 receptor alpha signaling to control skeletal muscle function and regeneration

Muscle-residing regulatory Tcells (Tregs) control local tissue integrity and function. However, the molecularinterface connecting Treg-based regulation with muscle function and regeneration remains largely unexplored. Here, we show that exercise fosters a stable induction of highly functional muscle-residing Tregs with increased expression of amphiregulin (Areg), EGFR, and ST2. Mechanistically, we find that mice lacking IL6Rα on Tcells (TKO) harbor significant reductions in muscle Treg functionality and satellite and fibro-adipogenic progenitor cells, which are required for muscle regeneration. Using exercise and sarcopenia models, IL6Rα TKO mice demonstrate deficits in Tregs, their functional maturation, and a more pronounced decline in muscle mass. Muscle injury models indicate that IL6Rα TKO mice have significant disabilities in muscle regeneration. Treg gain of function restores impaired muscle repair in IL6Rα TKO mice. Of note, pharmacological IL6R blockade in WT mice phenocopies deficits in muscle function identified in IL6Rα TKO mice, thereby highlighting the clinical implications of the findings.

Decoding the transcriptome of Duchenne muscular dystrophy to the single nuclei level reveals clinical-genetic correlations

Duchenne muscular dystrophy is a genetic disease produced by mutations in the dystrophin gene characterized by early onset muscle weakness leading to severe and irreversible disability. The cellular and molecular consequences of the lack of dystrophin in humans are only partially known, which is crucial for the development of new therapies aiming to slow or stop the progression of the disease. Here we have analyzed quadriceps muscle biopsies of seven DMD patients aged 2 to 4 years old and five age and gender matched controls using single nuclei RNA sequencing (snRNAseq) and correlated the results obtained with clinical data. SnRNAseq identified significant differences in the proportion of cell population present in the muscle samples, including an increase in the number of regenerative fibers, satellite cells, and fibro-adipogenic progenitor cells (FAPs) and a decrease in the number of slow fibers and smooth muscle cells. Muscle samples from the younger patients with stable mild weakness were characterized by an increase in regenerative fibers, while older patients with moderate and progressive weakness were characterized by loss of muscle fibers and an increase in FAPs. An analysis of the gene expression profile in muscle fibers identified a strong regenerative signature in DMD samples characterized by the upregulation of genes involved in myogenesis and muscle hypertrophy. In the case of FAPs, we observed upregulation of genes involved in the extracellular matrix regeneration but also several signaling pathways. Indeed, further analysis of the potential intercellular communication profile showed a dysregulation of the communication profile in DMD samples identifying FAPs as a key regulator of cell signaling in DMD muscle samples. In conclusion, our study has identified significant differences at the cellular and molecular levels in the different cell populations present in skeletal muscle samples of patients with DMD compared to controls.

Non-ammoniagenic proliferation and differentiation media for cultivated adipose tissue.

Ammonia (Amm), and its aqueous solved state, ammonium, which is produced from glutamine (Gln) metabolism, is a known inhibitor of stem cell proliferation in vitro. In the context of cultivated beef, primary bovine fibro-adipogenic progenitor cells (FAPs) need to be grown and differentiated for several weeks in vitro for the production of cultivated fat. In this study, the ammonium sensitivity of these cells was investigated by introducing ammonium chloride, which was found to inhibit their proliferation when above 5mM and their adipogenic differentiation when above 2mM. Novel serum-free proliferation and differentiation media were hence developed with the aim to suppress Amm production during expansion and adipogenesis. Glutamine substitutes, such as a-ketoglutarate (aKG), glutamate (Glt) and pyruvate (Pyr) were investigated. It was found that aKG based proliferation medium (PM) was the most effective in promoting and maintaining FAPs growth over several passages while the specific Amm production rate was reduced more than 5-fold. In terms of differentiation capacity, the substitution of glucose (Gluc) and Gln with galactose (Gal) and Pyr was shown to be the most effective in promoting FAPs differentiation into mature adipocytes, resulting in over 2-fold increase of fat volume per cell, while suppressing Amm production. Our findings suggest that FAPs do not require Gln as an essential nutrient but, on the contrary, possess all the necessary metabolic pathways to proliferate and subsequently differentiate in a Gln-free medium, resulting in decreased Amm production rates and seemingly synthesising glutamine de novo. These findings are important for prolonging the lifespan of culture medium, allowing for reduced costs and process interventions.

