Thrown for a loop: fibro-adipogenic progenitors in skeletal muscle fibrosis

Abstract

Fibro-adipogenic progenitors (FAPs) are key regulators of skeletal muscle regeneration and homeostasis. However, dysregulation of these cells leads to fibro-fatty infiltration across various muscle diseases. FAPs are the key source of extracellular matrix (ECM) deposition in muscle, and disruption to this process leads to a pathological accumulation of ECM, known as fibrosis. The replacement of contractile tissue with fibrotic ECM functionally impairs the muscle and increases muscle stiffness. FAPs and fibrotic muscle form a progressively degenerative feedback loop where, as a muscle becomes fibrotic, it induces a fibrotic FAP phenotype leading to further development of fibrosis. In this review, we summarize FAPs’ role in fibrosis in terms of their activation, heterogeneity, contributions to fibrotic degeneration, and role across musculoskeletal diseases. We also discuss current research on potential therapeutic avenues to attenuate fibrosis by targeting FAPs.

INTRODUCTION

Fibrosis is the pathological accumulation of extracellular matrix (ECM) components, predominantly fibrillar collagens, which impair tissue function (1, 2). Fibrosis is a consequence of a multitude of diseases across tissues where the regenerative processes are impaired (3–7). In skeletal muscle, the excessive accumulation of ECM in fibrosis takes the place of functional, contractile tissue, weakening the muscle and increasing muscle passive stiffness (8, 9). The weakened muscle becomes more prone to injury leading to further degeneration and fibrosis as the muscle becomes chronically injured.

The main cell driver of fibrosis in skeletal muscle is fibro-adipogenic progenitors (FAPs). FAPs are muscle-resident nonmyogenic cells that reside in the interstitial space (10). FAPs play an essential role in supporting regeneration. In uninjured muscle, FAPs exist at relatively low levels but rapidly proliferate after injury (11, 12). After rapid expansion in response to injury, FAPs deposit ECM components to replace the damaged matrix and release promyogenic factors to induce muscle differentiation (13, 14). FAPs then rapidly undergo apoptosis, returning to preinjury levels, which halts ECM deposition and allows for full regeneration of the muscle (12, 15). FAPs become resistant to apoptosis in fibrotic conditions, remaining at chronically high levels and continuously depositing ECM resulting in fibrosis (11, 12, 16). The development of fibrosis triggers FAPs to develop a fibrotic phenotype leading to a progressively degenerative feedback loop. As FAPs become activated into a fibrotic phenotype, they contribute to fibrotic ECM deposition and aberrant cell signaling, leading to further fibrotic activation and degeneration. Research on FAPs has rapidly expanded in the past couple of years, dramatically increasing our understanding. However, the mechanosensitivity of FAPs has only recently been defined and requires further research to understand the interplay between FAPs and the fibrotic ECM. The goal of this review is to highlight the positive feedback loop of FAPs and fibrosis; what contributes to this cyclic activation; and methods to disrupt the feedback loop to stop the progression of fibrosis.

ACTIVATION AND PERSISTENCE OF FIBROTIC PHENOTYPE

FAPs were first defined by their ability to spontaneously differentiate into adipocytes and activate into myofibroblasts (10). Notably, there is considerable overlap between FAPs and what may also be referred to as a muscle-resident fibroblast (17). Activation of myofibroblasts is often considered a marker of fibrosis with alpha-smooth muscle action (αSMA), a myofibroblast marker, significantly increasing in fibrotic conditions and strongly correlating with increased ECM deposition (18–23). A series of soluble factors and mechanical signaling from the ECM trigger FAP activation in myofibroblasts. These activation signals create a progressively degenerative feedback loop; as FAPs become activated into myofibroblasts, ECM deposition and release of profibrotic signals increase leading to additional induction of myofibroblast activation and fibrosis (Fig. 1).

Figure 1.

The profibrotic positive feedback loop between fibro-adipogenic progenitors (FAPs) and fibrotic muscle. As FAPs are activated into myofibroblasts, they produce fibrotic levels of extracellular matrix (ECM) leading to increased stiffness, which in turn activates FAPs further into myofibroblasts. Transforming growth factor-beta (TGF-β) contributes to myofibroblast activation and ECM production, furthering the fibrotic loop. Graph axis are stress (σ) and strain (ε) indicating the stiffness of a material. Pink line indicates the stiffness of healthy ECM, and red line indicates the stiffness of fibrotic ECM. Created with BioRender.com.

Soluble Signaling to FAPs

Various soluble factors induce FAP activation in myofibroblasts. The most established one being transforming growth factor-beta (TGF-β). In skeletal muscle, TGF-β is released by both FAPs and macrophages, which play a key role in modulating FAP behavior through soluble factors (24–26). TGF-β is widely regarded as a strong profibrotic signal, with its expression increased across fibrotic diseases (27). Exposure to TGF-β induces myofibroblast activation and is strongly correlated with fibrotic ECM deposition (12, 16, 27, 28). Other soluble factors, downstream of TGF-β, increase myofibroblast activation, further implicating TGF-β has a strong profibrotic factor. Connective tissue growth factor, also known as cellular communication network factor-2 (CTGF/CCN-2), downstream targets of TGF-β, increases αSMA when overexpressed in wildtype (wt) mice (29). In a rat model for an overuse fibrotic injury, TGF-β, αSMA, and CCN2 expression were all increased (30). Myostatin, which signals through the same SMAD pathway as TGF-β, increases FAP proliferation and αSMA expression (23). FAPs produce bone morphogenetic protein 1 (BMP1) and matrix metalloproteinase 14 (MMP14) that activate latent TGF-β into an active form (24). This leads to a feedforward loop as activated FAPs lead to the activation of TGF-β and therefore, more FAP activation.

