FAP clusters. For example, denervated C1 FAPs were associated with apoptotic p53 gene sets, C2 FAPs were associated with fibrosis and angiogenesis, and C3 FAPs were associated with adipogenesis. The denervation-mediated transcriptional changes in FAPs could be driven by specific regulons, such as TCF7L2 and SREBF1, which are activated in fibrotic C2 and adipogenic C3 FAPs. Our previous studies demonstrated that TCF7L2-expressing cells expand in response to sciatic denervation and in ALS-affected muscles (Contreras et al., 2016; Gonzalez et al., 2017). We also showed that the WNT-associated TCF7L2 transcription factor is downregulated by pro-fibrotic TGF-β signaling in FAPs and stromal cells. This provides a new cross-talk mechanism between TGF-β and WNT signaling pathways that may regulate fibrogenic fate and tissue fibrosis (Contreras et al., 2019a).

Interestingly, the C3 cluster was similar to that of Dpp4⁺ FAPs (Scott et al., 2019; Oprescu et al., 2020). Dpp4⁺ stromal cells have been shown to contribute to basal and de novo diet-induced adipogenesis in adipose tissue and bone marrow (Ambrosi et al., 2017; Merrick et al., 2019), suggesting that Dpp4-expressing FAPs could be adipogenic progenitors in skeletal muscles. However, recent evidence challenges this role, particularly in response to a HFD (Takada et al., 2022). Collectively, these findings suggest that tissue-resident macrophages, glial cells, and FAPs actively engage in cross-talk to modulate denervation onset, development, and degenerative outcomes.

### 5.6. Muscle disuse and deconditioning as triggers for FAP activation and IMAT accumulation

Reduced physical activity is associated with a loss of muscle mass, which is a strong determinant of IMAT accumulation, predicting up to 50% of its variance (Manini et al., 2007; Akima et al., 2015). Decreased muscle activity or deconditioning could cause muscle atrophy and FAP accumulation, even when neuromuscular function is preserved (Manini et al., 2007; Pagano et al., 2015, 2018). Dry immersion (to reproduce the effects of microgravity) promoted muscle wasting (~11% myofiber area reduction) in lean adult patients, accompanied by IMAT expansion and ~50% elevated protein levels of FAP and FAP-derived adipocyte markers (Pagano et al., 2018). Additionally, immobilization upregulated two senescence-associated genes in murine FAPs, *Il-1β* and *Cdkn2a*, suggesting that FAP senescence may play a novel role in the development of disuse muscle atrophy (Parker et al., 2022).

Diet-induced obesity has been associated with myofiber atrophy and sarcopenia, currently known as sarcopenic obesity (Song et al., 2004; Biolo et al., 2014; Fellner et al., 2014; Collins et al. 2016a, 2016b; Mogi et al., 2016; Buras et al., 2019). Genetic lineage tracing experiments using Pdgfrα-CreERT2 mice crossed with R26R-EYFP mice confirmed that HFD-induced IMAT was composed of adipocytes derived from FAPs (Takada et al., 2022), similar to what we and others have observed in multiple settings (Kopinke et al., 2017; Stumm et al., 2018; Hogarth et al., 2019) (Fig. 3). In the context of diet-induced obesity, muscles are characterized by a reduced myofiber cross-sectional area and a transient accumulation of senescent FAPs (Takada et al., 2022). Senescent FAPs in

<figure><img src="image_4.png" /><figcaption>**Fig. 3.** Adipocyte cell formation and IMAT deposition in vivo from muscle-resident PDGFRα⁺ fibro-adipogenic progenitors. (A) *Pdgfra* lineage tracing shows the complex cell structures and protrusions of muscle-resident FAPs and their close steady-state contact with healthy myofibers. (B) Lineage tracing reveals that IMAT-associated adipocytes (perilipin⁺) are derived from PDGFRα-expressing FAPs.</figcaption></figure>
FAP clusters. For example, denervated C1 FAPs were associated with apoptotic p53 gene sets, C2 FAPs were associated with fibrosis and angiogenesis, and C3 FAPs were associated with adipogenesis. The denervation-mediated transcriptional changes in FAPs could be driven by specific regulons, such as TCF7L2 and SREBF1, which are activated in fibrotic C2 and adipogenic C3 FAPs. Our previous studies demonstrated that TCF7L2-expressing cells expand in response to sciatic denervation and in ALS-affected muscles (Contreras et al., 2016; Gonzalez et al., 2017). We also showed that the WNT-associated TCF7L2 transcription factor is downregulated by pro-fibrotic TGF-β signaling in FAPs and stromal cells. This provides a new cross-talk mechanism between TGF-β and WNT signaling pathways that may regulate fibrogenic fate and tissue fibrosis (Contreras et al., 2019a).

Interestingly, the C3 cluster was similar to that of Dpp4⁺ FAPs (Scott et al., 2019; Oprescu et al., 2020). Dpp4⁺ stromal cells have been shown to contribute to basal and de novo diet-induced adipogenesis in adipose tissue and bone marrow (Ambrosi et al., 2017; Merrick et al., 2019), suggesting that Dpp4-expressing FAPs could be adipogenic progenitors in skeletal muscles. However, recent evidence challenges this role, particularly in response to a HFD (Takada et al., 2022). Collectively, these findings suggest that tissue-resident macrophages, glial cells, and FAPs actively engage in cross-talk to modulate denervation onset, development, and degenerative outcomes.

### 5.6. Muscle disuse and deconditioning as triggers for FAP activation and IMAT accumulation

Reduced physical activity is associated with a loss of muscle mass, which is a strong determinant of IMAT accumulation, predicting up to 50% of its variance (Manini et al., 2007; Akima et al., 2015). Decreased muscle activity or deconditioning could cause muscle atrophy and FAP accumulation, even when neuromuscular function is preserved (Manini et al., 2007; Pagano et al., 2015, 2018). Dry immersion (to reproduce the effects of microgravity) promoted muscle wasting (~11% myofiber area reduction) in lean adult patients, accompanied by IMAT expansion and ~50% elevated protein levels of FAP and FAP-derived adipocyte markers (Pagano et al., 2018). Additionally, immobilization upregulated two senescence-associated genes in murine FAPs, *Il-1β* and *Cdkn2a*, suggesting that FAP senescence may play a novel role in the development of disuse muscle atrophy (Parker et al., 2022).

Diet-induced obesity has been associated with myofiber atrophy and sarcopenia, currently known as sarcopenic obesity (Song et al., 2004; Biolo et al., 2014; Fellner et al., 2014; Collins et al. 2016a, 2016b; Mogi et al., 2016; Buras et al., 2019). Genetic lineage tracing experiments using Pdgfrα-CreERT2 mice crossed with R26R-EYFP mice confirmed that HFD-induced IMAT was composed of adipocytes derived from FAPs (Takada et al., 2022), similar to what we and others have observed in multiple settings (Kopinke et al., 2017; Stumm et al., 2018; Hogarth et al., 2019) (Fig. 3). In the context of diet-induced obesity, muscles are characterized by a reduced myofiber cross-sectional area and a transient accumulation of senescent FAPs (Takada et al., 2022). Senescent FAPs in

A

<figure><img src="image_5.png" /></figure>

Lineage-traced PDGFRα⁺ FAPs

PdgfraEYFP

Nuclei

Myofibers

<figure><img src="image_6.png" /></figure>

B

Pdgfra lineage-traced adipocytes after acute muscle damage

Perilipin Nuclei

Myofibers

PdgfraEYFP

Nuclei

<figure><img src="image_7.png" /></figure>

<figure><img src="image_8.png" /></figure>

<figure><img src="image_9.png" /><figcaption>**Fig. 3.** Adipocyte cell formation and IMAT deposition in vivo from muscle-resident PDGFRα⁺ fibro-adipogenic progenitors. (A) *Pdgfra* lineage tracing shows the complex cell structures and protrusions of muscle-resident FAPs and their close steady-state contact with healthy myofibers. (B) Lineage tracing reveals that IMAT-associated adipocytes (perilipin⁺) are derived from PDGFRα-expressing FAPs.</figcaption></figure>
HFD-fed mice exhibit upregulation of genes related to the PPARγ signaling pathway and adipogenesis, such as *perilipin-1* (PLIN1), *Fabp4*, and *Cd36* (Takada et al., 2022). These results suggest that a HFD could influence the obesogenic microenvironment and therefore promote muscle atrophy by inducing some FAPs to become adipogenic.

In relation to inflammation, inflammation-mediated IL-1β secretion by macrophages may inhibit FAP-derived adipogenesis, whereas macrophage depletion via clodronate-loaded liposome treatment increases IMAT deposition after CTX-induced injury (Marinkovic et al., 2019). Another study showed that macrophages activated with IL-1β-conditioned media secreted unidentified factors that inhibited FAP adipogenesis via paracrine signaling. Moreover, both the IL-1α and IL-1β isoforms effectively suppress FAP adipogenesis while not affecting their proliferation (Vumbaca et al., 2021). Conversely, macrophage activation via exposure to the anti-inflammatory cytokine IL-4 promoted FAP adipogenesis (Moratal et al., 2018). These results reflect the complex regulation of FAP activity in response to injury, inflammation and disease. FAPs may have evolved to self-regulate their cellular states by secreting both anti- and pro-adipogenic factors in a balanced manner, potentially as a safeguard mechanism. There are data that support this concept. Injury-induced intronic polyadenylation of *Pdgfra* results in the transcription of different *Pdgfra* isoforms, including one with a truncated kinase domain. These isoforms regulate FAP activity and may determine the fate of FAP proteins, thus impacting the development of muscle fibrosis (Mueller et al., 2016). Further research is warranted to unravel the complex and dynamic interplay between FAPs, macrophages, and cytokines in the context of muscle homeostasis and inactivity.

When muscle activity is chronically reduced, for example, due to exercise detraining or prolonged immobilization, the factors promoting IMAT formation become dominant, a situation that is exacerbated by aging (Taaffe et al., 2009; Popadic Gacesa et al., 2011; Sertie et al., 2019; Mathes et al., 2021). In this scenario, the initial recruitment of pro-regenerative FAPs and their secretion of pro-myogenic factors may no longer counteract the negative effects of muscle inactivity, thus leading to abnormal fibrogenic and adipogenic programs. In support of this, healthy young adults who underwent four weeks of unilateral limb suspension experienced a concurrent 8% decrease in muscle mass and a 17% increase in IMAT content in the suspended limb, as determined by MRI (Manini et al., 2007). Notably, muscle loss accounted for 26% of the variance in IMAT accumulation (Manini et al., 2007).