Nutrient supplementation of beef female calves at pre-weaning enhances the commitment of fibro-adipogenic progenitor cells to preadipocytes

We aimed to evaluate the impact of nutrient supplementation of beef female calves at pre-weaning on adipogenic determination. Thirty-four female calves were assigned to two experimental treatments: Control (CON, n = 17), where animals were supplemented only with mineral mixture; Supplemented (SUP, n = 17), where animals received energy-protein supplement containing minerals (5 g/kg of BW per day) of their body weight. Animals were supplemented from 100 to 250 days of age, and muscle samples were biopsied at the end of the supplementation period. Regarding the performance variables, there were no differences between treatments for initial body weight (P = 0.75). The final body weight (P = 0.07), average daily gain (P = 0.07), rib eye area (P = 0.03), and rib fat thickness (P = 0.08) were greater in SUP female calves compared with CON treatment. The number of fibro-adipogenic progenitor cells (P = 0.69) did not differ between treatments, while a greater number of intramuscular pre-adipocytes were observed in SUP than CON female calves (P = 0.01). The expression of miRNA-4429 (P = 0.20) did not differ between treatments, while the expression of miRNA-129-5p (P = 0.09) and miRNA-129-2-3p (P = 0.05) was greater in CON than SUP female calves. Our results suggest that nutrient supplementation at early postnatal stages of development enhances the commitment of fibro-adipogenic progenitor cells into the adipogenic lineages allowing to an increase in intramuscular fat deposition potential of the animals later in life.

Role of fibro-adipogenic progenitors in skeletal muscle aging

Maintaining muscle mass is of paramount importance from a clinical perspective as it supports the flexibility, strength, and essential daily tasks that the body requires. Furthermore, muscle plays a role in regulating the body’s metabolic system. Unfortunately, aging can lead to a loss of muscle mass, which can reduce personal independence and quality of life while increasing the risk of developing disease. Fibro-adipogenic progenitor cells (FAPs) are muscle-resident progenitor cells that are essential for maintaining skeletal muscle fiber size and muscle regeneration. These vital FAP functions are mediated by a complex secretome that interacts in a paracrine manner to promote the division and differentiation of muscle satellite cells. Dysregulated differentiation of FAPs can lead to fibrosis, fatty infiltration, muscle atrophy, and poor muscle regeneration. In this article, we review what is currently known about how FAPs function in aging muscle and how they may prevent the onset of muscle wasting and degeneration. Finally, we discuss how FAPs represent a population of cells that can be used as therapeutic targets to improve the health of skeletal and muscle tissues as they age.

Traumatic muscle injury promotes aberrant fibro-adipogenic progenitor cell population, leading to fibrosis and impaired muscle stem cell function

Skeletal muscle has an exceptional regenerative capacity that relies on the temporal activation and regulation of immune cells, fibro-adipogenic progenitors (FAPs), and muscle stem cells (MuSCs). However, following traumatic injury to skeletal muscle, such as in extensive muscle deficits caused by injury or surgery, also known as volumetric muscle loss (VML), this coordinated regenerative response is diminished, leading to fibrosis, inflammation, and chronic functional deficiencies. The goal of this study was to compare the temporal response and phenotype of key cell populations, including macrophages, FAPs, and MuSCs following critical and sub-critical VML injuries in the mouse quadriceps. We hypothesize that traumatic injury, specifically VML, drives cell phenotypic variability amongst these cell populations. Unilateral VML injuries were used to quantify cell type distribution via flow cytometry at 1, 3, and 7 days post-VML (pVML, n=4 per injury size and time point). At day 7 pVML, anti-inflammatory M2 macrophages, FAPs, and MuSCs were all present in significantly elevated numbers per tissue volume in critical (3-mm) injuries compared to sub-critical (2-mm) injuries (p<0.05). Using multi-dimensional flow cytometry analysis, a distinct sub-population of FAPs was identified in critically sized VML injuries which had larger cell content and highly expressed the β1-integrin cell-surface marker. FAPs from quadriceps at day 7 pVML (VML-FAPs) were purified using fluorescence-activated cell sorting and cultured in vitro. VML-FAPs exhibited increased proliferation, increased cell area, and showed elevated levels of β1-integrin expression. In co-culture experiments, VML-FAPs were also shown to significantly decrease the myogenic capacity of primary MuSCs by quantification of myotube formation (p<0.05). When cultured with the anti-inflammatory cytokine TGF-β1, VML-FAPs more readily differentiated down a fibroblastic lineage. Collectively, these results indicate that following traumatic injury, namely VML, there is an aberrant shift in the FAPs cell population, possibly driven by the accumulation of M2 macrophages, towards a lineage which is persistent, pro-fibrotic, and anti-myogenic. The identification of this shift in VML-FAPs will be key to the development of pro-regenerative therapeutics for VML, as success of these VML-FAP interventions will rely on the ability to downregulate the fibrotic lineage of FAPs while maintaining their traditional pro-myogenic qualities during regeneration. Furthermore, this study also provokes investigation of differing FAP phenotypes in other traumatic muscle injuries to identify possible trends or incongruencies that may exist. Funding: This work was supported by the National Institute of Health under award number R01AG072309, R01AR071708, and Department of Defense under award number W81XWH-20-1-0336. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Fibroadipogenic progenitor-derived Gdf10 regulates neuromuscular junction and fat content in tongue muscles

