Obesity impairs skeletal muscle repair through NID-1 mediated extracellular matrix remodeling by mesenchymal progenitors
Graphical abstract
A. Adult skeletal muscle mesenchymal progenitor fibro-adipogenic potential in regular diet conditions. Undifferentiated multipotent PDGFRα-expressing progenitors (FAPs) are able to generate pre-adipocytes (NID-1 low) and fibrocytes (NID-1 high) committed to become adipocytes and fibroblast-like cells, respectively, under homeostatic and regeneration condition. B. Adult MuSCs myogenic potential under regular diet. During homeostasis, MuSCs are quiescent Pax7-expressing cells. Their role is maintaining the number of skeletal muscle progenitors throughout life. However, certain stimuli, such as muscle injury, launch the muscle regeneration program. Pax7 expression is downregulated at the time MyoD is expressed. Activated myoblasts undergo several cell cycles (Ki67+) to generate abundant number of myoblasts. Following differentiation, myoblasts decrease MyoD expression, and upregulate the commitment marker MyoG. Finally, myocytes fuse to repair or generate new myotubes. C. Adult skeletal muscle mesenchymal progenitor fibro-adipogenic potential after High-Fat Diet (HFD). HFD promotes FAP infiltration into obese mouse skeletal muscle. The surplus of energy stimulates transformation of the NID-1 Low FAP subpopulation into adipocytes and therefore, intramuscular adipose tissue deposition. The overstimulation of low NID-1 expressing FAPs forces their differentiation or trans-differentiation to NID-1 high expression FAPs. In obese mice under homeostatic conditions, most FAPs are expressing high levels of NID-1 which give rise to fibroblast-like cells upon regeneration. This in turn leads to excessive extracellular matrix deposition once the muscle damage is repaired. D. Obese mice adipose tissue secretome enhances FAP's fibrogenic fate during regeneration. E. Adult MuSCs myogenic potential after HFD. The increase in NID-1 secreting-FAPs alters the MuSCs niche. In the steady state, NID-1 may maintain the quiescence of Pax7-expressing MuSCs, giving rise to a higher number of skeletal muscle progenitors. However, upon regeneration the persistence of NID-1 may blunt MuSCs clonal expansion and favor myoblast differentiation.
Introduction
The percentage of obese individuals has exponentially increased for over 50 years. According to the World Health Organization, 39% of the world population are overweight with a body mass index (BMI) over 25 kg/m2 whereas 13% are obese, with a BMI higher than 30 kg/m2. Excessive adipose tissue expansion is associated with chronic disorders such as insulin resistance, dyslipidemia, cardiovascular complications, premature senescence, and peripheral tissue lipotoxicity [15, 57].
Long-term obesogenic conditions lead to qualitative and quantitative muscle changes [74]. While Leptin has been shown to regulate skeletal muscle metabolism [14, 72], the cellular and additional molecular mechanisms underlying the systemic impact of adipose tissue on skeletal muscles remain poorly understood. Skeletal muscle integrity is fundamentally maintained by muscle stem cells, also known as muscle satellite cells (MuSCs). In adults, muscle stem cells constitute a pool of quiescent Pax7-expressing cells. Upon muscle damage, muscle satellite cells are activated, co-express Pax7 and MyoD and proliferate, a fundamental process to amplify the pool of myogenic progenitor cells (or myoblasts). While a subset of activated muscle progenitors will downregulate MyoD to restore the pool of muscle stem cells, the majority will activate Myogenin expression, and fuse to regenerate functional multinucleated muscle myofibers. Muscle stem cell status and function are tightly regulated by their niche. Skeletal muscle satellite cells are located between the myofiber sarcolemma and the basal lamina, a specialized extracellular matrix (ECM) that separates muscle stem cells from stroma [77]. The basal lamina is a hub for varied factors that include hormones, cytokines and structurally active ECM components secreted by the muscle fiber, satellite cells and endomysial cell populations [16].
Fibro-adipogenic progenitors (FAPs), also known as mesenchymal stromal cells or mesenchymal progenitors, are endomysial resident cells that contribute to muscle stem cell maintenance and repair [75]. Following muscle damage, FAPs are activated and proliferate, invading the damaged muscle and secreting trophic myogenic differentiation factors. For efficient regeneration, FAPs undergo a cell death program to restore their homeostatic number, a process required to resolve muscle repair [35, 43, 66]. However, in pathological conditions, FAPs fate and function is dysregulated, giving rise to deleterious intramuscular fatty-fibrotic scars, a hallmark of impaired muscle regeneration [35, 73].