In contrast to the causative role of muscle deconditioning in IMAT accumulation, studies in mice in which hindlimb unloading was used after acute GLY-induced muscle injury showed that by reducing muscle use, FAP accumulation and IMAT formation were prevented in regenerating muscles (Pagano et al. 2015, 2019). These effects were attributed to the maintenance of an inflammatory state post-injury that prevented FAP expansion by maintaining active TNF-α-mediated apoptotic signaling and reducing pro-proliferative TGF-β levels (Pagano et al., 2019). Accordingly, treating GLY-injured mice with decorin, an ECM and collagen-associated small leucine-rich proteoglycan that inhibits TGF-β, prevents IMAT accumulation (Pagano et al., 2019).

These findings suggest the occurrence of intricate, intercellular interactions between FAPs, immune cells, MuSCs, and mature myofibers that govern muscle mass maintenance and IMAT development. However, how myofibers communicate their activity state to nearby FAPs in the interstitial space is unknown. Despite being located in different compartments separated by the basal lamina, myofibers and MuSCs can inhibit FAP adipogenesis via cell-to-cell contact through NOTCH, retinoic acid signaling and Anx2 after muscle injury-induced basal lamina disruptions (Hogarth et al., 2019; Marinkovic et al., 2019). Reinforcing the importance of direct cell contact, neither conditioned media from differentiating myoblasts nor a transwell co-culture system (allowing media exchange but not direct contact) could block FAP adipogenesis, as previously described (Uezumi et al., 2010). However, whether uncharacterized secreted factors released by myofibers (i.e., myokines) are also involved in FAP adipogenesis is unknown. Whether physical activity at

the organismal level prevents the formation of IMAT by altering the phenotypical characteristics of FAPs via the release of exercise-induced myokines remains to be explored. Notably, combined advances in isolated FAP cell culture and optimization of differentiation media have enabled the generation of functional adipocytes from animal models and human FAPs (Johnson et al., 2022), establishing this approach as the gold standard for evaluating adipogenesis and FAP cell fate (Fig. 4A–C).

### 5.7. FAP adipogenesis and IMAT accumulation in aging and unhealthy aging

One of the most important physiological challenges affecting FAP function is aging. The proliferation and adipogenic capacities of muscle FAPs are significantly affected by aging. An early study by Mozzetta et al. (2013) revealed the enhanced adipogenic capacity of FAPs from the muscles of young *mdx* mice, which was reduced in FAPs from old *mdx* mice. Young FAPs also exert potent pro-myogenic effects via folliculin secretion on the activation of MuSCs and subsequent formation of myotubes (Mozzetta et al., 2013). Conversely, these effects were markedly reduced in aged *mdx*-FAPs. Interestingly, treatment with the histone deacetylase pan-inhibitor trichostatin A (TSA) reduced adipogenic potency while increasing the pro-myogenic effects of young *mdx*-FAPs; however, these effects were lost in aged *mdx*-FAPs. When young, pro-regenerative *mdx*-FAPs were transplanted into the muscles of old *mdx* mice, the capacity of TSA to promote regeneration was rescued (Mozzetta et al., 2013). This rescuing effect of the pan-HDAC inhibitor on the pro-regenerative capacity of aged FAPs suggested that over time, with the increase in degeneration/regeneration cycles, FAP properties change, coinciding with the accumulation of epigenetic marks on chromatin that can permanently impact the transcriptional output of FAPs, affecting their functional properties. In 15-month-old dystrophic *mdx* mice, the total number of muscle FAPs was reduced, while the proportion of cells expressing PPARγ was increased, suggesting that FAPs are more committed to adipogenic differentiation when exposed to the cell microenvironment of older dystrophic mice (Giuliani et al., 2021).

Aging impairs the paracrine pro-myogenic effect of FAPs to support MuSC activation and myogenesis (Lukjanenko et al., 2019). This effect is mediated by the FAP-derived matricellular protein WISP1. Like the findings of Mozzetta et al. (2013), transplantation of young FAPs or systemic treatment with WISP1 restored the myogenic capacity of MuSCs in aged mice, rescuing muscle regeneration (Lukjanenko et al., 2019). Aging markedly affects FAP proliferation and adipogenic fate in mice, as the total number of FAPs is lower in muscles from aged mice than in those from young mice, and FAPs from aged mice exhibit delayed injury-induced activation and expansion (Lukjanenko et al., 2019). Clonal analysis revealed a reduced capacity for expansion and formation of adipogenic clones, whereas fibrogenic differentiation was enhanced in FAPs from aged mice (Lukjanenko et al., 2019). These changes have been associated with age-related reductions in circulating fibroblast growth factor 2 (FGF2) and the SPARC myokine (Osteonectin) (Mathes et al., 2021). In this context, MuSCs and mature myofibers exert juxtacline anti-adipogenic effects on FAPs via the FGF2/miR-29a/SPARC signaling pathway (Mathes et al., 2021). FGF2 is actively secreted by FAPs, MuSCs and myofibers. This growth factor signals through the FGF receptor to activate MEK1/2 and ERK1/2, which upregulate miR-29a expression. Elevated miR-29a stimulates FAP adipogenesis through a reduction in SPARC, promoting IMAT formation (Mathes et al., 2021). Taken together, these findings support the idea that reduced muscle regeneration in aged muscles could be due to age-related changes in FAP chromatin dynamics, function and fate.

A recent study showed that atrophic muscles in aged mice were characterized by upregulation of hallmarks of senescence, including the cyclin-dependent kinase inhibitors *p16* and *p21*, together with telomere dysfunction, and loss of nuclear high mobility group box 1 protein (HMGB1) and Lamin B1 (Zhang et al., 2022). Interestingly, FAPs were
HFD-fed mice exhibit upregulation of genes related to the PPARγ signaling pathway and adipogenesis, such as *perilipin-1* (PLIN1), *Fabp4*, and *Cd36* (Takada et al., 2022). These results suggest that a HFD could influence the obesogenic microenvironment and therefore promote muscle atrophy by inducing some FAPs to become adipogenic.

In relation to inflammation, inflammation-mediated IL-1β secretion by macrophages may inhibit FAP-derived adipogenesis, whereas macrophage depletion via clodronate-loaded liposome treatment increases IMAT deposition after CTX-induced injury (Marinkovic et al., 2019). Another study showed that macrophages activated with IL-1β-conditioned media secreted unidentified factors that inhibited FAP adipogenesis via paracrine signaling. Moreover, both the IL-1α and IL-1β isoforms effectively suppress FAP adipogenesis while not affecting their proliferation (Vumbaca et al., 2021). Conversely, macrophage activation via exposure to the anti-inflammatory cytokine IL-4 promoted FAP adipogenesis (Moratal et al., 2018). These results reflect the complex regulation of FAP activity in response to injury, inflammation and disease. FAPs may have evolved to self-regulate their cellular states by secreting both anti- and pro-adipogenic factors in a balanced manner, potentially as a safeguard mechanism. There are data that support this concept. Injury-induced intronic polyadenylation of *Pdgfra* results in the transcription of different *Pdgfra* isoforms, including one with a truncated kinase domain. These isoforms regulate FAP activity and may determine the fate of FAP proteins, thus impacting the development of muscle fibrosis (Mueller et al., 2016). Further research is warranted to unravel the complex and dynamic interplay between FAPs, macrophages, and cytokines in the context of muscle homeostasis and inactivity.

When muscle activity is chronically reduced, for example, due to exercise detraining or prolonged immobilization, the factors promoting IMAT formation become dominant, a situation that is exacerbated by aging (Taaffe et al., 2009; Popadic Gacesa et al., 2011; Sertie et al., 2019; Mathes et al., 2021). In this scenario, the initial recruitment of pro-regenerative FAPs and their secretion of pro-myogenic factors may no longer counteract the negative effects of muscle inactivity, thus leading to abnormal fibrogenic and adipogenic programs. In support of this, healthy young adults who underwent four weeks of unilateral limb suspension experienced a concurrent 8% decrease in muscle mass and a 17% increase in IMAT content in the suspended limb, as determined by MRI (Manini et al., 2007). Notably, muscle loss accounted for 26% of the variance in IMAT accumulation (Manini et al., 2007).

In contrast to the causative role of muscle deconditioning in IMAT accumulation, studies in mice in which hindlimb unloading was used after acute GLY-induced muscle injury showed that by reducing muscle use, FAP accumulation and IMAT formation were prevented in regenerating muscles (Pagano et al. 2015, 2019). These effects were attributed to the maintenance of an inflammatory state post-injury that prevented FAP expansion by maintaining active TNF-α-mediated apoptotic signaling and reducing pro-proliferative TGF-β levels (Pagano et al., 2019). Accordingly, treating GLY-injured mice with decorin, an ECM and collagen-associated small leucine-rich proteoglycan that inhibits TGF-β, prevents IMAT accumulation (Pagano et al., 2019).

These findings suggest the occurrence of intricate, intercellular interactions between FAPs, immune cells, MuSCs, and mature myofibers that govern muscle mass maintenance and IMAT development. However, how myofibers communicate their activity state to nearby FAPs in the interstitial space is unknown. Despite being located in different compartments separated by the basal lamina, myofibers and MuSCs can inhibit FAP adipogenesis via cell-to-cell contact through NOTCH, retinoic acid signaling and Anx2 after muscle injury-induced basal lamina disruptions (Hogarth et al., 2019; Marinkovic et al., 2019). Reinforcing the importance of direct cell contact, neither conditioned media from differentiating myoblasts nor a transwell co-culture system (allowing media exchange but not direct contact) could block FAP adipogenesis, as previously described (Uezumi et al., 2010). However, whether uncharacterized secreted factors released by myofibers (i.e., myokines) are also involved in FAP adipogenesis is unknown. Whether physical activity at

the organismal level prevents the formation of IMAT by altering the phenotypical characteristics of FAPs via the release of exercise-induced myokines remains to be explored. Notably, combined advances in isolated FAP cell culture and optimization of differentiation media have enabled the generation of functional adipocytes from animal models and human FAPs (Johnson et al., 2022), establishing this approach as the gold standard for evaluating adipogenesis and FAP cell fate (Fig. 4A–C).