TGF-β is involved in activating the Hedgehog (Hh) signaling pathway. This signaling pathway is known to block FAP differentiation into adipocytes after injury and enhance regeneration (31). However, ectopic activation of the Hh pathway leads to increased activation of FAPs into myofibroblasts, increased collagen deposition, and impaired regeneration (32). As with TGF-β, Hh signaling plays a key role in promoting regeneration in muscle, however, excessive and sustained expression leads to overactivation of FAPs and initiates fibrotic degeneration.

Platelet-derived growth factor receptor A (PDGFRα) is a tyrosine kinase receptor that is used as the canonical marker for FAP isolation (13, 21, 33). PDGFRα expression is strongly correlated with collagen expression in FAPs suggesting that the surface marker plays a fibrogenic role. Exposure to PDGF-AA in vitro significantly inhibited FAP adipogenic potential while retaining fibrogenic potential and collagen expression (34, 35). Constitutively active PDGFRα expression in an injured mouse model resulted in increased collagen deposition and fibrosis (35, 36). Exposure to TGF-β decreases PDGFRα expression while increasing αSMA expression, suggesting FAPs lose their canonical marker as they commit to a fibrotic phenotype (19). PDGFRα modulates TGF-β-induced myofibroblast activation, with blockage of PDGFRα decreasing myofibroblast activation and fibrosis, highlighting the cross talk between these two play a key role in regulating FAPs’ activation and fate (19, 37). The persistence of FAPs in fibrosis leads to chronically high expression of PDGFRα that activates FAPs into a fibrotic phenotype, creating a positive feedback loop of fibrosis and fibrotic activation.

Excessive reactive oxygen species (ROS), particularly NADPH oxidase 4 (NOX4), have been implicated in myofibroblast activation in FAPs and other fibroblast cell types across tissues (38, 39). TGF-β induces NOX4 expression across different cell types, indicating it as a downstream target of the profibrotic pathway in FAPs (38–40). In an mdx mouse, a model for skeletal muscle fibrosis, NOX4 expression is significantly higher than in wt mice. In a NOX4-KO mdx mouse model, the number of myofibroblast is decreased suggesting NOX4 plays a role in FAP activation (39). ROS levels are elevated in fibrotic skeletal muscle, therefore leading to cyclic activation of FAPs in an already fibrotic condition (41, 42).

The persistence of a fibrotic phenotype is not only driven by FAP activation into myofibroblasts but also their resistance to apoptosis, resulting in the maintenance of chronically high levels of FAPs. Clearance of FAPs is an essential part of the regeneration and removes them from the profibrotic feedback loop. FAPs undergo apoptosis, mediated by tumor necrosis factor-alpha (TNFα) after rapidly proliferating and depositing ECM postinjury (12). This prevents excessive ECM deposition and keeps FAPs at relatively low levels in nonfibrotic and uninjured (11, 12, 43). TGF-β blocks TNFα-induced apoptosis, resulting in chronically high levels of FAPs and furthered fibrosis (12, 16).

Mechanical Signaling to FAPs

The mechanical properties and organization of the ECM are altered in fibrosis. Excessive ECM deposition leads to increased stiffness, crosslinking, and altered alignment (44–46). Similar to mesenchymal stem cells (MSCs), FAPs are responsive to substrate stiffness (47, 48). Increasing substrate stiffness dramatically increases αSMA expression in FAPs. Stiffness that is physiologically representative of fibrotic ECM has significantly more myofibroblast activation compared with stiffness representative of healthy ECM (48). In aged skeletal muscle, the increase in muscle stiffening leads to increased YAP/TAZ expression in muscle fibroblasts (49). YAP/TAZ are known to mediate TGF-β signaling to promote myofibroblast activation and collagen deposition (3, 4). Therefore, the increase in ECM stiffness due to fibrosis induces FAP activation into myofibroblasts through the YAP/TAZ pathway. As a muscle becomes fibrotic it leads to further myofibroblast activation in FAPs, contributing to the progressively degenerative positive feedback loop.

When cultured in standard conditions, FAPs largely spontaneously activate into myofibroblasts as measured by αSMA stress fibers (10, 13, 19). Given the mechanosensitivity of FAPs, this could be due to the excessive-high stiffness of tissue-cultured plastic. Therefore, culture conditions that are more physiologically representative of the ECM stiffness in vivo better represent FAP activation and behavior. A physiological substrate would thus include an elastic stiffness on the order of ∼10 kPa of healthy muscle (50). However, mimicking the mechanical environment would also dictate transitioning from standard two-dimensional (2-D) culture systems to three-dimensional (3-D) culture that can influence the response to substrate stiffness (51). Furthermore, MSCs respond not only to elastic stiffness but also viscoelasticity (52). The specific impact of these more complex mechanical features on FAPs is still currently unknown.