Nidogens/Entactins are a family of highly conserved proteoglycans composed by Nidogen-1 (NID-1) and Nidogen-2 (NID-2). Nidogens are mainly secreted by fibroblasts [19, 52] and their function was classically associated with wound healing and scarring due to their role as ECM organizers linking collagens and laminins [6]. In skeletal muscle, NID-1 is a ubiquitous component of the basal lamina, while NID-2 is involved in neuromuscular junction development [20]. NID-2 can replace NID-1 structural function in NID-1 mutant mice [48], whereas double mutants for NID-1 and -2 display limb development abnormalities [12] and perinatal lethality [3]. In skeletal muscle, loss of MuSCs quiescence and differentiation is associated with a reduction of NID-1 expression [50], while its overexpression promotes myotube stability in vitro [21, 28].
The effect of obesity on skeletal muscle has been previously linked with adipose tissue endocrine function [37]. Adipose tissue dysfunctionality and comorbidities are in part due to excessive connective tissue accumulation or fibrosis [44]. Here, we identify NID-1, a component of basal laminae, overexpressed in adipose tissue and skeletal muscle during obesity. We show that NID-1 is overexpressed by muscle FAPs in HFD-fed mice. Functionally, we demonstrate that NID-1 impairs myogenesis in cultured human myoblasts in vitro and in mouse skeletal muscle regeneration in vivo, linked with increased intra-muscular fibrosis. Finally, we show that intramuscular overexpression of NID-1 promotes FAPs fibrogenic fate at the expense of adipogenic differentiation. Altogether, we propose that obesity has a systemic impact on muscle homeostasis via NID-1 mediated remodeling of the skeletal muscle basal lamina by mesenchymal progenitor cells.
Section snippets
Human adipose tissue-secreted extracellular matrix components impair myogenesis
To determine whether adipose tissue has a direct impact on human skeletal muscle cells, we generated subcutaneous adipose tissue conditioned media (hSATCM) from twenty upper forelimb human adipose tissue biopsies. We tested these hSATCM by direct exposure of cultured human myoblasts (Sorted for CD56 expression) isolated from healthy adult donors. After 72h of culture, we analyzed the consequence of hSATCM exposure for the expression of Pax7 and MyoD by immunostaining (Fig. 1A). Exposure to
Discussion
Currently, the knowledge of molecular and cellular mechanisms by which adipose tissue drives tissue fibrosis is scarce [18]. Here we provide evidence supporting a model for NID-1 as a mediator of obesity leading to skeletal muscle fibrosis. We found that NID-1, previously described as highly secreted by subcutaneous adipose tissue during obesity in humans and mice [17, 64] is also expressed in excess by the skeletal muscle of obese patients and mice undergoing HFD, possibly as a consequence of
Author contribution
Experimental design: SPD, ZK, PL, GD, FJA, FR; Performed experiments: SPD, ZK, MB, FA, MP, BP, YBA; Analyzed data: SPD, ZK, MB, PL, FR; Writing: SPD, MB, PL, FR; Project supervision: PL, FR; Funding acquisition: GD, FJA, FR
Supplemental Figure 1
A. Experimental scheme for rNID-1-Anti NID-1 competitive treatments of MuSCs in vitro.
B. Top: MuSCs cultures were treated with 0µM of rNID-1 and 0 ng/ml of Anti NID-1 (Vehicle, black), 22µg/ml Anti NID-1 (Light orange), 200 µM rNID-1 (orange), or 200 µM rNID-1 and 22µg/ml Anti NID-1 (dark orange) and stained for Pax7 (green) and Ki67 (red). Nuclei are stained by Hoechst (blue). Lower panels represent higher magnification of the areas surrounded by a white dotted line in top images. Scale bars:
Supplemental Figure 2
A. Experimental scheme of CD56+ human myoblast culture.
B. Left, western blot analysis of Nidogen-1, MyoD and Myogenin expression by CD56+ myoblasts for 10 day of culture. Right, quantification of Nidogen-1, MyoD and Myogenin expression levels in densitometric units (D.U.). Actin immunoassay was used as a loading control. The results are presented as mean ± SEM. Every dot is the average of independent experiments (n=3).