### 5.7. FAP adipogenesis and IMAT accumulation in aging and unhealthy aging

One of the most important physiological challenges affecting FAP function is aging. The proliferation and adipogenic capacities of muscle FAPs are significantly affected by aging. An early study by Mozzetta et al. (2013) revealed the enhanced adipogenic capacity of FAPs from the muscles of young *mdx* mice, which was reduced in FAPs from old *mdx* mice. Young FAPs also exert potent pro-myogenic effects via folliculin secretion on the activation of MuSCs and subsequent formation of myotubes (Mozzetta et al., 2013). Conversely, these effects were markedly reduced in aged *mdx*-FAPs. Interestingly, treatment with the histone deacetylase pan-inhibitor trichostatin A (TSA) reduced adipogenic potency while increasing the pro-myogenic effects of young *mdx*-FAPs; however, these effects were lost in aged *mdx*-FAPs. When young, pro-regenerative *mdx*-FAPs were transplanted into the muscles of old *mdx* mice, the capacity of TSA to promote regeneration was rescued (Mozzetta et al., 2013). This rescuing effect of the pan-HDAC inhibitor on the pro-regenerative capacity of aged FAPs suggested that over time, with the increase in degeneration/regeneration cycles, FAP properties change, coinciding with the accumulation of epigenetic marks on chromatin that can permanently impact the transcriptional output of FAPs, affecting their functional properties. In 15-month-old dystrophic *mdx* mice, the total number of muscle FAPs was reduced, while the proportion of cells expressing PPARγ was increased, suggesting that FAPs are more committed to adipogenic differentiation when exposed to the cell microenvironment of older dystrophic mice (Giuliani et al., 2021).

Aging impairs the paracrine pro-myogenic effect of FAPs to support MuSC activation and myogenesis (Lukjanenko et al., 2019). This effect is mediated by the FAP-derived matricellular protein WISP1. Like the findings of Mozzetta et al. (2013), transplantation of young FAPs or systemic treatment with WISP1 restored the myogenic capacity of MuSCs in aged mice, rescuing muscle regeneration (Lukjanenko et al., 2019). Aging markedly affects FAP proliferation and adipogenic fate in mice, as the total number of FAPs is lower in muscles from aged mice than in those from young mice, and FAPs from aged mice exhibit delayed injury-induced activation and expansion (Lukjanenko et al., 2019). Clonal analysis revealed a reduced capacity for expansion and formation of adipogenic clones, whereas fibrogenic differentiation was enhanced in FAPs from aged mice (Lukjanenko et al., 2019). These changes have been associated with age-related reductions in circulating fibroblast growth factor 2 (FGF2) and the SPARC myokine (Osteonectin) (Mathes et al., 2021). In this context, MuSCs and mature myofibers exert juxtacline anti-adipogenic effects on FAPs via the FGF2/miR-29a/SPARC signaling pathway (Mathes et al., 2021). FGF2 is actively secreted by FAPs, MuSCs and myofibers. This growth factor signals through the FGF receptor to activate MEK1/2 and ERK1/2, which upregulate miR-29a expression. Elevated miR-29a stimulates FAP adipogenesis through a reduction in SPARC, promoting IMAT formation (Mathes et al., 2021). Taken together, these findings support the idea that reduced muscle regeneration in aged muscles could be due to age-related changes in FAP chromatin dynamics, function and fate.

A recent study showed that atrophic muscles in aged mice were characterized by upregulation of hallmarks of senescence, including the cyclin-dependent kinase inhibitors *p16* and *p21*, together with telomere dysfunction, and loss of nuclear high mobility group box 1 protein (HMGB1) and Lamin B1 (Zhang et al., 2022). Interestingly, FAPs were
<figure><img src="image_5.png" /><figcaption>**Fig. 4.** Formation of lipid droplet-rich adipocytes from fibro-adipogenic progenitors in cell culture. (A) Mouse muscle FAPs can be readily differentiated into functional adipocytes containing lipid droplets, as stained with Oil Red O. (B) PDGFRα lineage tracing shows that FAPs can generate adipocytes in culture with lipid droplets of different sizes and abundance. (C) Human muscle FAPs can also be differentiated into adipocytes that heavily accumulate lipid droplets. (D) Ventricle-resident fibroblasts can also generate perilipin⁺ adipocytes *in vitro*, although their adipogenic potential is known to be restrained *in vivo*.</figcaption></figure>

the most predominant cells expressing *p16*. Senescent *p16*<sup>high</sup> FAPs showed upregulation of senescence-related genes and processes including proliferation regulation, collagen processing, chemokine signaling, cytokine-cytokine receptor interaction, and MAK signaling (Zhang et al., 2022). Importantly, treatment with senolytic agents dasatinib and quercetin markedly attenuated the expression of senescence-related genes and pathways in skeletal muscle of old mice compared to untreated old counterparts and young mice (Zhang et al., 2022). Overall, these findings highlight a pathogenic role of senescent FAPs in age-related muscle sarcopenia.

## 5.8. FAP adipogenesis in rotator cuff tears

One key physiopathological example of FAP activation and proliferation and subsequent IMAT formation in musculoskeletal disorders is the extensive fibro-fatty infiltration of rotator cuff muscles after tearing (Liu et al., 2016). IMAT in response to rotator cuff tears (RCTs) compromises muscle-tendon repair, leading to poor functional outcomes, muscle atrophy, and tendon retearing (Goutallier et al., 2003; Gladstone et al., 2007; Klatte-Schulz et al., 2014). Following RCT, FAPs proliferate and undergo fibro-adipogenesis leading to rapid accumulation of fibrosis and IMAT near the site of the tendon tear. Here, IMAT is formed by a high number of small FAP-derived adipocytes clustering in large fat clumps rather than as enlarged hypertrophic adipocytes (Trudel et al., 2019). This adipocyte hyperplasia indicates continuous stimulation of adipogenesis in FAPs throughout the progression of muscle degeneration (Trudel et al., 2019). The association between IMAT and rotator cuff muscle atrophy may be related to the FAP-derived adipocyte secretome, which includes extracellular vesicles carrying microRNAs, fatty acids (FAs), and pro-inflammatory cytokines that can interfere with muscle signaling and metabolic pathways associated with muscle regeneration (Sachs et al., 2019; Sandona et al., 2020). This highlights the pathogenic role of FAP-derived adipocytes in muscle atrophy following RCT. Interestingly, treatment with retinoic acid receptor agonists (Shirasawa et al., 2021), inhibition of PDGFR signaling by imatinib (Shirasawa et al., 2017), the TGF-βR small molecule inhibitor SB431542 (Davies et al., 2016), and GDF10/BMP-3b inhibitors reduce FAP adipogenesis

and muscle atrophy in animal models of RCTs, opening up new therapeutic possibilities for debilitating RCTs.

Although the aberrant behavior of FAPs in chronic muscle damage and myopathy suggests that they play a major role in the pathogenesis of these diseases (Uezumi et al. 2010, 2021; Mozzetta et al., 2013; Buras et al., 2019; Hogarth et al., 2019; Eisner et al., 2020; Farup et al., 2021), this relationship is not necessarily causative. The associations can be divided into at least two components. First, chronic cycles of degeneration and regeneration can lead to abnormal FAP behavior, regardless of the initial trigger; second, after the FAP steady state is disrupted and fibro-adipogenesis is unleashed, this altered behavior can further contribute to the progression and worsening of subsequent pathologies. A better understanding of these components may shed light on the development and potential treatment of pathologies associated with aberrant FAP behavior.

## 5.9. Role of hypoxia and vascularization in FAP adipogenesis

### 5.9.1. Hypoxia inducible family (HIF) of transcription factors

Hypoxia refers to physiological or pathophysiological-induced states in which normal oxygen tension is reduced. The molecular and cellular adaptations to hypoxia are primarily mediated by the hypoxia inducible family (HIF) of the transcription factors alpha and beta (HIF-α and HIF-β). Under normal oxygen concentrations, HIF prolyl hydroxylase enzymes hydroxylate HIF-α subunits at conserved proline residues, which are recognized and ubiquitinated via the Von Hippel-Lindau E3 ubiquitin ligase and thus targeted for proteasomal degradation. However, hypoxia usually leads to the stabilization of HIF-α subunits via a mechanism involving low-oxygen-driven inhibition of the HIF prolyl-hydroxylase-Von Hippel-Lindau E3 ubiquitin ligase axis (Schofield and Ratcliffe 2004; Lee et al., 2020d). On the other hand, hypoxia blocks degradation and stabilizes HIF-α, which allows its cytoplasmic accumulation and translocation to the nucleus, where it heterodimerizes with one constitutively expressed HIF-β subunit to transactivate genes involved in the adaptation and sensing of hypoxia (Choudhry and Harris 2018).

HIF-1α and HIF-2α are essential for adult muscle regeneration but
<figure><img src="image_10.png" /><figcaption>**Fig. 4.** Formation of lipid droplet-rich adipocytes from fibro-adipogenic progenitors in cell culture. (A) Mouse muscle FAPs can be readily differentiated into functional adipocytes containing lipid droplets, as stained with Oil Red O. (B) PDGFRα lineage tracing shows that FAPs can generate adipocytes in culture with lipid droplets of different sizes and abundance. (C) Human muscle FAPs can also be differentiated into adipocytes that heavily accumulate lipid droplets. (D) Ventricle-resident fibroblasts can also generate perilipin⁺ adipocytes *in vitro*, although their adipogenic potential is known to be restrained *in vivo*.</figcaption></figure>

the most predominant cells expressing *p16*. Senescent *p16*<sup>high</sup> FAPs showed upregulation of senescence-related genes and processes including proliferation regulation, collagen processing, chemokine signaling, cytokine-cytokine receptor interaction, and MAK signaling (Zhang et al., 2022). Importantly, treatment with senolytic agents dasatinib and quercetin markedly attenuated the expression of senescence-related genes and pathways in skeletal muscle of old mice compared to untreated old counterparts and young mice (Zhang et al., 2022). Overall, these findings highlight a pathogenic role of senescent FAPs in age-related muscle sarcopenia.

