# Fibro-adipogenic progenitors in physiological adipogenesis and intermuscular adipose tissue remodeling

Marcelo Flores-Opazo, Daniel Kopinke, Françoise Helmbacher, Rodrigo Fernández- Verdejo, Mauro Tuñón-Suárez, Gordon S Lynch, Osvaldo Contreras

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## ► To cite this version:

Marcelo Flores-Opazo, Daniel Kopinke, Françoise Helmbacher, Rodrigo Fernández- Verdejo, Mauro Tuñón-Suárez, et al.. Fibro-adipogenic progenitors in physiological adipogenesis and inter-muscular adipose tissue remodeling. Molecular Aspects of Medicine, 2024, 97, pp.101277. 10.1016/j.mam.2024.101277. hal-04587011

HAL Id: hal-04587011

https://hal.science/hal-04587011

Submitted on 24 May 2024

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Review

Fibro-adipogenic progenitors in physiological adipogenesis and
intermuscular adipose tissue remodeling

Marcelo Flores-Opazo<sup>a, **</sup>, Daniel Kopinke<sup>b, c</sup>, Françoise Helmbacher<sup>d</sup>, Rodrigo Fernández-Verdejo<sup>e, f</sup>, Mauro Tuñón-Suárez<sup>f</sup>, Gordon S. Lynch<sup>g</sup>, Osvaldo Contreras<sup>h, i, *, 1</sup>

ARTICLE INFO

Keywords:

Skeletal muscle
FAPs
Adipocytes
Adipogenesis
Obesity
IMAT
Metabolism

A B S T R A C T

Excessive accumulation of intermuscular adipose tissue (IMAT) is a common pathological feature in various metabolic and health conditions and can cause muscle atrophy, reduced function, inflammation, insulin resistance, cardiovascular issues, and unhealthy aging. Although IMAT results from fat accumulation in muscle, the mechanisms underlying its onset, development, cellular components, and functions remain unclear. IMAT levels are influenced by several factors, such as changes in the tissue environment, muscle type and origin, extent and duration of trauma, and persistent activation of fibro-adipogenic progenitors (FAPs). FAPs are a diverse and transcriptionally heterogeneous population of stromal cells essential for tissue maintenance, neuromuscular stability, and tissue regeneration. However, in cases of chronic inflammation and pathological conditions, FAPs expand and differentiate into adipocytes, resulting in the development of abnormal and ectopic IMAT. This review discusses the role of FAPs in adipogenesis and how they remodel IMAT. It highlights evidence supporting FAPs and FAP-derived adipocytes as constituents of IMAT, emphasizing their significance in adipose tissue maintenance and development, as well as their involvement in metabolic disorders, chronic pathologies and diseases. We also investigated the intricate molecular pathways and cell interactions governing FAP behavior. adipogenesis, and IMAT accumulation in chronic diseases and muscle deconditioning. Finally, we hypothesize that impaired cellular metabolic flexibility in dysfunctional muscles impacts FAPs, leading to IMAT. A deeper understanding of the biology of IMAT accumulation and the mechanisms regulating FAP behavior and fate are essential for the development of new therapeutic strategies for several debilitating conditions.

1. Introduction

Normal physical activity and metabolic homeostasis rely on optimal skeletal muscle health and function. Intermuscular adipose tissue (IMAT) is an ectopic fat depot that grows between muscle fibers and

surrounding muscle groups. IMAT is a common feature of various chronic diseases in which muscle mass is progressively lost and replaced by adipose and fibrotic tissue. IMAT can be detected in individuals with chronic muscle disease and wasting, inflammatory muscle disorders, sarcopenia (Therkelsen et al. 2013, 2016; Addison et al., 2014), systemic and local immunometabolic and hormonal disturbances, metabolic

<footnote><sup>a</sup> Institute of Health Sciences, Universidad de O'Higgins, Rancagua, Chile</footnote>

<footnote><sup>b</sup> Department of Pharmacology and Therapeutics, University of Florida, Gainesville, 32610, FL, USA</footnote>

<footnote><sup>c</sup> Myology Institute, University of Florida College of Medicine, Gainesville, FL, USA</footnote>

<footnote><sup>d</sup> Aix Marseille Univ, CNRS, IBDM, UMR 7288, Marseille, France</footnote>

<footnote><sup>e</sup> Pennington Biomedical Research Center, Louisiana State University, Baton Rouge, LA, USA</footnote>

<footnote><sup>f</sup> Laboratorio de Fisiología Del Ejercicio y Metabolismo (LABFEM), Escuela de Kinesiología, Facultad de Medicina, Universidad Finis Terrae, Chile</footnote>

<footnote><sup>g</sup> Centre for Muscle Research, Department of Anatomy and Physiology, The University of Melbourne, Melbourne, Victoria, Parkville 3010, Australia</footnote>

<footnote><sup>h</sup> Developmental and Regenerative Biology Division, Victor Chang Cardiac Research Institute, Darlinghurst, NSW, 2010, Australia</footnote>

<footnote><sup>i</sup> School of Clinical Medicine, UNSW Sydney, Kensington, NSW 2052, Australia</footnote>

<footnote>* Corresponding author. Developmental and Regenerative Biology Division, Victor Chang Cardiac Research Institute, Darlinghurst, NSW, 2010, Australia.</footnote>

<footnote>** Corresponding author.</footnote>

<footnote>E-mail addresses: marcelo.flores@uoh.cl (M. Flores-Opazo), dkopinke@ufl.edu (D. Kopinke), francoise.helmbacher@univ-amu.fr (F. Helmbacher), rodrigo.fernandez@pbrc.edu (R. Fernández-Verdejo), maurotunonsuarez@gmail.com (M. Tuñón-Suárez), gsl@unimelb.edu.au (G.S. Lynch), o.contreras@victorchang.edu.au (O. Contreras).</footnote>

<footnote n="1">¹ Lead author.</footnote>
Review

Fibro-adipogenic progenitors in physiological adipogenesis and
intermuscular adipose tissue remodeling

Marcelo Flores-Opazo<sup>a, **</sup>, Daniel Kopinke<sup>b, c</sup>, Françoise Helmbacher<sup>d</sup>, Rodrigo Fernández-Verdejo<sup>e, f</sup>, Mauro Tuñón-Suárez<sup>f</sup>, Gordon S. Lynch<sup>g</sup>, Osvaldo Contreras<sup>h, i, *, 1</sup>

ARTICLE INFO

Keywords:

Skeletal muscle
FAPs
Adipocytes
Adipogenesis
Obesity
IMAT
Metabolism

A B S T R A C T

Excessive accumulation of intermuscular adipose tissue (IMAT) is a common pathological feature in various metabolic and health conditions and can cause muscle atrophy, reduced function, inflammation, insulin resistance, cardiovascular issues, and unhealthy aging. Although IMAT results from fat accumulation in muscle, the mechanisms underlying its onset, development, cellular components, and functions remain unclear. IMAT levels are influenced by several factors, such as changes in the tissue environment, muscle type and origin, extent and duration of trauma, and persistent activation of fibro-adipogenic progenitors (FAPs). FAPs are a diverse and transcriptionally heterogeneous population of stromal cells essential for tissue maintenance, neuromuscular stability, and tissue regeneration. However, in cases of chronic inflammation and pathological conditions, FAPs expand and differentiate into adipocytes, resulting in the development of abnormal and ectopic IMAT. This review discusses the role of FAPs in adipogenesis and how they remodel IMAT. It highlights evidence supporting FAPs and FAP-derived adipocytes as constituents of IMAT, emphasizing their significance in adipose tissue maintenance and development, as well as their involvement in metabolic disorders, chronic pathologies and diseases. We also investigated the intricate molecular pathways and cell interactions governing FAP behavior. adipogenesis, and IMAT accumulation in chronic diseases and muscle deconditioning. Finally, we hypothesize that impaired cellular metabolic flexibility in dysfunctional muscles impacts FAPs, leading to IMAT. A deeper understanding of the biology of IMAT accumulation and the mechanisms regulating FAP behavior and fate are essential for the development of new therapeutic strategies for several debilitating conditions.

1. Introduction

Normal physical activity and metabolic homeostasis rely on optimal skeletal muscle health and function. Intermuscular adipose tissue (IMAT) is an ectopic fat depot that grows between muscle fibers and

surrounding muscle groups. IMAT is a common feature of various chronic diseases in which muscle mass is progressively lost and replaced by adipose and fibrotic tissue. IMAT can be detected in individuals with chronic muscle disease and wasting, inflammatory muscle disorders, sarcopenia (Therkelsen et al. 2013, 2016; Addison et al., 2014), systemic and local immunometabolic and hormonal disturbances, metabolic

<footnote><sup>a</sup> Institute of Health Sciences, Universidad de O'Higgins, Rancagua, Chile</footnote>

<footnote><sup>b</sup> Department of Pharmacology and Therapeutics, University of Florida, Gainesville, 32610, FL, USA</footnote>

<footnote><sup>c</sup> Myology Institute, University of Florida College of Medicine, Gainesville, FL, USA</footnote>

<footnote><sup>d</sup> Aix Marseille Univ, CNRS, IBDM, UMR 7288, Marseille, France</footnote>

<footnote><sup>e</sup> Pennington Biomedical Research Center, Louisiana State University, Baton Rouge, LA, USA</footnote>

<footnote><sup>f</sup> Laboratorio de Fisiología Del Ejercicio y Metabolismo (LABFEM), Escuela de Kinesiología, Facultad de Medicina, Universidad Finis Terrae, Chile</footnote>

<footnote><sup>g</sup> Centre for Muscle Research, Department of Anatomy and Physiology, The University of Melbourne, Melbourne, Victoria, Parkville 3010, Australia</footnote>

<footnote><sup>h</sup> Developmental and Regenerative Biology Division, Victor Chang Cardiac Research Institute, Darlinghurst, NSW, 2010, Australia</footnote>

<footnote><sup>i</sup> School of Clinical Medicine, UNSW Sydney, Kensington, NSW 2052, Australia</footnote>

<footnote>* Corresponding author. Developmental and Regenerative Biology Division, Victor Chang Cardiac Research Institute, Darlinghurst, NSW, 2010, Australia.</footnote>

<footnote>** Corresponding author.</footnote>

<footnote>E-mail addresses: marcelo.flores@uoh.cl (M. Flores-Opazo), dkopinke@ufl.edu (D. Kopinke), francoise.helmbacher@univ-amu.fr (F. Helmbacher), rodrigo.fernandez@pbrc.edu (R. Fernández-Verdejo), maurotunonsuarez@gmail.com (M. Tuñón-Suárez), gsl@unimelb.edu.au (G.S. Lynch), o.contreras@victorchang.edu.au (O. Contreras).</footnote>

<footnote n="1">¹ Lead author.</footnote>
Abbreviations

<table><tr><td>AnxA2</td><td>Annexin-A2</td></tr><tr><td>C/EBPα</td><td>CCAAT/enhancer-binding protein alpha</td></tr><tr><td>CTX</td><td>Cardiotoxin</td></tr><tr><td>Drk-1</td><td>Delta-like Non-Canonical NOTCH Ligand 1</td></tr><tr><td>DMD</td><td>Duchenne Muscular Dystrophy</td></tr><tr><td>Dpp4</td><td>dipeptidyl peptidase 4</td></tr><tr><td>ECM</td><td>Extracellular matrix</td></tr><tr><td>FABP4</td><td>fatty-acid binding protein 4</td></tr><tr><td>FAPs</td><td>Fibro-adipogenic progenitor cells</td></tr><tr><td>FBN1</td><td>fibrillin-1</td></tr><tr><td>GLY</td><td>Glycerol</td></tr><tr><td>GSK</td><td>Glycogen synthase kinase</td></tr><tr><td>HDAC</td><td>Histone deacetylases</td></tr><tr><td>HFD</td><td>high-fat diet</td></tr><tr><td>Hh</td><td>Hedgehog</td></tr><tr><td>HIC1</td><td>Hypermethylated in Cancer protein 1</td></tr><tr><td>HIF</td><td>Hypoxia inducible family of transcription factors</td></tr><tr><td>IMAT</td><td>Intermuscular Adipose Tissue</td></tr><tr><td>IMTG</td><td>Intramyocellular triglycerides</td></tr><tr><td>LUM</td><td>lumicanMuSCs: Muscle stem cells</td></tr><tr><td>NECD</td><td>NOTCH extracellular domain</td></tr></table>

<table><tr><td>NICD</td><td>NOTCH intracellular domain</td></tr><tr><td>NOTCH</td><td>Neurogenic locus notch homolog protein</td></tr><tr><td>Osr1</td><td>Odd-Skipped Related Transcription Factor 1</td></tr><tr><td>PDGFRα</td><td>platelet-derived growth factor receptor alpha</td></tr><tr><td>PKCβ</td><td>protein kinase C-beta</td></tr><tr><td>PLIN-1</td><td>perilipin-1</td></tr><tr><td>PPARγ</td><td>Peroxisome Proliferator-Activated Receptor Gamma</td></tr><tr><td>RCT</td><td>rotator cuff tear</td></tr><tr><td>SCAT</td><td>Subcutaneous adipose tissue</td></tr><tr><td>sc-RNAseq</td><td>single-cell RNA sequencing</td></tr><tr><td>T2DM</td><td>type 2 diabetes mellitus</td></tr><tr><td>Tcf7l2</td><td>Transcription factor 7-like 2</td></tr><tr><td>TG</td><td>triglycerides</td></tr><tr><td>TGF-β:</td><td>transforming growth factor beta</td></tr><tr><td>TLE</td><td>Groucho/Transducin-Like Enhancer</td></tr><tr><td>TNF-α:</td><td>tumor necrosis factor-alpha</td></tr><tr><td>Trichostatin A</td><td>TSA</td></tr><tr><td>TSP-1</td><td>thrombospondin-1</td></tr><tr><td>UCP-1</td><td>uncoupling protein 1</td></tr><tr><td>VEGFA</td><td>Vascular endothelial growth factor A</td></tr><tr><td>WISP1</td><td>WNT1 Inducible Signaling Pathway Protein 1</td></tr><tr><td>WNT</td><td>Wingless-type MMTV integration site</td></tr></table>

disorders (i.e., obesity, insulin resistance, type 2 diabetes mellitus [T2DM]) and cardiovascular alterations, including hypertension and heart failure (Manini et al., 2007; Taaffe et al., 2009; Tuttle et al., 2011; Pagano et al., 2018; Burkhart et al., 2019). IMAT infiltration has also been associated with muscle deconditioning due to physical inactivity in the context of unhealthy aging, cancer, chronic kidney and lung disease, and fatty liver disease, where the presence of IMAT increases cardiometabolic mortality and morbidity risks (Fig. 1).

Physical activity is essential for maintaining skeletal muscle mass, oxidative capacity, metabolism, and function. In contrast, reduced physical activity can cause mitochondrial dysfunction and increase the accumulation of lipids in muscle tissue, both within myofibers (such as intramyocellular triglycerides [IMTG]) and in interstitial adipocytes, which are part of the IMAT (Bey and Hamilton 2003; Tarnopolsky et al., 2007; Meex et al., 2010; Leskinen et al., 2013; Hasegawa et al., 2016; Park et al., 2020; Seibert et al., 2020; Tunon-Suarez et al., 2021). Several studies have linked IMAT to impaired muscle functionality following a period of muscle disuse (such as immobilization or detraining) regardless of age, which can lead to disability and reduced quality of life (Manini et al., 2007; Brooks et al., 2008; Popadic Gacesa et al., 2011; Burkhart et al., 2019). Consequently, the economic burden of these conditions on families and healthcare systems can be significant. Despite the negative impacts of IMAT on muscular quality and function, the molecular and cellular mechanisms driving its accumulation are not well understood.

This review provides a comprehensive synthesis of the molecular pathways and cellular interactions regulating IMAT accumulation, with a focus on the role of fibro-adipogenic progenitors (FAPs), the main cell type of origin of IMAT, and their role in maintaining muscle health. We investigated the adipogenic commitment and differentiation of FAPs through consideration of factors such as muscle group-specific location, injury-specific cues, FAP ontogeny, epigenetic and chromatin remodeling, and muscle characteristics. Additionally, we discuss the role of reduced muscle contractile activity in the accretion of lipids into the IMAT and the potential involvement of the regulation of cellular metabolic flexibility within muscle in the expansion of FAPs leading to IMAT accumulation.

Given the high prevalence of chronic conditions, sarcopenia and unhealthy aging, understanding the physiological role of FAPs is crucial

for developing effective interventions for muscle tissue maintenance, regeneration and repair. In this context, promoting muscle repair cannot be effective without simultaneously counteracting IMAT development, as these two interventions are expected to act synergistically. Thus, targeting FAPs is a promising therapeutic strategy for mitigating the deleterious effects of chronic conditions and maintaining muscle health.

## 2. Intermuscular adipose tissue in muscle health, regeneration and disease

IMAT is characterized by the accumulation of adipocytes located beneath the outermost muscle fascia within muscle fascicles and between muscle fibers. Low levels of IMAT, in the form of small clusters of perivascular adipocytes, are normal components of muscle in adults without underlying pathology. However, IMAT is often associated with chronic diseases and muscle deconditioning. A severe increase in IMAT and fibrosis, known as fibro-fatty degeneration, is commonly observed in muscle diseases such as Duchenne muscular dystrophy (DMD), inflammatory myopathies, long-term denervation and trauma (Reimers and Finkenstaedt 1997; Chan and Liu 2002; Gladstone et al., 2007; Chang et al., 2018; Feeley et al., 2020).

IMAT accumulation has been linked to reduced muscle loading and physical activity, including prolonged bed rest, limb immobilization, zero-gravity spaceflight (Manini et al., 2007; Pagano et al., 2018; Burkhart et al., 2019), chronic musculoskeletal conditions or muscle trauma (i.e., knee osteoarthritis, low back pain, rotator cuff tears) (Ikemoto-Uezumi et al., 2017; Sions et al., 2017; Trudel et al., 2019), spinal cord injury (Elder et al., 2004; Gorgey and Dudley 2007; Gorgey et al., 2014), sarcopenia (Zoico et al., 2010; Yoshiko et al., 2018), and chronic diseases, including chronic obstructive pulmonary disease (Maddocks et al., 2014), chronic kidney disease (Cheema et al., 2010), alcoholic and non-alcoholic fatty liver disease (Montano-Loza et al., 2016), cancer (Fujiwara et al., 2015; Silva de Paula et al., 2018; Horii et al., 2020), HIV-related wasting syndrome (Torriani et al., 2003), obesity-related disorders, and T2DM (Goodpaster et al. 2000b, 2000c, 2023; Therkelsen et al., 2013). Finally, the accretion of IMAT has also been positively correlated with the progressive deterioration of muscle quality and physical performance in chronic inflammatory myopathies (i.e., inclusion body myositis) and rheumatoid arthritis (Khoja et al. 2018, 2020;
<table><thead><tr><th colspan="2">Abbreviations</th></tr></thead><tbody><tr><td>AnxA2</td><td>Annexin-A2</td></tr><tr><td>C/EBPα</td><td>CCAAT/enhancer-binding protein alpha</td></tr><tr><td>CTX</td><td>Cardiotoxin</td></tr><tr><td>Drk-1</td><td>Delta-like Non-Canonical NOTCH Ligand 1</td></tr><tr><td>DMD</td><td>Duchenne Muscular Dystrophy</td></tr><tr><td>Dpp4</td><td>dipeptidyl peptidase 4</td></tr><tr><td>ECM</td><td>Extracellular matrix</td></tr><tr><td>FABP4</td><td>fatty-acid binding protein 4</td></tr><tr><td>FAPs</td><td>Fibro-adipogenic progenitor cells</td></tr><tr><td>FBN1</td><td>fibrillin-1</td></tr><tr><td>GLY</td><td>Glycerol</td></tr><tr><td>GSK</td><td>Glycogen synthase kinase</td></tr><tr><td>HDAC</td><td>Histone deacetylases</td></tr><tr><td>HFD</td><td>high-fat diet</td></tr><tr><td>Hh</td><td>Hedgehog</td></tr><tr><td>HIC1</td><td>Hypermethylated in Cancer protein 1</td></tr><tr><td>HIF</td><td>Hypoxia inducible family of transcription factors</td></tr><tr><td>IMAT</td><td>Intermuscular Adipose Tissue</td></tr><tr><td>IMTG</td><td>Intramyocellular triglycerides</td></tr><tr><td>LUM</td><td>lumicanMuSCs: Muscle stem cells</td></tr><tr><td>NECD</td><td>NOTCH extracellular domain</td></tr></tbody></table><table><tbody><tr><td>NICD</td><td>NOTCH intracellular domain</td></tr><tr><td>NOTCH</td><td>Neurogenic locus notch homolog protein</td></tr><tr><td>Osr1</td><td>Odd-Skipped Related Transcription Factor 1</td></tr><tr><td>PDGFRα</td><td>platelet-derived growth factor receptor alpha</td></tr><tr><td>PKCβ</td><td>protein kinase C-beta</td></tr><tr><td>PLIN-1</td><td>perilipin-1</td></tr><tr><td>PPARγ</td><td>Peroxisome Proliferator-Activated Receptor Gamma</td></tr><tr><td>RCT</td><td>rotator cuff tear</td></tr><tr><td>SCAT</td><td>Subcutaneous adipose tissue</td></tr><tr><td>sc-RNAseq</td><td>single-cell RNA sequencing</td></tr><tr><td>T2DM</td><td>type 2 diabetes mellitus</td></tr><tr><td>Tcf7l2</td><td>Transcription factor 7-like 2</td></tr><tr><td>TG</td><td>triglycerides</td></tr><tr><td>TGF-β:</td><td>transforming growth factor beta</td></tr><tr><td>TLE</td><td>Groucho/Transducin-Like Enhancer</td></tr><tr><td>TNF-α:</td><td>tumor necrosis factor-alpha</td></tr><tr><td>Trichostatin A</td><td>TSA</td></tr><tr><td>TSP-1</td><td>thrombospondin-1</td></tr><tr><td>UCP-1</td><td>uncoupling protein 1</td></tr><tr><td>VEGFA</td><td>Vascular endothelial growth factor A</td></tr><tr><td>WISP1</td><td>WNT1 Inducible Signaling Pathway Protein 1</td></tr><tr><td>WNT</td><td>Wingless-type MMTV integration site</td></tr></tbody></table>

disorders (i.e., obesity, insulin resistance, type 2 diabetes mellitus [T2DM]) and cardiovascular alterations, including hypertension and heart failure (Manini et al., 2007; Taaffe et al., 2009; Tuttle et al., 2011; Pagano et al., 2018; Burkhart et al., 2019). IMAT infiltration has also been associated with muscle deconditioning due to physical inactivity in the context of unhealthy aging, cancer, chronic kidney and lung disease, and fatty liver disease, where the presence of IMAT increases cardiometabolic mortality and morbidity risks (Fig. 1).

Physical activity is essential for maintaining skeletal muscle mass, oxidative capacity, metabolism, and function. In contrast, reduced physical activity can cause mitochondrial dysfunction and increase the accumulation of lipids in muscle tissue, both within myofibers (such as intramyocellular triglycerides [IMTG]) and in interstitial adipocytes, which are part of the IMAT (Bey and Hamilton 2003; Tarnopolsky et al., 2007; Meex et al., 2010; Leskinen et al., 2013; Hasegawa et al., 2016; Park et al., 2020; Seibert et al., 2020; Tunon-Suarez et al., 2021). Several studies have linked IMAT to impaired muscle functionality following a period of muscle disuse (such as immobilization or detraining) regardless of age, which can lead to disability and reduced quality of life (Manini et al., 2007; Brooks et al., 2008; Popadic Gacesa et al., 2011; Burkhart et al., 2019). Consequently, the economic burden of these conditions on families and healthcare systems can be significant. Despite the negative impacts of IMAT on muscular quality and function, the molecular and cellular mechanisms driving its accumulation are not well understood.

This review provides a comprehensive synthesis of the molecular pathways and cellular interactions regulating IMAT accumulation, with a focus on the role of fibro-adipogenic progenitors (FAPs), the main cell type of origin of IMAT, and their role in maintaining muscle health. We investigated the adipogenic commitment and differentiation of FAPs through consideration of factors such as muscle group-specific location, injury-specific cues, FAP ontogeny, epigenetic and chromatin remodeling, and muscle characteristics. Additionally, we discuss the role of reduced muscle contractile activity in the accretion of lipids into the IMAT and the potential involvement of the regulation of cellular metabolic flexibility within muscle in the expansion of FAPs leading to IMAT accumulation.