Obstructive sleep apnea (OSA) is the most prevalent sleep disorder globally 4.5% in men and 2.5% in women. OSA increases the risk of more severe diseases such as heart attack, high blood pressure, type 2 diabetes, obesity, mild cognitive impairment, and depression. The known craniofacial anatomical changes leading to OSA are relaxed muscles and/or enlarged soft tissues by fat infiltration in the soft palate, pharynx, and particularly tongue, which blocks the upper airway during sleep. Although obesity is one of the main causes to induce OSA, the underlying mechanism of those changes has not been researched. The objective of our study is to understand the mechanism leading to those craniofacial changes that generate foundations to develop potential therapeutics for OSA. We hypothesize that high-fat diet-induced obesity regulates the fibroadipogenic progenitors, which are critical for neuromuscular interaction and is mainly responsible for increased fat content in skeletal muscles. To test this hypothesis, we used a mouse model fed with a high-fat diet to induce obesity or a normal diet (control group) for 6 months. Then we isolated tongue muscles for histology analysis, such as muscle size, neuromuscular junction integrity, and adipocyte content.We found that a high-fat diet leaded adipocyte hypertrophy and reduced intact neuromuscular junction in tongue muscles. To investigate the role of fibroadipogenic progenitors for tongue phenotypes by a high-fat diet, we isolated fibroadipogenic progenitor cells to measure transcript levels of Gdf10, also known as Bmp3b, since fibroadipogenic progenitor-derived Gdf10 has been known to stabilize Schwann cells for neuromuscular integrity in muscles. The role of Gdf10 has been suggested to protect from obesity by limiting adipocyte hypertrophy. Using single-cell RNA sequencing analysis, Gdf10 is specifically expressed in fibroadipogenic progenitors but not adipocyte or Schwann cells. We found that Gdf10 transcript levels are significantly reduced by a high-fat diet in tongue fibroadipose progenitors. Taken together, the reduced Gdf10 of fibroadipogenic progenitors by a high-fat diet reduces neuromuscular integrity but induces fat infiltration in tongue muscles in a paracrine manner. Therefore, targeting to increase Gdf10 expression is a potential therapeutic to reduce OSA by maintaining healthy tongue muscle. NIH R01AR071397 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Single cell analysis reveals satellite cell heterogeneity for proinflammatory chemokine expression

Background: The expression of proinflammatory signals at the site of muscle injury are essential for efficient tissue repair and their dysregulation can lead to inflammatory myopathies. Macrophages, neutrophils, and fibroadipogenic progenitor cells residing in the muscle are significant sources of proinflammatory cytokines and chemokines. However, the inducibility of the myogenic satellite cell population and their contribution to proinflammatory signaling is less understood. Methods: Mouse satellite cells were isolated and exposed to lipopolysaccharide (LPS) to mimic sterile skeletal muscle injury and changes in the expression of proinflammatory genes was examined by RT-qPCR and single cell RNA sequencing. Expression patterns were validated in skeletal muscle injured with cardiotoxin by RT-qPCR and immunofluorescence. Results: Satellite cells in culture were able to express Tnfa, Ccl2, and Il6, within 2h of treatment with LPS. Single cell RNA-Seq revealed seven cell clusters representing the continuum from activation to differentiation. LPS treatment led to a heterogeneous pattern of induction of C-C and C-X-C chemokines (e.g., Ccl2, Ccl5, and Cxcl0) and cytokines (e.g., Tgfb1, Bmp2, Il18, and Il33) associated with innate immune cell recruitment and satellite cell proliferation. One cell cluster was enriched for expression of the antiviral interferon pathway genes under control conditions and LPS treatment. Activation of this pathway in satellite cells was also detectable at the site of cardiotoxin induced muscle injury. Conclusion: These data demonstrate that satellite cells respond to inflammatory signals and secrete chemokines and cytokines. Further, we identified a previously unrecognized subset of satellite cells that may act as sensors for muscle infection or injury using the antiviral interferon pathway.