## 5.8. FAP adipogenesis in rotator cuff tears

One key physiopathological example of FAP activation and proliferation and subsequent IMAT formation in musculoskeletal disorders is the extensive fibro-fatty infiltration of rotator cuff muscles after tearing (Liu et al., 2016). IMAT in response to rotator cuff tears (RCTs) compromises muscle-tendon repair, leading to poor functional outcomes, muscle atrophy, and tendon retearing (Goutallier et al., 2003; Gladstone et al., 2007; Klatte-Schulz et al., 2014). Following RCT, FAPs proliferate and undergo fibro-adipogenesis leading to rapid accumulation of fibrosis and IMAT near the site of the tendon tear. Here, IMAT is formed by a high number of small FAP-derived adipocytes clustering in large fat clumps rather than as enlarged hypertrophic adipocytes (Trudel et al., 2019). This adipocyte hyperplasia indicates continuous stimulation of adipogenesis in FAPs throughout the progression of muscle degeneration (Trudel et al., 2019). The association between IMAT and rotator cuff muscle atrophy may be related to the FAP-derived adipocyte secretome, which includes extracellular vesicles carrying microRNAs, fatty acids (FAs), and pro-inflammatory cytokines that can interfere with muscle signaling and metabolic pathways associated with muscle regeneration (Sachs et al., 2019; Sandona et al., 2020). This highlights the pathogenic role of FAP-derived adipocytes in muscle atrophy following RCT. Interestingly, treatment with retinoic acid receptor agonists (Shirasawa et al., 2021), inhibition of PDGFR signaling by imatinib (Shirasawa et al., 2017), the TGF-βR small molecule inhibitor SB431542 (Davies et al., 2016), and GDF10/BMP-3b inhibitors reduce FAP adipogenesis

and muscle atrophy in animal models of RCTs, opening up new therapeutic possibilities for debilitating RCTs.

Although the aberrant behavior of FAPs in chronic muscle damage and myopathy suggests that they play a major role in the pathogenesis of these diseases (Uezumi et al. 2010, 2021; Mozzetta et al., 2013; Buras et al., 2019; Hogarth et al., 2019; Eisner et al., 2020; Farup et al., 2021), this relationship is not necessarily causative. The associations can be divided into at least two components. First, chronic cycles of degeneration and regeneration can lead to abnormal FAP behavior, regardless of the initial trigger; second, after the FAP steady state is disrupted and fibro-adipogenesis is unleashed, this altered behavior can further contribute to the progression and worsening of subsequent pathologies. A better understanding of these components may shed light on the development and potential treatment of pathologies associated with aberrant FAP behavior.

## 5.9. Role of hypoxia and vascularization in FAP adipogenesis

### 5.9.1. Hypoxia inducible family (HIF) of transcription factors

Hypoxia refers to physiological or pathophysiological-induced states in which normal oxygen tension is reduced. The molecular and cellular adaptations to hypoxia are primarily mediated by the hypoxia inducible family (HIF) of the transcription factors alpha and beta (HIF-α and HIF-β). Under normal oxygen concentrations, HIF prolyl hydroxylase enzymes hydroxylate HIF-α subunits at conserved proline residues, which are recognized and ubiquitinated via the Von Hippel-Lindau E3 ubiquitin ligase and thus targeted for proteasomal degradation. However, hypoxia usually leads to the stabilization of HIF-α subunits via a mechanism involving low-oxygen-driven inhibition of the HIF prolyl-hydroxylase-Von Hippel-Lindau E3 ubiquitin ligase axis (Schofield and Ratcliffe 2004; Lee et al., 2020d). On the other hand, hypoxia blocks degradation and stabilizes HIF-α, which allows its cytoplasmic accumulation and translocation to the nucleus, where it heterodimerizes with one constitutively expressed HIF-β subunit to transactivate genes involved in the adaptation and sensing of hypoxia (Choudhry and Harris 2018).

HIF-1α and HIF-2α are essential for adult muscle regeneration but
dispensable for embryonic skeletal muscle development (Yang et al., 2017). Acute injury induces hypoxia and inhibits FAP adipogenesis (Drouin et al., 2019) by disrupting the vascular network (Morton et al., 2019). Furthermore, hypobaric hypoxia compromises skeletal muscle regeneration after notexin-induced damage in rats (Chaillou et al., 2014). Muscle hypoxia-like states are also observed after denervation in ALS-affected hSOD1G93A mutant mice and dystrophic mdx mice and are correlated with reduced endothelial cell density (Valle-Tenney et al., 2020). However, bulk HIF-1α protein levels vary in these models of muscle disease (Valle-Tenney et al., 2020), suggesting differential regulation of HIF-1α in distinct myo-degenerative conditions. Increased HIF expression was linked to IMAT and atrophy after tendon retraction in humans (Lakemeier et al., 2010), where HIF expression was proportional to the magnitude and extent of damage. On the other hand, Lee et al. (2017b) showed that hypoxia increases HIF-1α and HIF-1β binding to the FABP4 promoter in C3H10T1/2 cells, leading to increased FABP4 mRNA and protein levels (Lee et al., 2017b). Complete supraspinatus tendon transection, a mouse model of RCT, is related to hypoxia-induced IMAT accumulation, which is reversed by treatment with either HIF-1 or FABP4 inhibitors (Lee et al., 2017b). These findings contrast with the hypoxia-driven anti-adipogenic effects of FAPs (Drouin et al., 2019) and reveal the association of fat infiltration after RCT with hypoxia-mediated HIF induction and its effect on adipogenic FABP4 expression (Lee et al., 2017b).

A recent preprint revealed that muscle FAPs expressed more HIF-1α than MuSCs did, and CTX injury-induced hypoxia promoted the proliferation of FAPs through a mechanism mediated by mTORC and HIF-1α (Ollitrault et al., 2021). The authors also confirmed that hypoxia blocks FAP adipogenesis, as previously described (Drouin et al., 2019). These findings could explain the environment-driven differentiation of FAPs toward an activated fibroblast rather than an adipogenic phenotype upon acute injury-induced hypoxia. We recently explored the role of HIF-1α in cardiac fibroblasts (likely FAPs) in response to myocardial infarction. These cells had an elevated hypoxic status together with increased levels of HIF-1α compared with those in the total interstitial cell fraction (Janbandhu et al., 2022). Conditional knockout of *Hif-1α* enhances the proliferation of PDGFRα⁺ cells postcardiac injury, likely driven by priming cell cycle entry after myocardial infarction (Janbandhu et al., 2022). These findings indicated that tissue challenges, hypoxia, and disease conditions play pivotal roles in controlling the behavior and dynamics of fibroblast lineages. Potential therapeutic interventions utilizing oxygen tension and the HIF axis in diseased muscles have yet to be demonstrated.

### 5.9.2. Vascular endothelial growth factor a (VEGFA)

A recent study revealed that FAPs, immune cells, and MuSCs secrete the pro-angiogenic factor VEGFA (Groppa et al., 2023). First, hematopoietic cell-derived VEGFA is essential for macrophage infiltration and vascular remodeling in response to damage; therefore, immune-derived VEGFA is critical for muscle regeneration (Groppa et al., 2023). On the one hand, MuSC-derived but not FAP-derived VEGFA was necessary for efficient muscle regeneration following notexin-induced damage (Groppa et al., 2023). On the other hand, following femoral artery ligation to induce ischemic damage, VEGFA secreted by FAPs is critical for inducing endothelial cell proliferation and angiogenesis. Conversely, the lack of this signal induces a pro-inflammatory phenotype, mostly via IL-6 upregulation in FAPs. Notably, FAP adipogenesis was upregulated in the absence of MuSC-derived VEGFA but markedly reduced in mice lacking FAP-derived VEGFA, demonstrating an autocrine anti-adipogenic effect of FAP-derived IL-6 secretion (Groppa et al., 2023). Ischemic damage to skeletal muscle reorganizes the basement membrane, an ECM-based physical barrier that separates MuSCs, endothelial cells, and FAPs into different compartments within the interstitial space and myofibers. Ischemia-induced basement membrane rupture allows FAPs and endothelial cells to interact. This reorganization allows FAP-derived VEGFA to be delivered directly to endothelial

cells, thus providing the required signal for EC proliferation and angiogenesis (Groppa et al., 2023).

Aging alters VEGFA signaling in muscles. Compared with young mice, aged mice have reduced levels of VEGFA in skeletal muscle and an impaired regeneration capacity after injury (Endo et al., 2023). This effect can be rescued via systemic administration of the small molecule ML228, which induces HIF-1α nuclear translocation and stimulates VEGF production (Endo et al., 2023). Genetically mediated global reduction in VEGFA (probably affecting global VEGFA production by different cell types, including immune cells, MPs and FAPs) led to marked impairment of regeneration and a threefold increase in IMAT deposition in mice (Endo et al., 2023). This finding suggested compromised crosstalk of VEGFA between different muscle compartments within muscle tissue. Overall, these findings revealed a VEGF-mediated intricate and promiscuous intercellular crosstalk network that regulates vascular remodeling and myogenesis, as well as FAP activation, proliferation, and fate.

## 6. Reduced muscle substrate buffering capacity as a driver of FAP adipogenesis and IMAT: a hypothetical framework

Given that IMAT is a fat depot, it is susceptible to alterations based on an individual's dietary intake. Daily macronutrient and alcohol intake can influence body fat distribution and the IMAT content in young and old individuals (Sjöholm et al., 2016). Protein and alcohol intake, as well as serum cholesterol levels, were correlated positively with IMAT, whereas carbohydrate intake exerted the opposite effect (Sjöholm et al., 2016). In line with these findings, a recent multi-ethnic study showed that heavy alcohol intake and binge alcohol consumption significantly increase the accumulation of ectopic fat in different tissues, including IMAT (Kazibwe et al., 2023). However, in the case of IMAT, this association was not significant once adjusted for other health-related habits, including physical activity (Kazibwe et al., 2023). Studies using short-term metabolic challenges, such as a HFD *in vivo*, glucose-lowering agents, and fatty acid treatments in FAP cultures, have demonstrated a shift toward a pathological state involving the remodeling of FAP oxidative metabolism (Aguiari et al., 2008; Agley et al., 2013; Buras et al., 2019; Reggio et al., 2020a; Farup et al., 2021). In this section, we elaborate on a hypothetical framework for how reduced muscle activity may impair metabolic flexibility within muscles and discuss the evidence for a link between these phenomena and adipogenesis.