Apoptosis provides a mechanism for FAPs to exit the profibrotic feedback loop and restore homeostatic conditions in skeletal muscle. However, profibrotic signaling, such as TGF-β and increased stiffness, block this exit pathway leading to a cyclic process of profibrotic activation, further fibrotic signaling and activation, and fibrotic development. This profibrotic feedback loop leads to continuous fibrotic activation of FAPs and the progressively degenerative nature of fibrosis.

HETEROGENEITY WITHIN FAPs

FAPs overall are one of the main cell drivers of fibrosis. Recent studies have identified key subpopulations in FAPs that affect their fibrotic and regenerative capacities through single-cell RNA sequencing (scRNAseq). Studies have divided these subpopulations by different markers, but overall, there is significant heterogeneity within FAPs that is altered in injury and disease.

Malecova et al. identified distinct subpopulations of FAPs that changed in injury and fibrosis. These subpopulations were defined by the relative expression of two surface markers, vascular cell adhesion molecule 1 (Vcam1) and tyrosine kinase receptor (Tie2), that have unique transcriptional profiles. Vcam1 expression was significantly increased after acute injury and in mdx mice, but relatively low in wt mice. Tie2 expression levels were the opposite, most FAPs expressed some level of Tie2 in healthy muscle and those levels significantly decreased after injury and in fibrosis (11). High expression of Vcam1+ FAPs was observed in other fibrotic conditions. In oculopharyngeal muscular dystrophy (OPMD), a genetic disorder affecting the muscles in the eyelids and pharynx, there was a higher abundance of Vcam1+ FAPs in the fibrotic muscle compared with nonfibrotic muscle (43). Human FAPs isolated from nonfibrotic muscles clustered differently from those isolated from fibrotic muscles, suggesting changes in gene expression in injury and disease (43). Vcam1 levels are upregulated in other tissue-type diseases and associated with increased proliferation including in idiopathic pulmonary fibrosis and lung cancer (5, 53). In lung fibroblasts, TGFβ significantly increased Vcam1 expression and fibroblast proliferation (5). If TGFβ triggers Vcam1 expression in FAPs as well, it further implicates this subpopulation as the more fibrotic phenotype.

Type 2 diabetes (T2D) is a disease that is associated with fibro-fatty infiltration of skeletal muscle, with contractile tissue being replaced by both excessive ECM deposition and adipocytes (34). Farup et al. analyzed human FAPs isolated from patients with T2D clustered into four distinct subpopulations. A population high in αSMA expression, also differentially expressed collagens and other ECM proteins, indicating that the myofibroblast population of FAPs is primarily responsible for ECM deposition (6, 34). FAPs from patients with T2D clustered based on CD90+/− expression, with genes upregulated in CD90+ FAPs were also upregulated in T2D muscle. CD90+ FAPs had higher proliferative capacity and clonal formation, suggesting a progenitor phenotype while CD90− FAPs expressed a more adipogenic phenotype. CD90 positive and negative FAPs had distinct transcriptomes. CD90− FAPs were enriched for angiogenesis and regulation of endothelial proliferation whereas CD90+ subset was enriched for ECM proteins, collagen synthesis, and TGFβ response, suggesting this subset is a more fibrotic phenotype. Patients with T2D had a significantly higher population of CD90+ FAPs, exclusively, implying this population is primarily responsible for the fibrotic degeneration seen in T2D (34).

Davies et al. (54) divided murine FAPs into subpopulations based on their expression of uncoupling protein 1 (UCP1), a precursor marker for beige adipose. UCP1+ FAPs released promyogenic exosomes whereas UCP1− FAPs were defined by profibrotic exosomes. FAPs isolated from humans with rotator cuff tears, an injury associated with fatty infiltration and fibrosis, clustered into six distinct subpopulations. These distinct subpopulations had differential expression of fibrogenic and adipogenic genes including COL1A1, PDGRFA, ADIPOQ, and UCP1 (54).

In cells isolated from patients with limb-girdle muscular dystrophy (LGMD), Depuydt et al. divided FAPS based on lumican (LUM+) and proteoglycan 4 (PRG4+) expression. LUM+ expression positively correlated with disease severity (55). In nondiseased human muscle, Rubenstein et al. also found FAPs to cluster based on LUM+ and PRG4+ markers. After using mouse samples to validate the human sequencing, however, FAPs were divided into two subpopulations based on lumican (LUM+) and fibrillin 1 (FBN1+) expression, highlighting the heterogeneity between species. Both of these subpopulations had high expression of fibrillar collagens I and III, the predominant proteins in fibrosis, but LUM+ FAPs differentially expressed types IV and XV collagens whereas FBN1+ FAPs had heightened expression of collage XIV.