Cellular interplay in skeletal muscle regeneration and wasting: insights from animal models.

Skeletal muscle wasting, whether related to physiological ageing, muscle disuse or to an underlying chronic disease, is a key determinant to quality of life and mortality. However, cellular basis responsible for increased catabolism in myocytes often remains unclear. Although myocytes represent the vast majority of skeletal muscle cellular population, they are surrounded by numerous cells with various functions. Animal models, mostly rodents, can help to decipher the mechanisms behind this highly dynamic process, by allowing access to every muscle as well as time-course studies. Satellite cells (SCs) play a crucial role in muscle regeneration, within a niche also composed of fibroblasts and vascular and immune cells. Their proliferation and differentiation is altered in several models of muscle wasting such as cancer, chronic kidney disease or chronic obstructive pulmonary disease (COPD). Fibro-adipogenic progenitor cells are also responsible for functional muscle growth and repair and are associated in disease to muscle fibrosis such as in chronic kidney disease. Other cells have recently proven to have direct myogenic potential, such as pericytes. Outside their role in angiogenesis, endothelial cells and pericytes also participate to healthy muscle homoeostasis by promoting SC pool maintenance (so-called myogenesis-angiogenesis coupling). Their role in chronic diseases muscle wasting has been less studied. Immune cells are pivotal for muscle repair after injury: Macrophages undergo a transition from the M1 to the M2 state along with the transition between the inflammatory and resolutive phase of muscle repair. T regulatory lymphocytes promote and regulate this transition and are also able to activate SC proliferation and differentiation. Neural cells such as terminal Schwann cells, motor neurons and kranocytes are notably implicated in age-related sarcopenia. Last, newly identified cells in skeletal muscle, such as telocytes or interstitial tenocytes could play a role in tissular homoeostasis. We also put a special focus on cellular alterations occurring in COPD, a chronic and highly prevalent respiratory disease mainly linked to tobacco smoke exposure, where muscle wasting is strongly associated with increased mortality, and discuss the pros and cons of animal models versus human studies in this context. Finally, we discuss resident cells metabolism and present future promising leads for research, including the use of muscle organoids.

Extracellular matrix content and remodeling markers do not differ in college-aged men classified as higher and lower responders to resistance training.

We determined if skeletal muscle extracellular matrix (ECM) content and remodeling markers adapted with resistance training or were associated with hypertrophic outcomes. Thirty-eight untrained males (21 ± 3 yr) participated in whole body resistance training (10 wk, 2 × weekly). Participants completed testing [ultrasound, peripheral quantitative computed tomography (pQCT)] and donated a vastus lateralis (VL) biopsy 1 wk before training and 72 h following the last training bout. Higher responders (HR, n = 10) and lower responders (LR, n = 10) were stratified based on a composite score considering changes in pQCT-derived mid-thigh cross-sectional area (mCSA), ultrasound-derived VL thickness, and mean fiber cross-sectional area (fCSA). In all participants, training reduced matrix metalloprotease (MMP)-14 protein (P < 0.001) and increased satellite cell abundance (P < 0.001); however, VL fascial thickness, ECM protein content per myofiber, MMP-2/-9 protein content, tissue inhibitor of metalloproteinase (TIMP)-1/-2 protein content, collagen-1/-4 protein content, macrophage abundance, or fibroadipogenic progenitor cell abundance were not altered. Regarding responder analysis, MMP-14 exhibited an interaction (P = 0.007), and post hoc analysis revealed higher protein content in HR versus LR before training (P = 0.026) and a significant decrease from pre to posttraining in HR only (P = 0.002). In summary, basal skeletal muscle ECM markers are minimally affected with 10 wk of resistance training, and these findings could be related to not capturing more dynamic alterations in the assayed markers earlier in training. However, the downregulation in MMP-14 in college-aged men classified as HR is a novel finding and warrants continued investigation, and further research is needed to delineate muscle connective tissue strength attributes between HR and LR.NEW & NOTEWORTHY Although past studies have examined aspects of extracellular matrix remodeling in relation to mechanical overload or resistance training, this study serves to expand our knowledge on a multitude of extracellular matrix markers and whether these markers adapt to resistance training or are associated with differential hypertrophic responses.