Given the high prevalence of chronic conditions, sarcopenia and unhealthy aging, understanding the physiological role of FAPs is crucial

for developing effective interventions for muscle tissue maintenance, regeneration and repair. In this context, promoting muscle repair cannot be effective without simultaneously counteracting IMAT development, as these two interventions are expected to act synergistically. Thus, targeting FAPs is a promising therapeutic strategy for mitigating the deleterious effects of chronic conditions and maintaining muscle health.

## 2. Intermuscular adipose tissue in muscle health, regeneration and disease

IMAT is characterized by the accumulation of adipocytes located beneath the outermost muscle fascia within muscle fascicles and between muscle fibers. Low levels of IMAT, in the form of small clusters of perivascular adipocytes, are normal components of muscle in adults without underlying pathology. However, IMAT is often associated with chronic diseases and muscle deconditioning. A severe increase in IMAT and fibrosis, known as fibro-fatty degeneration, is commonly observed in muscle diseases such as Duchenne muscular dystrophy (DMD), inflammatory myopathies, long-term denervation and trauma (Reimers and Finkenstaedt 1997; Chan and Liu 2002; Gladstone et al., 2007; Chang et al., 2018; Feeley et al., 2020).

IMAT accumulation has been linked to reduced muscle loading and physical activity, including prolonged bed rest, limb immobilization, zero-gravity spaceflight (Manini et al., 2007; Pagano et al., 2018; Burkhart et al., 2019), chronic musculoskeletal conditions or muscle trauma (i.e., knee osteoarthritis, low back pain, rotator cuff tears) (Ikemoto-Uezumi et al., 2017; Sions et al., 2017; Trudel et al., 2019), spinal cord injury (Elder et al., 2004; Gorgey and Dudley 2007; Gorgey et al., 2014), sarcopenia (Zoico et al., 2010; Yoshiko et al., 2018), and chronic diseases, including chronic obstructive pulmonary disease (Maddocks et al., 2014), chronic kidney disease (Cheema et al., 2010), alcoholic and non-alcoholic fatty liver disease (Montano-Loza et al., 2016), cancer (Fujiwara et al., 2015; Silva de Paula et al., 2018; Horii et al., 2020), HIV-related wasting syndrome (Torriani et al., 2003), obesity-related disorders, and T2DM (Goodpaster et al. 2000b, 2000c, 2023; Therkelsen et al., 2013). Finally, the accretion of IMAT has also been positively correlated with the progressive deterioration of muscle quality and physical performance in chronic inflammatory myopathies (i.e., inclusion body myositis) and rheumatoid arthritis (Khoja et al. 2018, 2020;
<figure><img src="image_2.png" /><figcaption>**Fig. 1.** IMAT deposition, a hallmark of chronic fatty infiltration, is exacerbated in a variety of debilitating and chronic human diseases and injuries. This illus - tration highlights the diverse range of pathologies in which IMAT deposition is implicated, including unhealthy aging, obesity, diabetes and metabolic syn - drome, car- diovascular disease, neurode - generative diseases, cancer, and chronic lung and kidney disease.</figcaption></figure>

Laurent et al., 2022) (Fig. 1, Table 1).

In animal models, extensive IMAT formation occurs after a single round of glycerol (GLY) muscle injury (Kawai et al., 1990; Pisani et al., 2010a; Lukjanenko et al., 2013; Mahdy et al., 2015; Pagano et al. 2015, 2019; Biltz and Meyer 2017; Biltz et al., 2020; Xu et al., 2021) (Fig. 2A). Recently, we demonstrated that the accumulation of adipocytes after GLY-induced injury to the tibialis anterior muscle was five times greater than that caused by gold-standard cardiotoxin (CTX) injury in CD1 wild-type mice (Waisman et al., 2021). Tracking with increased IMAT, we also found that myofiber regeneration in the tibialis anterior was reduced with GLY-induced injury compared to CTX injury (Waisman et al., 2021). Notably, because IMAT development following GLY-induced injury tends to persist beyond the expected completion of the regeneration process in 21 days, it shares some of the characteristics of a chronic model. Taken together, these data highlight the potential of the GLY injury model as an effective tool for inducing chronic IMAT infiltration and thus investigating the underlying mechanisms of muscle disease and aging. In summary, IMAT development seems to be a hall- mark of acute and chronic conditions, suggesting that all these condi- tions interfere with FAP homeostasis.

3. Fibro-adipogenic progenitors as the main progenitor cells of adipocytes in IMAT

**3.1. Identification and main characteristics of FAPs**

FAPs are a diverse group of stromal non-myogenic cells that play
crucial roles in maintaining and repairing muscle tissues. FAP

identification was assessed by the expression of mesenchymal cell sur-
face markers such as Sca-1, CD34, and platelet-derived growth factor
receptor alpha (PDGFRα). The latter is considered the gold-standard
marker for identifying FAPs (Joe et al., 2010; Pisani et al., 2010a;
Uezumi et al., 2010; Uezumi et al., 2011). FAPs can differentiate into
fibroblasts and adipocytes both *in vitro* and *in vivo* and are responsible for
depositing fibrotic and adipose tissues in response to muscle injury (Joe
et al., 2010; Uezumi et al. 2010, 2014).

Later studies further expanded our understanding and revealed
several important characteristics of FAPs. First, FAPs were identified as
the primary cell type source of fibrosis in a widely used model of DMD,
the mdx mouse (Uezumi et al., 2011). Second, despite being precursors
derived from a Pax3-nonmyogenic lineage (Pisani et al., 2010b; Liu
et al., 2012), the ablation of FAPs significantly impaired the regenera-
tion of acutely injured muscles, which demonstrated a crucial role in
muscle regeneration and homeostasis (Pisani et al., 2010b; Roberts
et al., 2013; Uezumi et al. 2014, 2021; Wosczyna et al., 2019). Third,
adipogenic FAPs exhibit a cell surface marker profile similar to that
previously characterized for progenitor cells derived from white and
brown adipose tissues (Rodeheffer et al., 2008; Uezumi et al., 2010;
Pannérec et al., 2013; Arrighi et al., 2015). A second set of studies
investigated the regulatory mechanisms controlling FAP activity and
behavior. FAPs are highly responsive to the type 2 innate cytokine
IL-4/IL-13 released by eosinophils (Heredia et al., 2013), and FAP sur-
vival is tightly controlled by cross-talk with inflammatory macrophages,
which involves induction of apoptosis via tumor necrosis factor-alpha
(TNF-α) and release of the mitogenic cytokine transforming growth
factor-beta 1 (TGF-β1) (Lemos et al., 2015). Additionally, FAP fate
<figure><img src="image_3.png" /><figcaption>**Fig. 1.** IMAT deposition, a hallmark of chronic fatty infiltration, is exacerbated in a variety of debilitating and chronic human diseases and injuries. This illus - tration highlights the diverse range of pathologies in which IMAT deposition is implicated, including unhealthy aging, obesity, diabetes and metabolic syn - drome, car- diovascular disease, neurode - generative diseases, cancer, and chronic lung and kidney disease.</figcaption></figure>

Laurent et al., 2022) (Fig. 1, Table 1).

In animal models, extensive IMAT formation occurs after a single round of glycerol (GLY) muscle injury (Kawai et al., 1990; Pisani et al., 2010a; Lukjanenko et al., 2013; Mahdy et al., 2015; Pagano et al. 2015, 2019; Biltz and Meyer 2017; Biltz et al., 2020; Xu et al., 2021) (Fig. 2A). Recently, we demonstrated that the accumulation of adipocytes after GLY-induced injury to the tibialis anterior muscle was five times greater than that caused by gold-standard cardiotoxin (CTX) injury in CD1 wild-type mice (Waisman et al., 2021). Tracking with increased IMAT, we also found that myofiber regeneration in the tibialis anterior was reduced with GLY-induced injury compared to CTX injury (Waisman et al., 2021). Notably, because IMAT development following GLY-induced injury tends to persist beyond the expected completion of the regeneration process in 21 days, it shares some of the characteristics of a chronic model. Taken together, these data highlight the potential of the GLY injury model as an effective tool for inducing chronic IMAT infiltration and thus investigating the underlying mechanisms of muscle disease and aging. In summary, IMAT development seems to be a hall- mark of acute and chronic conditions, suggesting that all these condi- tions interfere with FAP homeostasis.

**3. Fibro-adipogenic progenitors as the main progenitor cells of adipocytes in IMAT**

**3.1. Identification and main characteristics of FAPs**

FAPs are a diverse group of stromal non-myogenic cells that play
crucial roles in maintaining and repairing muscle tissues. FAP

identification was assessed by the expression of mesenchymal cell sur-
face markers such as Sca-1, CD34, and platelet-derived growth factor
receptor alpha (PDGFRα). The latter is considered the gold-standard
marker for identifying FAPs (Joe et al., 2010; Pisani et al., 2010a;
Uezumi et al., 2010; Uezumi et al., 2011). FAPs can differentiate into
fibroblasts and adipocytes both *in vitro* and *in vivo* and are responsible for
depositing fibrotic and adipose tissues in response to muscle injury (Joe
et al., 2010; Uezumi et al. 2010, 2014).

Later studies further expanded our understanding and revealed
several important characteristics of FAPs. First, FAPs were identified as
the primary cell type source of fibrosis in a widely used model of DMD,
the mdx mouse (Uezumi et al., 2011). Second, despite being precursors
derived from a Pax3-nonmyogenic lineage (Pisani et al., 2010b; Liu
et al., 2012), the ablation of FAPs significantly impaired the regenera-
tion of acutely injured muscles, which demonstrated a crucial role in
muscle regeneration and homeostasis (Pisani et al., 2010b; Roberts
et al., 2013; Uezumi et al. 2014, 2021; Wosczyna et al., 2019). Third,
adipogenic FAPs exhibit a cell surface marker profile similar to that
previously characterized for progenitor cells derived from white and
brown adipose tissues (Rodeheffer et al., 2008; Uezumi et al., 2010;
Pannérec et al., 2013; Arrighi et al., 2015). A second set of studies
investigated the regulatory mechanisms controlling FAP activity and
behavior. FAPs are highly responsive to the type 2 innate cytokine
IL-4/IL-13 released by eosinophils (Heredia et al., 2013), and FAP sur-
vival is tightly controlled by cross-talk with inflammatory macrophages,
which involves induction of apoptosis via tumor necrosis factor-alpha
(TNF-α) and release of the mitogenic cytokine transforming growth
factor-beta 1 (TGF-β1) (Lemos et al., 2015). Additionally, FAP fate
<caption>**Table 1**</caption>

IMAT accumulation and its relationship with metabolic and functional markers in human chronic diseases, and immunometabolic and musculoskeletal disorders.

<table><thead><tr><th>Diagnosis</th><th>IMAT</th><th>IMTG</th><th>Lean mass</th><th>Association with circulating and functional markers</th><th>Reference</th></tr></thead><tbody><tr><td>Aging</td><td>↑</td><td>?</td><td>↓ or =</td><td>↓ Insulin sensitivity, ↓ Adiponectin, ↓↑Leptin, ↑ IL-6, ↑ Myostatin, ↑ skeletal muscle fibrosis, mobility limitations, ↑ risk of fall</td><td>(Visser et al., 2005; Zoico et al., 2013, Kim, Dunville et al. 2017; Konopka et al., 2018; Yoshiko et al., 2018; Vitale et al., 2021)</td></tr><tr><td>Reduced muscle contractile activity</td><td>↑</td><td>↑</td><td>↓</td><td>↑, = Thigh-SCAT</td><td>(Manini et al., 2007; Brooks et al., 2008; Taaffe et al., 2009; Popadic Gacesa, Kozic et al., 2011, Burkhart et al., 2019)</td></tr><tr><td>Hormonal Disorders (acromegaly, PCOS, Post menopause)</td><td>↑, =</td><td>↑?</td><td>↓, =</td><td>↓Insulin sensitivity ↓HDLc, ↑TG, ↓vit-D</td><td>(Freda et al., 2008; Freda et al., 2009; Reyes-Vidal et al., 2015; Goss et al., 2014; Scott et al., 2016; Goss et al., 2014; Scott et al., 2016)</td></tr><tr><td>Immunometabolic and cardiovascular disorders</td><td>↑</td><td>↑</td><td>↓, =</td><td>↓ Insulin sensitivity, Dyslipidemia ↓GH, ↑MCP-1, ↑FGF21, ↓FGFR1, ↑adiponectin, ↓Leptin, ↑ creatinine, ↑RCP, ↑ Hypertension Blood pressure, ↑T2DM risk & prevalence, ↑ diabetic peripheral neuropathy</td><td>(Yim et al., 2007; Therkelsen et al., 2013)<br>(Hasegawa et al., 2015; Hasegawa et al., 2016; Komiya et al., 2006; Bergia et al., 2018; Lim et al., 2019; Park et al., 2020)<br>(Goodpaster et al., 2023; Boettcher et al., 2009; Register et al., 2013; Hong et al., 2019; Lo et al., 2007; Yoshimura et al., 2011; Yaskolka Meir et al., 2016; Khoja et al., 2018)</td></tr><tr><td>Advanced cardiovascular disease</td><td>↑</td><td>↑?</td><td>↓, =</td><td>↑CMR, ↑ muscle wasting, ↑ RCP, ↑ major cardiovascular events, ↑ coronary artery calcification</td><td>(Terry et al., 2017; Matsubara et al., 2018; Sugai et al., 2018; Yamashita et al., 2019)</td></tr><tr><td>Musculoskeletal disorders: KOA, CLBP, RCT, SCI</td><td>↑</td><td>↑, =</td><td>↓</td><td>↑ cartilage loss, ↑sarcopenia & ↓ functional capacity, ↑ muscle PDGFRα/PLIN expression, predictor of disease prognosis</td><td>(Ruhdorfer et al., 2015; Teichtahl et al., 2015; Ikemoto-Uezumi et al., 2017; Hebert et al., 2014; Sions et al., 2017; Hebert et al., 2020; Goutallier et al., 2003; Gladstone et al., 2007; Klatte-Schulz et al., 2014; Davies et al., 2022; Davies et al., 2022)</td></tr><tr><td>Muscular dystrophies: BMD, DMD, LGMD2A, Sodium channelopathies</td><td>↑↑</td><td>?</td><td>↓↓</td><td>↓ Irisin, ↑ leptin, ↑ FABP4, ↑ NAMPT, ↑ CNDP1, ↓ PLA2G2A, ↓ MYBPC1, ↓ CKM. Myocardial fibrosis and cardiomyopathy</td><td>(Leroy-Willig et al., 1997; Gong et al., 2000; Mankodi et al., 2016; Marty et al., 2018; Spitali et al., 2018; Schlaeger et al., 2019; Henson et al., 2021; Sun et al., 2021; Barp et al., 2022)</td></tr><tr><td>Neuromuscular diseases and inflammatory myopathies:</td><td>↑↑</td><td>?</td><td>↓, =</td><td>Progressive functional deficits, ↑ nerve damage & muscle edema, Hypermetabolic state, ↑ resistin, visfatin, PAI-1, GLP1, GIP, C-peptide, adipsin, adiponectin, dyslipidemia, ↑</td><td>(Lichtenstein et al., 2018; Kim et al., 2022; Morrow et al., 2016; Cornett et al., 2019; Kim et al., 2019; Diamanti et al., 2019; Hackett et al., 2019; Li et al., 2022; Li et al., 2022; Vernikouskaya et al., 2022; Steyn et al., 2018; Laurent et al., 2022)</td></tr><tr><td>Chronic organ disease: COPD, CKD, CLD</td><td>↑</td><td>↑</td><td>↓ or =</td><td>↓Thiol/protein ratio, ↓Endothelial function, ↑ disease progression and morbidity, ↑IL-8, ↓albumin & pre-albumin, ↓phosphates, ↑Amonnia, ↑creatinine, ↓insulin sensitivity</td><td>(Maddocks et al., 2014; Shields et al., 2015; Coats et al., 2018; Awano et al., 2020; Cheema et al., 2010; Keddar et al., 2020; Kitajima et al., 2013; Montano-Loza et al., 2016; Bhanji et al., 2018; Nardelli et al., 2019)</td></tr><tr><td>Muscle wasting: Cancer, HIV,</td><td>↑, =</td><td>?</td><td>↓, =</td><td>↓ survival, ↑ poor prognosis, ↑ frailty, ↑dyslipidemia, ↓ insulin sensitivity</td><td>(Fujiwara et al., 2015; Silva de Paula, de Aguiar Bruno et al., 2018; Williams et al., 2018; Mardian et al., 2019; Albu et al., 2007; Scherzer et al., 2011)<br>(Mayer et al., 2005; Gill et al., 2016)</td></tr><tr><td>Anorexia Nervosa</td><td>↓</td><td>?</td><td>↓</td><td>↑ Estradiol</td><td></td></tr></tbody></table>

HDL-c: high-density lipoprotein cholesterol, TG: triglycerides, MCP-1: monocyte chemoattractant protein-1, FGF21: fibroblast growth factor 21, FGFR1: fibroblast growth factor receptor 1, CLBP: chronic low back pain, CMR: cardiometabolic risk, RCP: reactive C-protein, MCP-1, FGF21, FGFR1, NAMPT: nicotinamide phosphoribosyltransferase, CNDP1: Carnosine dipeptidase 1, PLA2G2A: phospholipase A2 group IIA, MYBPC1: Myosin Binding Protein C1 CKM: muscle creatine kinase.

depends on balanced cross-talk between pro-mitogenic and pro-fibrotic signals, TGF-β1 and platelet-derived growth factors (Contreras et al. 2019b, 2020). Other mechanisms regulating FAP behavior include cili- ation dynamics and activation of hedgehog intercellular signaling (Kopinke et al., 2017); cell-to-cell contact signals, such as the neurogenic locus notch homolog protein (NOTCH) (Marinkovic et al., 2019); the juxtafine effect of factors released by damaged myofibers (Hogarth et al., 2019); the regulation of the adipogenic master regulator peroxi- some proliferator-activated receptor-gamma (PPARγ) transcriptional activity (Reggio et al., 2019); the autocrine and paracrine effects of Wingless-type MMTV integration site (WNT) ligands; the subsequent activation of the WNT/β-catenin signaling cascade and metabolic cues (Reggio et al., 2020a); and microheterogeneity regulation of FAP dif- ferentiation and cell states (Giuliani et al., 2021).

In parallel, several studies have demonstrated that FAPs are neces- sary for the activation and differentiation of myogenic progenitors. This critical role is exerted through the secretion of pro-myogenic paracrine and juxtafine factors (Joe et al., 2010; Murphy et al., 2011; Mozzetta et al., 2013; Roberts et al., 2013; Lukjanenko et al., 2019; Uezumi et al., 2021). In vitro co-culture and conditioned media from activated FAPs have been shown to increase the myoblast fusion rate and myotube diameter (Mathew et al., 2011; Mozzetta et al., 2013; Madaro et al., 2018). Moreover, ablation of FAPs has been associated with muscle

atrophy, muscle weakness, a reduction in muscle stem cells (MuSCs), a fast-to-slow shift in fiber type, neuromuscular junctions and Schwann cell degeneration (Murphy et al., 2011; Liu et al., 2012; Roberts et al., 2013; Wosczyna et al., 2019; Uezumi et al., 2021). Overall, research on FAPs is still in its infancy, and further studies are needed to fully un- derstand their complex biology.

## 3.2. Differentiation routes and fates of FAPs

FAPs represent a population of multipotent progenitor cells in the muscle interstitium, and a significant proportion reside in the vicinity of blood vessels, where they act as perivascular cells rather than pericytes (Joe et al., 2010; Uezumi et al., 2010; Santini et al., 2020; Sono et al., 2020; Theret et al., 2021). Although their main differentiation routes guide them toward fibrogenic and adipogenic lineages, they can acquire other cell fates under specific stimuli. Potentially, a small subset of injury-activated FAPs may differentiate into endothelial cells in response to hypoxia and glucose deprivation (Ollitrault et al., 2021). In severe inflammatory and chronic disease conditions, muscle FAPs ac- quire an osteogenic phenotype, as observed in mouse models of het- erotopic ossification and severe muscle disease (Lees-Shepard et al., 2018; Eisner et al., 2020; Mázala et al., 2020; Julien et al., 2021). Despite their multipotent differentiation capacity, FAPs traditionally
<caption>**Table 1**</caption>

IMAT accumulation and its relationship with metabolic and functional markers in human chronic diseases, and immunometabolic and musculoskeletal disorders.

<table><thead><tr><th>Diagnosis</th><th>IMAT</th><th>IMTG</th><th>Lean mass</th><th>Association with circulating and functional markers</th><th>Reference</th></tr></thead><tbody><tr><td>Aging</td><td>↑</td><td>?</td><td>↓ or =</td><td>↓ Insulin sensitivity, ↓ Adiponectin, ↓↑Leptin, ↑ IL-6, ↑ Myostatin, ↑ skeletal muscle fibrosis, mobility limitations, ↑ risk of fall</td><td>(Visser et al., 2005; Zoico et al., 2013, Kim, Dunville et al. 2017; Konopka et al., 2018; Yoshiko et al., 2018; Vitale et al., 2021)</td></tr><tr><td>Reduced muscle contractile activity</td><td>↑</td><td>↑</td><td>↓</td><td>↑, = Thigh-SCAT</td><td>(Manini et al., 2007; Brooks et al., 2008; Taaffe et al., 2009; Popadic Gacesa, Kozic et al., 2011, Burkhart et al., 2019)</td></tr><tr><td>Hormonal Disorders (acromegaly, PCOS, Post menopause)</td><td>↑, =</td><td>↑?</td><td>↓, =</td><td>↓Insulin sensitivity ↓HDLc, ↑TG, ↓vit-D</td><td>(Freda et al., 2008; Freda et al., 2009; Reyes-Vidal et al., 2015; Goss et al., 2014; Scott et al., 2016; Goss et al., 2014; Scott et al., 2016)</td></tr><tr><td>Immunometabolic and cardiovascular disorders</td><td>↑</td><td>↑</td><td>↓, =</td><td>↓ Insulin sensitivity, Dyslipidemia ↓GH, ↑MCP-1, ↑FGF21, ↓FGFR1, ↑adiponectin, ↓Leptin, ↑ creatinine, ↑RCP, ↑ Hypertension Blood pressure, ↑T2DM risk & prevalence, ↑ diabetic peripheral neuropathy</td><td>(Yim et al., 2007; Therkelsen et al., 2013)<br>(Hasegawa et al., 2015; Hasegawa et al., 2016; Komiya et al., 2006; Bergia et al., 2018; Lim et al., 2019; Park et al., 2020)<br>(Goodpaster et al., 2023; Boettcher et al., 2009; Register et al., 2013; Hong et al., 2019; Lo et al., 2007; Yoshimura et al., 2011; Yaskolka Meir et al., 2016; Khoja et al., 2018)</td></tr><tr><td>Advanced cardiovascular disease</td><td>↑</td><td>↑?</td><td>↓, =</td><td>↑CMR, ↑ muscle wasting, ↑ RCP, ↑ major cardiovascular events, ↑ coronary artery calcification</td><td>(Terry et al., 2017; Matsubara et al., 2018; Sugai et al., 2018; Yamashita et al., 2019)</td></tr><tr><td>Musculoskeletal disorders: KOA, CLBP, RCT, SCI</td><td>↑</td><td>↑, =</td><td>↓</td><td>↑ cartilage loss, ↑sarcopenia & ↓ functional capacity, ↑ muscle PDGFRα/PLIN expression, predictor of disease prognosis</td><td>(Ruhdorfer et al., 2015; Teichtahl et al., 2015; Ikemoto-Uezumi et al., 2017; Hebert et al., 2014; Sions et al., 2017; Hebert et al., 2020; Goutallier et al., 2003; Gladstone et al., 2007; Klatte-Schulz et al., 2014; Davies et al., 2022; Davies et al., 2022)</td></tr><tr><td>Muscular dystrophies: BMD, DMD, LGMD2A, Sodium channelopathies</td><td>↑↑</td><td>?</td><td>↓↓</td><td>↓ Irisin, ↑ leptin, ↑ FABP4, ↑ NAMPT, ↑ CNDP1, ↓ PLA2G2A, ↓ MYBPC1, ↓ CKM. Myocardial fibrosis and cardiomyopathy</td><td>(Leroy-Willig et al., 1997; Gong et al., 2000; Mankodi et al., 2016; Marty et al., 2018; Spitali et al., 2018; Schlaeger et al., 2019; Henson et al., 2021; Sun et al., 2021; Barp et al., 2022)</td></tr><tr><td>Neuromuscular diseases and inflammatory myopathies:</td><td>↑↑</td><td>?</td><td>↓, =</td><td>Progressive functional deficits, ↑ nerve damage & muscle edema, Hypermetabolic state, ↑ resistin, visfatin, PAI-1, GLP1, GIP, C-peptide, adipsin, adiponectin, dyslipidemia, ↑</td><td>(Lichtenstein et al., 2018; Kim et al., 2022; Morrow et al., 2016; Cornett et al., 2019; Kim et al., 2019; Diamanti et al., 2019; Hackett et al., 2019; Li et al., 2022; Li et al., 2022; Vernikouskaya et al., 2022; Steyn et al., 2018; Laurent et al., 2022)</td></tr><tr><td>Chronic organ disease: COPD, CKD, CLD</td><td>↑</td><td>↑</td><td>↓ or =</td><td>↓Thiol/protein ratio, ↓Endothelial function, ↑ disease progression and morbidity, ↑IL-8, ↓albumin & pre-albumin, ↓phosphates, ↑Amonnia, ↑creatinine, ↓insulin sensitivity</td><td>(Maddocks et al., 2014; Shields et al., 2015; Coats et al., 2018; Awano et al., 2020; Cheema et al., 2010; Keddar et al., 2020; Kitajima et al., 2013; Montano-Loza et al., 2016; Bhanji et al., 2018; Nardelli et al., 2019)</td></tr><tr><td>Muscle wasting: Cancer, HIV,</td><td>↑, =</td><td>?</td><td>↓, =</td><td>↓ survival, ↑ poor prognosis, ↑ frailty, ↑dyslipidemia, ↓ insulin sensitivity</td><td>(Fujiwara et al., 2015; Silva de Paula, de Aguiar Bruno et al., 2018; Williams et al., 2018; Mardian et al., 2019; Albu et al., 2007; Scherzer et al., 2011)<br>(Mayer et al., 2005; Gill et al., 2016)</td></tr><tr><td>Anorexia Nervosa</td><td>↓</td><td>?</td><td>↓</td><td>↑ Estradiol</td><td></td></tr></tbody></table>

HDL-c: high-density lipoprotein cholesterol, TG: triglycerides, MCP-1: monocyte chemoattractant protein-1, FGF21: fibroblast growth factor 21, FGFR1: fibroblast growth factor receptor 1, CLBP: chronic low back pain, CMR: cardiometabolic risk, RCP: reactive C-protein, MCP-1, FGF21, FGFR1, NAMPT: nicotinamide phosphoribosyltransferase, CNDP1: Carnosine dipeptidase 1, PLA2G2A: phospholipase A2 group IIA, MYBPC1: Myosin Binding Protein C1 CKM: muscle creatine kinase.

depends on balanced cross-talk between pro-mitogenic and pro-fibrotic signals, TGF-β1 and platelet-derived growth factors (Contreras et al. 2019b, 2020). Other mechanisms regulating FAP behavior include cili- ation dynamics and activation of hedgehog intercellular signaling (Kopinke et al., 2017); cell-to-cell contact signals, such as the neurogenic locus notch homolog protein (NOTCH) (Marinkovic et al., 2019); the juxtafine effect of factors released by damaged myofibers (Hogarth et al., 2019); the regulation of the adipogenic master regulator peroxi- some proliferator-activated receptor-gamma (PPARγ) transcriptional activity (Reggio et al., 2019); the autocrine and paracrine effects of Wingless-type MMTV integration site (WNT) ligands; the subsequent activation of the WNT/β-catenin signaling cascade and metabolic cues (Reggio et al., 2020a); and microheterogeneity regulation of FAP dif- ferentiation and cell states (Giuliani et al., 2021).