Several studies have looked at FAP heterogeneity in the context of regeneration. Although these do not give direct insights into FAP subpopulations in fibrosis, the subpopulations of FAPs responsible for ECM deposition and TGF-β signaling in regeneration likely share characteristics with FAPs that persist in fibrosis (56, 57). Scott et al. (58) classified FAPs into two subpopulations that differentially expressed genes for ECM components and cell signaling pathways. The temporal expression of these genes after injury with notexin (NTX) suggests a subset of the FAP population is primarily responsible for ECM deposition after injury. This same subpopulation, defined by ECM expression, may become persistent in chronic injury and disease leading to fibrosis. Oprescu et al. identified subpopulations of murine FAPs that are dynamically altered in homeostasis and cardiotoxin (CTX)-induced injury. After muscle damage, FAPs diverged into subpopulations, the markers of which are time-dependent. At 21 days postinjury (DPI), FAPs could be divided into Osr1+ FAPs and fibroblasts enriched with collagen-I, which might represent the more persistent, fibrotic subset of FAPs (57). Osr1+ is associated with pro-regenerative signaling in FAPs and is upregulated in FAPs after acute injury (14, 15). Leinroth et al. described six distinct subpopulations of FAPs with varying adipogenic potential and ability to respond to BaCl₂-induced injury. Osr1+ defined one subpopulation that expressed genes related to stemness and development, indicating a precursor population (15, 59). Two clusters defined by Adam12 and Gap43 had enhanced enrichment of fibrotic genes including TGFβ and Fibronectin 1 (Fn1) and downregulation of Ltbp4, a negative regulator of TGF-β (59).

FAPs are the primary contributors of fibrotic ECM deposition and play essential roles in regeneration and homeostasis. Although ablating FAPs reduces excessive ECM deposition, it results in muscle atrophy and muscle satellite cell (MuSC) depletion, even under homeostatic conditions (60). Recent illumination of FAP subpopulations implies an opportunity for more targeted approaches to reduce fibrosis. Inhibiting a profibrotic subpopulation of FAPs while leaving a pro-regenerative subpopulation intact offers an avenue to maintain the supportive role of FAPs while attenuating fibrosis. However, there seems to be little consensus about how to define these subpopulations in both the context of regeneration and fibrosis. This is likely due to biological and technical variability that exists across samples, disease models, and analysis techniques. Despite different markers, these subpopulations share similarities across studies. For example, in healthy mice, Rubenstein et al. (61) defined a subpopulation by FBN1+ expression, notably, the FBN1+ subset coexpressed TEK, the gene for Tie2, the defining marker for a subpopulation in the Malecova et al. (11) study. Although αSMA is the canonical marker of myofibroblasts it does not have a distinguishing factor in profibrotic FAPs at the transcriptional level across studies in mice and humans (Table 1). Although the identifying markers can shift, a consistent feature of profibrotic FAPs is increased expression of fibrillar collagens I and III as well as collagen crosslinking enzymes.

Table 1.

Summary of FAP heterogeneity and potential markers for profibrotic populations, determined by high fibrillar collagen expression and relative expression in fibrotic conditions

Profibrotic Subpopulation(s)	Other Subpopulation(s)	Sample	Condition	Ref.
Vcam1+	Tie2+	Mouse	Fibrosis (mdx)	Malecova et al. (11)
UCP1−	UCP1+	Mouse	Rotator cuff tear	Davies et al. (54)
CD90+	CD90−	Human	T2D	Farup et al. (34)
Lumican+	Proteoglycan 4+	Human	LGMD	Depuydt et al. (55)
Lumican+Fibrillin 1+		Mouse, human	Healthy, resting	Rubenstein et al. (61)
Dlk1+	Dpp4+	Mouse	CTX-injury	Oprescu et al. (57)
Fibroblasts	Cxcl14+
	Ors1+
	Wisp1+
FAP1	FAP2	Mouse	NTX-injury	Scott et al. (58)
	Gli1 +	Mouse	NTX-injury	Yao et al. (56)
	Gli1−
Adam12+	Osr1+	Mouse	BaCl2 injury	Leinroth et al. (59)
Gap43+	Gli1+
	Hsd11b1+
	Clu+
	Osr1+	Mouse	Freeze injury, glycerol injury	Stumm et al. (15)
	Osr1−
MME+	MME−	Human	HOA	Fitzgerald et al. (62)

CTX, cardiotoxin; FAP, fibro-adipogenic progenitor; LGMD, limb-girdle muscular dystrophy; NTX, notexin; T2D, type 2 diabetes.

CONTRIBUTIONS TO FIBROSIS

In acute injury, FAPs play a key role in supporting healthy regeneration by both depositing ECM to replace the damaged matrix and signaling to MuSCs through the release of promyogenic signals. In a fibrotic state, these pathways get disrupted leading to excessive ECM deposition, fatty infiltration, and aberrant cell signaling. The fibrotic contributions of FAPs lead to further profibrotic activation, continually disrupting the proregenerative pathways and leading to progressively degenerative fibrosis.