Supplemental Figure 3
A. Left: TA sections were stained for Type IIa (green) and Type IIb (red) myosins. The absence of color represents fiber type I. Nuclei are stained by Hoechst (blue). Scale bars: 50 µm. Right: Graph showing the percentage of muscle fiber type IIa, IIb and type I in CD- (black) and HFD- (orange) fed mice. The results are presented as mean ± SEM. Dots are individual values of independent animals (n=5 for each condition).
Supplemental Figure 4
A. Left: Human skeletal muscles were stained for Nidogen-1 (NID-1, red). Nuclei are stained by Hoechst (blue). Bottom panels represent higher magnification of the areas surrounded by a white dotted line in top images. Scale bar: 50µm. Right: Quantification of NID-1 deposition per area of human skeletal muscle sections of individuals with BMI<25m2/kg (black) and >25m2/kg (red). The results are presented as mean ± SEM. Dots are individual values of independent donors (n=11 for each condition).
B.
Supplemental Figure 5
Principal Component analysis (PCA) of HFD- (n=3) and CD-fed mice MuSC RNA-seq (n=3).
Supplemental Figure 6
A. Gene expression analysis of Nid-1, Pdgfrα, Pax7, MyoD and Myogenin at 7 days post-injury of CD- (black) and HFD- (orange) fed mice TA muscles. The results are presented as mean ± SEM relative to β-Actin. Dots are individual values of independent animals (n=5 for each condition).
B. Left: Immunodetection of Fibronectin (red) in TA muscle sections of CD and HFD -fed mice at 30 days post-injury. Nuclei were stained with Hoechst (blue). Bottom panels represent higher magnification of the areas
Supplemental Figure 7
A. TA sections were stained for PDGFRα (red) on infected muscle areas (GFP, green) 7 days post-injury. Nuclei are stained by Hoechst (blue). Rightmost panels represent higher magnification of the areas surrounded by a white dotted line in Merge images. Scale bars: 100 µm.
B. Left: Western blot immunoassay against Nidogen-1 7 days-post injury on protein lysates isolated from Control and NID-1 TA muscles. Right: Quantification of NID-1 expression in densitometric units (D.U.). Actin immunoassay
Supplemental Figure 8
A. Experimental scheme of MuSCs treatments with conditioned media from FAPs infected with NID-1-GFP viral particles (FAPNID-1) or Control-GFP viral particles (FAPControl).
B. Left: MuSCs cultures were exposed to FAPNID-1 and FAPControl and stained for Pax7 (green) and Ki67 (red) by immunofluorescence. Nuclei were stained by Hoechst (blue). Bottom panels represent higher magnification of the areas surrounded by a white dotted line in top images. Scale bars: 50 µm. Right: Quantification of Pax7+
STAR METHODS
KEY RESOURCES TABLE
See key resources table
CONTACT FOR REAGENT AND RESOURCE SHARING
For further information and requesting of reagents and resources please contact the lead contact, Frederic Relaix ([email protected])
METHOD DETAILS
Human samples
Supra-deltoid (Subcutaneous) adipose tissues from individuals without previously described chronic diseases undergoing routine muscle evaluation were collected in DMEM high glucose (Thermo Fisher Scientific. Ref:31966021) supplemented with 1 %
Declaration of Interests
The authors declare no competing interests.
Acknowledgements
This work was supported by funding to FR from: Agence Nationale pour la Recherche (ANR) grant Satnet (ANR-15-CE13-0011-01), RHU CARMMA (ANR-15-RHUS-0003), Labex REVIVE (ANR-10-LABX-73), Association Française contre les Myopathies (AFM) via TRANSLAMUSCLE (PROJECT 19507 and 22946). GD and FJA work was supported by RHU CARMMA (ANR-15-RHUS-0003) funding.
REFERENCES (77)
- et al.
Impaired wound healing in mice lacking the basement membrane protein nidogen 1
Matrix Biol
(2010) - et al.
Fibronectin regulates Wnt7a signaling and satellite cell expansion
Cell Stem Cell
(2013) - et al.