In parallel, several studies have demonstrated that FAPs are neces- sary for the activation and differentiation of myogenic progenitors. This critical role is exerted through the secretion of pro-myogenic paracrine and juxtafine factors (Joe et al., 2010; Murphy et al., 2011; Mozzetta et al., 2013; Roberts et al., 2013; Lukjanenko et al., 2019; Uezumi et al., 2021). In vitro co-culture and conditioned media from activated FAPs have been shown to increase the myoblast fusion rate and myotube diameter (Mathew et al., 2011; Mozzetta et al., 2013; Madaro et al., 2018). Moreover, ablation of FAPs has been associated with muscle

atrophy, muscle weakness, a reduction in muscle stem cells (MuSCs), a fast-to-slow shift in fiber type, neuromuscular junctions and Schwann cell degeneration (Murphy et al., 2011; Liu et al., 2012; Roberts et al., 2013; Wosczyna et al., 2019; Uezumi et al., 2021). Overall, research on FAPs is still in its infancy, and further studies are needed to fully un- derstand their complex biology.

## 3.2. Differentiation routes and fates of FAPs

FAPs represent a population of multipotent progenitor cells in the muscle interstitium, and a significant proportion reside in the vicinity of blood vessels, where they act as perivascular cells rather than pericytes (Joe et al., 2010; Uezumi et al., 2010; Santini et al., 2020; Sono et al., 2020; Theret et al., 2021). Although their main differentiation routes guide them toward fibrogenic and adipogenic lineages, they can acquire other cell fates under specific stimuli. Potentially, a small subset of injury-activated FAPs may differentiate into endothelial cells in response to hypoxia and glucose deprivation (Ollitrault et al., 2021). In severe inflammatory and chronic disease conditions, muscle FAPs ac-quire an osteogenic phenotype, as observed in mouse models of het- erotopic ossification and severe muscle disease (Lees-Shepard et al., 2018; Eisner et al., 2020; Mázala et al., 2020; Julien et al., 2021). Despite their multipotent differentiation capacity, FAPs traditionally
<figure><img src="image_3.png" /><figcaption>**Fig. 2.** Deposition of IMAT and adipocyte accumulation in mouse models of acute and chronic muscle damage. (A) Three-dimensional rendering illustrating IMAT deposition and enhanced adipocyte accumulation in between regenerating myofibers (left panel), alongside a transverse section imaged via laser confocal microscopy displaying accumulated Perilipin⁺ adipocytes following glycerol-induced damage (right panel). (B) Confocal microscopy images demonstrating the abundance of Perilipin adipocytes within the muscle stroma of dystrophic mdx limb skeletal muscles.</figcaption></figure>

were not thought to contribute to the myogenic lineage under normal conditions. This fate limitation has been related to the expression and activity of the nuclear envelope protein Prdm16 in FAPs (Biferali et al., 2021). This transcription factor is typically expressed in Myf5⁺/Pax7⁺ myogenic and brown-fat progenitors and acts as a fate switch regulating the choice of these cells to become either muscle or brown fat cells (Seale et al., 2008; An et al., 2017). Moreover, knockdown of *Prdm16* in brown fat precursors induced myogenesis, whereas its overexpression promoted brown adipocyte differentiation in myogenic cells. In FAPs, Prdm16 inhibits the expression of a myogenic-like gene profile, an activity exerted in conjunction with the histone methyltransferases G9a and GLP, to confine myogenic genomic loci within a silent heterochromatin-like compartment underneath the nuclear lamina (Biferali et al., 2021). In line with these findings, the selective inhibition of G9a induced an anti-adipogenic effect on FAPs and promoted myotube formation (Randazzo et al., 2022). These results suggested that Prdm16 and G9a-mediated H3K9 histone methylation switch off myogenic differentiation in FAPs, thus confirming their stable non-myogenic fate. While most FAPs are generally not involved in muscle cell formation, recent research suggests that certain types of FAPs may contribute to creating muscle cells. Specifically, a study in 2023 by Flynn and colleagues identified a group of that express the gene

Homeobox A11 (Hoxa11). These cells have been shown to help form new myogenic cells that then become part of the muscle fibers (Flynn et al., 2023). This finding supports the emerging idea that some interstitial progenitor cells, rather than the traditional Pax7-expressing MuSCs, serve as a significant source for adult muscle tissue maintenance *in vivo* (Flynn et al., 2023).

Regarding FAP adipogenic capacity, comparative *in vitro* and *ex vivo* studies have shown that FAPs share similar adipogenicity and functional properties with adipocyte precursors (APCs) from subcutaneous and visceral fat depots (Arrighi et al., 2015; Laurens et al., 2016; Sachs et al., 2019). Arrighi et al. (2015) demonstrated that adipogenesis, expression of adipogenic markers, triglyceride synthesis and degradation were comparable between muscle FAP-derived adipocytes and APC-derived adipocytes. However, insulin failed to stimulate glucose uptake or insulin signaling in FAPs. Hence, the authors concluded that the IMAT behaves as a fat compartment that is insensitive to insulin (Arrighi et al., 2015). Notably, the differentiation media used in the study contained 860 nM insulin (0.5 µg/ml), which is 43-fold greater than that of standard adipogenic cocktails (20 nM or 11.6 ng/ml). This high concentration of insulin may have yielded FAP-derived adipocytes unresponsive to its effect since a previous study revealed that prolonged insulin treatment dose-dependently induced insulin resistance and impaired
<figure><img src="image_4.png" /><figcaption>**Fig. 2.** Deposition of IMAT and adipocyte accumulation in mouse models of acute and chronic muscle damage. (A) Three-dimensional rendering illustrating IMAT deposition and enhanced adipocyte accumulation in between regenerating myofibers (left panel), alongside a transverse section imaged via laser confocal microscopy displaying accumulated Perilipin⁺ adipocytes following glycerol-induced damage (right panel). (B) Confocal microscopy images demonstrating the abundance of Perilipin adipocytes within the muscle stroma of dystrophic mdx limb skeletal muscles.</figcaption></figure>

were not thought to contribute to the myogenic lineage under normal conditions. This fate limitation has been related to the expression and activity of the nuclear envelope protein Prdm16 in FAPs (Biferali et al., 2021). This transcription factor is typically expressed in Myf5⁺/Pax7⁺ myogenic and brown-fat progenitors and acts as a fate switch regulating the choice of these cells to become either muscle or brown fat cells (Seale et al., 2008; An et al., 2017). Moreover, knockdown of *Prdm16* in brown fat precursors induced myogenesis, whereas its overexpression promoted brown adipocyte differentiation in myogenic cells. In FAPs, Prdm16 inhibits the expression of a myogenic-like gene profile, an activity exerted in conjunction with the histone methyltransferases G9a and GLP, to confine myogenic genomic loci within a silent heterochromatin-like compartment underneath the nuclear lamina (Biferali et al., 2021). In line with these findings, the selective inhibition of G9a induced an anti-adipogenic effect on FAPs and promoted myotube formation (Randazzo et al., 2022). These results suggested that Prdm16 and G9a-mediated H3K9 histone methylation switch off myogenic differentiation in FAPs, thus confirming their stable non-myogenic fate. While most FAPs are generally not involved in muscle cell formation, recent research suggests that certain types of FAPs may contribute to creating muscle cells. Specifically, a study in 2023 by Flynn and colleagues identified a group of that express the gene

Homeobox A11 (Hoxa11). These cells have been shown to help form new myogenic cells that then become part of the muscle fibers (Flynn et al., 2023). This finding supports the emerging idea that some interstitial progenitor cells, rather than the traditional Pax7-expressing MuSCs, serve as a significant source for adult muscle tissue maintenance *in vivo* (Flynn et al., 2023).

Regarding FAP adipogenic capacity, comparative *in vitro* and *ex vivo* studies have shown that FAPs share similar adipogenicity and functional properties with adipocyte precursors (APCs) from subcutaneous and visceral fat depots (Arrighi et al., 2015; Laurens et al., 2016; Sachs et al., 2019). Arrighi et al. (2015) demonstrated that adipogenesis, expression of adipogenic markers, triglyceride synthesis and degradation were comparable between muscle FAP-derived adipocytes and APC-derived adipocytes. However, insulin failed to stimulate glucose uptake or insulin signaling in FAPs. Hence, the authors concluded that the IMAT behaves as a fat compartment that is insensitive to insulin (Arrighi et al., 2015). Notably, the differentiation media used in the study contained 860 nM insulin (0.5 µg/ml), which is 43-fold greater than that of standard adipogenic cocktails (20 nM or 11.6 ng/ml). This high concentration of insulin may have yielded FAP-derived adipocytes unresponsive to its effect since a previous study revealed that prolonged insulin treatment dose-dependently induced insulin resistance and impaired
mitochondrial function in contractile cells of lymphatic vessels (Lee et al., 2017a). Moreover, the idea of FAPs being insensitive to insulin was further refuted by transcriptomic analyses revealing the expression of the insulin receptor and downstream insulin effectors, such as the serine-threonine protein kinase AKT and the glucose transporter GLUT4 (SLC2A4), in isolated human IMAT explants (Sachs et al., 2019). In fact, the expression of these markers correlated with the insulin sensitivity of the donor and was downregulated in patients with low insulin sensitivity (Sachs et al., 2019).

Additionally, FAPs have been shown to differentiate into brown-like adipocytes expressing uncoupling protein 1 (UCP-1) in response to β-adrenergic stimulation or cold exposure in both mice and humans (Uezumi et al., 2014; Gorski et al., 2018; Lee et al. 2020b, 2020c). However, not all studies have evaluated this ability (Pisani et al., 2010b; Woszczyna et al., 2012; Arrighi et al., 2015), and the results appear dependent on cell culture conditions and differentiation protocols (Gorski et al., 2018). Nonetheless, the specific conditions favoring white or brown/beige fat cell differentiation in FAPs in vivo, have yet to be determined. In a translational setting, transplantation of UCP-1⁺ FAPs, described as beige-FAPs, seems to improve muscle function and reduce fatty degeneration in a mouse model of rotator cuff tear (Lee et al., 2020c).

Despite the well-established roles of FAPs in muscle maintenance and regeneration, excessive activation and subsequent differentiation into adipocytes lead to pathological formation of IMAT and fibro-fatty degeneration in muscle diseases (Uezumi et al., 2011; Paylor et al., 2014; Lemos et al., 2015; Contreras et al. 2016, 2019c; Ikemoto-Uezumi et al., 2017; Hogarth et al., 2019; Reggio et al. 2019, 2020b; Farup et al., 2021; Liu et al., 2021; Shirasawa et al., 2021; Vumbaca et al., 2021; Davies et al., 2022). FAPs also contribute to IMAT formation in response to chronic systemic diseases and muscle injury (Fig. 1), and this adipogenic potential is influenced by the interaction between FAPs, surrounding cells, and the prevailing cellular environment, as demonstrated in transplantation studies (Joe et al., 2010; Uezumi et al., 2010; Liu et al., 2012; Uezumi et al., 2014; Kopinke, Roberson et al. 2017; Stumm et al., 2018; Hogarth et al., 2019; Contreras and Harvey 2023).

Nonetheless, immune cells may also contribute to ectopic IMAT adipogenesis, perhaps to a small degree. For instance, single-cell RNA-seq analysis revealed a subpopulation of muscle-resident myeloid-derived cells that expressed adipocyte-enriched genes and accumulated lipids upon adipogenic induction in vitro (Xu et al., 2021). Future studies that include parabiosis and detailed lineage tracing should help to answer these questions.

### 3.3. Cellular and functional heterogeneity of muscle FAPs

Muscle FAPs are not homogeneous but rather constitute a group of cells with varying characteristics and roles in vivo (Collins and Kardon 2021; Contreras et al., 2021b). The diversity of FAPs relies on the anatomical location of the muscle in which they reside (Muhl et al., 2020; Contreras et al., 2021b) and dynamic changes in the microenvironment after muscle injury and in diseased states (Malecova et al., 2018; Marinkovic et al., 2019; Zhang et al., 2019; Contreras et al., 2020; Rubenstein et al., 2020; Giuliani et al., 2021). The FAP secretome and its functions are highly dynamic and ensure that the regenerative microenvironment sustains muscle health and integrity. This section explores the factors that influence the emergence of diverse FAP cellular states and fates and how this adaptive response fosters a favorable pro-regenerative microenvironment that supports muscle regeneration or may lead to debilitating IMAT formation in muscle diseases. Additionally, we describe the transcriptional profiles and functional characteristics of distinct FAP states from embryonic FAPs to quiescent FAPs in adult muscles and their activation to a promyogenic state after injury and the presence of other FAP subtypes under normal and pathological conditions and in diseases.

#### 3.3.1. Heterogeneity of tissue of origin of FAPs

During embryonic development, muscle connective tissue progenitor cells, including FAPs, originate from different embryonic structures (reviewed previously (Helmbacher and Stricker 2020)). Three major embryonic origins have been identified for muscle-resident FAPs: 1) the neural crest for craniofacial and neck muscles, and most of the cartilage, bone, dente and other connective tissues of the head (Diogo et al., 2015); 2) the lateral plate mesoderm for trunk and limb muscles; and 3) the pleuroperitoneal folds for the diaphragm (Contreras et al., 2021b). Cells emerging from the cranial neural crest participate in the formation and shape of muscles on the shoulder girdle, neck, and face and are the source of FAPs in these regions. Subsequently, these FAPs may eventually form IMATs in rotator cuff tears and craniofacial muscle fibro-fatty infiltrating disorders (Paylor et al., 2014; Lee et al., 2020a). Lateral plate mesoderm and somites form axial trunk muscles and muscles of the limbs and are the source of FAPs, contributing to the formation of IMAT within these muscle groups (Lemos et al., 2012; Paylor et al., 2014). Finally, pleuroperitoneal folds are transient non-myogenic structures that develop in conjunction with the diaphragm and give rise to the central tendon and FAPs in mice (Merrell et al., 2015). They are an important source of myogenic promoting signals during diaphragm development. In contrast, FAPs contribute to IMAT in the diaphragm in adult mice in response to a high-fat diet (HFD) (Buras et al., 2019).

A population of embryonic FAPs expressing the odd-skipped related transcription factor 1 (Osr1) zinc-finger transcription factor was identified (Vallecillo-García et al., 2017). These Osr1⁺ FAPs exhibited a transcriptional program associated with extracellular matrix (ECM) biogenesis and matrisome assembly; this program included collagen chains (Col6a1, Col6a2, and Col6a3), lumican, matrillin, decorin, fibromodulin, and nidogen 2. Osr1⁺ FAPs express the chemokine Cxcl12 and the growth factor bone morphogenetic protein 4 (Bmp4), both of which are known to promote myogenesis (Vallecillo-García et al., 2017). Thus, Osr1⁺ FAPs create a favorable environment supporting the proliferation and survival of myogenic progenitors during development by expressing ECM components and promyogenic factors (Vallecillo-García et al., 2017). In a recent study, scRNA-seq revealed six clusters of embryonic PDGFRα⁺ cells in developing muscle, from which cluster 4 showed high Osr1 expression and other genes associated with stemness and developmental processes (Leinroth et al., 2022). The other five clusters followed three distinctive differentiation trajectories. First, an immune responsive population with high fibrogenic and adipogenic potency. Second, highly osteogenic cells expressing clusterin and hemicentin-1. Finally, neuromuscular junction (NMJ)-associated cells with no adipogenic potential (Leinroth et al., 2022). Additionally, although the number of embryonic Osr1⁺ FAPs declines in the muscle interstitium soon after birth, their progeny contributes to FAP populations in adult muscle, giving rise to Sca1⁺ and PDGFRα⁺ FAPs (Vallecillo-García et al., 2017; Stumm et al., 2018).

Regarding their location, adult FAPs from different muscle locations differ in their number, functional proliferation, and adipogenic properties. Lee et al. (2020d) demonstrated that the masseter, rotator cuff, and paraspinal muscles have a greater proportion of FAPs per gram of tissue than does the gastrocnemius and tibialis anterior muscles of the hindlimb in mice (Lee et al., 2020a). Furthermore, FAPs isolated from these muscles, especially from the shoulder muscles, exhibit differential proliferative activity and adipogenic potency (Lee et al., 2020a), which is relevant considering the rapid onset of IMAT after rotator cuff tears. However, whether the differences in the functional capabilities of FAPs are related to their heterogeneous embryonic origin or whether changes occur later in their developmental history has not been studied. Apart from FAP ontogeny, this functional variability could also be attributed to variable pro-adipogenic patterns across muscle groups, in situ injury-specific cues, intrinsic epigenetic mechanisms, and differences in muscle fiber type composition, innervation, vascularity, and biomechanical function. Given the complexity of the adipogenic fate of muscle FAPs, we propose that all these variables could determine the fate of
mitochondrial function in contractile cells of lymphatic vessels (Lee et al., 2017a). Moreover, the idea of FAPs being insensitive to insulin was further refuted by transcriptomic analyses revealing the expression of the insulin receptor and downstream insulin effectors, such as the serine-threonine protein kinase AKT and the glucose transporter GLUT4 (SLC2A4), in isolated human IMAT explants (Sachs et al., 2019). In fact, the expression of these markers correlated with the insulin sensitivity of the donor and was downregulated in patients with low insulin sensitivity (Sachs et al., 2019).

Additionally, FAPs have been shown to differentiate into brown-like adipocytes expressing uncoupling protein 1 (UCP-1) in response to β-adrenergic stimulation or cold exposure in both mice and humans (Uezumi et al., 2014; Gorski et al., 2018; Lee et al. 2020b, 2020c). However, not all studies have evaluated this ability (Pisani et al., 2010b; Woszczyna et al., 2012; Arrighi et al., 2015), and the results appear dependent on cell culture conditions and differentiation protocols (Gorski et al., 2018). Nonetheless, the specific conditions favoring white or brown/beige fat cell differentiation in FAPs in vivo, have yet to be determined. In a translational setting, transplantation of UCP-1⁺ FAPs, described as beige-FAPs, seems to improve muscle function and reduce fatty degeneration in a mouse model of rotator cuff tear (Lee et al., 2020c).

Despite the well-established roles of FAPs in muscle maintenance and regeneration, excessive activation and subsequent differentiation into adipocytes lead to pathological formation of IMAT and fibro-fatty degeneration in muscle diseases (Uezumi et al., 2011; Paylor et al., 2014; Lemos et al., 2015; Contreras et al. 2016, 2019c; Ikemoto-Uezumi et al., 2017; Hogarth et al., 2019; Reggio et al. 2019, 2020b; Farup et al., 2021; Liu et al., 2021; Shirasawa et al., 2021; Vumbaca et al., 2021; Davies et al., 2022). FAPs also contribute to IMAT formation in response to chronic systemic diseases and muscle injury (Fig. 1), and this adipogenic potential is influenced by the interaction between FAPs, surrounding cells, and the prevailing cellular environment, as demonstrated in transplantation studies (Joe et al., 2010; Uezumi et al., 2010; Liu et al., 2012; Uezumi et al., 2014; Kopinke, Roberson et al. 2017; Stumm et al., 2018; Hogarth et al., 2019; Contreras and Harvey 2023).

Nonetheless, immune cells may also contribute to ectopic IMAT adipogenesis, perhaps to a small degree. For instance, single-cell RNA-seq analysis revealed a subpopulation of muscle-resident myeloid-derived cells that expressed adipocyte-enriched genes and accumulated lipids upon adipogenic induction in vitro (Xu et al., 2021). Future studies that include parabiosis and detailed lineage tracing should help to answer these questions.

### 3.3. Cellular and functional heterogeneity of muscle FAPs

Muscle FAPs are not homogeneous but rather constitute a group of cells with varying characteristics and roles in vivo (Collins and Kardon 2021; Contreras et al., 2021b). The diversity of FAPs relies on the anatomical location of the muscle in which they reside (Muhl et al., 2020; Contreras et al., 2021b) and dynamic changes in the microenvironment after muscle injury and in diseased states (Malecova et al., 2018; Marinkovic et al., 2019; Zhang et al., 2019; Contreras et al., 2020; Rubenstein et al., 2020; Giuliani et al., 2021). The FAP secretome and its functions are highly dynamic and ensure that the regenerative microenvironment sustains muscle health and integrity. This section explores the factors that influence the emergence of diverse FAP cellular states and fates and how this adaptive response fosters a favorable pro-regenerative microenvironment that supports muscle regeneration or may lead to debilitating IMAT formation in muscle diseases. Additionally, we describe the transcriptional profiles and functional characteristics of distinct FAP states from embryonic FAPs to quiescent FAPs in adult muscles and their activation to a promyogenic state after injury and the presence of other FAP subtypes under normal and pathological conditions and in diseases.