ECM Deposition

The defining characteristic of fibrosis is a pathological excess of ECM deposition, which stiffens the muscle and impairs contractile function (2, 7, 43–45, 63). FAPs account for the majority of collagen expression in skeletal muscle both in regenerating injuries and fibrotic conditions (10, 12, 13, 19–21, 31, 64). The number of FAPs strongly correlates with muscle collagen levels (22, 23). This is supported by the increase in the expression of ECM proteins, particularly fibrillar collagens I and III, from FAPs derived from fibrotic conditions (58, 65). Increased synthesis of various proteoglycans, including decorin and biglycan, found within the ECM is seen in both patients with Duchenne muscular dystrophy (DMD) and mdx mice (65, 66). Lumican and fibrillin, proteoglycans upregulated in FAPs are known to accumulate with fibrillar collagens, promote thicker collagen fibril formation, and induce myofibroblast activation through the TGF-β pathway in other fibrotic diseases (67–70). The increase in ECM deposition stiffens the muscle, leading to further myofibroblast activation in FAPs and fibrotic development (45, 48).

The skeletal muscle ECM not only increases in content throughout fibrosis but also the organization of the ECM and collagen fibrils is altered. Collagen crosslinking and alignment are increased in fibrotic muscle, indicating a change in how FAPs deposit and organize the ECM (44, 71). FAPs express high levels of not only ECM genes but also regulatory genes that control ECM degradation and organization. FAPs have increased expression of lysyl oxidases (LOXs), which promote collagen crosslinking (65, 72, 73). This makes the ECM stiffer and resistant to degradation, contributing to the continuous buildup of ECM and stiffening of the muscle (44, 45, 71). Fibrotic FAPs express CD147, an inducer of matrix metalloproteinases (MMPs), at higher levels than nonfibrotic FAPs (43, 74, 75). MMPs are a large class of proteinases, many of which degrade ECM and are considered antifibrotic. However, MMPs affect a large range of biological processes, and some have been identified as having profibrotic properties. Particularly mdx FAPs had increased expression of MMP2 and MMP14, both of which are upregulated in the presence of TGF-β and have been implicated in the furthering of fibrosis (76–78). Although it may be counterintuitive that MMPs that breakdown ECM are increased in fibrosis, the expression of tissue inhibitors of MMPs (TIMPs) are also overexpressed in fibrotic conditions that shift the balance toward ECM accumulation (79).

FAPs contribution to the fibrotic ECM perpetuates the positive feedback loop of FAPs and fibrosis. FAPs are sensitive to both the ECM stiffness and architecture, inducing myofibroblast activation (48). Fibrotic FAPs have altered the expression of regulatory ECM genes affecting ECM organization and remodeling. As FAPs produce a more fibrotic ECM in terms of stiffness, content, and architecture, it induces further myofibroblast activation in the FAPs leading to more fibrotic ECM deposition. This cycle continues leading to continuing stiffer and more disorganized ECM as fibrosis progresses.

Fatty Infiltration

Fibrosis is, by definition, a pathological accumulation of ECM, which is a result of FAPs profibrotic activation into myofibroblasts. However, the dysregulation of FAPs that occurs in fibrotic diseases, often results in fatty infiltration of the muscle too, as FAPs both differentiate into adipocytes and activate into myofibroblasts, highlighting the multipotency of these cells. The persistently high levels of FAPs due to their apoptotic resistance during fibrosis can lead to high levels of both fibroblastic and adipogenic activation.

Fibro-fatty infiltration is seen in various chronic injuries including rotator cuff tears and DMD (80–82). MRI assessment of skeletal muscles reveals a high-fat fraction in DMD, predominantly in the hip and thigh muscles (83, 84). The amount of fatty infiltration negatively correlates with muscle function and strength (85). Therefore, FAPs impairment of muscle function in fibrotic diseases is in part through the replacement of contractile tissue with not only ECM but also fatty connective tissue.

FAP activation into myofibroblasts and differentiation into adipocytes are often viewed as opposing sides of a coin with profibrotic factors, such as TGF-β, inhibiting adipogenesis whereas promoting fibrogenesis and proadipogenic factors having the opposite effect (86, 87). However, the existence of fibro-fatty infiltration across various skeletal muscle diseases suggests a dual capacity of FAPs to activate in both directions under the same conditions. This is attributed in part to the heterogeneity of FAPs, with certain subsets of FAPs accounting for almost all adipogenic differentiation in muscle (54, 62). The dysregulation of signaling in fibrotic conditions may induce subsets of FAPs to undertake a fibrotic or adipogenic phenotype.

Aberrant Cell Signaling

The altered ECM provides a nondirect way in which FAPs signal to surrounding muscle cells. MuSCs are sensitive to their mechanical and architectural environment and therefore, are affected by the FAP-produced ECM (88). The increased collagen cross-linking and stiffness, seen in fibrosis, impair MuSC differentiation (50, 88, 89). Since the fibrotic response and the regenerative response are opposing pathways to dealing with damage, the inhibition of myogenesis propagates the progressive nature of fibrosis.