### 6.1. Substrate-driven metabolic reprogramming of FAPs

FAPs respond to metabolic challenges in skeletal muscle by remodeling the metabolic machinery in accordance with the prevailing cellular environment. At rest, FAPs rely mainly on mitochondrial fatty acid (FA) oxidation for energy production (Reggio et al., 2020a). Proteomic profiling of FAPs revealed that proteins involved in key metabolic processes are differentially regulated upon activation and differentiation in muscular dystrophy (Marinkovic et al., 2019; Reggio et al., 2020a). The expression of enzymes involved in FA β-oxidation, the tricarboxylic acid cycle, oxidative phosphorylation and lipid biosynthesis is downregulated, thus reducing oxygen consumption, the mitochondrial potential, and ATP production (Reggio et al., 2020a). Conversely, enzymes that participate in glycolysis and the pentose phosphate cycle, including pyruvate kinase M2 and glucose-6-phosphate dehydrogenase, are upregulated (Marinkovic et al., 2019; Reggio et al., 2020a). These findings are consistent with a metabolic switch of FAPs in a dystrophic milieu, with upregulated glycolytic, anabolic-like metabolism (Reggio et al., 2020a). A reliance on glycolysis, with subsequent increased lactate production, has been reported in human fibrogenic CD90⁺ FAPs (Farup et al., 2021). This metabolic switch was associated with a pro-fibrogenic phenotype required to synthesize ECM (Farup et al., 2021). Interestingly, adipogenic CD90⁻ FAPs also rely on glycolysis when exposed to PDGF-AA (Farup et al., 2021). In line with these
dispensable for embryonic skeletal muscle development (Yang et al., 2017). Acute injury induces hypoxia and inhibits FAP adipogenesis (Drouin et al., 2019) by disrupting the vascular network (Morton et al., 2019). Furthermore, hypobaric hypoxia compromises skeletal muscle regeneration after notexin-induced damage in rats (Chaillou et al., 2014). Muscle hypoxia-like states are also observed after denervation in ALS-affected hSOD1G93A mutant mice and dystrophic mdx mice and are correlated with reduced endothelial cell density (Valle-Tenney et al., 2020). However, bulk HIF-1α protein levels vary in these models of muscle disease (Valle-Tenney et al., 2020), suggesting differential regulation of HIF-1α in distinct myo-degenerative conditions. Increased HIF expression was linked to IMAT and atrophy after tendon retraction in humans (Lakemeier et al., 2010), where HIF expression was proportional to the magnitude and extent of damage. On the other hand, Lee et al. (2017b) showed that hypoxia increases HIF-1α and HIF-1β binding to the FABP4 promoter in C3H10T1/2 cells, leading to increased FABP4 mRNA and protein levels (Lee et al., 2017b). Complete supraspinatus tendon transection, a mouse model of RCT, is related to hypoxia-induced IMAT accumulation, which is reversed by treatment with either HIF-1 or FABP4 inhibitors (Lee et al., 2017b). These findings contrast with the hypoxia-driven anti-adipogenic effects of FAPs (Drouin et al., 2019) and reveal the association of fat infiltration after RCT with hypoxia-mediated HIF induction and its effect on adipogenic FABP4 expression (Lee et al., 2017b).

A recent preprint revealed that muscle FAPs expressed more HIF-1α than MuSCs did, and CTX injury-induced hypoxia promoted the proliferation of FAPs through a mechanism mediated by mTORC and HIF-1α (Ollitrault et al., 2021). The authors also confirmed that hypoxia blocks FAP adipogenesis, as previously described (Drouin et al., 2019). These findings could explain the environment-driven differentiation of FAPs toward an activated fibroblast rather than an adipogenic phenotype upon acute injury-induced hypoxia. We recently explored the role of HIF-1α in cardiac fibroblasts (likely FAPs) in response to myocardial infarction. These cells had an elevated hypoxic status together with increased levels of HIF-1α compared with those in the total interstitial cell fraction (Janbandhu et al., 2022). Conditional knockout of *Hif-1α* enhances the proliferation of PDGFRα⁺ cells postcardiac injury, likely driven by priming cell cycle entry after myocardial infarction (Janbandhu et al., 2022). These findings indicated that tissue challenges, hypoxia, and disease conditions play pivotal roles in controlling the behavior and dynamics of fibroblast lineages. Potential therapeutic interventions utilizing oxygen tension and the HIF axis in diseased muscles have yet to be demonstrated.

### 5.9.2. Vascular endothelial growth factor a (VEGFA)

A recent study revealed that FAPs, immune cells, and MuSCs secrete the pro-angiogenic factor VEGFA (Groppa et al., 2023). First, hematopoietic cell-derived VEGFA is essential for macrophage infiltration and vascular remodeling in response to damage; therefore, immune-derived VEGFA is critical for muscle regeneration (Groppa et al., 2023). On the one hand, MuSC-derived but not FAP-derived VEGFA was necessary for efficient muscle regeneration following notexin-induced damage (Groppa et al., 2023). On the other hand, following femoral artery ligation to induce ischemic damage, VEGFA secreted by FAPs is critical for inducing endothelial cell proliferation and angiogenesis. Conversely, the lack of this signal induces a pro-inflammatory phenotype, mostly via IL-6 upregulation in FAPs. Notably, FAP adipogenesis was upregulated in the absence of MuSC-derived VEGFA but markedly reduced in mice lacking FAP-derived VEGFA, demonstrating an autocrine anti-adipogenic effect of FAP-derived IL-6 secretion (Groppa et al., 2023). Ischemic damage to skeletal muscle reorganizes the basement membrane, an ECM-based physical barrier that separates MuSCs, endothelial cells, and FAPs into different compartments within the interstitial space and myofibers. Ischemia-induced basement membrane rupture allows FAPs and endothelial cells to interact. This reorganization allows FAP-derived VEGFA to be delivered directly to endothelial

cells, thus providing the required signal for EC proliferation and angiogenesis (Groppa et al., 2023).

Aging alters VEGFA signaling in muscles. Compared with young mice, aged mice have reduced levels of VEGFA in skeletal muscle and an impaired regeneration capacity after injury (Endo et al., 2023). This effect can be rescued via systemic administration of the small molecule ML228, which induces HIF-1α nuclear translocation and stimulates VEGF production (Endo et al., 2023). Genetically mediated global reduction in VEGFA (probably affecting global VEGFA production by different cell types, including immune cells, MPs and FAPs) led to marked impairment of regeneration and a threefold increase in IMAT deposition in mice (Endo et al., 2023). This finding suggested compromised crosstalk of VEGFA between different muscle compartments within muscle tissue. Overall, these findings revealed a VEGF-mediated intricate and promiscuous intercellular crosstalk network that regulates vascular remodeling and myogenesis, as well as FAP activation, proliferation, and fate.

## 6. Reduced muscle substrate buffering capacity as a driver of FAP adipogenesis and IMAT: a hypothetical framework

Given that IMAT is a fat depot, it is susceptible to alterations based on an individual's dietary intake. Daily macronutrient and alcohol intake can influence body fat distribution and the IMAT content in young and old individuals (Sjöholm et al., 2016). Protein and alcohol intake, as well as serum cholesterol levels, were correlated positively with IMAT, whereas carbohydrate intake exerted the opposite effect (Sjöholm et al., 2016). In line with these findings, a recent multi-ethnic study showed that heavy alcohol intake and binge alcohol consumption significantly increase the accumulation of ectopic fat in different tissues, including IMAT (Kazibwe et al., 2023). However, in the case of IMAT, this association was not significant once adjusted for other health-related habits, including physical activity (Kazibwe et al., 2023). Studies using short-term metabolic challenges, such as a HFD *in vivo*, glucose-lowering agents, and fatty acid treatments in FAP cultures, have demonstrated a shift toward a pathological state involving the remodeling of FAP oxidative metabolism (Aguiari et al., 2008; Agley et al., 2013; Buras et al., 2019; Reggio et al., 2020a; Farup et al., 2021). In this section, we elaborate on a hypothetical framework for how reduced muscle activity may impair metabolic flexibility within muscles and discuss the evidence for a link between these phenomena and adipogenesis.

### 6.1. Substrate-driven metabolic reprogramming of FAPs

FAPs respond to metabolic challenges in skeletal muscle by remodeling the metabolic machinery in accordance with the prevailing cellular environment. At rest, FAPs rely mainly on mitochondrial fatty acid (FA) oxidation for energy production (Reggio et al., 2020a). Proteomic profiling of FAPs revealed that proteins involved in key metabolic processes are differentially regulated upon activation and differentiation in muscular dystrophy (Marinkovic et al., 2019; Reggio et al., 2020a). The expression of enzymes involved in FA β-oxidation, the tricarboxylic acid cycle, oxidative phosphorylation and lipid biosynthesis is downregulated, thus reducing oxygen consumption, the mitochondrial potential, and ATP production (Reggio et al., 2020a). Conversely, enzymes that participate in glycolysis and the pentose phosphate cycle, including pyruvate kinase M2 and glucose-6-phosphate dehydrogenase, are upregulated (Marinkovic et al., 2019; Reggio et al., 2020a). These findings are consistent with a metabolic switch of FAPs in a dystrophic milieu, with upregulated glycolytic, anabolic-like metabolism (Reggio et al., 2020a). A reliance on glycolysis, with subsequent increased lactate production, has been reported in human fibrogenic CD90⁺ FAPs (Farup et al., 2021). This metabolic switch was associated with a pro-fibrogenic phenotype required to synthesize ECM (Farup et al., 2021). Interestingly, adipogenic CD90⁻ FAPs also rely on glycolysis when exposed to PDGF-AA (Farup et al., 2021). In line with these
findings, an increase in glycolytic flux was associated with increased proliferation and adipogenic differentiation of FAPs isolated from the muscles of *mdx* mice (Reggio et al., 2020a). Interestingly, a short-term HFD regimen restored the expression of mitochondrial proteins; rescued FA oxidation and oxygen consumption; and increased ATP production while reducing the proliferation of dystrophic FAPs. Together, these effects ameliorate the dystrophic muscle phenotype in mice (Reggio et al., 2020a).

High glucose reprograms the adipogenic potential of precursor cells by upregulating adipogenic transcription factor expression and inhibiting anti-adipogenic WNT/β-catenin signaling (Keats et al., 2014; Kolodziej et al., 2019). This effect may be mediated by the production of ROS and subsequent activation of conventional protein kinase C-beta (PKCβ) (Aguiari et al., 2008). PKCβ synergistically potentiates PPARγ transcriptional activity during adipogenesis (Zhou et al., 2006), which likely explains these effects.

Cardiac FAPs exhibit increased glycolytic metabolism in association with increased proliferation, scar formation and reactive oxygen species (ROS) release, leading to contractile dysfunction after myocardial infarction (Janbandhu et al., 2022). Importantly, by fractionating cardiac FAPs based on mitochondrial mass, we reported that Mitohigh FAPs have greater fibrogenesis potential but lacks adipogenic potential in vitro. In contrast, ~15% of the Mitolow and Mitomedium FAP fractions differentiated into PLIN1+ adipocytes upon adipogenic stimulation, indicating that FAPs are metabolically heterogeneous, with progenitor cells maintaining a lower mitochondrial mass, and reinforcing the notion that mitochondrial dysfunction could drive the adipogenic fate of FAPs (Janbandhu et al., 2022) (Fig. 4D).