#### 3.3.1. Heterogeneity of tissue of origin of FAPs

During embryonic development, muscle connective tissue progenitor cells, including FAPs, originate from different embryonic structures (reviewed previously (Helmbacher and Stricker 2020)). Three major embryonic origins have been identified for muscle-resident FAPs: 1) the neural crest for craniofacial and neck muscles, and most of the cartilage, bone, dente and other connective tissues of the head (Diogo et al., 2015); 2) the lateral plate mesoderm for trunk and limb muscles; and 3) the pleuroperitoneal folds for the diaphragm (Contreras et al., 2021b). Cells emerging from the cranial neural crest participate in the formation and shape of muscles on the shoulder girdle, neck, and face and are the source of FAPs in these regions. Subsequently, these FAPs may eventually form IMATs in rotator cuff tears and craniofacial muscle fibro-fatty infiltrating disorders (Paylor et al., 2014; Lee et al., 2020a). Lateral plate mesoderm and somites form axial trunk muscles and muscles of the limbs and are the source of FAPs, contributing to the formation of IMAT within these muscle groups (Lemos et al., 2012; Paylor et al., 2014). Finally, pleuroperitoneal folds are transient non-myogenic structures that develop in conjunction with the diaphragm and give rise to the central tendon and FAPs in mice (Merrell et al., 2015). They are an important source of myogenic promoting signals during diaphragm development. In contrast, FAPs contribute to IMAT in the diaphragm in adult mice in response to a high-fat diet (HFD) (Buras et al., 2019).

A population of embryonic FAPs expressing the odd-skipped related transcription factor 1 (Osr1) zinc-finger transcription factor was identified (Vallecillo-García et al., 2017). These Osr1⁺ FAPs exhibited a transcriptional program associated with extracellular matrix (ECM) biogenesis and matrisome assembly; this program included collagen chains (Col6a1, Col6a2, and Col6a3), lumican, matrillin, decorin, fibromodulin, and nidogen 2. Osr1⁺ FAPs express the chemokine Cxcl12 and the growth factor bone morphogenetic protein 4 (Bmp4), both of which are known to promote myogenesis (Vallecillo-García et al., 2017). Thus, Osr1⁺ FAPs create a favorable environment supporting the proliferation and survival of myogenic progenitors during development by expressing ECM components and promyogenic factors (Vallecillo-García et al., 2017). In a recent study, scRNA-seq revealed six clusters of embryonic PDGFRα⁺ cells in developing muscle, from which cluster 4 showed high Osr1 expression and other genes associated with stemness and developmental processes (Leinroth et al., 2022). The other five clusters followed three distinctive differentiation trajectories. First, an immune responsive population with high fibrogenic and adipogenic potency. Second, highly osteogenic cells expressing clusterin and hemicentin-1. Finally, neuromuscular junction (NMJ)-associated cells with no adipogenic potential (Leinroth et al., 2022). Additionally, although the number of embryonic Osr1⁺ FAPs declines in the muscle interstitium soon after birth, their progeny contributes to FAP populations in adult muscle, giving rise to Sca1⁺ and PDGFRα⁺ FAPs (Vallecillo-García et al., 2017; Stumm et al., 2018).

Regarding their location, adult FAPs from different muscle locations differ in their number, functional proliferation, and adipogenic properties. Lee et al. (2020d) demonstrated that the masseter, rotator cuff, and paraspinal muscles have a greater proportion of FAPs per gram of tissue than does the gastrocnemius and tibialis anterior muscles of the hindlimb in mice (Lee et al., 2020a). Furthermore, FAPs isolated from these muscles, especially from the shoulder muscles, exhibit differential proliferative activity and adipogenic potency (Lee et al., 2020a), which is relevant considering the rapid onset of IMAT after rotator cuff tears. However, whether the differences in the functional capabilities of FAPs are related to their heterogeneous embryonic origin or whether changes occur later in their developmental history has not been studied. Apart from FAP ontogeny, this functional variability could also be attributed to variable pro-adipogenic patterns across muscle groups, in situ injury-specific cues, intrinsic epigenetic mechanisms, and differences in muscle fiber type composition, innervation, vascularity, and biomechanical function. Given the complexity of the adipogenic fate of muscle FAPs, we propose that all these variables could determine the fate of
FAPs and, therefore, need to be considered when designing in-
terventions to enhance muscle tissue regeneration and repair. Regard-
less of their embryonic origin and location, FAPs are the source of signals
necessary to promote myogenic growth during development and
regeneration, metabolic alterations, and systemic nutritional cues
(Paylor et al., 2014; Mogi et al., 2016; Buras et al., 2019), which dem-
onstrates functional convergence in supporting muscle homeostasis.

3.4. *The potential pro-regenerative role of FAP-derived adipocytes in homeostatic muscle*

Although IMAT accumulation is observed in various pathological conditions, even healthy humans and rodents have small amounts of IMAT in most muscles. However, the physiological role of IMAT has not been determined. Small numbers of adipocytes can be observed in adult muscles, suggesting they may play a supportive and homeostatic role in muscle (Pagano et al., 2018; Sachs et al., 2019; Jakobsen et al., 2021). An example of the potential homeostatic role of FAP-derived adipocytes is illustrated by the perivascular adipocytes found in the myotendinous junction and lining the venous arcade in the diaphragm muscle of mice (Stuelsatz et al., 2012; Sono et al., 2020)). These observations were recently corroborated in different muscle groups of healthy humans and mice (Zhang and Wang 2015; Jakobsen et al., 2021), suggesting an unappreciated role in regulating muscle-tendon transitional zones and myotendinous junctions. In muscle regeneration, genetic elimination of committed/differentiating Ap2/Fabp4⁺ muscle adipocytes results in impaired regeneration (Liu et al., 2012; Dammone et al., 2018). Mice lacking Pparg exhibit no ectopic IMAT formation during muscle regeneration. This absence of IMAT disrupted muscle regeneration and injury-induced MuSC expansion and myogenesis, potentially through alterations in the inflammatory response (Dammone et al., 2018). However, further research is needed to determine the role of FAP-derived adipocytes and their potential contributions to these physiological processes, as well as potential therapeutic applications in muscle maintenance, regeneration and repair.

**3.5. Challenges to the ectopic IMAT dogma: subcutaneous adipose stromal cells as a novel source of IMAT**

Muscle-resident FAPs are considered to be the main cell type of origin for ectopic adipogenesis, yet Sengenès' group challenged this idea by showing that an 8-week HFD can mobilize CXCR4-expressing subcutaneous adipose stromal cells into skeletal muscle (Girousse et al., 2019). This involved the CXCL12/CXCR4 axis, where high CXCL12 levels facilitate retention of adipose stromal cells in subcutaneous adipose tissue (SCAT), whereas low CXCL12 levels favor egress from SCAT. To visualize the trafficking and relocation of these cells in response to a HFD, a piece of subcutaneous adipose tissue from CD34<sup>EGFP</sup> and Ad-Cre/Zs1Green mice was grafted into the SCAT of wild-type mice that were subsequently fed a HFD for 8 weeks (Girousse et al., 2019). First, CD34<sup>+</sup>/CD45<sup>-</sup>/CD31<sup>-</sup>/EGFP<sup>+</sup>/SCA1<sup>+</sup> progenitor cells relocated from the SCAT graft into the quadriceps muscle, together with Zs1Green-labeled adipocytes found in the same muscle group. Remarkably, they found that promoting adipose stromal cell egress from SCAT with the CXCR4 antagonist AMD3100 was sufficient to increase IMAT deposition and to impair glucose tolerance to levels comparable to those of HFD (Girousse et al., 2019). These findings suggest that blocking the mobilization of these stromal cells might prevent ectopic IMAT deposition and metabolic disturbances. However, the function of AMD3100 in muscle-resident FAPs and MuSCs was not further evaluated.

Recently, the same group showed that SCAT-derived stromal cells serve, not only as a source of muscle FAPs in HFD-fed mice, but also after muscle injury. In this situation, SCAT-derived FAPs promoted muscle regeneration in a manner similar to FAPs (Sastourné-Arrey et al., 2023). The authors found that the number of FAPs significantly increased 24 h after CTX or GLY-induced injury in the quadriceps muscle

(Sastourné-Arrey et al., 2023). CD34<sup>EGFP</sup> grafting experiments suggested that the increase in FAP content occurred via injury-induced stromal cell migration from SCAT into muscle and not solely by proliferation of muscle-resident FAPs. They proposed a model in which the podoplanin-mediated interaction between adipose stromal cells and platelets influences the infiltration of SCAT-derived stromal cells into damaged muscles (Sastourné-Arrey et al., 2023). Blocking stromal cell egression by depleting platelets resulted in impaired expression of late myogenic markers and muscle regeneration, suggesting SCAT-derived stromal cells play an early pro-regenerative and supportive role in the damaged muscle microenvironment, as is known for muscle-resident FAPs (Sastourné-Arrey et al., 2023). These findings suggest that SCAT interacts with skeletal muscle through the mobilization of adipogenic progenitors. However, identifying muscle-resident or adipose tissue-exported FAPs using lineage tracing is challenging because these cells share cell-specific markers, such as CD34. Moreover, CD34 is expressed in MuSCs, circulatory cells, and the endothelium (Dooley et al., 2004; Rodeheffer et al., 2008; Pisani et al., 2010b; Mitterberger et al., 2012).

**3.5.1. In search of true adipogenic progenitor cells: a dual fate**

Single-cell and single-nucleus transcriptomics together with mass cytometry advances have enabled an unprecedented description of the transcriptional heterogeneity and protein dynamic transitions of various FAP subpopulations and cell states. For instance, Petrilli et al. (2020) provided a compendium of mass cytometry FAP markers in mice and humans, validating previously described markers. Additional studies have expanded this information and detailed how FAP subpopulations change their transcriptional signatures and secretome depending on their activation and progression during the regenerative process and in response to a HFD or exercise (Malecova et al., 2018; Scott et al., 2019; Muhl et al., 2020; Oprescu et al., 2020; Yang et al., 2021; Fitzgerald et al., 2023). In this section, we summarize the main identity markers and functional properties of different FAP subtypes and states during adult muscle regeneration, repair and aging.

**3.5.1.1. Quiescent FAPs.** In unperturbed adult muscle, quiescent FAPs are identifiable based on their lack of protein markers of endothelial cells (CD31<sup>-</sup>), hematopoietic cells (CD45<sup>-</sup>), myogenic cells (SM/C-2.6<sup>-</sup>, CD56<sup>-</sup>, Pax7<sup>-</sup> and α7-integrin<sup>-</sup>), and erythroid cells (Ter119<sup>-</sup>). Most importantly, FAPs are characterized by high expression levels of mesenchymal stromal cell surface markers, including PDGFRα, SCA1, CD34, the angiopoietin receptor Tie2, and THY-1 (known as CD90), and the transcription factors Osr1 and TCF7L2. Malecova et al. (2018) were the first to utilize single-cell approaches to identify distinct subpopulations of SCA1<sup>+</sup>CD34<sup>+</sup> murine FAPs. These subpopulations were characterized by the expression of the vascular cell adhesion molecule Vcam1 and the angiopoietin-1 receptor Tie2 (Malecova et al., 2018). In healthy adult muscle, most quiescent FAPs expressed low levels of Tie2 (Tie2<sup>low</sup>). Next, based on the differential expression of SCA1 and CD34 at the single-cell level, a follow-up multiparametric mass cytometry study identified distinct subpopulations of muscle FAPs from juvenile (6-week-old) WT mice, CTX-injured muscles, and muscles from mdx mice (Marinkovic et al., 2019), where quiescent FAPs expressed low levels of both antigens (SCA1<sup>low</sup>CD34<sup>low</sup>). In parallel, we distinguished the activation and differentiation states of pro-fibrotic FAPs based on the expression of PDGFRα and TCF7L2, with both markers being highly expressed in a quiescent state (PDGFRα<sup>high</sup>TCF7L2<sup>high</sup> FAPs) (Contreras et al. 2019b, 2020). Upon trauma, mouse and human TCF7L2<sup>+</sup> FAPs are located preferentially in areas surrounding regenerative muscle fibers (Contreras et al., 2016; Mackey et al., 2017). Although many of the aforementioned markers are enriched in FAPs, they are not exclusive to these cells, and their expression level varies along non-homeostatic conditions (reviewed by (Contreras et al., 2021b)). Another two distinct FAP subpopulations were identified in humans using scRNAseq
FAPs and, therefore, need to be considered when designing in-
terventions to enhance muscle tissue regeneration and repair. Regard-
less of their embryonic origin and location, FAPs are the source of signals
necessary to promote myogenic growth during development and
regeneration, metabolic alterations, and systemic nutritional cues
(Paylor et al., 2014; Mogi et al., 2016; Buras et al., 2019), which dem-
onstrates functional convergence in supporting muscle homeostasis.

3.4. The potential pro-regenerative role of FAP-derived adipocytes in homeostatic muscle

Although IMAT accumulation is observed in various pathological conditions, even healthy humans and rodents have small amounts of IMAT in most muscles. However, the physiological role of IMAT has not been determined. Small numbers of adipocytes can be observed in adult muscles, suggesting they may play a supportive and homeostatic role in muscle (Pagano et al., 2018; Sachs et al., 2019; Jakobsen et al., 2021). An example of the potential homeostatic role of FAP-derived adipocytes is illustrated by the perivascular adipocytes found in the myotendinous junction and lining the venous arcade in the diaphragm muscle of mice (Stuelsatz et al., 2012; Sono et al., 2020)). These observations were recently corroborated in different muscle groups of healthy humans and mice (Zhang and Wang 2015; Jakobsen et al., 2021), suggesting an unappreciated role in regulating muscle-tendon transitional zones and myotendinous junctions. In muscle regeneration, genetic elimination of committed/differentiating Ap2/Fabp4⁺ muscle adipocytes results in impaired regeneration (Liu et al., 2012; Dammone et al., 2018). Mice lacking Pparg exhibit no ectopic IMAT formation during muscle regeneration. This absence of IMAT disrupted muscle regeneration and injury-induced MuSC expansion and myogenesis, potentially through alterations in the inflammatory response (Dammone et al., 2018). However, further research is needed to determine the role of FAP-derived adipocytes and their potential contributions to these physiological processes, as well as potential therapeutic applications in muscle maintenance, regeneration and repair.

**3.5. Challenges to the ectopic IMAT dogma: subcutaneous adipose stromal cells as a novel source of IMAT**

Muscle-resident FAPs are considered to be the main cell type of origin for ectopic adipogenesis, yet Sengenès' group challenged this idea by showing that an 8-week HFD can mobilize CXCR4-expressing subcutaneous adipose stromal cells into skeletal muscle (Girousse et al., 2019). This involved the CXCL12/CXCR4 axis, where high CXCL12 levels facilitate retention of adipose stromal cells in subcutaneous adipose tissue (SCAT), whereas low CXCL12 levels favor egress from SCAT. To visualize the trafficking and relocation of these cells in response to a HFD, a piece of subcutaneous adipose tissue from CD34<sup>EGFP</sup> and Ad-Cre/Zs1Green mice was grafted into the SCAT of wild-type mice that were subsequently fed a HFD for 8 weeks (Girousse et al., 2019). First, CD34<sup>+</sup>/CD45<sup>-</sup>/CD31<sup>-</sup>/EGFP<sup>+</sup>/SCA1<sup>+</sup> progenitor cells relocated from the SCAT graft into the quadriceps muscle, together with Zs1Green-labeled adipocytes found in the same muscle group. Remarkably, they found that promoting adipose stromal cell egress from SCAT with the CXCR4 antagonist AMD3100 was sufficient to increase IMAT deposition and to impair glucose tolerance to levels comparable to those of HFD (Girousse et al., 2019). These findings suggest that blocking the mobilization of these stromal cells might prevent ectopic IMAT deposition and metabolic disturbances. However, the function of AMD3100 in muscle-resident FAPs and MuSCs was not further evaluated.

Recently, the same group showed that SCAT-derived stromal cells serve, not only as a source of muscle FAPs in HFD-fed mice, but also after muscle injury. In this situation, SCAT-derived FAPs promoted muscle regeneration in a manner similar to FAPs (Sastourné-Arrey et al., 2023). The authors found that the number of FAPs significantly increased 24 h after CTX or GLY-induced injury in the quadriceps muscle

(Sastourné-Arrey et al., 2023). CD34<sup>EGFP</sup> grafting experiments suggested that the increase in FAP content occurred via injury-induced stromal cell migration from SCAT into muscle and not solely by proliferation of muscle-resident FAPs. They proposed a model in which the podoplanin-mediated interaction between adipose stromal cells and platelets influences the infiltration of SCAT-derived stromal cells into damaged muscles (Sastourné-Arrey et al., 2023). Blocking stromal cell egression by depleting platelets resulted in impaired expression of late myogenic markers and muscle regeneration, suggesting SCAT-derived stromal cells play an early pro-regenerative and supportive role in the damaged muscle microenvironment, as is known for muscle-resident FAPs (Sastourné-Arrey et al., 2023). These findings suggest that SCAT interacts with skeletal muscle through the mobilization of adipogenic progenitors. However, identifying muscle-resident or adipose tissue-exported FAPs using lineage tracing is challenging because these cells share cell-specific markers, such as CD34. Moreover, CD34 is expressed in MuSCs, circulatory cells, and the endothelium (Dooley et al., 2004; Rodeheffer et al., 2008; Pisani et al., 2010b; Mitterberger et al., 2012).

**3.5.1. In search of true adipogenic progenitor cells: a dual fate**

Single-cell and single-nucleus transcriptomics together with mass cytometry advances have enabled an unprecedented description of the transcriptional heterogeneity and protein dynamic transitions of various FAP subpopulations and cell states. For instance, Petrilli et al. (2020) provided a compendium of mass cytometry FAP markers in mice and humans, validating previously described markers. Additional studies have expanded this information and detailed how FAP subpopulations change their transcriptional signatures and secretome depending on their activation and progression during the regenerative process and in response to a HFD or exercise (Malecova et al., 2018; Scott et al., 2019; Muhl et al., 2020; Oprescu et al., 2020; Yang et al., 2021; Fitzgerald et al., 2023). In this section, we summarize the main identity markers and functional properties of different FAP subtypes and states during adult muscle regeneration, repair and aging.

**3.5.1.1. Quiescent FAPs.** In unperturbed adult muscle, quiescent FAPs are identifiable based on their lack of protein markers of endothelial cells (CD31<sup>-</sup>), hematopoietic cells (CD45<sup>-</sup>), myogenic cells (SM/C-2.6<sup>-</sup>, CD56<sup>-</sup>, Pax7<sup>-</sup> and α7-integrin<sup>-</sup>), and erythroid cells (Ter119<sup>-</sup>). Most importantly, FAPs are characterized by high expression levels of mesenchymal stromal cell surface markers, including PDGFRα, SCA1, CD34, the angiopoietin receptor Tie2, and THY-1 (known as CD90), and the transcription factors Osr1 and TCF7L2. Malecova et al. (2018) were the first to utilize single-cell approaches to identify distinct subpopulations of SCA1<sup>+</sup>CD34<sup>+</sup> murine FAPs. These subpopulations were characterized by the expression of the vascular cell adhesion molecule Vcam1 and the angiopoietin-1 receptor Tie2 (Malecova et al., 2018). In healthy adult muscle, most quiescent FAPs expressed low levels of Tie2 (Tie2<sup>low</sup>). Next, based on the differential expression of SCA1 and CD34 at the single-cell level, a follow-up multiparametric mass cytometry study identified distinct subpopulations of muscle FAPs from juvenile (6-week-old) WT mice, CTX-injured muscles, and muscles from mdx mice (Marinkovic et al., 2019), where quiescent FAPs expressed low levels of both antigens (SCA1<sup>low</sup>CD34<sup>low</sup>). In parallel, we distinguished the activation and differentiation states of pro-fibrotic FAPs based on the expression of PDGFRα and TCF7L2, with both markers being highly expressed in a quiescent state (PDGFRα<sup>high</sup>TCF7L2<sup>high</sup> FAPs) (Contreras et al. 2019b, 2020). Upon trauma, mouse and human TCF7L2<sup>+</sup> FAPs are located preferentially in areas surrounding regenerative muscle fibers (Contreras et al., 2016; Mackey et al., 2017). Although many of the aforementioned markers are enriched in FAPs, they are not exclusive to these cells, and their expression level varies along non-homeostatic conditions (reviewed by (Contreras et al., 2021b)). Another two distinct FAP subpopulations were identified in humans using scRNAseq
based on the expression of *FIBRILLIN-1* (FBN1⁺) and *LUMICAN* (LUM⁺) in unperturbed muscle obtained from donors aged 15–75 years (Rubenstein et al., 2020). These subsets differed in their location within muscle and expressed different collagen genes. Whereas FBN1⁺ FAPs are located preferentially in the perimysium, LUM⁺ FAPs are located closer to the endomysium (Rubenstein et al., 2020). Finally, the transcription factor hypermethylated in cancer 1 protein (HIC1) is expressed in quiescent FAPs in muscles and the heart (Scott et al., 2019; Soliman et al., 2020; Contreras et al., 2021b). *Hic1* deletion experiments suggest that HIC1 downregulation is required for the activation of cardiac FAPs post myocardial infarction (Soliman et al., 2020).

### 3.5.1.2. Injury-induced activation of FAPs.
In adult muscles, FAP cell dynamics involve the activation of a quiescent pool of FAPs in response to various myotrauma-related autocrine, paracrine and juxtacrine factors, especially those linked to the rapid acute inflammatory response. In response to acute muscle damage, the composition of the interstitial milieu changes, allowing previously quiescent FAPs to be rapidly activated, enter the cell cycle and proliferate, resulting in a significant increase in their number. This proliferative wave peaks between day 3 and day 5 after injury (Joe et al., 2010; Lemos et al., 2015; Contreras et al. 2019c, 2020) (reviewed by (Theret et al., 2021)). Notably, gene expression analysis revealed that the majority of muscle cycling FAPs exhibit upregulated expression of genes associated with cell cycle progression as early as day 2 following acute injury, including *Mki67*, *Top2a*, *Stmn1*, and *Birc5* (Contreras and Harvey 2023). These findings further support the findings of previous studies using mass spectrometry-proteomic profiling, which revealed increased expression of proteins involved in the cell cycle and DNA replication in FAPs in response to acute muscle damage and muscular dystrophy (Marinkovic et al., 2019; Reggio et al., 2020a).

The expression of mesenchymal cell markers, such as PDGFRα and TCF7L2, which is characteristically high in quiescent cells, is down-regulated during FAP activation in response to tissue damage (Contreras et al. 2019b, 2020). In contrast, the number of SCA1⁺CD34⁺ FAPs and Osr1⁺ FAPs is increased (Vallecillo-García et al., 2017; Stumm et al., 2018; Marinkovic et al., 2019). Together, these transcriptional and proteomic changes indicate the activation of quiescent FAPs. Upon injury, FAPs are also recognized by gene expression patterns that are essential for adult myogenesis, ECM remodeling, and immune cell attraction, leading to efficient muscle regeneration (Scott et al., 2019; De Micheli et al., 2020a; Oprescu et al., 2020; Contreras and Harvey 2023).

Although predominant during embryonic development, Osr1⁺ FAPs are rare in mature muscles, but they rapidly increase in number following acute muscle injury and undergo apoptosis shortly after this stage. Moreover, the decrease in survival of injury-activated Osr1⁺ FAPs can give rise to a portion of TCF7L2⁺ FAPs and interstitial adipocytes after injury (Stumm et al., 2018). These findings suggest that adult Osr1⁺ FAPs can reactivate a developmental program that supports muscle tissue regeneration (Vallecillo-García et al., 2017; Stumm et al., 2018). The pro-regenerative activity of Osr1⁺ FAPs was confirmed to be functionally linked to *Osr1* gene function, as conditional deletion of *Osr1* in FAPs drives excessive FAP fibrogenesis, favoring muscle fibrosis and impairing muscle regeneration (Kotsaris et al., 2023).