FAPs also signal to MuSCs through the release of soluble factors. In a regenerative state, FAPs release promyogenic exosomes that promote MuSC migration to the injury site and differentiation into muscle (54, 56). In fibrosis, these signaling pathways are disrupted, with FAPs impairing rather than promoting myogenesis (90, 91). MuSCs cultured in conditioned media obtained from fibrotic myofibroblasts in skeletal muscle had declined proliferation and increased apoptosis, resulting in poor wound healing (37). Coculturing of FAPs isolated from fibrotic conditions with MuSCs had a negative effect on fusion index compared with coculture of nonfibrotic FAPs, both in direct coculture and in indirect transwell experiments, indicating at least a portion of the negative effect comes from FAP soluble factor release rather than direct cell contact (14, 43).

Fibrotic degeneration is a complex process involving various cell types, but FAPs play a main role in promoting deterioration through multiple pathways. The proregenerative nature of FAPs is switched to a degenerative phenotype of fibro-fatty infiltration and impaired myogenesis advancing fibrotic development across skeletal muscle diseases. FAPs also receive signaling from MuSCs, differentiated muscle fibers play a role in determining FAPs’ adipogenic or fibrogenic fate (92, 93). As FAPs’ signaling to MuSCs becomes dysregulated in fibrosis so does MuSCs signaling to FAPs as a result. Therefore, the cyclic nature of fibrosis continues leading to fibrotic activation of FAPs and aberrant cell signaling.

FAPs IN FIBROTIC CONDITIONS

Fibrosis is a consequence of nearly all skeletal muscle diseases and chronic injuries. Although the causes of the disease may differ, a common feature is the persistent presence of FAPs and their contributions to fibrosis.

Muscular Dystrophies

One of the more commonly studied groups of fibrotic diseases in skeletal muscles is muscular dystrophies, a group of genetic diseases that progressively weaken muscles. The most studied one being DMD. DMD is an x-linked recessive disorder resulting from a mutation in the dystrophin gene, a structural protein that provides mechanical stability and strength to the muscle. The muscles in DMD are more prone to injury without a fully functioning dystrophin protein and are significantly weaker leading to chronic damage and fibrosis. FAPs in DMD muscle are more highly activated into myofibroblasts and exist in larger numbers than in nondiseased muscle driving the progressively degenerative fibrosis (94). Profibrotic markers are enhanced in DMD FAPs including TGF-β, αSMA, and fibrillar collagens (11, 34). Profibrotic miRNAs are elevated in FAPs isolated from DMD versus control muscle. These profibrotic miRNAs strongly correlate with TGF-β and ECM protein expression levels (94).

Limb-girdle muscular dystrophy (LGMD) is a disease caused by a mutation in the dysferlin gene, impairing muscle repair and leading to chronic injury. FAPs in LGMD, specifically the subpopulation expressing lumican (LUM), progressively increase with disease severity (55, 95). LGMD muscle had upregulation of genes related to fibro-fatty infiltration whereas genes related to protein synthesis and regeneration were downregulated (55). Particularly, FAPs contribute significantly to adipogenic replacement of contractile tissue in LGMD, impairing muscle function and furthering disease progression (95). FAPs isolated from patients with OPMD have the significant increased proliferative capacity and were more motile compared with control FAPs. OPMD FAPs have increased the expression of ECM proteins and altered the expression of genes related to cell signaling, which resulted in an impaired fusion index of MuSCs (43).

Skeletal Muscle Diseases and Chronic Injuries

Skeletal muscle fibrosis is most commonly studied in the context of muscular dystrophies, however, almost any chronic skeletal muscle injury or disease results in some level of fibrosis. Across these diseases, FAP levels are elevated and highly activated leading to increased ECM deposition and fibrosis. In cerebral palsy, a group of motor disorders, collagen expression is elevated in FAPs compared with typically developing controls (22, 46, 96). In a mouse model for amyotrophic lateral sclerosis (ALS), a motor neuron disease, αSMA expression is increased over wild-type controls, indicating enhanced FAP activation into myofibroblasts (20). Chronic kidney disease (CKD) and kidney dysfunction are strongly correlated with a progressively fibrotic muscle phenotype. This is purportedly due to decreased TNF-α expression leading to a persistently expanding pool of FAPs resulting in the number of FAPs nearly doubling in CKD muscle (8), or increased myostatin in CKD stimulating the differentiation of FAPs to myofibroblasts (23). In a mouse model for CKD, αSMA and ECM protein expressions are elevated (23). T2D is associated with fibro-fatty degeneration of skeletal muscle and increased PDGFRα expression. Genes related to ECM turnover and remodeling are differently expressed in FAPs, including increased collagen-I deposition (34).

FAPs are responsible for the high levels of fatty infiltration and fibrotic deposition observed in rotator cuff tears (82, 97). FAP levels are elevated and the apoptotic index is decreased in a mouse model for rotator cuff tears (81). TGF-β levels are increased after rotator cuff injury and increase FAP survival (81, 97). Rotator cuff injuries develop fatty infiltration at a high rate and concentration than other muscles across diseases. This may in part be due to the heterogeneity of FAPs discussed earlier not only within a muscle but across anatomical regions. FAPs from rotator cuff muscles reside at a higher concentration and demonstrate greater adipogenic potential than lower limb muscle FAPs (98).