In individuals with T2DM, both arterial and interstitial glucose concentrations are increased, reducing the transcapillary glucose gradient (Frossard et al., 2005). This can be attributed to reduced muscle insulin signaling transduction, which leads to insulin resistance and reduced insulin-stimulated glucose uptake (~30–40%) in individuals with T2DM (Sjöstrand et al., 2000; Middelbeek et al., 2013). Taken together, these findings suggest that impaired insulin signaling transduction and reduced local blood flow may increase muscle interstitial glucose and insulin concentrations, resulting in their accumulation in the muscle interstitium. This could affect FAP differentiation capacity, particularly in FAP subtypes enriched in lipid and adipogenesis pathways, such as FBN1 FAPs in humans and Prg4 FAPs in mice (Rubenstein et al., 2020; Yang et al., 2021). Insulin is the primary hormonal regulator of adipogenesis in progenitor cells and preadipocytes (Czech et al., 2013) and of lipid turnover in mature adipocytes (Shao et al., 2016; Krycer et al., 2020). In patients with diabetes, a larger arterial-interstitial insulin gradient and transcapillary transport time are required for insulin to reach and stimulate glucose uptake (Sandqvist et al., 2011). The accumulation of IMAT has been suggested to impair insulin diffusion through the muscle interstitial space, leading to insulin resistance in dogs (Kolka et al., 2010). These findings suggest that reduced insulin clearance from the interstitial space under metabolic conditions may generate proper medium for stimulating FAP adipogenesis. In addition to glucose serving as a source for de novo lipogenesis, lactate can also be utilized to synthesize glycerol-3-phosphate and increase TG synthesis, thus supporting the storage of excess energy in conditions of impaired glucose metabolism (Nye et al. 2008a, 2008b). Plasma lactate is increased in patients with obesity, metabolic syndrome, or T2DM and is inversely correlated with insulin sensitivity (reviewed by (Broskey et al., 2020)). An increased interstitial lactate concentration can modulate vascular tone in muscle arterioles, reducing muscle blood flow and preventing lactate clearance from the interstitial space (Qvisth et al., 2007). This interstitial lactate may be taken up by FAP-derived adipocytes and utilized as a lipogenic precursor. Muscle-derived lactate stimulates TGF-β2 expression in adipocytes (Takahashi et al., 2019). High levels of lactate in the muscle interstitial space can induce the expression and secretion of TGF-β2 from FAPs, favoring their proliferation and survival. Overall, these findings suggest

that impaired glucose metabolism within muscle, characterized by increased interstitial levels of glucose, lactate, and insulin, may provide a favorable cellular environment for the adipogenic differentiation of FAPs.

Long-chain fatty acids are ligands of PPARγ and can induce adipogenesis of precursor cells in adipose tissue (Amri et al., 1995; Shao et al., 2016). Physiological doses of FAs increase the intracellular lipid content and upregulate the expression of *Pparγ* and *C/ebpα* in both human FAPs and MuSCs in culture, but adipocyte formation occurs only in FAPs (Agley et al., 2013). A 4-week short-term HFD intervention improved mitochondrial function and FA oxidation in muscular dystrophy FAPs and upregulated anti-adipogenic WNT/β-catenin signaling and the expression of the pro-myogenic factor *follistatin*. Overall, these changes led to an improved dystrophic phenotype in adult *mdx* mice (Reggio et al., 2020a).

Long-term (20-week) HFD-induced obesity led to interstitial accumulation of the lectin family protein galectin-3 (Gal-3), a potent che-moattractant (Takada et al., 2022) involved in cell adhesion, cell cycle progression, apoptosis, inflammation, cell proliferation, and differentiation (Henderson and Sethi 2009). Gal-3 is predominantly secreted by immune cells, mature adipocytes, and stromal cells in visceral and subcutaneous adipose tissues, and its expression is upregulated in individuals with obesity and T2DM (Weigert et al., 2010; Rhodes et al., 2013). Conversely, Gal-3 knockdown reduced adipocyte differentiation via downregulation of the transcriptional activity of PPARγ, C/EBPα, and C/EBPβ and the expression of lipogenic genes in visceral adipose tissue and the liver (Kiwaki et al., 2007; Baek et al., 2015).

A chronic HFD has been shown to increase FAP adipogenesis and muscle dysfunction (Buras et al., 2019; White et al., 2021; Zhang et al., 2021). Likewise, a 20-week HFD induced FAP adipogenic differentiation and IMAT accumulation in hindlimb muscles, with more accumulation in the quadriceps than in the gastrocnemius and *tibialis anterior* muscles (Takada et al., 2022). Moreover, a HFD impaired muscle regeneration after CTX-induced injury by accelerating IMAT formation and promoting myofiber atrophy; similar responses were observed in genetically obese *ob/ob* and *db/db* mice (Takada et al., 2022). These findings indicate that obesity induces IMAT formation independently of diet composition, similar to what has been described in obese, insulin-resistant *db/db* and KK-A^Y mice, a genetic model of obesity and diabetes characterized by altered adipokine expression, dyslipidemia, and insulin resistance (Mogi et al., 2016).

Finally, impaired circulating levels of hormones such as insulin, GH, IGF-1, estradiol, and testosterone T3 (Freda et al., 2008; Gill et al., 2016; Farup et al., 2021); various cytokines, including TSP-1, DKK1, CCN1, IL-8, MCP1, TNF-α, FGF2, FGF21 and SPARC (Poehlman et al., 2000; Varma et al., 2008; Cheema et al., 2010; Haam et al., 2016; Ali et al., 2019; Buras et al., 2019; Hong et al., 2019; Hu et al., 2019; Mathes et al., 2021; Yang et al., 2021); circulating TG; lipoproteins; and metabolites such as creatinine and ammonia (Gilsanz et al., 2010; Kitajima et al., 2013; Montano-Loza et al., 2016; Nardelli et al., 2019), have all been associated with increased IMAT content in chronic diseases (Table 1).

## 7. Exercise as a treatment against pathological IMAT accumulation

Investigating the impact of exercise training on ectopic IMAT in adults with chronic diseases, we found that moderate-intensity aerobic exercise, alone or in combination with resistance training, effectively reduced IMAT (Tunon-Suarez et al., 2021). Exercise may affect FAPs and FAP-derived adipocytes, although the mechanisms underlying this effect have not been fully elucidated. For example, exercise can reduce muscle glucose interstitial concentration through contraction-stimulated glucose uptake, independent of insulin-mediated capillary vasodilation (McConell et al., 2020), and downregulate ECM deposition and FAP adipogenic differentiation in insulin-resistant states (Yang et al., 2021). Hence, exercise has the potential to counteract the impact of impaired
findings, an increase in glycolytic flux was associated with increased proliferation and adipogenic differentiation of FAPs isolated from the muscles of *mdx* mice (Reggio et al., 2020a). Interestingly, a short-term HFD regimen restored the expression of mitochondrial proteins; rescued FA oxidation and oxygen consumption; and increased ATP production while reducing the proliferation of dystrophic FAPs. Together, these effects ameliorate the dystrophic muscle phenotype in mice (Reggio et al., 2020a).

High glucose reprograms the adipogenic potential of precursor cells by upregulating adipogenic transcription factor expression and inhibiting anti-adipogenic WNT/β-catenin signaling (Keats et al., 2014; Kolodziej et al., 2019). This effect may be mediated by the production of ROS and subsequent activation of conventional protein kinase C-beta (PKCβ) (Aguiari et al., 2008). PKCβ synergistically potentiates PPARγ transcriptional activity during adipogenesis (Zhou et al., 2006), which likely explains these effects.

Cardiac FAPs exhibit increased glycolytic metabolism in association with increased proliferation, scar formation and reactive oxygen species (ROS) release, leading to contractile dysfunction after myocardial infarction (Janbandhu et al., 2022). Importantly, by fractionating cardiac FAPs based on mitochondrial mass, we reported that Mitohigh FAPs have greater fibrogenesis potential but lacks adipogenic potential in vitro. In contrast, ~15% of the Mitolow and Mitomedium FAP fractions differentiated into PLIN1+ adipocytes upon adipogenic stimulation, indicating that FAPs are metabolically heterogeneous, with progenitor cells maintaining a lower mitochondrial mass, and reinforcing the notion that mitochondrial dysfunction could drive the adipogenic fate of FAPs (Janbandhu et al., 2022) (Fig. 4D).

In individuals with T2DM, both arterial and interstitial glucose concentrations are increased, reducing the transcapillary glucose gradient (Frossard et al., 2005). This can be attributed to reduced muscle insulin signaling transduction, which leads to insulin resistance and reduced insulin-stimulated glucose uptake (~30-40%) in individuals with T2DM (Sjöstrand et al., 2000; Middelbeek et al., 2013). Taken together, these findings suggest that impaired insulin signaling transduction and reduced local blood flow may increase muscle interstitial glucose and insulin concentrations, resulting in their accumulation in the muscle interstitium. This could affect FAP differentiation capacity, particularly in FAP subtypes enriched in lipid and adipogenesis pathways, such as FBN1 FAPs in humans and Prg4 FAPs in mice (Rubenstein et al., 2020; Yang et al., 2021). Insulin is the primary hormonal regulator of adipogenesis in progenitor cells and preadipocytes (Czech et al., 2013) and of lipid turnover in mature adipocytes (Shao et al., 2016; Krycer et al., 2020). In patients with diabetes, a larger arterial-interstitial insulin gradient and transcapillary transport time are required for insulin to reach and stimulate glucose uptake (Sandqvist et al., 2011). The accumulation of IMAT has been suggested to impair insulin diffusion through the muscle interstitial space, leading to insulin resistance in dogs (Kolka et al., 2010). These findings suggest that reduced insulin clearance from the interstitial space under metabolic conditions may generate proper medium for stimulating FAP adipogenesis. In addition to glucose serving as a source for de novo lipogenesis, lactate can also be utilized to synthesize glycerol-3-phosphate and increase TG synthesis, thus supporting the storage of excess energy in conditions of impaired glucose metabolism (Nye et al. 2008a, 2008b). Plasma lactate is increased in patients with obesity, metabolic syndrome, or T2DM and is inversely correlated with insulin sensitivity (reviewed by (Broskey et al., 2020)). An increased interstitial lactate concentration can modulate vascular tone in muscle arterioles, reducing muscle blood flow and preventing lactate clearance from the interstitial space (Qvisth et al., 2007). This interstitial lactate may be taken up by FAP-derived adipocytes and utilized as a lipogenic precursor. Muscle-derived lactate stimulates TGF-β2 expression in adipocytes (Takahashi et al., 2019). High levels of lactate in the muscle interstitial space can induce the expression and secretion of TGF-β2 from FAPs, favoring their proliferation and survival. Overall, these findings suggest

that impaired glucose metabolism within muscle, characterized by increased interstitial levels of glucose, lactate, and insulin, may provide a favorable cellular environment for the adipogenic differentiation of FAPs.