### 3.5.1.3. Role of FAPs in early post-injury stage.
During the very early stages of muscle regeneration, activated FAPs dynamically express immunoreactive factors such as CC motif chemokine ligands (Ccl2, Ccl5, and Ccl11), C-X-C motif ligands (Cxcl3, Cxcl5, and Cxcl14), CCN2/CTGF, and Timp1, among other factors necessary for the recruitment, expansion, and maturation of immune cells (Lukjanenko et al., 2019; Scott et al., 2019; De Micheli et al., 2020a; Oprescu et al., 2020; Rubenstein et al., 2020; Contreras et al., 2021b; Contreras and Harvey 2023). This immune-like signature suggests that activated FAPs act as

immunomodulatory cells that engage in active cross-talk with each other and with other muscle-resident and non-resident cells to regenerate muscle and restore function (Contreras et al., 2021b; Theret et al., 2021). Following this immune-regulatory phase, FAPs produce several trophic and myogenic factors that generate a proper cellular milieu for muscle repair and maintenance. First, FAPs produce collagen VI, which helps to maintain MuSC quiescence and self-renewal during muscle regeneration (Urciuolo et al., 2013). Second, in mature muscles, the pro-myogenic role of FAPs favors a pro-regenerative MuSC behavior and fate via the secretion of factors such as insulin-like growth factor-1 (IGF-1), interleukin-6 (IL-6), WNT ligands, follistatin, and bone morphogenetic protein-3b (Bmp3b, or growth differentiation factor 10, Gdf10) (Joe et al., 2010; Mozzetta et al., 2013; Roberts et al., 2013; Farup et al., 2015; Scott et al., 2019; Oprescu et al., 2020; Reggio et al., 2020b; Rubenstein et al., 2020; Uezumi et al., 2021; Norris et al., 2023). Activated FAPs and their progeny actively secrete a plethora of matrix-cellular factors (e.g., CCN2/CTGF, WNT1 inducible signaling pathway protein 1 [WISP1]) and matrisome components, including laminins, collagens, fibronectin, lumican and decorin, among others (Contreras et al., 2016; Mackey et al., 2017; Scott et al., 2019). The secretion of FAP-derived WISP1 is required to induce MuSC proliferation, commitment, and myogenic differentiation (Lukjanenko et al., 2019). This promotes MuSC activation and differentiation, myocyte fusion, and the formation and maintenance of NMJ (Mackey et al., 2017; Uezumi et al., 2021). These are critical steps for maintaining a regenerative and pro-myogenic niche (Heredia et al., 2013; Contreras et al. 2019c, 2020; Scott et al., 2019; Mázala et al., 2020).

### 3.5.1.4. Role of FAPs in advanced post-injury stage.
Three different subsets of FAPs have been described to exist at an advanced postinjury timepoint. First, a subgroup of Tie2<sup>high</sup> FAPs, which exhibit a gene signature critical for muscle regeneration, including genes involved in the chemotaxis of blood cells, maturation of dendritic cells, and neoangiogenesis, was found (Malecova et al., 2018; Endo et al., 2023). Second, a population of FAPs expressing Delta-like Noncanonical NOTCH Ligand 1 (Dlk1, also known as preadipocyte factor-1; Pref-1) appears. This subset of genes exhibited a signature characterized by the expression of genes associated with the complex imprinting pattern, including *B830012L14*, *Rik*, *Meg3*, *Ainr*, *Peg3* [Pw1], *Zim1*, *H19*, and *Igf2*. Whether imprinting plays a role in a specific functional transition has not been studied. Notably, Dlk1/Pref-1 has been identified as a marker of adipose precursors, and ablation of Pref-1-expressing cells was shown to prevent adipose tissue development and expansion, thus demonstrating the requirement of Pref-1⁺ cells for adipogenesis (Hudak et al., 2014). However, whether the Dlk1⁺FAP population represents a pre-adipocyte state during muscle regeneration has yet to be determined. Third, a small fraction of Osr1⁺ FAPs has been described (Stumm et al., 2018; Oprescu et al., 2020). These cells are characterized by the expression of cell signaling-related genes, such as *Ccl1*, *Bmp4*, *Bmp5*, and the anti-adipogenic factor *Wnt5a* (Oprescu et al., 2020). Interestingly, based on the anti-adipogenic effect of recombinant WNT5A and *in vitro* treatment with an inhibitor of the upstream β-catenin kinase glycogen synthase kinase (GSK), Reggio et al. (2020a) suggested that WNT5A serves as an autocrine adipogenic break and that FAPs secrete WNT5A to restrict the development of IMAT at a later time point during regeneration (Reggio et al., 2020b). At the end of regeneration, some Osr1⁺ FAPs diverged into two populations, identified as the dipeptidyl peptidase 4 (Dpp4)⁺ FAP and Cxcl14⁺ FAP subpopulations (Oprescu et al., 2020). Previously, Dpp4-expressing cells were identified as bona fide adipogenic progenitor cells located in the reticular interstitium of white adipose tissue (Merrick et al., 2019). The adipogenic potential of Dpp4⁺ and Cxcl14⁺ FAP subpopulations in skeletal muscle remains to be determined, though we speculate that Dpp4⁺FAPs may exhibit greater adipogenic properties compared to Cxcl14⁺FAPs. Additionally, beyond their differentiation capabilities, Dpp4⁺FAPs appear to possess
based on the expression of *FIBRILLIN-1* (FBN1⁺) and *LUMICAN* (LUM⁺) in unperturbed muscle obtained from donors aged 15–75 years (Rubenstein et al., 2020). These subsets differed in their location within muscle and expressed different collagen genes. Whereas FBN1⁺ FAPs are located preferentially in the perimysium, LUM⁺ FAPs are located closer to the endomysium (Rubenstein et al., 2020). Finally, the transcription factor hypermethylated in cancer 1 protein (HIC1) is expressed in quiescent FAPs in muscles and the heart (Scott et al., 2019; Soliman et al., 2020; Contreras et al., 2021b). *Hic1* deletion experiments suggest that HIC1 downregulation is required for the activation of cardiac FAPs post myocardial infarction (Soliman et al., 2020).

### 3.5.1.2. Injury-induced activation of FAPs.
In adult muscles, FAP cell dynamics involve the activation of a quiescent pool of FAPs in response to various myotrauma-related autocrine, paracrine and juxtacrine factors, especially those linked to the rapid acute inflammatory response. In response to acute muscle damage, the composition of the interstitial milieu changes, allowing previously quiescent FAPs to be rapidly activated, enter the cell cycle and proliferate, resulting in a significant increase in their number. This proliferative wave peaks between day 3 and day 5 after injury (Joe et al., 2010; Lemos et al., 2015; Contreras et al. 2019c, 2020) (reviewed by (Theret et al., 2021)). Notably, gene expression analysis revealed that the majority of muscle cycling FAPs exhibit upregulated expression of genes associated with cell cycle progression as early as day 2 following acute injury, including *Mki67*, *Top2a*, *Stmn1*, and *Birc5* (Contreras and Harvey 2023). These findings further support the findings of previous studies using mass spectrometry-proteomic profiling, which revealed increased expression of proteins involved in the cell cycle and DNA replication in FAPs in response to acute muscle damage and muscular dystrophy (Marinkovic et al., 2019; Reggio et al., 2020a).

The expression of mesenchymal cell markers, such as PDGFRα and TCF7L2, which is characteristically high in quiescent cells, is down-regulated during FAP activation in response to tissue damage (Contreras et al. 2019b, 2020). In contrast, the number of SCA1⁺CD34⁺ FAPs and Osr1⁺ FAPs is increased (Vallecillo-García et al., 2017; Stumm et al., 2018; Marinkovic et al., 2019). Together, these transcriptional and proteomic changes indicate the activation of quiescent FAPs. Upon injury, FAPs are also recognized by gene expression patterns that are essential for adult myogenesis, ECM remodeling, and immune cell attraction, leading to efficient muscle regeneration (Scott et al., 2019; De Micheli et al., 2020a; Oprescu et al., 2020; Contreras and Harvey 2023).

Although predominant during embryonic development, Osr1⁺ FAPs are rare in mature muscles, but they rapidly increase in number following acute muscle injury and undergo apoptosis shortly after this stage. Moreover, the decrease in survival of injury-activated Osr1⁺ FAPs can give rise to a portion of TCF7L2⁺ FAPs and interstitial adipocytes after injury (Stumm et al., 2018). These findings suggest that adult Osr1⁺ FAPs can reactivate a developmental program that supports muscle tissue regeneration (Vallecillo-García et al., 2017; Stumm et al., 2018). The pro-regenerative activity of Osr1⁺ FAPs was confirmed to be functionally linked to *Osr1* gene function, as conditional deletion of *Osr1* in FAPs drives excessive FAP fibrogenesis, favoring muscle fibrosis and impairing muscle regeneration (Kotsaris et al., 2023).

### 3.5.1.3. Role of FAPs in early post-injury stage.
During the very early stages of muscle regeneration, activated FAPs dynamically express immunoreactive factors such as CC motif chemokine ligands (Ccl2, Ccl5, and Ccl11), C-X-C motif ligands (Cxcl3, Cxcl5, and Cxcl14), CCN2/CTGF, and Timp1, among other factors necessary for the recruitment, expansion, and maturation of immune cells (Lukjanenko et al., 2019; Scott et al., 2019; De Micheli et al., 2020a; Oprescu et al., 2020; Rubenstein et al., 2020; Contreras et al., 2021b; Contreras and Harvey 2023). This immune-like signature suggests that activated FAPs act as

immunomodulatory cells that engage in active cross-talk with each other and with other muscle-resident and non-resident cells to regenerate muscle and restore function (Contreras et al., 2021b; Theret et al., 2021). Following this immune-regulatory phase, FAPs produce several trophic and myogenic factors that generate a proper cellular milieu for muscle repair and maintenance. First, FAPs produce collagen VI, which helps to maintain MuSC quiescence and self-renewal during muscle regeneration (Urciuolo et al., 2013). Second, in mature muscles, the pro-myogenic role of FAPs favors a pro-regenerative MuSC behavior and fate via the secretion of factors such as insulin-like growth factor-1 (IGF-1), interleukin-6 (IL-6), WNT ligands, follistatin, and bone morphogenetic protein-3b (Bmp3b, or growth differentiation factor 10, Gdf10) (Joe et al., 2010; Mozzetta et al., 2013; Roberts et al., 2013; Farup et al., 2015; Scott et al., 2019; Oprescu et al., 2020; Reggio et al., 2020b; Rubenstein et al., 2020; Uezumi et al., 2021; Norris et al., 2023). Activated FAPs and their progeny actively secrete a plethora of matrix-cellular factors (e.g., CCN2/CTGF, WNT1 inducible signaling pathway protein 1 [WISP1]) and matrisome components, including laminins, collagens, fibronectin, lumican and decorin, among others (Contreras et al., 2016; Mackey et al., 2017; Scott et al., 2019). The secretion of FAP-derived WISP1 is required to induce MuSC proliferation, commitment, and myogenic differentiation (Lukjanenko et al., 2019). This promotes MuSC activation and differentiation, myocyte fusion, and the formation and maintenance of NMJ (Mackey et al., 2017; Uezumi et al., 2021). These are critical steps for maintaining a regenerative and pro-myogenic niche (Heredia et al., 2013; Contreras et al. 2019c, 2020; Scott et al., 2019; Mázala et al., 2020).

### 3.5.1.4. Role of FAPs in advanced post-injury stage.
Three different subsets of FAPs have been described to exist at an advanced postinjury timepoint. First, a subgroup of Tie2<sup>high</sup> FAPs, which exhibit a gene signature critical for muscle regeneration, including genes involved in the chemotaxis of blood cells, maturation of dendritic cells, and neoangiogenesis, was found (Malecova et al., 2018; Endo et al., 2023). Second, a population of FAPs expressing Delta-like Noncanonical NOTCH Ligand 1 (Dlk1, also known as preadipocyte factor-1; Pref-1) appears. This subset of genes exhibited a signature characterized by the expression of genes associated with the complex imprinting pattern, including *B830012L14*, *Rik*, *Meg3*, *Ainr*, *Peg3* [Pw1], *Zim1*, *H19*, and *Igf2*. Whether imprinting plays a role in a specific functional transition has not been studied. Notably, Dlk1/Pref-1 has been identified as a marker of adipose precursors, and ablation of Pref-1-expressing cells was shown to prevent adipose tissue development and expansion, thus demonstrating the requirement of Pref-1⁺ cells for adipogenesis (Hudak et al., 2014). However, whether the Dlk1⁺FAP population represents a pre-adipocyte state during muscle regeneration has yet to be determined. Third, a small fraction of Osr1⁺ FAPs has been described (Stumm et al., 2018; Oprescu et al., 2020). These cells are characterized by the expression of cell signaling-related genes, such as *Ccl1*, *Bmp4*, *Bmp5*, and the anti-adipogenic factor *Wnt5a* (Oprescu et al., 2020). Interestingly, based on the anti-adipogenic effect of recombinant WNT5A and *in vitro* treatment with an inhibitor of the upstream β-catenin kinase glycogen synthase kinase (GSK), Reggio et al. (2020a) suggested that WNT5A serves as an autocrine adipogenic break and that FAPs secrete WNT5A to restrict the development of IMAT at a later time point during regeneration (Reggio et al., 2020b). At the end of regeneration, some Osr1⁺ FAPs diverged into two populations, identified as the dipeptidyl peptidase 4 (Dpp4)⁺ FAP and Cxcl14⁺ FAP subpopulations (Oprescu et al., 2020). Previously, Dpp4-expressing cells were identified as bona fide adipogenic progenitor cells located in the reticular interstitium of white adipose tissue (Merrick et al., 2019). The adipogenic potential of Dpp4⁺ and Cxcl14⁺ FAP subpopulations in skeletal muscle remains to be determined, though we speculate that Dpp4⁺FAPs may exhibit greater adipogenic properties compared to Cxcl14⁺FAPs. Additionally, beyond their differentiation capabilities, Dpp4⁺FAPs appear to possess
neuroprotective effects, playing a critical role in the maturation and maintenance of the neuromuscular system (Kim et al., 2022). This neuroprotective function is mediated through the expression of Survival of Motor Neuron protein (SMN), which is essential for the proper function and survival of motor neurons. Through the expression of the SMN-stabilizing deubiquitinase Bap1, Dpp4<sup>+</sup> FAPs influence NMJ maturation and motor neuron homeostasis (Kim et al., 2022).

### 3.5.1.5. Characteristics of pathology-associated FAP subtypes.
In pathological settings, FAP states, subtypes, and selective evolution are usually modulated by the microenvironment. In contrast to the critical transition to a pro-myogenic supporting role in muscle regeneration, the persistence of activated and proliferative FAPs beyond a critical period during muscle repair allows the survival of (an) inducible subpopulation (s) of FAPs capable of responding to prevailing inflammatory, profibrotic and/or pro-adipogenic signals, which should define their functional capabilities that may ultimately compromise muscle regeneration and metabolic homeostasis through the formation of fibrosis and IMAT. Malecova et al. (2018) first described a pro-fibrotic-like Vcam1<sup>+</sup> FAP subgroup appearing in association with a widespread inflammatory response to acute muscle damage and in dystrophic muscles from 2- to 3-month-old wild-type and *mdx* mice (Malcova et al., 2018). Additionally, FAPs from dystrophic mice exhibited a robust adipogenic phenotype *in vivo* and *in vitro*, a phenotype linked to the loss of NOTCH-mediated repression of adipogenesis (Mozzetta et al., 2013; Marinkovic et al., 2019).

Giuliani et al. (2021) described dynamic SCA1 protein expression within FAPs, enabling a SCA1-driven micro-heterogeneity that influences their fate (Giuliani et al., 2021). SCA1<sup>high</sup> FAPs exhibit greater potential for proliferation and adipogenesis than SCA1<sup>low</sup> FAPs. Additionally, adiponectin expression was highly upregulated in SCA1<sup>high</sup> FAPs, suggesting that the autocrine factor adiponectin was responsible for their greater adipogenesis. Additionally, *Dpp4* expression was increased in SCA1<sup>high</sup> FAPs (Giuliani et al., 2021). Several studies have demonstrated that immune cells play a crucial role in regulating FAP function and fate. Giuliani et al. (2021) showed that conditioned media from CD45<sup>+</sup> leucocytes isolated from young dystrophic mice inhibited the adipogenic differentiation of SCA1<sup>low</sup> and SCA1<sup>high</sup> FAPs. However, leukocyte-conditioned media from old dystrophic mice was less effective at limiting SCA1<sup>high</sup> FAP adipogenesis, which could help explain the appearance of IMAT in muscles of *mdx* mice, dystrophic patients and chronic diseased individuals (Fig. 2B).

The frequency of TCF7L2<sup>medium/low</sup> differentiated FAPs increases concomitantly with ECM deposition and fibrosis in dystrophic and chronically inflamed muscle (Contreras et al., 2020). *In vitro* passaging of muscle FAPs causes a reduction in PDGFRα protein expression, likely resulting in myofibroblastic phenotypic changes that lead to the downregulation of FAPs as they lose their progenitor identity (Contreras et al., 2019c). This dynamic downregulation of PDGFRα and TCF7L2 can also be mediated by the pro-fibrotic TGF-β axis (Contreras et al. 2019a, 2020), which results in FAP activation under pathological fibrotic conditions and a loss of progenitor identity and myofibroblast transition. Interestingly, a recent study showed that fibrogenic potency of FAPs is partially regulated by ECM architecture and stiffness, and that FAPs can sense mechanic constraints via activation of the Yes-associated protein YAP (product of the *Yap1* gene) signaling (Loomis et al., 2022).

In human skeletal muscles from patients with T2DM, two FAP subpopulations with distinct phenotypic and molecular signatures were identified based on CD90 levels (Farup et al., 2021). A subset of Lin<sup>-</sup>/CD56<sup>-</sup>/CD82<sup>-</sup>/CD34<sup>+</sup>/CD90<sup>+</sup> FAPs was associated with muscle fibrosis and insulin resistance in degenerative T2DM settings (Farup et al., 2021). Compared with CD90<sup>-</sup> FAPs, CD90<sup>+</sup> FAPs are larger, proliferate faster, express more ECM genes, and exhibit greater *in vitro* glycolytic flux and oxygen consumption (Farup et al., 2021). After PDGF-AA treatment, CD90<sup>+</sup> FAPs exhibit enhanced fibrogenesis but

reduced adipogenesis, whereas CD90<sup>-</sup> FAPs exhibit enhanced adipogenesis (Farup et al., 2021). Additionally, FAPs isolated from muscle biopsies of patients with oculopharyngeal muscular dystrophy showed high proliferative and pro-fibrotic capacities in response to TGF-β profibrotic signal and endothelin. Furthermore, in co-culture experiments, fibrotic FAPs impair the formation of myotubes by interfering with fusion of myogenic cells (Bensalah et al., 2022).

From a therapeutic perspective, metformin reduced proliferation, oxygen consumption, and adipogenesis but increased the glycolytic flux of CD90<sup>+</sup> FAPs (Farup et al., 2021). Metformin is widely used as a frontline pharmacological approach for treating T2DM, mainly due to its glucose- and lipid-lowering effects, which reduce overall cardiovascular morbidity and mortality (Zhou et al., 2001; Gormsen et al., 2018). The effects of metformin are primarily attributed to the activation of AMP-dependent protein kinase (AMPK) (Rena et al., 2017). A recent study demonstrated that selective deletion of the AMPK subunit α1 in FAPs impaired muscle regeneration following injury and induced FAP fibrogenesis, suggesting metformin exerts an anti-fibrotic effect through AMPK signaling (Liu et al., 2023). Thus, identifying distinct FAP subsets based on their sensitivity to metformin could lead to the identification of a novel druggable cell-based target for reducing fibrosis and IMAT deposition in T2DM patients. Using mass cytometry, Petrilli et al. (2020) showed that CD90.2 identifies murine FAPs together with SCA1, CD34, PDGFRα, and vimentin (Petrilli et al., 2020) and that CD90 levels are associated with a pro-adipogenic Vcam1<sup>+</sup> FAP subpopulation (Joe et al., 2010; Pisani et al., 2010b; Roberts et al., 2013).

In relation to obesity, Goodpaster et al. (2023) used scRNA-seq to profile 22 major subtypes of mesenchymal stromal cells from subcutaneous and visceral white adipose tissues, and skeletal muscle in response to HFD-induced obesity and exercise (Yang et al., 2022). Among these subtypes, seven muscle FAPs were identified: multipotent IPC_SkM, Cxcl14<sup>+</sup> FAPs, proteoglycan-4<sup>+</sup> FAPs (Prg4<sup>+</sup>), adipogenesis-regulating CD142<sup>+</sup> FAPs, inflammatory post-injury-like FAPs, mesoangioblasts Alpl<sup>+</sup> FAPs (MAB), and previously unreported MSC-derived pro-fibrotic Sca1<sup>-</sup> FAPs. Among these cells, IPC_SkM and CD142<sup>+</sup> FAPs were highly responsive to a HFD, exercise and the combination of these intervention, namely rescue intervention, (Yang et al., 2022). In single-cell transcriptomics, the rescue intervention led to 203 DEG versus 74 in exercise and 23 in HFD alone, mainly compromising IPC_SkM and FAP_CD142<sup>+</sup> cells. In these cells, HFD upregulated the expression of genes involved in processes such as ECM organization, fat cell differentiation, inflammatory signaling pathways (IL-18 and cytokine, NF-κB), regulation of proteolysis, and circadian rhythm. On the other hand, training, with or without HFD, downregulated adipocyte differentiation preferentially in CD142<sup>+</sup> FAPs, together with reducing ECM organization, inflammation-related pathways, while upregulating circadian pathways (Yang et al., 2022).

In the context of muscle atrophy and cachexia, the activation of the canonical WNT-β-catenin pathway is increased and leads to FAP-mediated fibrotic degeneration of muscle and myofiber atrophy (Kajabadi et al., 2023). Moreover, in transgenic mice engineered to harbor degradation-resistant β-catenin in FAPs, the constitutive accumulation of β-catenin led to fibrosis and rapid loss of muscle mass and function (Kajabadi et al., 2023). Indeed, activated FAPs had increased expression of atrogenic genes such as *Foxo1*, and secretion of the atrogenic factors of NOGGIN and ACTIVIN-A, a member of the TGF-β family ligands. Mechanistically, ACTIVIN-A binds to the type II activin receptor ACTVR2, potentially leading to increased SMAD2/3 activity in muscle fibers and ultimately, myofiber atrophy (Kajabadi et al., 2023).

Finally, a recent study by Fitzgerald et al. (2023) used single-nuclei RNAseq to identify three FAP subpopulations in the hip muscles from patients who underwent total hip replacement. These cells were MME<sup>+</sup>, GPC3<sup>+</sup> and CD55<sup>+</sup> FAPs (Fitzgerald et al., 2023), and the MME<sup>+</sup> subset differed from bona fide adipose tissue progenitors such as ICAM1<sup>+</sup> and CD142<sup>+</sup>. MME<sup>+</sup> FAPs expressed higher levels of *Cebpa* and *Ppary* along with downregulated WNT ligand biogenesis, trafficking, and canonical
neuroprotective effects, playing a critical role in the maturation and maintenance of the neuromuscular system (Kim et al., 2022). This neuroprotective function is mediated through the expression of Survival of Motor Neuron protein (SMN), which is essential for the proper function and survival of motor neurons. Through the expression of the SMN-stabilizing deubiquitinase Bap1, Dpp4<sup>+</sup> FAPs influence NMJ maturation and motor neuron homeostasis (Kim et al., 2022).

### 3.5.1.5. Characteristics of pathology-associated FAP subtypes.
In pathological settings, FAP states, subtypes, and selective evolution are usually modulated by the microenvironment. In contrast to the critical transition to a pro-myogenic supporting role in muscle regeneration, the persistence of activated and proliferative FAPs beyond a critical period during muscle repair allows the survival of (an) inducible subpopulation (s) of FAPs capable of responding to prevailing inflammatory, profibrotic and/or pro-adipogenic signals, which should define their functional capabilities that may ultimately compromise muscle regeneration and metabolic homeostasis through the formation of fibrosis and IMAT. Malecova et al. (2018) first described a pro-fibrotic-like Vcam1<sup>+</sup> FAP subgroup appearing in association with a widespread inflammatory response to acute muscle damage and in dystrophic muscles from 2- to 3-month-old wild-type and *mdx* mice (Malcova et al., 2018). Additionally, FAPs from dystrophic mice exhibited a robust adipogenic phenotype *in vivo* and *in vitro*, a phenotype linked to the loss of NOTCH-mediated repression of adipogenesis (Mozzetta et al., 2013; Marinkovic et al., 2019).

Giuliani et al. (2021) described dynamic SCA1 protein expression within FAPs, enabling a SCA1-driven micro-heterogeneity that influences their fate (Giuliani et al., 2021). SCA1<sup>high</sup> FAPs exhibit greater potential for proliferation and adipogenesis than SCA1<sup>low</sup> FAPs. Additionally, adiponectin expression was highly upregulated in SCA1<sup>high</sup> FAPs, suggesting that the autocrine factor adiponectin was responsible for their greater adipogenesis. Additionally, *Dpp4* expression was increased in SCA1<sup>high</sup> FAPs (Giuliani et al., 2021). Several studies have demonstrated that immune cells play a crucial role in regulating FAP function and fate. Giuliani et al. (2021) showed that conditioned media from CD45<sup>+</sup> leucocytes isolated from young dystrophic mice inhibited the adipogenic differentiation of SCA1<sup>low</sup> and SCA1<sup>high</sup> FAPs. However, leukocyte-conditioned media from old dystrophic mice was less effective at limiting SCA1<sup>high</sup> FAP adipogenesis, which could help explain the appearance of IMAT in muscles of *mdx* mice, dystrophic patients and chronic diseased individuals (Fig. 2B).