Skeletal muscle diseases and chronic injuries vary in their cause, symptoms, and development. However, a common factor across diseases is the role FAPs play in the progressively degenerative nature of fibrosis. Therefore, targeting FAPs through various antifibrotic therapies offers an avenue to attenuate a broad range of fibrotic conditions (Fig. 2).

Figure 2.

Signaling pathways involved in fibro-adipogenic progenitor (FAP) fibrotic activation and potential therapeutic targets. Therapeutics to block fibrotic pathways are marked by the red boxes. ECM, extracellular matrix; TGF-β, transforming growth factor-beta; TNFα, tumor necrosis factor alpha Created with BioRender.com.

ATTENUATION OF FIBROSIS THROUGH TARGETING FAPs

The positive feedback loop of FAPs and fibrosis causes muscles to progressively degenerate. To attenuate fibrosis and improve muscle function, this cycle needs to be disrupted otherwise FAPs will continually activate and contribute to fibrosis. Methods to break this cycle include blocking the soluble and mechanical signaling within the cycle as well as promoting apoptosis, to remove FAPs from the fibrotic feedback loop.

Blockage of TGF-β Signaling

TGF-β plays a significant role in driving FAPs’ fibrotic behavior by inducing proliferation, myofibroblast activation, and blocking apoptosis. Methods that inhibit TGF-β signaling to FAPs help to promote apoptosis, restoring a key aspect of the regenerative cycle, and limiting myofibroblast activation and ECM deposition. Small molecule inhibitors of TGF-β reduce FAP number and fibrotic signaling in mice (81, 99). In a mouse model of a rotator cuff injury, SB431542, a small molecule TGF-β inhibitor reduced αSMA expression, collagen deposition, the number of FAP cells, and increased FAPs’ apoptotic index (81). ITD-1, which targets TGF-β receptors, reduced TGF-β activity, FAP accumulation, and fibrotic area in mdx mice (99). Nilotinib, a tyrosine kinase inhibitor, blocks TGF-β signaling, restoring FAP apoptosis and as a result, reducing collagen expression (12, 100).

The benefits of blocking TGF-β to attenuate fibrosis have been widely studied and proven successful in promoting FAP apoptosis and reduction of ECM deposition. However, these techniques need to be applied with caution as total inhibition can have adverse side effects. TGF-β plays a role in many different biological signaling pathways including myogenesis (101). Therefore, complete inhibition could impair regeneration, rendering the reduction in fibrosis functionally inconsequential. Targeting downstream targets of TGF-β specifically involved in myofibroblast activation provides a more specific method to attenuating fibrosis while leaving most broad signaling pathways intact. TGF-β acts on many different signaling pathways, with most fibrotic upregulation occurring through the SMAD2/3 pathway (4, 38, 43). Therefore, blocking downstream targets of TGF-β related to this pathway allows for the inhibition of fibrotic signaling while leaving other important signaling pathways intact. Blocking CTGF/CCN-2, a downstream target of TGF-β/SMAD pathway, reduces αSMA expression and fibrosis in a rat overuse injury model (30). ATA 842, a drug that inhibits the myostatin/SMAD pathway, prevents fibrosis progression, promotes apoptosis, and reduces myofibroblast activation in fibrotic FAPs (23, 102).

Blocking Mechanosensing

The mechanical environment of the muscle is altered in fibrosis with stiffness increasing as fibrosis progresses. FAPs are sensitive to these increases in stiffness, inducing higher myofibroblast activation on increased stiffness (48). Blocking FAPs’ ability to sense fibrotic stiffness provides an avenue to reduce myofibroblast activation in fibrotic muscle by stopping the profibrotic feedback loop between myofibroblast activation, ECM deposition, and stiffness. YAP/TAZ are proteins involved in mechanosensing, translocating to the nucleus on stiffer substrates, and are linked to myofibroblast activation in FAPs and other fibroblast-like cells (3, 48, 103). Inhibiting YAP/TAZ translocation to the nucleus prevents myofibroblast activation on stiffer substrates and therefore, could be applied to fibrotic conditions where the muscles have elevated stiffness (4, 48, 104). Drugs that block YAP translocation to the nucleus have shown preclinical efficacy as antifibrotic drugs including verteporfin (105) and a dopamine receptor agonist (106). However, to our knowledge, YAP inhibitors have not been applied in the context of skeletal muscle tissue.

A stiff mechanical environment, also, inhibits apoptosis of profibrotic myofibroblasts. Despite the presence of apoptotic signals being present, stiffness induces the expression of antiapoptotic protein BCL-X_L (107). To combat this pathway and reduce the number of myofibroblasts, strategies to block BCL-X_L can be applied. The drug ABT-263 inhibits BCL-X_L and has demonstrated antifibrotic activity in the lung (108), liver (109), and heart (110), but similarly has not been applied to skeletal muscle.