Long-chain fatty acids are ligands of PPARγ and can induce adipogenesis of precursor cells in adipose tissue (Amri et al., 1995; Shao et al., 2016). Physiological doses of FAs increase the intracellular lipid content and upregulate the expression of *Pparγ* and *C/ebpα* in both human FAPs and MuSCs in culture, but adipocyte formation occurs only in FAPs (Agley et al., 2013). A 4-week short-term HFD intervention improved mitochondrial function and FA oxidation in muscular dystrophy FAPs and upregulated anti-adipogenic WNT/β-catenin signaling and the expression of the pro-myogenic factor *follistatin*. Overall, these changes led to an improved dystrophic phenotype in adult *mdx* mice (Reggio et al., 2020a).

Long-term (20-week) HFD-induced obesity led to interstitial accumulation of the lectin family protein galectin-3 (Gal-3), a potent che-moattractant (Takada et al., 2022) involved in cell adhesion, cell cycle progression, apoptosis, inflammation, cell proliferation, and differentiation (Henderson and Sethi 2009). Gal-3 is predominantly secreted by immune cells, mature adipocytes, and stromal cells in visceral and subcutaneous adipose tissues, and its expression is upregulated in individuals with obesity and T2DM (Weigert et al., 2010; Rhodes et al., 2013). Conversely, Gal-3 knockdown reduced adipocyte differentiation via downregulation of the transcriptional activity of PPARγ, C/EBPα, and C/EBPβ and the expression of lipogenic genes in visceral adipose tissue and the liver (Kiwaki et al., 2007; Baek et al., 2015).

A chronic HFD has been shown to increase FAP adipogenesis and muscle dysfunction (Buras et al., 2019; White et al., 2021; Zhang et al., 2021). Likewise, a 20-week HFD induced FAP adipogenic differentiation and IMAT accumulation in hindlimb muscles, with more accumulation in the quadriceps than in the gastrocnemius and *tibialis anterior* muscles (Takada et al., 2022). Moreover, a HFD impaired muscle regeneration after CTX-induced injury by accelerating IMAT formation and promoting myofiber atrophy; similar responses were observed in genetically obese *ob/ob* and *db/db* mice (Takada et al., 2022). These findings indicate that obesity induces IMAT formation independently of diet composition, similar to what has been described in obese, insulin-resistant *db/db* and KK-A^Y mice, a genetic model of obesity and diabetes characterized by altered adipokine expression, dyslipidemia, and insulin resistance (Mogi et al., 2016).

Finally, impaired circulating levels of hormones such as insulin, GH, IGF-1, estradiol, and testosterone T3 (Freda et al., 2008; Gill et al., 2016; Farup et al., 2021); various cytokines, including TSP-1, DKK1, CCN1, IL-8, MCP1, TNF-α, FGF2, FGF21 and SPARC (Poehlman et al., 2000; Varma et al., 2008; Cheema et al., 2010; Haam et al., 2016; Ali et al., 2019; Buras et al., 2019; Hong et al., 2019; Hu et al., 2019; Mathes et al., 2021; Yang et al., 2021); circulating TG; lipoproteins; and metabolites such as creatinine and ammonia (Gilsanz et al., 2010; Kitajima et al., 2013; Montano-Loza et al., 2016; Nardelli et al., 2019), have all been associated with increased IMAT content in chronic diseases (Table 1).

## 7. Exercise as a treatment against pathological IMAT accumulation

Investigating the impact of exercise training on ectopic IMAT in adults with chronic diseases, we found that moderate-intensity aerobic exercise, alone or in combination with resistance training, effectively reduced IMAT (Tunon-Suarez et al., 2021). Exercise may affect FAPs and FAP-derived adipocytes, although the mechanisms underlying this effect have not been fully elucidated. For example, exercise can reduce muscle glucose interstitial concentration through contraction-stimulated glucose uptake, independent of insulin-mediated capillary vasodilation (McConell et al., 2020), and downregulate ECM deposition and FAP adipogenic differentiation in insulin-resistant states (Yang et al., 2021). Hence, exercise has the potential to counteract the impact of impaired
insulin signaling transduction and diminished local blood flow on
interstitial glucose availability. This may lead to a reduction in the
adipogenic differentiation capacity of FAPs. In a mouse model of chronic
inflammation, exercise training combined with pharmacological acti-
vation of AMP-dependent protein kinase prevented muscle fatty
degeneration by rescuing apoptosis-mediated clearance of FAPs (Saito
et al., 2020). *In vitro* treatment of human pro-adipogenic CD90<sup>-</sup> FAPs
with the exercise mimetic drug metformin completely blunted adipo-
genesis (Farup et al., 2021). Notably, bona fide myokines such as IL-6
and IL-15 (Kang et al., 2018; Madaro et al., 2018) and other factors
associated with exercise responses, including histamine and nitric oxide
(Cordani et al., 2014; Kasai et al., 2017), are involved in the regulation
of FAP proliferation and fat cell differentiation. Although these findings
have not been described in the context of exercise, they suggest potential
effects of the milieu and circulating factors produced by active muscles
(also known as exerkines) on FAP homeostasis and fate. For a review of
drug strategies used to target muscle fibro-adipogenic progenitor dif-
ferentiation and adipogenic differentiation, see (Contreras et al., 2021b;
Giuliani et al., 2022).

A recent study analyzed the transcriptomic responses of young mice to 4 weeks of voluntary wheel running exercise and a HFD in subcutaneous and visceral adipose tissues and skeletal muscle (Yang et al., 2022). In these three metabolic tissues, whole-tissue and single-cell transcriptomics revealed cell-state- and cell-type-specific gene expression changes in response to diet-induced obesity with or without exercise training. In skeletal muscle, exercise increased the content of FAPs and myeloid and endothelial cells, and the combined HFD and exercise intervention had a greater effect on these cells. The main effects observed in FAPs were related to the differential expression of genes involved in FA processing, mitochondrial respiration, the antioxidant response, the immune response, the downregulation of ECM remodeling (synthesis, organization) and adipogenic differentiation, and the upregulation of rhythmic processes and circadian behavior. The anti-adipogenic effect was suggested to be related to an increase in CD142⁺ FAPs (Yang et al., 2022).

Exercise training was shown to increase FAP numbers in mouse
models of cancer, including rhabdomyosarcoma, preneoplastic colo-
rectal lesions, and radiation-induced muscle damage (Roubos et al.,
2021; Collao et al., 2023). Additionally, D'Souza et al. (2019) reported
that in irradiated muscles, a HFD reduced the FAP content but increased
the level of markers of fibro-fatty infiltration in muscle (D'Souza et al.,
2019). Furthermore, exercise training upregulated FAP-tenocyte and
FAP-macrophage cross-talk, and the combination of exercise and HFD
increased fibrosis and muscle adiposity in irradiated muscles (D'Souza
et al., 2019). Together, these findings shed light on the molecular
mechanisms underlying the beneficial effects of exercise on IMAT,
which can inform the development of exercise-based interventions for
disorders characterized by increased IMAT accumulation.

To elucidate the potential effect of exercise on reducing IMAT, studies have quantified the amount of ectopic IMAT in muscles using noninvasive imaging techniques (e.g., computed tomography, magnetic resonance imaging, and ultrasound) (Goodpaster et al., 2008; Cadore et al., 2014; de Almeida et al., 2020). Unfortunately, these methods cannot be used to distinguish between intramyocellular triglycerides and extramyocellular (i.e., IMAT) lipid compartments. Early studies analyzing the effect of intramuscular lipids by biochemical determination of total TG also failed to distinguish intramyocellular lipids from extramyocellular lipids (Pan et al., 1997; Goodpaster et al., 2000a). These technical limitations make it difficult to establish whether the FAPs released from contracting muscles into the bloodstream are derived from the IMTG or IMAT pools. Studies combining different methods to measure IMAT, such as muscle biopsy (Pagano et al., 2018; Sachs et al., 2019) and ¹H-nuclear magnetic resonance (Hasegawa et al. 2015, 2016; Park et al., 2020), are required to elucidate the exercise-induced cellular and molecular mechanisms involved in the reduction in IMAT after exercise training.

8. Concluding remarks

FAPs play a crucial role in muscle homeostasis and regeneration. However, their progressive activation, survival, and differentiation also contribute to metabolic disturbances leading to loss of muscle mass, fibro-fatty replacement, IMAT and insulin resistance. In this review, we discussed the current knowledge and highlight research gaps related to the identity, characteristics, and roles of FAPs in the ectopic accumulation of adipocytes and IMAT. The activation, commitment, and terminal differentiation of pro-adipogenic subpopulation(s) of FAPs are consistently regulated by crosstalk mechanisms and substrate availability within the muscle niche. Overall, the recruitment and expansion of FAP subgroups in muscles are not only closely linked but also constantly adapt once homeostasis has been disrupted. This plasticity enables muscles to adjust to metabolic and biomechanical disruptions. However, the available evidence strongly suggests that the muscle environment, immune milieu, and developmental origin play crucial roles in determining the identity and fate of FAPs. Herein, we propose a mechanism in which reduced muscle contractile activity impairs the capacity of myofibers to buffer energy substrates, leading to cellular metabolic inflexibility, which is sensed by FAPs, after which the cells differentiate into adipocytes. Although this hypothesis highlights the importance of FAPs in the development of ectopic fat accumulation, many questions remain unanswered, including how FAPs sense myofiber contractile activity, and what metabolic cues from surrounding muscle cells are involved. Additionally, the athlete's paradox, which refers to the similarity of the density of lipid droplets in muscle cells between well-trained individuals and patients with obesity, raises further questions regarding the relationship between exercise, FAPs, and lipid compartmentalization within skeletal muscle. A better understanding of the contribution of FAPs to metabolic disorders will provide insights into new therapeutic strategies for these conditions.

FUNDING

This work was supported by the EMCR GO4740 Victor Chang Cardiac Research Institute Outstanding Early and Mid-Career Researcher Grant Program to O.C.; by Fondecyt Iniciación 11190971, ANID, Chile to M.F-O; by the US National Institutes of Health (NIH) grant 1R01AR079449 to DK; by the CNRS (French National Center for Scientific Research); by a grant (incentive action) from the Marseille Institute for Rare Diseases MARMARA to F.H.; and by the Australian Research Council (DP230102652; DP240102721), National Health & Medical Research Council (Australia, GNT2013615), and Muscular Dystrophy Association (USA, 964509) to GSL. The funding agencies had no role in the review's design, reference collection and analysis, decision to publish, or preparation of the manuscript.