The frequency of TCF7L2<sup>medium/low</sup> differentiated FAPs increases concomitantly with ECM deposition and fibrosis in dystrophic and chronically inflamed muscle (Contreras et al., 2020). *In vitro* passaging of muscle FAPs causes a reduction in PDGFRα protein expression, likely resulting in myofibroblastic phenotypic changes that lead to the downregulation of FAPs as they lose their progenitor identity (Contreras et al., 2019c). This dynamic downregulation of PDGFRα and TCF7L2 can also be mediated by the pro-fibrotic TGF-β axis (Contreras et al. 2019a, 2020), which results in FAP activation under pathological fibrotic conditions and a loss of progenitor identity and myofibroblast transition. Interestingly, a recent study showed that fibrogenic potency of FAPs is partially regulated by ECM architecture and stiffness, and that FAPs can sense mechanic constraints via activation of the Yes-associated protein YAP (product of the *Yap1* gene) signaling (Loomis et al., 2022).

In human skeletal muscles from patients with T2DM, two FAP subpopulations with distinct phenotypic and molecular signatures were identified based on CD90 levels (Farup et al., 2021). A subset of Lin<sup>-</sup>/CD56<sup>-</sup>/CD82<sup>-</sup>/CD34<sup>+</sup>/CD90<sup>+</sup> FAPs was associated with muscle fibrosis and insulin resistance in degenerative T2DM settings (Farup et al., 2021). Compared with CD90<sup>-</sup> FAPs, CD90<sup>+</sup> FAPs are larger, proliferate faster, express more ECM genes, and exhibit greater *in vitro* glycolytic flux and oxygen consumption (Farup et al., 2021). After PDGF-AA treatment, CD90<sup>+</sup> FAPs exhibit enhanced fibrogenesis but

reduced adipogenesis, whereas CD90<sup>-</sup> FAPs exhibit enhanced adipogenesis (Farup et al., 2021). Additionally, FAPs isolated from muscle biopsies of patients with oculopharyngeal muscular dystrophy showed high proliferative and pro-fibrotic capacities in response to TGF-β profibrotic signal and endothelin. Furthermore, in co-culture experiments, fibrotic FAPs impair the formation of myotubes by interfering with fusion of myogenic cells (Bensalah et al., 2022).

From a therapeutic perspective, metformin reduced proliferation, oxygen consumption, and adipogenesis but increased the glycolytic flux of CD90<sup>+</sup> FAPs (Farup et al., 2021). Metformin is widely used as a frontline pharmacological approach for treating T2DM, mainly due to its glucose- and lipid-lowering effects, which reduce overall cardiovascular morbidity and mortality (Zhou et al., 2001; Gormsen et al., 2018). The effects of metformin are primarily attributed to the activation of AMP-dependent protein kinase (AMPK) (Rena et al., 2017). A recent study demonstrated that selective deletion of the AMPK subunit α1 in FAPs impaired muscle regeneration following injury and induced FAP fibrogenesis, suggesting metformin exerts an anti-fibrotic effect through AMPK signaling (Liu et al., 2023). Thus, identifying distinct FAP subsets based on their sensitivity to metformin could lead to the identification of a novel druggable cell-based target for reducing fibrosis and IMAT deposition in T2DM patients. Using mass cytometry, Petrilli et al. (2020) showed that CD90.2 identifies murine FAPs together with SCA1, CD34, PDGFRα, and vimentin (Petrilli et al., 2020) and that CD90 levels are associated with a pro-adipogenic Vcam1<sup>+</sup> FAP subpopulation (Joe et al., 2010; Pisani et al., 2010b; Roberts et al., 2013).

In relation to obesity, Goodpaster et al. (2023) used scRNA-seq to profile 22 major subtypes of mesenchymal stromal cells from subcutaneous and visceral white adipose tissues, and skeletal muscle in response to HFD-induced obesity and exercise (Yang et al., 2022). Among these subtypes, seven muscle FAPs were identified: multipotent IPC_SkM, Cxcl14<sup>+</sup> FAPs, proteoglycan-4<sup>+</sup> FAPs (Prg4<sup>+</sup>), adipogenesis-regulating CD142<sup>+</sup> FAPs, inflammatory post-injury-like FAPs, mesoangioblasts Alpl<sup>+</sup> FAPs (MAB), and previously unreported MSC-derived pro-fibrotic Sca1<sup>-</sup> FAPs. Among these cells, IPC_SkM and CD142<sup>+</sup> FAPs were highly responsive to a HFD, exercise and the combination of these intervention, namely rescue intervention, (Yang et al., 2022). In single-cell transcriptomics, the rescue intervention led to 203 DEG versus 74 in exercise and 23 in HFD alone, mainly compromising IPC_SkM and FAP_CD142<sup>+</sup> cells. In these cells, HFD upregulated the expression of genes involved in processes such as ECM organization, fat cell differentiation, inflammatory signaling pathways (IL-18 and cytokine, NF-κB), regulation of proteolysis, and circadian rhythm. On the other hand, training, with or without HFD, downregulated adipocyte differentiation preferentially in CD142<sup>+</sup> FAPs, together with reducing ECM organization, inflammation-related pathways, while upregulating circadian pathways (Yang et al., 2022).

In the context of muscle atrophy and cachexia, the activation of the canonical WNT-β-catenin pathway is increased and leads to FAP-mediated fibrotic degeneration of muscle and myofiber atrophy (Kajabadi et al., 2023). Moreover, in transgenic mice engineered to harbor degradation-resistant β-catenin in FAPs, the constitutive accumulation of β-catenin led to fibrosis and rapid loss of muscle mass and function (Kajabadi et al., 2023). Indeed, activated FAPs had increased expression of atrogenic genes such as *Foxo1*, and secretion of the atrogenic factors of NOGGIN and ACTIVIN-A, a member of the TGF-β family ligands. Mechanistically, ACTIVIN-A binds to the type II activin receptor ACTVR2, potentially leading to increased SMAD2/3 activity in muscle fibers and ultimately, myofiber atrophy (Kajabadi et al., 2023).

Finally, a recent study by Fitzgerald et al. (2023) used single-nuclei RNAseq to identify three FAP subpopulations in the hip muscles from patients who underwent total hip replacement. These cells were MME<sup>+</sup>, GPC3<sup>+</sup> and CD55<sup>+</sup> FAPs (Fitzgerald et al., 2023), and the MME<sup>+</sup> subset differed from bona fide adipose tissue progenitors such as ICAM1<sup>+</sup> and CD142<sup>+</sup>. MME<sup>+</sup> FAPs expressed higher levels of *Cebpa* and *Ppary* along with downregulated WNT ligand biogenesis, trafficking, and canonical
WNT signaling (Fitzgerald et al., 2023). Notably, the MME<sup>+</sup> FAPs had a markedly greater adipogenic capacity under minimal *in vitro* adipogenesis (only insulin). Cell tracing experiments demonstrated that MME<sup>+</sup> FAPs readily contribute to IMAT after GLY-induced muscle injury, and also express genes involved in vascular processes and angiogenesis. These characteristics resembled those of previously characterized LUM<sup>+</sup> FAPs and muscle adipocytes (De Micheli et al., 2020b; Rubenstein et al., 2020). Interestingly, compared with MME<sup>-</sup> FAPs, MME<sup>+</sup> FAPs underwent selective apoptosis under regenerative conditions. In addition, at a later timepoint during regeneration, the basal number of MME<sup>+</sup> FAPs was replenished by the transition of MME<sup>-</sup> FAPs to MME<sup>+</sup> FAPs (Fitzgerald et al., 2023). Finally, in muscles of the hip affected with OA that were chronically infiltrated with IMAT, there was a reduced number of MME<sup>+</sup> FAPs, suggesting that FAP heterogeneity and composition are reduced, most likely due to their differentiation to fibro-fatty lineages (Fitzgerald et al., 2023).

# 4. Mechanisms regulating FAP activation, proliferation and survival

## 4.1. Immune cross-talk

The activation and proliferation of FAPs are primarily orchestrated by cell autonomous and cross-talk mechanisms with other stromal cells, endothelial cells, myofibers, and immune cells (Heredia et al., 2013; Mackey et al., 2017; Moratal et al., 2018; Mathes et al., 2021). Following muscle injury, infiltrating eosinophils secrete the type 2 innate cytokines interleukin-4 (IL-4) and interleukin-13 (IL-13), which induce the expression of IL-4 receptor-alpha (IL-4Rα) in FAPs. Activation of the IL-4Rα/STAT6 axis promotes FAP proliferation but inhibits adipogenic differentiation (Heredia et al., 2013). Moreover, IL-4-deficient mice exhibit increased IMAT in regenerating muscle, while treatment with IL-4 prevents the pro-adipogenic effects of adipogenic media on isolated FAPs (Heredia et al., 2013; Dong et al., 2014). Notably, initial immune infiltration and tissue granulation could be mediated by cytokines and chemokines secreted by FAPs one day after injury, indicating that FAPs are immunomodulatory cells (Scott et al., 2019; Theret et al., 2021).

## 4.2. FAP death is required for proper muscle regeneration

While the initial phase of muscle regeneration involves FAP expansion, the later phase requires freeing the space to replenish muscle with de novo myofibers. This requires eliminating a pool of the expanded FAPs after they accomplish their early pro-regenerative role, followed by the clearance of FAP debris. This progression relies partly on the cross-talk between FAPs and macrophages and the opposing effects of TNF-α and TGF-β signaling (Lemos et al., 2015; Tidball 2017). TNF-α signaling triggers FAP apoptosis, which relies on the upregulation of TNF-receptor superfamily members (*Tnfrsf*) and the downregulation of TGF-β signaling (Lemos et al., 2015; Fiore et al., 2016; Tidball 2017). Two studies have dissected this mechanism in fibroblasts (Wynes et al., 2011; Liu et al., 2017). First, TNF-α upregulated the expression of the *Tnfrsf* member cell surface death receptor Fas (Wynes et al., 2011). Second, upon TNF-α-induced activation, Fas trimerizes and translocates to lipid raft micro-domains at the plasma membrane, where it interacts with Thy1/CD90 and triggers apoptosis by blocking the anti-apoptotic effects of Bcl2 and Bcl-xL and by increasing caspase activation (Liu et al., 2017). Nonetheless, it is unknown whether CD90<sup>-</sup> FAPs are resistant to apoptosis.

## 4.3. Pro-survival signals regulating apoptosis-dependent FAP clearance

The accumulation of apoptosis-resistant FAPs has been shown in a mouse model of chronic inflammatory myopathy (Saito et al., 2020). Moreover, anti-apoptotic FAPs reduce gene expression of *Tnfrsf*, including *Fas*; increase gene expression of all TGF-β isoforms; and

upregulate *Tgfbr1* and *Tgfbr2* (Saito et al., 2020). In contrast, acute muscle injury triggered a senescence-like phenotype characterized by upregulation of *Cdkn2a* and *Trp53* expression and the histone variant γH2A.X (102). There is additional evidence for impaired FAP apoptosis in chronic conditions, as observed in *mdx* mice. FAPs isolated from dystrophic muscles exhibit a reduced apoptosis signaling pathway along with an upregulation of fibrogenic genes (Malecova et al., 2018). These findings support the notion that the microenvironment of chronic muscle disease favors anti-apoptotic FAP phenotypes that ultimately drive pathology.

Chronic diseases increase the proliferation of FAPs through chronic activation of mitogenic factors. TGF-β and PDGF ligands are potent mitogens that both induce FAP activation, proliferation, and differentiation (Lemos et al., 2015; Mueller et al., 2016; Contreras et al., 2019a; Mázala et al., 2020; Theret et al., 2021). These mitogens may be potentiated by interaction with the ECM glycoprotein thrombospondin-1 (TSP-1), product of the *Thsb1* gene (Belotti et al., 2016). TSP-1 expression is increased in the blood and muscle tissue of patients affected by brachio-cervical inflammatory myopathy and systemic sclerosis (BCIM-SSc), where muscle *Thbs1* expression is positively correlated with tissue fibrosis and infiltration of macrophages in the muscle (Suárez-Calvet et al., 2020). In addition, *in vitro* treatment with recombinant TSP-1, serum from BCIM-SSc patients or the supernatant of previously treated cells increased the proliferation of isolated human muscle-derived fibroblasts and TGF-β levels (Suárez-Calvet et al., 2020). Recent findings uncovered that FAPs secrete TSP-1 in response to the mechanical stress of surrounding ECM. This response is mediated by the transcriptional activation of YAP/TAZ (TAZ being the gene product of *Wwtr1*, encoding a transcriptional coactivator). In contrast to the known pro-fibrotic effect of TSP-1, this study describes a new role of FAP-secreted TSP-1, which acts by activating signaling by the CD47 receptor in MuSC, thereby driving their proliferation, and a pro-myogenic effect. This mechanism is critical to supply new myogenic nuclei for the enlargement of muscle fibers during load-related muscle hypertrophy (Kaneshige et al., 2022).

TGF-β is initially secreted in its latent, non-active pro-cytokine form, which is activated through a complex mechanism involving binding to TSP-1 and other factors (Crawford et al., 1998; Murphy-Ullrich and Suto 2018; Contreras et al., 2021b). TSP-1 binds to latent TGF-β through its type 1 repeats (KRFK sequence) and subsequently disrupts a conserved inhibitory sequence (LSKL) on TGF-β, thereby leading to TGF-β activation (Murphy-Ullrich and Suto 2018). Interestingly, hypertrophied visceral fat adipocytes have been identified as the primary source of circulating TSP-1 in individuals with insulin resistance or obesity and in HFD-fed mice (Varma et al., 2008; Buras et al., 2019). Elevated circulating levels of TSP-1 have also been associated with an increased risk of coronary and peripheral artery disease, T2DM, all-cause mortality, and adverse outcomes in chronic kidney failure patients (Smadja et al., 2011; Choi et al., 2012). Conversely, *Thbs1*-null mice are protected against obesity-induced metabolic alterations, largely by preventing fibrotic muscle degeneration (Inoue et al., 2013). In addition, dystrophic muscles from juvenile D2-*mdx* mice, a murine model of DMD, are characterized by a tonic increased in TGF-β and inflammatory responses to muscle damage, which is associated with worsening of histopathological changes (i.e., fibrosis and bone-like areas) and reduced regeneration (Mázala et al., 2023). Conversely, adult D2-*mdx* mice, despite exhibiting increased fibrosis, showed an improved muscle regeneration capacity. The differences between muscles of juvenile and adult dystrophic mice in terms of regenerative potential were associated with a predominant inflammatory niche in juvenile, characterized by increased expression of TNF-α, increased pro-inflammatory versus pro-regenerative macrophage activation, an impaired myogenic response, and elevated expression of pro-fibrotic markers such as Periostin and Osteopontin (Mázala et al., 2023). Interestingly, *in vitro* MuSC fusion and myotube formation were impaired when co-cultured with FAPs isolated from muscles of juvenile D2-*mdx* dystrophic mice (Mázala et al., 2023).
WNT signaling (Fitzgerald et al., 2023). Notably, the MME<sup>+</sup> FAPs had a markedly greater adipogenic capacity under minimal *in vitro* adipogenesis (only insulin). Cell tracing experiments demonstrated that MME<sup>+</sup> FAPs readily contribute to IMAT after GLY-induced muscle injury, and also express genes involved in vascular processes and angiogenesis. These characteristics resembled those of previously characterized LUM<sup>+</sup> FAPs and muscle adipocytes (De Micheli et al., 2020b; Rubenstein et al., 2020). Interestingly, compared with MME<sup>-</sup> FAPs, MME<sup>+</sup> FAPs underwent selective apoptosis under regenerative conditions. In addition, at a later timepoint during regeneration, the basal number of MME<sup>+</sup> FAPs was replenished by the transition of MME<sup>-</sup> FAPs to MME<sup>+</sup> FAPs (Fitzgerald et al., 2023). Finally, in muscles of the hip affected with OA that were chronically infiltrated with IMAT, there was a reduced number of MME<sup>+</sup> FAPs, suggesting that FAP heterogeneity and composition are reduced, most likely due to their differentiation to fibro-fatty lineages (Fitzgerald et al., 2023).

# 4. Mechanisms regulating FAP activation, proliferation and survival

## 4.1. Immune cross-talk

The activation and proliferation of FAPs are primarily orchestrated by cell autonomous and cross-talk mechanisms with other stromal cells, endothelial cells, myofibers, and immune cells (Heredia et al., 2013; Mackey et al., 2017; Moratal et al., 2018; Mathes et al., 2021). Following muscle injury, infiltrating eosinophils secrete the type 2 innate cytokines interleukin-4 (IL-4) and interleukin-13 (IL-13), which induce the expression of IL-4 receptor-alpha (IL-4Rα) in FAPs. Activation of the IL-4Rα/STAT6 axis promotes FAP proliferation but inhibits adipogenic differentiation (Heredia et al., 2013). Moreover, IL-4-deficient mice exhibit increased IMAT in regenerating muscle, while treatment with IL-4 prevents the pro-adipogenic effects of adipogenic media on isolated FAPs (Heredia et al., 2013; Dong et al., 2014). Notably, initial immune infiltration and tissue granulation could be mediated by cytokines and chemokines secreted by FAPs one day after injury, indicating that FAPs are immunomodulatory cells (Scott et al., 2019; Theret et al., 2021).

## 4.2. FAP death is required for proper muscle regeneration

While the initial phase of muscle regeneration involves FAP expansion, the later phase requires freeing the space to replenish muscle with de novo myofibers. This requires eliminating a pool of the expanded FAPs after they accomplish their early pro-regenerative role, followed by the clearance of FAP debris. This progression relies partly on the cross-talk between FAPs and macrophages and the opposing effects of TNF-α and TGF-β signaling (Lemos et al., 2015; Tidball 2017). TNF-α signaling triggers FAP apoptosis, which relies on the upregulation of TNF-receptor superfamily members (*Tnfrsf*) and the downregulation of TGF-β signaling (Lemos et al., 2015; Fiore et al., 2016; Tidball 2017). Two studies have dissected this mechanism in fibroblasts (Wynes et al., 2011; Liu et al., 2017). First, TNF-α upregulated the expression of the *Tnfrsf* member cell surface death receptor Fas (Wynes et al., 2011). Second, upon TNF-α-induced activation, Fas trimerizes and translocates to lipid raft micro-domains at the plasma membrane, where it interacts with Thy1/CD90 and triggers apoptosis by blocking the anti-apoptotic effects of Bcl2 and Bcl-xL and by increasing caspase activation (Liu et al., 2017). Nonetheless, it is unknown whether CD90<sup>-</sup> FAPs are resistant to apoptosis.

## 4.3. Pro-survival signals regulating apoptosis-dependent FAP clearance

The accumulation of apoptosis-resistant FAPs has been shown in a mouse model of chronic inflammatory myopathy (Saito et al., 2020). Moreover, anti-apoptotic FAPs reduce gene expression of *Tnfrsf*, including *Fas*; increase gene expression of all TGF-β isoforms; and

upregulate *Tgfbr1* and *Tgfbr2* (Saito et al., 2020). In contrast, acute muscle injury triggered a senescence-like phenotype characterized by upregulation of *Cdkn2a* and *Trp53* expression and the histone variant γH2A.X (102). There is additional evidence for impaired FAP apoptosis in chronic conditions, as observed in *mdx* mice. FAPs isolated from dystrophic muscles exhibit a reduced apoptosis signaling pathway along with an upregulation of fibrogenic genes (Malecova et al., 2018). These findings support the notion that the microenvironment of chronic muscle disease favors anti-apoptotic FAP phenotypes that ultimately drive pathology.

Chronic diseases increase the proliferation of FAPs through chronic activation of mitogenic factors. TGF-β and PDGF ligands are potent mitogens that both induce FAP activation, proliferation, and differentiation (Lemos et al., 2015; Mueller et al., 2016; Contreras et al., 2019a; Mázala et al., 2020; Theret et al., 2021). These mitogens may be potentiated by interaction with the ECM glycoprotein thrombospondin-1 (TSP-1), product of the *Thsb1* gene (Belotti et al., 2016). TSP-1 expression is increased in the blood and muscle tissue of patients affected by brachio-cervical inflammatory myopathy and systemic sclerosis (BCIM-SSc), where muscle *Thbs1* expression is positively correlated with tissue fibrosis and infiltration of macrophages in the muscle (Suárez-Calvet et al., 2020). In addition, *in vitro* treatment with recombinant TSP-1, serum from BCIM-SSc patients or the supernatant of previously treated cells increased the proliferation of isolated human muscle-derived fibroblasts and TGF-β levels (Suárez-Calvet et al., 2020). Recent findings uncovered that FAPs secrete TSP-1 in response to the mechanical stress of surrounding ECM. This response is mediated by the transcriptional activation of YAP/TAZ (TAZ being the gene product of *Wwtr1*, encoding a transcriptional coactivator). In contrast to the known pro-fibrotic effect of TSP-1, this study describes a new role of FAP-secreted TSP-1, which acts by activating signaling by the CD47 receptor in MuSC, thereby driving their proliferation, and a pro-myogenic effect. This mechanism is critical to supply new myogenic nuclei for the enlargement of muscle fibers during load-related muscle hypertrophy (Kaneshige et al., 2022).

TGF-β is initially secreted in its latent, non-active pro-cytokine form, which is activated through a complex mechanism involving binding to TSP-1 and other factors (Crawford et al., 1998; Murphy-Ullrich and Suto 2018; Contreras et al., 2021b). TSP-1 binds to latent TGF-β through its type 1 repeats (KRFK sequence) and subsequently disrupts a conserved inhibitory sequence (LSKL) on TGF-β, thereby leading to TGF-β activation (Murphy-Ullrich and Suto 2018). Interestingly, hypertrophied visceral fat adipocytes have been identified as the primary source of circulating TSP-1 in individuals with insulin resistance or obesity and in HFD-fed mice (Varma et al., 2008; Buras et al., 2019). Elevated circulating levels of TSP-1 have also been associated with an increased risk of coronary and peripheral artery disease, T2DM, all-cause mortality, and adverse outcomes in chronic kidney failure patients (Smadja et al., 2011; Choi et al., 2012). Conversely, *Thbs1*-null mice are protected against obesity-induced metabolic alterations, largely by preventing fibrotic muscle degeneration (Inoue et al., 2013). In addition, dystrophic muscles from juvenile D2-*mdx* mice, a murine model of DMD, are characterized by a tonic increased in TGF-β and inflammatory responses to muscle damage, which is associated with worsening of histopathological changes (i.e., fibrosis and bone-like areas) and reduced regeneration (Mázala et al., 2023). Conversely, adult D2-*mdx* mice, despite exhibiting increased fibrosis, showed an improved muscle regeneration capacity. The differences between muscles of juvenile and adult dystrophic mice in terms of regenerative potential were associated with a predominant inflammatory niche in juvenile, characterized by increased expression of TNF-α, increased pro-inflammatory versus pro-regenerative macrophage activation, an impaired myogenic response, and elevated expression of pro-fibrotic markers such as Periostin and Osteopontin (Mázala et al., 2023). Interestingly, *in vitro* MuSC fusion and myotube formation were impaired when co-cultured with FAPs isolated from muscles of juvenile D2-*mdx* dystrophic mice (Mázala et al., 2023).
Secreted PDGFs are retained in the ECM, where they form complexes with ECM glycoproteins such as TSP-1. TSP-1 stabilizes PDGF-BB, thus facilitating its binding to membrane receptors on target cells (Tokunaga et al., 2008). PDGF signaling activation by PDGF-AA and PDGF-BB stimulates the proliferation and differentiation of FAPs (Uezumi et al., 2014; Mueller et al., 2016), which has been linked to pathological increases in PDGF ligand levels and PDGFRα overactivation, both of which are promoters of muscle fibrosis in chronically damaged muscles (Ieronimakis et al., 2016). Additionally, the cross-talk between PDGF and TGF-β modulates the fate of FAPs. Treatment of FAPs with TGF-β downregulates PDGFRα and induces fibrotic differentiation and migration while inhibiting FAP adipogenicity (Uezumi et al., 2014; Contreras et al., 2019a). Whether TSP-1 can mediate the cross-talk between TGF-β and PDGFRα warrants further investigation. Remarkably, inflammatory cells, endothelial cells, MuSCs, myofibers, and FAPs express *Pdgf* genes in adult muscles (Contreras et al., 2021a).