Loss of nidogen-1 and -2 results in syndactyly and changes in limb development
J. Biol. Chem.
(2006) - et al.
Short-term effects of leptin on skeletal muscle protein metabolism in the rat
J. Nutr. Biochem.
(2000) - et al.
The epidemiology of obesity
Metabolism
(2019) - et al.
Changes in adipose tissue gene expression with energy-restricted diets in obese women
Am. J. Clin. Nutr.
(2005) - et al.
Skin fibroblasts are the only source of nidogen during early basal lamina formation in vitro
J. Invest. Dermatol.
(1995) - et al.
Niche Cadherins Control the Quiescence-to-Activation Transition in Muscle Stem Cells
Cell Rep
(2017) - et al.
Niche Cadherins Control the Quiescence-to-Activation Transition in Muscle Stem Cells
Cell Rep
(2017) - et al.
Intramuscular lipid content is increased in obesity and decreased by weight loss
Metabolism
(2000)
Impaired primary mouse myotube formation on crosslinked type i collagen films is enhanced by laminin and entactin
Acta Biomater
Discovering Cell-Adhesion Peptides in Tissue Engineering: Beyond RGD
Trends Biotechnol
Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCT method
Methods
A PDGFRα-Mediated Switch toward CD9high Adipocyte Progenitors Controls Obesity-Induced Adipose Tissue Fibrosis
Cell Metab
Differential expression of entactin-1/nidogen-1 and entactin-2/nidogen-2 in myogenic differentiation
Differentiation
Differential expression of entactin-1/nidogen-1 and entactin-2/nidogen-2 in myogenic differentiation
Differentiation
Lack of nidogen-1 and -2 prevents basement membrane assembly in skin-organotypic coculture
J. Invest. Dermatol.
Temporal Dynamics and Heterogeneity of Cell Populations during Skeletal Muscle Regeneration
IScience
Efficient and Rapid Induction of a Chronic Myelogenous Leukemia-Like Myeloproliferative Disease in Mice Receiving P210 bcr/abl-Transduced Bone Marrow
Blood
The basement membrane/basal lamina of skeletal muscle
J. Biol. Chem.
Mechanical stretch induces activation of skeletal muscle satellite cells in vitro
Exp. Cell Res.
The Galaxy platform for accessible, reproducible and collaborative biomedical analyses: 2016 update
Nucleic Acids Res.
Implication of the satellite cell in dystrophic muscle fibrosis: A self-perpetuating mechanism of collagen overproduction
Am. J. Physiol. - Cell Physiol.
Compound Genetic Ablation of Nidogen 1 and 2 Causes Basement Membrane Defects and Perinatal Lethality in Mice
Mol. Cell. Biol.
Reciprocal signalling by Notch/Collagen V/CALCR retains muscle stem cells in their niche
Nature
Nidogen-1 Contributes to the Interaction Network Involved in Pro-B Cell Retention in the Peri-sinusoidal Hematopoietic Stem Cell Niche
Cell Rep
Expression of Cd34 and Myf5 Defines the Majority of Quiescent Adult Skeletal Muscle Satellite Cells
J. Cell Biol.
Cellular dynamics in the muscle satellite cell niche
EMBO Rep
Fibro–Adipogenic Progenitors Cross-Talk in Skeletal Muscle: The Social Network
Front. Physiol.
Manipulation of FASTQ data with Galaxy
Bioinformatics
Fibro-adipogenic remodeling of the diaphragm in obesity-associated respiratory dysfunction
Diabetes
Skeletal Muscle Extracellular Matrix – What Do We Know About Its Composition, Regulation, and Physiological Roles?
A Narrative Review. Front. Physiol.
Fat fibrosis: friend or foe?
JCI Insight
A synaptic nidogen: developmental regulation and role of nidogen-2 at the neuromuscular junction
Neural Dev
Entactin promotes adhesion and long-term maintenance of cultured regenerated skeletal myotubes
J. Cell. Physiol.
Total adiponectin in overweight and obese subjects and its response to visceral fat loss
BMC Endocr. Disord.
Dual effects of obesity on satellite cells and muscle regeneration
Physiol. Rep.
High-Dimensional Single-Cell Cartography Reveals Novel Skeletal Muscle-Resident Cell Populations
Mol. Cell
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