CRediT authorship contribution statement

Marcelo Flores-Opazo: Writing – review & editing, Writing – original draft, Visualization, Validation, Supervision, Methodology, Investigation, Funding acquisition, Conceptualization. Daniel Kopinke: Writing – review & editing, Writing – original draft, Visualization, Validation, Funding acquisition. Françoise Helmbacher: Writing – review & editing, Writing – original draft, Visualization, Validation, Investigation, Funding acquisition. Rodrigo Fernández-Verdejo: Writing – review & editing, Writing – original draft, Visualization, Validation. Mauro Tuñón-Suárez: Writing – review & editing. Gordon S. Lynch: Writing – review & editing, Writing – original draft, Visualization, Validation, Funding acquisition. Osvaldo Contreras: Writing – review & editing, Writing – original draft, Visualization, Validation, Supervision, Project administration, Methodology, Investigation, Funding acquisition, Conceptualization.
insulin signaling transduction and diminished local blood flow on
interstitial glucose availability. This may lead to a reduction in the
adipogenic differentiation capacity of FAPs. In a mouse model of chronic
inflammation, exercise training combined with pharmacological acti-
vation of AMP-dependent protein kinase prevented muscle fatty
degeneration by rescuing apoptosis-mediated clearance of FAPs (Saito
et al., 2020). *In vitro* treatment of human pro-adipogenic CD90<sup>-</sup> FAPs
with the exercise mimetic drug metformin completely blunted adipo-
genesis (Farup et al., 2021). Notably, bona fide myokines such as IL-6
and IL-15 (Kang et al., 2018; Madaro et al., 2018) and other factors
associated with exercise responses, including histamine and nitric oxide
(Cordani et al., 2014; Kasai et al., 2017), are involved in the regulation
of FAP proliferation and fat cell differentiation. Although these findings
have not been described in the context of exercise, they suggest potential
effects of the milieu and circulating factors produced by active muscles
(also known as exerkines) on FAP homeostasis and fate. For a review of
drug strategies used to target muscle fibro-adipogenic progenitor dif-
ferentiation and adipogenic differentiation, see (Contreras et al., 2021b;
Giuliani et al., 2022).

A recent study analyzed the transcriptomic responses of young mice to 4 weeks of voluntary wheel running exercise and a HFD in subcutaneous and visceral adipose tissues and skeletal muscle (Yang et al., 2022). In these three metabolic tissues, whole-tissue and single-cell transcriptomics revealed cell-state- and cell-type-specific gene expression changes in response to diet-induced obesity with or without exercise training. In skeletal muscle, exercise increased the content of FAPs and myeloid and endothelial cells, and the combined HFD and exercise intervention had a greater effect on these cells. The main effects observed in FAPs were related to the differential expression of genes involved in FA processing, mitochondrial respiration, the antioxidant response, the immune response, the downregulation of ECM remodeling (synthesis, organization) and adipogenic differentiation, and the upregulation of rhythmic processes and circadian behavior. The anti-adipogenic effect was suggested to be related to an increase in CD142⁺ FAPs (Yang et al., 2022).

Exercise training was shown to increase FAP numbers in mouse
models of cancer, including rhabdomyosarcoma, preneoplastic colo-
rectal lesions, and radiation-induced muscle damage (Roubos et al.,
2021; Collao et al., 2023). Additionally, D'Souza et al. (2019) reported
that in irradiated muscles, a HFD reduced the FAP content but increased
the level of markers of fibro-fatty infiltration in muscle (D'Souza et al.,
2019). Furthermore, exercise training upregulated FAP-tenocyte and
FAP-macrophage cross-talk, and the combination of exercise and HFD
increased fibrosis and muscle adiposity in irradiated muscles (D'Souza
et al., 2019). Together, these findings shed light on the molecular
mechanisms underlying the beneficial effects of exercise on IMAT,
which can inform the development of exercise-based interventions for
disorders characterized by increased IMAT accumulation.

To elucidate the potential effect of exercise on reducing IMAT, studies have quantified the amount of ectopic IMAT in muscles using noninvasive imaging techniques (e.g., computed tomography, magnetic resonance imaging, and ultrasound) (Goodpaster et al., 2008; Cadore et al., 2014; de Almeida et al., 2020). Unfortunately, these methods cannot be used to distinguish between intramyocellular triglycerides and extramyocellular (i.e., IMAT) lipid compartments. Early studies analyzing the effect of intramuscular lipids by biochemical determination of total TG also failed to distinguish intramyocellular lipids from extramyocellular lipids (Pan et al., 1997; Goodpaster et al., 2000a). These technical limitations make it difficult to establish whether the FAPs released from contracting muscles into the bloodstream are derived from the IMTG or IMAT pools. Studies combining different methods to measure IMAT, such as muscle biopsy (Pagano et al., 2018; Sachs et al., 2019) and ¹H-nuclear magnetic resonance (Hasegawa et al. 2015, 2016; Park et al., 2020), are required to elucidate the exercise-induced cellular and molecular mechanisms involved in the reduction in IMAT after exercise training.

8. Concluding remarks

FAPs play a crucial role in muscle homeostasis and regeneration. However, their progressive activation, survival, and differentiation also contribute to metabolic disturbances leading to loss of muscle mass, fibro-fatty replacement, IMAT and insulin resistance. In this review, we discussed the current knowledge and highlight research gaps related to the identity, characteristics, and roles of FAPs in the ectopic accumulation of adipocytes and IMAT. The activation, commitment, and terminal differentiation of pro-adipogenic subpopulation(s) of FAPs are consistently regulated by crosstalk mechanisms and substrate availability within the muscle niche. Overall, the recruitment and expansion of FAP subgroups in muscles are not only closely linked but also constantly adapt once homeostasis has been disrupted. This plasticity enables muscles to adjust to metabolic and biomechanical disruptions. However, the available evidence strongly suggests that the muscle environment, immune milieu, and developmental origin play crucial roles in determining the identity and fate of FAPs. Herein, we propose a mechanism in which reduced muscle contractile activity impairs the capacity of myofibers to buffer energy substrates, leading to cellular metabolic inflexibility, which is sensed by FAPs, after which the cells differentiate into adipocytes. Although this hypothesis highlights the importance of FAPs in the development of ectopic fat accumulation, many questions remain unanswered, including how FAPs sense myofiber contractile activity, and what metabolic cues from surrounding muscle cells are involved. Additionally, the athlete's paradox, which refers to the similarity of the density of lipid droplets in muscle cells between well-trained individuals and patients with obesity, raises further questions regarding the relationship between exercise, FAPs, and lipid compartmentalization within skeletal muscle. A better understanding of the contribution of FAPs to metabolic disorders will provide insights into new therapeutic strategies for these conditions.

FUNDING

This work was supported by the EMCR GO4740 Victor Chang Cardiac Research Institute Outstanding Early and Mid-Career Researcher Grant Program to O.C.; by Fondecyt Iniciación 11190971, ANID, Chile to M.F-O; by the US National Institutes of Health (NIH) grant 1R01AR079449 to DK; by the CNRS (French National Center for Scientific Research); by a grant (incentive action) from the Marseille Institute for Rare Diseases MARMARA to F.H.; and by the Australian Research Council (DP230102652; DP240102721), National Health & Medical Research Council (Australia, GNT2013615), and Muscular Dystrophy Association (USA, 964509) to GSL. The funding agencies had no role in the review's design, reference collection and analysis, decision to publish, or preparation of the manuscript.

CRediT authorship contribution statement

Marcelo Flores-Opazo: Writing – review & editing, Writing – original draft, Visualization, Validation, Supervision, Methodology, Investigation, Funding acquisition, Conceptualization. **Daniel Kopinke:** Writing – review & editing, Writing – original draft, Visualization, Validation, Funding acquisition. **Françoise Helmbacher:** Writing – review & editing, Writing – original draft, Visualization, Validation, Investigation, Funding acquisition. **Rodrigo Fernández-Verdejo:** Writing – review & editing, Writing – original draft, Visualization, Validation. **Mauro Tuñón-Suárez:** Writing – review & editing. **Gordon S. Lynch:** Writing – review & editing, Writing – original draft, Visualization, Validation, Funding acquisition. **Osvaldo Contreras:** Writing – review & editing, Writing – original draft, Visualization, Validation, Supervision, Project administration, Methodology, Investigation, Funding acquisition, Conceptualization.
# Declaration of competing interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as potential conflicts of interest.

# Acknowledgments

We are thankful to Felipe Serrano (www.illustrative-science.com) for designing Fig. 1 and the graphical abstract.

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# Declaration of competing interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as potential conflicts of interest.

# Acknowledgments

We are thankful to Felipe Serrano (www.illustrative-science.com) for designing Fig. 1 and the graphical abstract.

# References

Addison, O., Marcus, R.L., Lastayo, P.C., Ryan, A.S., 2014. Intermuscular fat: a review of the consequences and causes. Internet J. Endocrinol. 2014, 309570.

Agarwal, A.K., Tunison, K., Mitsche, M.A., McDonald, J.G., Garg, A., Insights into lipid accumulation in skeletal muscle in dysferlin-deficient mice. J. Lipid Res. 60 (12), 2057-2073.

Agley, C.C., Rowlerson, A.M., Velloso, C.P., Lazarus, N.R., Harridge, S.D., 2013. Human skeletal muscle fibroblasts, but not myogenic cells, readily undergo adipogenic differentiation. J. Cell Sci. 126 (Pt 24), 5610-5625.

Aguiari, P., Leo, S., Zavan, B., Vindigni, V., Rimessi, A., Bianchi, K., Franzin, C., Cortivo, R., Rossato, M., Vettor, R., Abatangelo, G., Pozzan, T., Pinton, P., Rizzuto, R., 2008. High glucose induces adipogenic differentiation of muscle-derived stem cells. Proc. Natl. Acad. Sci. U. S. A. 105 (4), 1226-1231.

Aiki, H., Yoshiko, A., Hioki, M., Kanehira, N., Shimaoka, K., Koike, T., Sakakibara, H., Oshida, Y., 2015. Skeletal muscle size is a major predictor of intramuscular fat content regardless of age. Eur. J. Appl. Physiol. 115 (8), 1627-1635.

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