PDGF signaling regulates the activation, cell cycle, and fate of myogenic progenitors (Contreras et al., 2021a). Owing to this, the PDGF signaling pathway has emerged as a promising molecular target for promoting skeletal muscle regeneration and boosting tissue repair. From a clinical perspective, increased PDGF-AA serum concentrations correlate with several muscle functional tests in DMD patients (Alonso-Jiménez et al., 2021). Conversely, decreased levels of serum PDGF-AA at advanced stages of the disease are associated with muscle loss and weakness and increased IMAT (Alonso-Jiménez et al., 2021). These findings suggest that PDGF-AA can serve as a biomarker for monitoring disease progression in DMD patients.

PDGF-AA exerts a pro-migratory and proliferative effects on FAPs, a response that has been recently corroborated in human DMD FAPs (Fernández-Simón et al., 2022). Proteomic analysis of these cells revealed that upregulation of the RhoA/ROCK2 axis modulates PDGF-AA-promoted migration, proliferation, and fibrogenic differentiation (Fernández-Simón et al., 2022). This mechanism may be relevant for preventing FAP accumulation, fibrogenic differentiation, and subsequent fibrotic muscle degeneration observed in T2DM patients (Farup et al., 2021). To test this hypothesis, Fernández-Simón et al. (2022) treated DBA/2 J-*mdx* mice with fasudil, a pan-Rho-kinase inhibitor, and found reduced FAP numbers, decreased Collagen I staining, and increased grip strength in the treated animals (Fernández-Simón et al., 2022). These results demonstrated that the PDGF-PDGFR-RhoA/ROCK2 axis is a targetable pathway through which FAPs enhance muscle regeneration in disease settings. However, further studies are needed to fully understand the therapeutic potential of manipulating this pathway.

**5. Cellular and molecular regulatory mechanisms controlling FAP adipogenesis**

Under homeostatic conditions, several processes work together to restrain FAP adipogenesis while maintaining its steady state. These processes include, but are not restricted to, cell-autonomous signals such as WNT/β-catenin, NOTCH, Annexin-A2 (AnxA2), and Hedgehog (Hh) (Defour et al., 2017; Kopinke et al., 2017; Marinkovic et al., 2019). Understanding how FAP adipogenic commitment and adipocyte maturation regulate chronic diseases could lead to new treatments for muscle degeneration. Therefore, in this review, we have explored the role of these cues in both normal and diseased conditions.

**5.1. WNT/β-catenin signaling and TCF/LEF transcription factors**

FAPs are relevant sources of WNT ligands in muscle (Reggio et al., 2020b). Proliferative FAPs have a high content of cytoplasmic β-catenin, but adipogenic induction reduces the β-catenin protein level, while the β-catenin mRNA level remains stable. This suggests that post-transcriptional degradation of β-catenin is involved in adipocyte commitment (Reggio et al., 2020b). In addition to FAPs, macrophages produce a significant amount of WNT ligands in skeletal muscle in

response to acute injury (Tusavitz et al., 2020). Additionally, muscle fibers actively secrete WNT4A, which helps maintain MuSC quiescence (Eliazer et al., 2019).

WNT signaling activation is well known to inhibit adipogenesis (Ross et al., 2000; Bennett et al., 2002; Longo et al., 2004). Overexpression of WNT1, a phosphorylation-resistant β-catenin, or inhibition of β-catenin phosphorylation with lithium chloride prevented the adipogenic differentiation of 3T3-L1 pre-adipocytes (Ross et al., 2000).

In murine models, selective overexpression of WNT10B from the fatty-acid binding protein 4 (FABP4) promoter repressed the expression of the adipogenic program in adipocytes, thus impairing adipose tissue development and preventing obesity in mice (Longo et al., 2004; Wright et al., 2007). In a study by Fu et al. (2023), the WNT7A protein inhibited FAP adipogenesis *in vitro* and reduced adipocyte formation in response to GLY-induced injury without affecting myofiber size. Furthermore, a positive feedback loop was suggested in which WNT-Rho-YAP/TAZ upregulates canonical WNT signaling in activated FAPs (Fu et al., 2023), supporting the idea of manipulating WNT signaling as a way to reduce IMAT.

WNT TCF/LEF transcription factors also play a key role in adipocyte cell fate determination. Early evidence showed that overexpression of a dominant-negative form of TCF7L2 caused pre-adipocytes differentiation to adipocytes (Ross et al., 2000). In terms of adipogenic commitment, TCF7L1, another TCF/LEF member, acts as a transcriptional repressor that elicits adipogenic differentiation (Cristancho et al., 2011). Interestingly, TCF7L1 inhibited adipogenic differentiation, whereas its overexpression led to the opposite effects (Cristancho et al., 2011). Although *Tcf7l1* is highly expressed in FAPs (Contreras et al., 2020), the role of TCF7L1 in FAPs has not been addressed. Regarding TCF7L2, Geoghegan et al. (2019) showed that adipose-specific *Tcf7l2* knockout mice exhibited altered glucose metabolism, insulin responsiveness, weight gain, and increased adipose tissue mass in response to a HFD (Geoghegan et al., 2019). Thus, TCF7L2 regulates energy consumption, insulin homeostasis, lipolysis and lipogenesis in adipose tissue (Geoghegan et al., 2019). Notably, *Tcf7l2* deletion in pre-adipocytes enhanced adipocyte differentiation and lipid accumulation (Geoghegan et al., 2019). Using RNA-seq and TCF7L2 ChIP-seq integration analysis of adipocytes, TCF7L2 was shown to occupy and regulate genes involved in metabolic homeostasis and the cell cycle. These findings showed that the WNT effector TCF7L2 modulates adipocyte hypertrophy and links this phenotype to genome-wide occupancy of metabolic-associated genes. However, the specific mechanisms by which TCF7L2 regulates lipid and metabolic genes have yet to be determined, although previous evidence has shown that co-factors regulate the ability of TCF7L2 to bind DNA and regulate gene expression (Norton et al., 2014).

In limb muscles, the loss of *Tcf7l2* in FAPs reduced the expression of myosin heavy chain type I and embryonic myosin, thus affecting the formation of large multinucleated myofibers (Mathew et al., 2011). We demonstrated that TGF-β-mediated TCF7L2 downregulation coincides with fibrogenic differentiation of FAPs (Contreras et al., 2020). TGF-β promoted TCF7L2 protein degradation via the ubiquitin-proteasome system and repressed *Tcf7l2* gene expression through histone deacetylases (Contreras et al., 2020). In parallel, selective inhibition of GSK-3, an upstream β-catenin kinase, reversed adipogenic differentiation of FAPs and IMAT degeneration in *mdx* mice (Reggio et al., 2020b), demonstrating that β-catenin mediates FAP adipogenesis.

Si et al. (2006) showed that the matricellular protein cysteine-rich protein 61 (Cyr61, also known as CCN1) was a WNT/β-catenin target and that moreover, CCN1 was required for WNT-mediated regulation of mesenchymal stromal cell fate (Si et al., 2006). Ccn1 silencing in bone marrow stromal cells and C3H10T1/2 mesenchymal cells reduced β-catenin and TCF7L2 protein levels in the nucleus and activated mTORC1, thereby driving adipogenic differentiation. Conversely, CCN1 overexpression inhibited adipogenic differentiation (Yang et al., 2018). However, elevated CCN1 levels in muscle and serum have been
Secreted PDGFs are retained in the ECM, where they form complexes with ECM glycoproteins such as TSP-1. TSP-1 stabilizes PDGF-BB, thus facilitating its binding to membrane receptors on target cells (Tokunaga et al., 2008). PDGF signaling activation by PDGF-AA and PDGF-BB stimulates the proliferation and differentiation of FAPs (Uezumi et al., 2014; Mueller et al., 2016), which has been linked to pathological increases in PDGF ligand levels and PDGFRα overactivation, both of which are promoters of muscle fibrosis in chronically damaged muscles (Ieronimakis et al., 2016). Additionally, the cross-talk between PDGF and TGF-β modulates the fate of FAPs. Treatment of FAPs with TGF-β downregulates PDGFRα and induces fibrotic differentiation and migration while inhibiting FAP adipogenicity (Uezumi et al., 2014; Contreras et al., 2019a). Whether TSP-1 can mediate the cross-talk between TGF-β and PDGFRα warrants further investigation. Remarkably, inflammatory cells, endothelial cells, MuSCs, myofibers, and FAPs express *Pdgf* genes in adult muscles (Contreras et al., 2021a).

PDGF signaling regulates the activation, cell cycle, and fate of myogenic progenitors (Contreras et al., 2021a). Owing to this, the PDGF signaling pathway has emerged as a promising molecular target for promoting skeletal muscle regeneration and boosting tissue repair. From a clinical perspective, increased PDGF-AA serum concentrations correlate with several muscle functional tests in DMD patients (Alonso-Jiménez et al., 2021). Conversely, decreased levels of serum PDGF-AA at advanced stages of the disease are associated with muscle loss and weakness and increased IMAT (Alonso-Jiménez et al., 2021). These findings suggest that PDGF-AA can serve as a biomarker for monitoring disease progression in DMD patients.

PDGF-AA exerts a pro-migratory and proliferative effects on FAPs, a response that has been recently corroborated in human DMD FAPs (Fernández-Simón et al., 2022). Proteomic analysis of these cells revealed that upregulation of the RhoA/ROCK2 axis modulates PDGF-AA-promoted migration, proliferation, and fibrogenic differentiation (Fernández-Simón et al., 2022). This mechanism may be relevant for preventing FAP accumulation, fibrogenic differentiation, and subsequent fibrotic muscle degeneration observed in T2DM patients (Farup et al., 2021). To test this hypothesis, Fernández-Simón et al. (2022) treated DBA/2 J-*mdx* mice with fasudil, a pan-Rho-kinase inhibitor, and found reduced FAP numbers, decreased Collagen I staining, and increased grip strength in the treated animals (Fernández-Simón et al., 2022). These results demonstrated that the PDGF-PDGFR-RhoA/ROCK2 axis is a targetable pathway through which FAPs enhance muscle regeneration in disease settings. However, further studies are needed to fully understand the therapeutic potential of manipulating this pathway.

## 5. Cellular and molecular regulatory mechanisms controlling FAP adipogenesis

Under homeostatic conditions, several processes work together to restrain FAP adipogenesis while maintaining its steady state. These processes include, but are not restricted to, cell-autonomous signals such as WNT/β-catenin, NOTCH, Annexin-A2 (AnxA2), and Hedgehog (Hh) (Defour et al., 2017; Kopinke et al., 2017; Marinkovic et al., 2019). Understanding how FAP adipogenic commitment and adipocyte maturation regulate chronic diseases could lead to new treatments for muscle degeneration. Therefore, in this review, we have explored the role of these cues in both normal and diseased conditions.

### 5.1. WNT/β-catenin signaling and TCF/LEF transcription factors

FAPs are relevant sources of WNT ligands in muscle (Reggio et al., 2020b). Proliferative FAPs have a high content of cytoplasmic β-catenin, but adipogenic induction reduces the β-catenin protein level, while the β-catenin mRNA level remains stable. This suggests that post-transcriptional degradation of β-catenin is involved in adipocyte commitment (Reggio et al., 2020b). In addition to FAPs, macrophages produce a significant amount of WNT ligands in skeletal muscle in

response to acute injury (Tusavitz et al., 2020). Additionally, muscle fibers actively secrete WNT4A, which helps maintain MuSC quiescence (Eliazer et al., 2019).

WNT signaling activation is well known to inhibit adipogenesis (Ross et al., 2000; Bennett et al., 2002; Longo et al., 2004). Overexpression of WNT1, a phosphorylation-resistant β-catenin, or inhibition of β-catenin phosphorylation with lithium chloride prevented the adipogenic differentiation of 3T3-L1 pre-adipocytes (Ross et al., 2000).

In murine models, selective overexpression of WNT10B from the fatty-acid binding protein 4 (FABP4) promoter repressed the expression of the adipogenic program in adipocytes, thus impairing adipose tissue development and preventing obesity in mice (Longo et al., 2004; Wright et al., 2007). In a study by Fu et al. (2023), the WNT7A protein inhibited FAP adipogenesis *in vitro* and reduced adipocyte formation in response to GLY-induced injury without affecting myofiber size. Furthermore, a positive feedback loop was suggested in which WNT-Rho-YAP/TAZ upregulates canonical WNT signaling in activated FAPs (Fu et al., 2023), supporting the idea of manipulating WNT signaling as a way to reduce IMAT.

WNT TCF/LEF transcription factors also play a key role in adipocyte cell fate determination. Early evidence showed that overexpression of a dominant-negative form of TCF7L2 caused pre-adipocytes differentiation to adipocytes (Ross et al., 2000). In terms of adipogenic commitment, TCF7L1, another TCF/LEF member, acts as a transcriptional repressor that elicits adipogenic differentiation (Cristancho et al., 2011). Interestingly, TCF7L1 inhibited adipogenic differentiation, whereas its overexpression led to the opposite effects (Cristancho et al., 2011). Although *Tcf7l1* is highly expressed in FAPs (Contreras et al., 2020), the role of TCF7L1 in FAPs has not been addressed. Regarding TCF7L2, Geoghegan et al. (2019) showed that adipose-specific *Tcf7l2* knockout mice exhibited altered glucose metabolism, insulin responsiveness, weight gain, and increased adipose tissue mass in response to a HFD (Geoghegan et al., 2019). Thus, TCF7L2 regulates energy consumption, insulin homeostasis, lipolysis and lipogenesis in adipose tissue (Geoghegan et al., 2019). Notably, *Tcf7l2* deletion in pre-adipocytes enhanced adipocyte differentiation and lipid accumulation (Geoghegan et al., 2019). Using RNA-seq and TCF7L2 ChIP-seq integration analysis of adipocytes, TCF7L2 was shown to occupy and regulate genes involved in metabolic homeostasis and the cell cycle. These findings showed that the WNT effector TCF7L2 modulates adipocyte hypertrophy and links this phenotype to genome-wide occupancy of metabolic-associated genes. However, the specific mechanisms by which TCF7L2 regulates lipid and metabolic genes have yet to be determined, although previous evidence has shown that co-factors regulate the ability of TCF7L2 to bind DNA and regulate gene expression (Norton et al., 2014).

In limb muscles, the loss of *Tcf7l2* in FAPs reduced the expression of myosin heavy chain type I and embryonic myosin, thus affecting the formation of large multinucleated myofibers (Mathew et al., 2011). We demonstrated that TGF-β-mediated TCF7L2 downregulation coincides with fibrogenic differentiation of FAPs (Contreras et al., 2020). TGF-β promoted TCF7L2 protein degradation via the ubiquitin-proteasome system and repressed *Tcf7l2* gene expression through histone deacetylases (Contreras et al., 2020). In parallel, selective inhibition of GSK-3, an upstream β-catenin kinase, reversed adipogenic differentiation of FAPs and IMAT degeneration in *mdx* mice (Reggio et al., 2020b), demonstrating that β-catenin mediates FAP adipogenesis.

Si et al. (2006) showed that the matricellular protein cysteine-rich protein 61 (Cyr61, also known as CCN1) was a WNT/β-catenin target and that moreover, CCN1 was required for WNT-mediated regulation of mesenchymal stromal cell fate (Si et al., 2006). Ccn1 silencing in bone marrow stromal cells and C3H10T1/2 mesenchymal cells reduced β-catenin and TCF7L2 protein levels in the nucleus and activated mTORC1, thereby driving adipogenic differentiation. Conversely, CCN1 overexpression inhibited adipogenic differentiation (Yang et al., 2018). However, elevated CCN1 levels in muscle and serum have been
associated with increased FAP adipogenic differentiation in humans and in mice with chronic kidney disease (Hu et al., 2019). Additionally, increased serum levels of Dickkopf-related protein 1 (DKK1), a known inhibitor of WNT signaling, have been associated with increased IMAT deposition in individuals with obesity and insulin resistance (Ali et al., 2019).

Reciprocal regulation occurs between adipogenic signals and WNT-mediated adipogenesis repression. Mechanistically, pharmacological induction or conditional expression of CCAAT/enhancer-binding protein alpha (C/EBPα) suppresses nuclear WNT/β-catenin-TCF/LEF signaling during adipogenesis of 3T3-L1 pre-adipocytes (Moldes et al., 2003). This decrease in the nuclear level of β-catenin occurred in a dose-dependent manner following ligand-induced PPARγ activation (Moldes et al., 2003). The inhibitory effect of PPARγ on WNT/β-catenin-TCF/LEF signaling has been attributed to TLE3, a potent adipogenic factor that facilitates PPARγ activity on its target genes while repressing TCF/LEF activity (Villanueva et al., 2011).

In contrast to the anti-adipogenic effect of canonical WNT/β-catenin signaling, activation of the non-canonical pathway seems to induce adipogenesis. Keats et al. (2014) reported that the adipogenic effect of a high-glucose environment was mediated by autogenous activation of the non-canonical WNT11/Ca²⁺/protein kinase C signaling. High glucose triggered adipogenesis by upregulating C/EBPβ and C/EBPδ. This effect was mediated by increased Wnt11 expression and subsequent autocrine WNT11-mediated activation of non-canonical PKC signaling (Keats et al., 2014). Therefore, the balance between WNT canonical and non-canonical mechanisms that regulate FAP adipogenesis has yet to be fully elucidated.

## 5.2. Notch signaling

NOTCH signaling regulates FAP adipogenesis in an autocrine/juxta-crine manner (Marinkovic et al., 2019). Treatment with the γ-secretase inhibitor DAPT, which inhibits proteolytic cleavage of NICD, abrogates the anti-adipogenic effect of NOTCH signaling in FAPs cultured in DLL1-coated plates. Similarly, knockdown of the NOTCH2 receptor in FAPs increased adipogenesis and PPARγ expression (Marinkovic et al., 2019).

MuSCs and myotubes inhibit FAP adipogenic differentiation *in vitro* when these cells are co-cultured. This effect is mediated by NOTCH signaling activation, as exposure to DAPT increased FAP adipogenesis, lipid droplet accumulation and PPARγ expression (Marinkovic et al., 2019). In addition, mice treated with intraperitoneal injections of DAPT following CTX-induced muscle injury developed IMAT (Marinkovic et al., 2019). Taken together, these findings revealed the critical role of NOTCH signaling in limiting the adipogenic potency of FAPs. Intriguingly, FAPs isolated from dystrophic mice were insensitive to NOTCH signaling (Marinkovic et al., 2019). This desensitization was attributed to proteomic changes, including downregulation of components of the SWI/SNF chromatin-remodeling complex, upregulation of CDK5, an upstream kinase that increases PPARγ transcriptional activity, and upregulation of the retinoic X receptor alpha, which forms a heterodimer with PPARγ to drive adipogenesis (Marinkovic et al., 2019). Remarkably, signals associated with the inflammatory response to muscle injury, mainly TNF-α and NFκB, potentiate NOTCH signaling and increase its anti-adipogenic effect (Marinkovic et al., 2019). These findings corroborate prior studies suggesting that FAPs from uninjured and dystrophic muscle exist in different cell states.

## 5.3. Annexin-A2

Limb girdle muscular dystrophy type 2 B (LGMB2B) is caused by disease-causing variants in the *DYSFERLIN* gene and characterized by late-onset muscle weakness and muscle wasting associated with IMAT (Grounds et al., 2014; Hogarth et al., 2019). Dysferlin is a membrane-associated protein involved in Ca²⁺-dependent sarcolemmal

stability, vesicle trafficking, and membrane fusion (Grounds et al., 2014). Thus, the absence of dysferlin impairs sarcolemmal repair and disrupts Ca²⁺ homeostasis, leading to chronic inflammation, muscle degeneration, and gradual IMAT development (Grounds et al., 2014; Defour et al., 2017; Hogarth et al., 2019). The appearance of IMAT in the dysferlinopathic muscles of mouse models has been associated with the accumulation of Annexin-A2 (AnxA2), a protein that interacts with Dysferlin to facilitate sarcolemma repair after injury (Defour et al., 2017; Agarwal et al., 2019; Hogarth et al., 2019). As a Ca²⁺-dependent phospholipid-binding protein with intracellular and extracellular roles, intracellular AnxA2 interacts with dysferlin and actin to facilitate plasma membrane repair (McDade et al., 2014). Extracellular AnxA2 is produced by several cell types, including myofibers, monocytes and macrophages, and is secreted as both a soluble protein and a membrane-bound protein via two mechanisms: oxidative stress-mediated p38 MAPK activation and insulin/IGF-1 receptor activation (Zhao et al., 2003; Brownstein et al., 2004; Kita et al., 2016).

Interstitial AnxA2 levels positively correlate with clinical severity and myodegeneration in Dysferlin-deficient muscles (Cagliani et al., 2005; Hogarth et al., 2019). In dysferlinopathic mice, the severity of symptoms, inflammation and adipogenesis are reduced upon AnxA2 deletion, despite poor sarcolemmal repair (Defour et al., 2017). Remarkably, Hogarth and colleagues identified FAPs as the driving cells of fatty degeneration in the B6A/J mouse, a murine model of LGMB2B (Hogarth et al., 2019). FAPs accumulate and differentiate into adipocytes in dysferlin-deficient old mice after repeated rounds of injury-regeneration. FAPs and macrophages are enriched in interstitial regions where AnxA2 levels increase, suggesting that both cell types are recruited to the injured area in response to increased AnxA2 (Hogarth et al., 2019). In fact, compared with dysferlin-deficient mice, double knockout Dysferlin/AnxA2 mice (A2-B6A/J) exhibited reversal of macrophage infiltration and FAP-driven fatty degeneration (Hogarth et al., 2019). Remarkably, *in vitro* spontaneous adipogenesis is increased in dysferlinopathic FAPs but reduced in A2-B6A/J FAPs. Furthermore, treatment of dysferlinopathic FAPs with 100 µM AnxA2 drastically increased spontaneous adipogenic differentiation, and notexin-induced injury in the presence of exogenous AnxA2 increased the number of FAPs and adipocytes. Hence, in individuals with Dysferlin deficiency, impaired membrane repair and chronic inflammation create a loop in which damaged muscle fibers amplify AnxA2 production, which further fuels adipocyte accumulation, leading to excessive IMAT deposits in individuals with dysferlinopathy.

## 5.4. Ciliary hedgehog signaling

Another pathway that controls the adipogenic fate of FAPs is the Hedgehog (Hh) pathway (Kopinke et al., 2017). Hh signaling relies on microtubule-based ‘antennae’ called primary cilia to receive and interpret external cues (Goetz and Anderson 2010; Anvarian et al., 2019; Kopinke et al., 2021). Ciliary-controlled signaling events are crucial for tissue patterning because they mediate individual cell fate decisions and coordinate cell-cell communication. In addition to Hh signaling, multiple signaling pathways, such as the TGF-β, PDGF, WNT, GPCR and NOTCH signaling pathways, have been shown to utilize cilia for their functions (Goetz and Anderson 2010; Arrighi et al., 2017; Kopinke et al. 2017, 2021; Malicki and Johnson 2017; Anvarian et al., 2019; Hilgendorf et al., 2019). Hh signaling initiates with the production and secretion of Hh ligands: Sonic (Shh), Indian (Ihh) and Desert (Dhh) hedgehog proteins. In the off-state, primary cilia repress Hh target genes such as *Gli1* and *Ptch1* via the formation of the transcriptional repressor GLI3 (Liu et al., 2005). The binding of a Hh ligand to the receptor Patched-1 (PTCH1) allows the G protein-coupled receptor-like transmembrane protein Smoothened (Smo) to enter the cilium (Liu et al., 2005; Ruiz i Altaba 2011; Kopinke et al., 2017). Once inside the cilium, Smo activity promotes the formation of the transcriptional activator GLI2 and prevents GLI3 repressor formation (Liu et al., 2005).
associated with increased FAP adipogenic differentiation in humans and in mice with chronic kidney disease (Hu et al., 2019). Additionally, increased serum levels of Dickkopf-related protein 1 (DKK1), a known inhibitor of WNT signaling, have been associated with increased IMAT deposition in individuals with obesity and insulin resistance (Ali et al., 2019).