Restoring Cell Signaling Pathways

In fibrosis, FAP signaling to MuSCs is disrupted with FAPs releasing inhibitory exosomes rather than promyogenic exosomes. Histone deacetylase inhibitors (HDACis) have emerged as a therapeutic method to tune FAP signaling to a more pro-regenerative state. Extracellular vesicles (EVs) released from both human DMD FAPs and mouse mdx FAPs had altered expressions of miRNAs. HDACis increased the expression of pro-regenerative miR-206 from FAPs which in turn increased differentiation in MuSCs and decreased the amount of collagen in the muscle (90, 111). HDACis also inhibit FAPs adipogenic potential and increase FAP expression of follistatin, a glycoprotein known to inhibit myostatin, therefore blocking myofibroblast activation (112, 113). Givinostat, an HDACi, has been used in clinical trials for DMD with successful reduction of fibrotic and fatty areas (114, 115).

However, experiments in mdx mice showed HDACis were only effective in restoring FAPs’ pro-regenerative signaling in young mice, whereas old mdx FAPs were resistant to HDACi treatment (112). Similarly, antibody treatments against myostatin increased muscle size and specific force, only when administered to young mdx mice, and had no effect on adult mdx mice (102). Fibrosis is largely considered irreversible, although there is some evidence to the contrary (106, 116). Treatments that prevent the development of fibrosis rather than reversing already existing fibrosis tend to be more efficacious. Therefore, targeting FAPs to prevent profibrotic activation before excessive fibrotic development provides the best method for improving muscle function.

PERSPECTIVES AND FUTURE STUDIES

Fibrosis is a progressively degenerative consequence of nearly all skeletal muscle diseases as a result of the profibrotic feedback loop that, not only activates FAPs into a profibrotic state but keeps them there increasing fibrotic contributions (7, 80, 117). Soluble and mechanical signaling contribute to the activation of FAPs and block their exit from the profibrotic feedback loop (12, 16, 48, 118). Investigations into methods to attenuate fibrosis must address ways to disrupt this loop otherwise the cyclic nature of FAPs and fibrosis will continue.

Two interesting avenues to break the profibrotic feedback loop are the relatively new discoveries of FAPs’ mechanosensitivity and heterogeneity. FAPs’ mechanosensitivity has only recently been defined and adds insight into the cyclic nature of fibrotic tissue stiffening and the activation of myofibroblasts (48). However, the signaling pathways that induce myofibroblast activation of FAPs on stiff substrates are not fully defined and further investigation could provide more insight and therapeutic targets for disrupting FAP activation and fibrosis. Methods to block YAP/TAZ or other mechanosensing pathways in vivo have proved effective in other fibrotic tissue (104, 119). Another prospective strategy to break the fibrotic feedback loop would be to target the ECM structure directly. Direct injection of collagenase to digest fibrotic collagen is performed in Dupuytren’s contracture (120) and has been proposed as a method of reducing muscle stiffness in muscle contractures of children with cerebral palsy (121, 122). Blocking collagen crosslinking is a potential target as well to increase the turnover by endogenous collagenase and decrease the stiffness of the collagen network (123). A range of collagen crosslinking inhibitors have been explored in other tissues (124) although a common collagen crosslinking inhibitor was found to be ineffective in blocking crosslinking in skeletal muscle. Methods to disrupt the profibrotic cycle through mechanosensing or altering the mechanics should be further developed in the skeletal muscle environment to determine their efficacy in treating skeletal muscle fibrosis.

The heterogeneity of FAPs in the context of regeneration and fibrosis offers various avenues for future studies to parse out the individual contributions of each subpopulation. Experimentation isolating out individual subpopulations and defining their matrisome and secretome would better define the profibrotic and proregenerative nature of the subpopulations. This could be explored through depletion experiments to ablate a specific subpopulation of FAPs and assess changes in skeletal muscle function and fibrosis in vivo.

CONCLUSIONS

FAPs play an essential role in the regeneration and homeostasis of skeletal muscle through their contributions to the ECM and myogenic signaling. However, in disease, these pathways get disrupted and FAPs quickly take on a pathogenic phenotype. The transient nature of FAPs after injury is essential for maintaining their pro-regenerative role. The persistence of FAPs and resistance to apoptosis leads to the chronic cycle of ECM deposition, fibrosis, and fibrotic activation of FAPs. Recent progress in RNAseq and transcriptomics has allowed insights into the vast heterogeneity of FAPs in healthy and diseased muscle. The importance of these subpopulations is not fully understood but may provide insights into the multidirectional nature of FAPs and more specific targets for antifibrotic therapies.

FAPs and fibrosis contribute to a progressively degenerative positive feedback loop. The development of fibrosis disrupts cell signaling leading to fibrogenic activation of FAPs, furthering fibrosis and, as a result, FAP activation and contributions to fibrosis. Disrupting this feedforward pathway is necessary to attenuate fibrosis and restore muscle function. Targeting the various signaling pathways that induce myofibroblast activation and inhibit apoptosis in FAPs will disrupt the fibrotic feedback loop.

GRANTS

This work was supported by grants from the National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS) R01AR079545 (to L.R. Smith) and 1F31AR082700 (to T. Loomis) and Congressionally Directed Medical Research Programs Grant MD210110 (to L.R. Smith).

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

T.L. prepared figures; T.L. drafted manuscript; T.L. and L.R.S. edited and revised manuscript; T.L. and L.R.S. approved final version of manuscript.