Reciprocal regulation occurs between adipogenic signals and WNT-mediated adipogenesis repression. Mechanistically, pharmacological induction or conditional expression of CCAAT/enhancer-binding protein alpha (C/EBPα) suppresses nuclear WNT/β-catenin-TCF/LEF signaling during adipogenesis of 3T3-L1 pre-adipocytes (Moldes et al., 2003). This decrease in the nuclear level of β-catenin occurred in a dose-dependent manner following ligand-induced PPARγ activation (Moldes et al., 2003). The inhibitory effect of PPARγ on WNT/β-catenin-TCF/LEF signaling has been attributed to TLE3, a potent adipogenic factor that facilitates PPARγ activity on its target genes while repressing TCF/LEF activity (Villanueva et al., 2011).

In contrast to the anti-adipogenic effect of canonical WNT/β-catenin signaling, activation of the non-canonical pathway seems to induce adipogenesis. Keats et al. (2014) reported that the adipogenic effect of a high-glucose environment was mediated by autogenous activation of the non-canonical WNT11/Ca²⁺/protein kinase C signaling. High glucose triggered adipogenesis by upregulating C/EBPβ and C/EBPδ. This effect was mediated by increased Wnt11 expression and subsequent autocrine WNT11-mediated activation of non-canonical PKC signaling (Keats et al., 2014). Therefore, the balance between WNT canonical and non-canonical mechanisms that regulate FAP adipogenesis has yet to be fully elucidated.

## 5.2. Notch signaling

NOTCH signaling regulates FAP adipogenesis in an autocrine/juxta-crine manner (Marinkovic et al., 2019). Treatment with the γ-secretase inhibitor DAPT, which inhibits proteolytic cleavage of NICD, abrogates the anti-adipogenic effect of NOTCH signaling in FAPs cultured in DLL1-coated plates. Similarly, knockdown of the NOTCH2 receptor in FAPs increased adipogenesis and PPARγ expression (Marinkovic et al., 2019).

MuSCs and myotubes inhibit FAP adipogenic differentiation *in vitro* when these cells are co-cultured. This effect is mediated by NOTCH signaling activation, as exposure to DAPT increased FAP adipogenesis, lipid droplet accumulation and PPARγ expression (Marinkovic et al., 2019). In addition, mice treated with intraperitoneal injections of DAPT following CTX-induced muscle injury developed IMAT (Marinkovic et al., 2019). Taken together, these findings revealed the critical role of NOTCH signaling in limiting the adipogenic potency of FAPs. Intriguingly, FAPs isolated from dystrophic mice were insensitive to NOTCH signaling (Marinkovic et al., 2019). This desensitization was attributed to proteomic changes, including downregulation of components of the SWI/SNF chromatin-remodeling complex, upregulation of CDK5, an upstream kinase that increases PPARγ transcriptional activity, and upregulation of the retinoic X receptor alpha, which forms a heterodimer with PPARγ to drive adipogenesis (Marinkovic et al., 2019). Remarkably, signals associated with the inflammatory response to muscle injury, mainly TNF-α and NFκB, potentiate NOTCH signaling and increase its anti-adipogenic effect (Marinkovic et al., 2019). These findings corroborate prior studies suggesting that FAPs from uninjured and dystrophic muscle exist in different cell states.

## 5.3. Annexin-A2

Limb girdle muscular dystrophy type 2 B (LGMB2B) is caused by disease-causing variants in the *DYSFERLIN* gene and characterized by late-onset muscle weakness and muscle wasting associated with IMAT (Grounds et al., 2014; Hogarth et al., 2019). Dysferlin is a membrane-associated protein involved in Ca²⁺-dependent sarcolemmal

stability, vesicle trafficking, and membrane fusion (Grounds et al., 2014). Thus, the absence of dysferlin impairs sarcolemmal repair and disrupts Ca²⁺ homeostasis, leading to chronic inflammation, muscle degeneration, and gradual IMAT development (Grounds et al., 2014; Defour et al., 2017; Hogarth et al., 2019). The appearance of IMAT in the dysferlinopathic muscles of mouse models has been associated with the accumulation of Annexin-A2 (AnxA2), a protein that interacts with Dysferlin to facilitate sarcolemma repair after injury (Defour et al., 2017; Agarwal et al., 2019; Hogarth et al., 2019). As a Ca²⁺-dependent phospholipid-binding protein with intracellular and extracellular roles, intracellular AnxA2 interacts with dysferlin and actin to facilitate plasma membrane repair (McDade et al., 2014). Extracellular AnxA2 is produced by several cell types, including myofibers, monocytes and macrophages, and is secreted as both a soluble protein and a membrane-bound protein via two mechanisms: oxidative stress-mediated p38 MAPK activation and insulin/IGF-1 receptor activation (Zhao et al., 2003; Brownstein et al., 2004; Kita et al., 2016).

Interstitial AnxA2 levels positively correlate with clinical severity and myodegeneration in Dysferlin-deficient muscles (Cagliani et al., 2005; Hogarth et al., 2019). In dysferlinopathic mice, the severity of symptoms, inflammation and adipogenesis are reduced upon AnxA2 deletion, despite poor sarcolemmal repair (Defour et al., 2017). Remarkably, Hogarth and colleagues identified FAPs as the driving cells of fatty degeneration in the B6A/J mouse, a murine model of LGMB2B (Hogarth et al., 2019). FAPs accumulate and differentiate into adipocytes in dysferlin-deficient old mice after repeated rounds of injury-regeneration. FAPs and macrophages are enriched in interstitial regions where AnxA2 levels increase, suggesting that both cell types are recruited to the injured area in response to increased AnxA2 (Hogarth et al., 2019). In fact, compared with dysferlin-deficient mice, double knockout Dysferlin/AnxA2 mice (A2-B6A/J) exhibited reversal of macrophage infiltration and FAP-driven fatty degeneration (Hogarth et al., 2019). Remarkably, *in vitro* spontaneous adipogenesis is increased in dysferlinopathic FAPs but reduced in A2-B6A/J FAPs. Furthermore, treatment of dysferlinopathic FAPs with 100 µM AnxA2 drastically increased spontaneous adipogenic differentiation, and notexin-induced injury in the presence of exogenous AnxA2 increased the number of FAPs and adipocytes. Hence, in individuals with Dysferlin deficiency, impaired membrane repair and chronic inflammation create a loop in which damaged muscle fibers amplify AnxA2 production, which further fuels adipocyte accumulation, leading to excessive IMAT deposits in individuals with dysferlinopathy.

## 5.4. Ciliary hedgehog signaling

Another pathway that controls the adipogenic fate of FAPs is the Hedgehog (Hh) pathway (Kopinke et al., 2017). Hh signaling relies on microtubule-based ‘antennae’ called primary cilia to receive and interpret external cues (Goetz and Anderson 2010; Anvarian et al., 2019; Kopinke et al., 2021). Ciliary-controlled signaling events are crucial for tissue patterning because they mediate individual cell fate decisions and coordinate cell-cell communication. In addition to Hh signaling, multiple signaling pathways, such as the TGF-β, PDGF, WNT, GPCR and NOTCH signaling pathways, have been shown to utilize cilia for their functions (Goetz and Anderson 2010; Arrighi et al., 2017; Kopinke et al. 2017, 2021; Malicki and Johnson 2017; Anvarian et al., 2019; Hilgendorf et al., 2019). Hh signaling initiates with the production and secretion of Hh ligands: Sonic (Shh), Indian (Ihh) and Desert (Dhh) hedgehog proteins. In the off-state, primary cilia repress Hh target genes such as *Gli1* and *Ptch1* via the formation of the transcriptional repressor GLI3 (Liu et al., 2005). The binding of a Hh ligand to the receptor Patched-1 (PTCH1) allows the G protein-coupled receptor-like transmembrane protein Smoothened (Smo) to enter the cilium (Liu et al., 2005; Ruiz i Altaba 2011; Kopinke et al., 2017). Once inside the cilium, Smo activity promotes the formation of the transcriptional activator GLI2 and prevents GLI3 repressor formation (Liu et al., 2005).
Within skeletal muscle, FAPs are the major ciliated cell type (Kopinke et al., 2017). During muscle repair, FAP ciliation dynamically changes. While few FAPs are ciliated prior to injury, their ciliation frequently increases immediately after acute injury before returning to pre-injury levels (Kopinke et al., 2017). Interestingly, FAP cilia are remodeled and lost early during differentiation into adipocytes (Kopinke et al., 2017; Hilgendorf et al., 2019). To probe the function of FAP cilia, we conditionally deleted intraflagellar transport 88 (Ift88), a gene required for ciliogenesis and ciliary maintenance, specifically from FAPs. Loss of FAP cilia prevents the differentiation of FAPs into Plin1⁺/FABP4⁺ adipocytes following acute muscle injury (Kopinke et al., 2017). Similarly, the removal of cilia in FAPs drastically reduced FAP adipogenesis and, consequently, IMAT infiltration in dystrophic muscles of *mdx* mice (Fig. 2B). In parallel, the loss of FAP cilia enhanced muscle regeneration and increased myofiber size, thus preserving muscle regenerative capacity in mice after GLY-induced muscle injury as well as in muscles of dystrophic *mdx* mice.

Mechanistically, we found that FAP cilia loss increased the expression of Hh target genes due to loss of the GLI3 repressor, resulting in Hh derepression. This low-level Hh activation upon loss of cilia was sufficient to repress IMAT formation. Similarly, we found that directly activating Hh signaling via conditional deletion of *Ptch1* in FAPs also repressed the ability of FAPs to differentiate into adipocytes (Kopinke et al., 2017). Likewise, the activation of Smo with a small-molecule agonist (SAG) replicated the upstream anti-adipogenic effects of Hh signaling activation and cilia removal. We further determined that ciliary Hh signaling promotes adipogenesis non-cell-autonomously via upregulation of the expression of the secreted anti-adipogenic protein tissue inhibitor of metalloproteinase 3 (TIMP3). Loss of FAP cilia and activation of Hh signaling via removal of *Ptch1* upregulated *Timp3* expression (Kopinke et al., 2017). TIMP-3 is involved in the regulation of essential early inducers of adipogenesis, including *C/ebpβ*, and its down-regulation is mandatory for proper implementation of the adipocyte differentiation program (Bernot et al., 2010). Furthermore, TIMP3 modulates the shedding of the NOTCH ligand DLK1 and ECM remodeling via collagen 1 degradation and inhibition of the metalloproteinases ADAM17 and MMP14 to allow adipose expansion (i.e., adipocyte hypertrophy) (Fenech et al., 2015). The anti-adipogenic effect of ciliary Hh signaling activation through TIMP3 relies on its effect on MMP14 activity (Kopinke et al., 2017). Pharmacological inhibition of metalloproteinases with batimastat or specific inhibition of MMP14 by NSC405020 mimicked the effects of TIMP3 and robustly inhibited adipogenesis of FAPs *in vitro* and *in vivo* after GLY-induced injury (Kopinke et al., 2017).

Our recent findings demonstrated that DHH, which is sensed by FAPs, is the key Hh ligand involved in muscle regeneration. Genetic loss of *Dhh* resulted in accelerated IMAT formation due to repression of TIMP3 (Norris et al., 2023). Moreover, myofiber regeneration was severely impacted by the absence of Hh activity due to the lack of induction of GDF10 (Bmp3b), a potent myogenic factor (Uezumi et al., 2021), within FAPs. Notably, the adipo-regulatory CD142⁺ FAPs subpopulation inhibits adipogenesis via the secretion of GDF10. Interestingly, CD142⁺ FAPs are almost completely lost in dystrophic mice, thus leading to increased adipogenesis (Camps et al., 2020).

Together, our findings reveal an undescribed role for cilia-mediated Hh signaling in FAP adipogenesis and IMAT accumulation and identify Hh signaling as an endogenous adipogenic brake. In this context, a previous study provided further evidence that FAPs are the major Hh responders. Yao et al. (2021) described a subpopulation of Gli1⁺ FAPs (Gli1 is a direct Hh target) that preferentially expands upon injury-mediated Hh signaling activation (Yao et al., 2021). These cells retain greater clonogenic capabilities at the expense of reduced adipogenic differentiation capacity than Gli1⁻ FAPs. Remarkably, both the depletion of Gli1⁺ FAPs and the pharmacological inhibition of Hh signaling by GANT61 led to the increased formation of Perilipin⁺ adipocytes after GLY-induced injury (Yao et al., 2021). In summary,

pharmacological strategies aimed at reducing fatty degeneration and improving the regeneration of skeletal muscle should consider the modulation of ciliary Hh and its effect on TIMP3-mediated MMP14 activity.

### 5.5. FAP dynamics and adipogenic potency in neuromuscular trauma and disease

Sciatic nerve denervation causes pronounced muscle atrophy, ECM deposition, and accumulation of FAPs (Contreras et al., 2016; Madaro et al., 2018; Rebolledo et al., 2019). Similarly, FAPs expand in response to chronic neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS). Specifically, the loss of neuromuscular junction integrity caused by denervation or ALS has been related to the activation, expansion, and differentiation of FAPs and the accumulation of fibro-fatty tissues (Contreras et al. 2016, 2019c; Gonzalez et al., 2017; Madaro et al., 2018). Thus, excessive activation and accumulation of FAPs may contribute to the progression of muscle wasting (Theret et al., 2021). FAPs from denervated muscles exhibit pro-fibrotic properties and express genes associated with inflammation and pro-atrophic and catabolic factors (Madaro et al., 2018). For instance, persistent secretion of IL-6 and subsequent IL-6-mediated activation of STAT3 signaling in FAPs from denervated muscles induced muscle fiber atrophy and fibrosis (Madaro et al., 2018). *In vivo* blockade of IL-6 signaling via a neutralizing antibody or a selective STAT3 inhibitor prevents FAP-mediated muscle atrophy and fibrosis (Madaro et al., 2018). Remarkably, the PDGFRα⁺/CD34⁺/Sca-1⁻ FAP subtype is particularly prone to upregulating IL-6 secretion and initiating an oxidative stress response that contributes to denervation-induced muscle atrophy (Yang et al., 2021). These findings highlight the importance of IL-6 signaling in FAP-mediated muscle loss and suggest that targeting this pathway has therapeutic potential for muscle wasting diseases.

Two recent studies utilized single-cell RNA sequencing (scRNA-seq) to investigate the response of muscle-resident cells to transfection of the sciatic nerve and identified two cell populations responsive to denervation: muscle glial cells (likely activated Schwann cells) and FAP-derived activated fibroblasts (Pdgfra<sup>low</sup> and Sca1<sup>low</sup>) (Nicoletti et al., 2020; Proietti et al., 2021). Both cell populations quickly respond to denervation by expanding their numbers and upregulating transcription (Nicoletti et al., 2023). Interestingly, Schwann cells and FAPs closely interact in muscle nerve- and neuromuscular junction (NMJ)-associated niches, where FAPs regulate Schwann cell dynamics and homeostasis (Theret et al., 2021; Uezumi et al., 2021). These and previous studies reporting FAP and fibroblast activation after denervation (Contreras et al., 2016; Gonzalez et al., 2017; Madaro et al., 2018; Rebolledo et al., 2019), indicate that denervation drives crosstalk between activated glial cells and FAPs (Wei et al., 2021). Overall, these studies revealed the cellular responses to denervation in muscle, highlighting the complex interplay between nerve cells and FAPs, which promotes a non-regenerative environment in response to denervation-induced NMJ catabolic stimuli.

Acute myotrauma leads to the early expansion of non-resident and muscle-resident immune cells, whereas denervation does not immediately induce significant immune cell infiltration (Madaro et al., 2018; Nicoletti et al., 2020; Oprescu et al., 2020; Theret et al., 2021; Wei et al., 2021). These findings were supported by a study in which single-nucleus RNA sequencing was utilized to decode the transcriptome of the gastrocnemius muscle after sciatic nerve denervation (Lin et al., 2021). In this context, C1, C2, and C3 FAP clusters were described with transcriptional trajectories that expanded to form three main branches, with C2 forming the fibrotic branch and C3 the adipogenic branch (Lin et al., 2021), corroborating that the FAP transcriptome responds to denervation, as previously shown (Madaro et al., 2018). Using gene set enrichment analysis, the authors found that denervation downregulated myogenesis, hypoxia, and metabolism-related gene expression in all the FAP cells, with differentially upregulated gene sets in each of the three
Within skeletal muscle, FAPs are the major ciliated cell type (Kopinke et al., 2017). During muscle repair, FAP ciliation dynamically changes. While few FAPs are ciliated prior to injury, their ciliation frequently increases immediately after acute injury before returning to pre-injury levels (Kopinke et al., 2017). Interestingly, FAP cilia are remodeled and lost early during differentiation into adipocytes (Kopinke et al., 2017; Hilgendorf et al., 2019). To probe the function of FAP cilia, we conditionally deleted intraflagellar transport 88 (Ift88), a gene required for ciliogenesis and ciliary maintenance, specifically from FAPs. Loss of FAP cilia prevents the differentiation of FAPs into Plin1⁺/FABP4⁺ adipocytes following acute muscle injury (Kopinke et al., 2017). Similarly, the removal of cilia in FAPs drastically reduced FAP adipogenesis and, consequently, IMAT infiltration in dystrophic muscles of *mdx* mice (Fig. 2B). In parallel, the loss of FAP cilia enhanced muscle regeneration and increased myofiber size, thus preserving muscle regenerative capacity in mice after GLY-induced muscle injury as well as in muscles of dystrophic *mdx* mice.

Mechanistically, we found that FAP cilia loss increased the expression of Hh target genes due to loss of the GLI3 repressor, resulting in Hh derepression. This low-level Hh activation upon loss of cilia was sufficient to repress IMAT formation. Similarly, we found that directly activating Hh signaling via conditional deletion of *Ptch1* in FAPs also repressed the ability of FAPs to differentiate into adipocytes (Kopinke et al., 2017). Likewise, the activation of Smo with a small-molecule agonist (SAG) replicated the upstream anti-adipogenic effects of Hh signaling activation and cilia removal. We further determined that ciliary Hh signaling promotes adipogenesis non-cell-autonomously via upregulation of the expression of the secreted anti-adipogenic protein tissue inhibitor of metalloproteinase 3 (TIMP3). Loss of FAP cilia and activation of Hh signaling via removal of *Ptch1* upregulated *Timp3* expression (Kopinke et al., 2017). TIMP-3 is involved in the regulation of essential early inducers of adipogenesis, including *C/ebpβ*, and its down-regulation is mandatory for proper implementation of the adipocyte differentiation program (Bernot et al., 2010). Furthermore, TIMP3 modulates the shedding of the NOTCH ligand DLK1 and ECM remodeling via collagen 1 degradation and inhibition of the metalloproteinases ADAM17 and MMP14 to allow adipose expansion (i.e., adipocyte hypertrophy) (Fenech et al., 2015). The anti-adipogenic effect of ciliary Hh signaling activation through TIMP3 relies on its effect on MMP14 activity (Kopinke et al., 2017). Pharmacological inhibition of metalloproteinases with batimastat or specific inhibition of MMP14 by NSC405020 mimicked the effects of TIMP3 and robustly inhibited adipogenesis of FAPs *in vitro* and *in vivo* after GLY-induced injury (Kopinke et al., 2017).

Our recent findings demonstrated that DHH, which is sensed by FAPs, is the key Hh ligand involved in muscle regeneration. Genetic loss of *Dhh* resulted in accelerated IMAT formation due to repression of TIMP3 (Norris et al., 2023). Moreover, myofiber regeneration was severely impacted by the absence of Hh activity due to the lack of induction of GDF10 (Bmp3b), a potent myogenic factor (Uezumi et al., 2021), within FAPs. Notably, the adipo-regulatory CD142⁺ FAPs subpopulation inhibits adipogenesis via the secretion of GDF10. Interestingly, CD142⁺ FAPs are almost completely lost in dystrophic mice, thus leading to increased adipogenesis (Camps et al., 2020).

Together, our findings reveal an undescribed role for cilia-mediated Hh signaling in FAP adipogenesis and IMAT accumulation and identify Hh signaling as an endogenous adipogenic brake. In this context, a previous study provided further evidence that FAPs are the major Hh responders. Yao et al. (2021) described a subpopulation of Gli1⁺ FAPs (Gli1 is a direct Hh target) that preferentially expands upon injury-mediated Hh signaling activation (Yao et al., 2021). These cells retain greater clonogenic capabilities at the expense of reduced adipogenic differentiation capacity than Gli1⁻ FAPs. Remarkably, both the depletion of Gli1⁺ FAPs and the pharmacological inhibition of Hh signaling by GANT61 led to the increased formation of Perilipin⁺ adipocytes after GLY-induced injury (Yao et al., 2021). In summary,

pharmacological strategies aimed at reducing fatty degeneration and improving the regeneration of skeletal muscle should consider the modulation of ciliary Hh and its effect on TIMP3-mediated MMP14 activity.

### 5.5. FAP dynamics and adipogenic potency in neuromuscular trauma and disease

Sciatic nerve denervation causes pronounced muscle atrophy, ECM deposition, and accumulation of FAPs (Contreras et al., 2016; Madaro et al., 2018; Rebolledo et al., 2019). Similarly, FAPs expand in response to chronic neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS). Specifically, the loss of neuromuscular junction integrity caused by denervation or ALS has been related to the activation, expansion, and differentiation of FAPs and the accumulation of fibro-fatty tissues (Contreras et al. 2016, 2019c; Gonzalez et al., 2017; Madaro et al., 2018). Thus, excessive activation and accumulation of FAPs may contribute to the progression of muscle wasting (Theret et al., 2021). FAPs from denervated muscles exhibit pro-fibrotic properties and express genes associated with inflammation and pro-atrophic and catabolic factors (Madaro et al., 2018). For instance, persistent secretion of IL-6 and subsequent IL-6-mediated activation of STAT3 signaling in FAPs from denervated muscles induced muscle fiber atrophy and fibrosis (Madaro et al., 2018). *In vivo* blockade of IL-6 signaling via a neutralizing antibody or a selective STAT3 inhibitor prevents FAP-mediated muscle atrophy and fibrosis (Madaro et al., 2018). Remarkably, the PDGFRα⁺/CD34⁺/Sca-1⁻ FAP subtype is particularly prone to upregulating IL-6 secretion and initiating an oxidative stress response that contributes to denervation-induced muscle atrophy (Yang et al., 2021). These findings highlight the importance of IL-6 signaling in FAP-mediated muscle loss and suggest that targeting this pathway has therapeutic potential for muscle wasting diseases.

Two recent studies utilized single-cell RNA sequencing (scRNA-seq) to investigate the response of muscle-resident cells to transfection of the sciatic nerve and identified two cell populations responsive to denervation: muscle glial cells (likely activated Schwann cells) and FAP-derived activated fibroblasts (Pdgfra<sup>low</sup> and Sca1<sup>low</sup>) (Nicoletti et al., 2020; Proietti et al., 2021). Both cell populations quickly respond to denervation by expanding their numbers and upregulating transcription (Nicoletti et al., 2023). Interestingly, Schwann cells and FAPs closely interact in muscle nerve- and neuromuscular junction (NMJ)-associated niches, where FAPs regulate Schwann cell dynamics and homeostasis (Theret et al., 2021; Uezumi et al., 2021). These and previous studies reporting FAP and fibroblast activation after denervation (Contreras et al., 2016; Gonzalez et al., 2017; Madaro et al., 2018; Rebolledo et al., 2019), indicate that denervation drives crosstalk between activated glial cells and FAPs (Wei et al., 2021). Overall, these studies revealed the cellular responses to denervation in muscle, highlighting the complex interplay between nerve cells and FAPs, which promotes a non-regenerative environment in response to denervation-induced NMJ catabolic stimuli.

Acute myotrauma leads to the early expansion of non-resident and muscle-resident immune cells, whereas denervation does not immediately induce significant immune cell infiltration (Madaro et al., 2018; Nicoletti et al., 2020; Oprescu et al., 2020; Theret et al., 2021; Wei et al., 2021). These findings were supported by a study in which single-nucleus RNA sequencing was utilized to decode the transcriptome of the gastrocnemius muscle after sciatic nerve denervation (Lin et al., 2021). In this context, C1, C2, and C3 FAP clusters were described with transcriptional trajectories that expanded to form three main branches, with C2 forming the fibrotic branch and C3 the adipogenic branch (Lin et al., 2021), corroborating that the FAP transcriptome responds to denervation, as previously shown (Madaro et al., 2018). Using gene set enrichment analysis, the authors found that denervation downregulated myogenesis, hypoxia, and metabolism-related gene expression in all the FAP cells, with differentially upregulated gene sets in each of the three