Fibro-adipogenic progenitors prevent skeletal muscle degeneration at acute phase upon tendon rupture in a murine tibialis anterior tenotomy model

Abstract

BACKGROUND

Fibro-adipogenic progenitors (FAPs) are a group of mesenchymal stem cells that cause fibro-fatty degeneration in skeletal muscle in various chronic disease models. FAPs also play a role in preventing muscle degeneration at acute stages during disease progression. However, few studies have reported the changes in and function of FAPs in the acute phase after tendon rupture.

AIM

To clarify the changes in the number of FAPs and their impact on skeletal muscle soon after tendon rupture to facilitate future studies targeting FAPs to treat muscle degeneration.

METHODS

We utilized Pdgfra-H2B::eGFP mice to trace and quantify FAPs in a tibialis anterior tenotomy (TAT) model at 0 and 3 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, and 6 weeks post-injury, and the results were further validated using fluorescence-activated cell sorting analysis with C57BL/6 mice at the same post-injury timepoints. We subsequently used PdgfraCre^ERT::Rosa^DTA mice, and evaluated the severity of post-TAT skeletal muscle degeneration with or without FAP-depletion.

RESULTS

The number of FAPs peaked at 1 week post-TAT before gradually declining to a level comparable to that pre-TAT. The change in the number of FAPs was potentially temporally correlated with the progression of skeletal muscle degeneration after TAT. FAP-depletion led to more severe degeneration early after TAT, indicating that FAPs potentially alleviate muscle degeneration after tendon rupture in the early post-injury phase.

CONCLUSION

FAPs potentially alleviate the degeneration of skeletal muscle in the acute stage after tendon rupture.

Key Words: Fibro-adipogenic progenitors; Skeletal muscle degeneration; Tibialis anterior tenotomy; Tendon rupture; Acute phase

Core Tip: In this study, we demonstrate that fibro-adipogenic progenitors (FAPs) may actually attenuate early-stage degenerative processes following tendon rupture. To our knowledge, this represents the first investigation into the role of FAPs in early-stage skeletal muscle degeneration post-tendon rupture. Our work elucidates the dynamic changes in FAPs population and function during the early phases of muscle degeneration following tendon rupture, providing a more comprehensive understanding of FAPs biology in this context. These findings offer valuable insights for future research on therapeutic interventions and preventive strategies for post-rupture muscle degeneration.

INTRODUCTION

After tendon rupture, fibro-fatty degeneration within the muscle, including muscle atrophy, fibrosis and fatty infiltration[1], progresses and can become a key factor hindering functional recovery of the muscle-tendon unit, even if the tendon is repaired[2,3]. Recently, fibro-adipogenic progenitors (FAPs), a group of mesenchymal stem cells, were identified in skeletal muscle[4,5] and were found to function as a ‘double-edged sword’ in skeletal muscle regeneration after injury[6-9].

There were only a few quiescent FAPs in situ among the intact skeletal muscle fibers. In acute injuries such as cardiotoxin injection, as well as acute phase in disease progression such as aging, FAPs proliferate, facilitating muscle regeneration and delaying muscle degeneration through the secretion of Bmp3b, interleukin-33, etc[5,7,10,11]. In contrast, in the chronic pathologies such as denervation, FAPs differentiate and lead to fibro-fatty degeneration of the muscle upon the activation of signal transducer and activator of transcription 3/interleukin-6 signaling pathways, etc[6,7,9,12,13].

Although FAPs are a popular research topic, few studies have focused on FAPs in the context of tendon rupture, which is one of the most prevalent musculotendinous conditions. It has been reported that FAPs cause fibro-fatty degeneration of the muscle in the chronic stage after tendon rupture[12,14]; therefore, researchers have suggested targeting FAPs to prevent or treat muscle degeneration after tendon rupture. However, how FAPs change during the muscle degeneration that occurs after tendon rupture remains unknown. In addition, the role FAPs play in the acute phase after tendon rupture has not been clarified. This study was performed with the aim of investigating how the number of FAPs in the muscle is altered after tendon rupture, and determining the influence of FAPs on muscle degeneration in the acute phase after tendon rupture.

MATERIALS AND METHODS

Mice

All of the procedures performed on mice in this study were approved by the Institutional Animal Care and Use Committee of Shanghai General Hospital. We adhered to the ARRIVE guidelines and used the ARRIVE Checklist. Enhanced green fluorescence protein-labeled Pdgfra⁺ cells were used to trace FAPs in Pdgfra-H2B::eGFP mice (Jackson Laboratory, Shanghai, China). Eight-week-old male Pdgfra-H2B::eGFP mice were sacrificed, muscle samples were collected and sectioned, and then were observed via microscopy for pre-tibialis anterior tenotomy (TAT) 0 day results. Eight-week-old male Pdgfra-H2B::eGFP mice were subjected to TAT before being sacrificed for muscle collection and their samples sectioned for observation via microscopy at 3 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, and 6 weeks after TAT (n = 3). Additionally, 8-week-old male C57BL/6 (wild-type, WT) mice underwent TAT before being sacrificed for fluorescence-activated cell sorting (FACS), and histological tests of the muscle at 0 day and 1 week, 2 weeks, 4 weeks, and 6 weeks after TAT (n = 5).

To generate mice with depleted FAPs, PdgfraCre^ERT::Rosa^DTA mice were generated by breeding PdgfraCre^ERT mice with Rosa^DTA mice (both from Cyagen Biosciences, CA, United States). Six-week-old male PdgfraCre^ERT::Rosa^DTA mice were injected intraperitoneal with either tamoxifen (n = 3) dissolved in corn oil or plain corn oil (n = 3) every 24 hours for 7 consecutive days to generate FAP-depleted mice and control mice. Then, the mice were subjected to TAT before being sacrificed for further histological tests of the muscle at 0 day, 1 week, and 6 weeks after TAT.

TAT model

The mice were anesthetized, and the skin and hair of the lower limbs were prepared before an incision was made directly beneath the lateral ankle. Then, the tibialis anterior (TA) and insertion into the ankle were exposed. The TA tendon was clamped adjacent to the insertion and transected. After complete transection of the TA tendon, the incision was sterilized and closed. In both Pdgfra-H2B::eGFP mice and PdgfraCre^ERT::Rosa^DTA mice, TAT was performed both hind limbs. WT mice underwent TAT on the left hind limb and sham surgery (SS) on the right hind limb as a control treatment; in the SS, the same procedures for TAT were performed, except the TA tendon was not transected.

FACS analysis

The TA muscle was collected and minced before being digested with 0.2% type II collagenase (Biofroxx, Germany) for 1 hour at 37 °C. Muscle slurries were filtered through 100 μm and 40 μm cell strainers (BD Bioscience, NJ, United States) in sequence. After erythrocytes were removed, the cells were resuspended in phosphate buffered saline containing 2% foetal bovine serum. The cells were subsequently stained with the following antibodies: Alexa Fluor 488-conjugated anti-mouse CD31 antibody (Biolegend, CA, United States), Alexa Fluor 488-conjugated anti-mouse CD45 antibody (Biolegend, CA, United States), APC-conjugated anti-mouse integrin α7 antibody (R&D, MN, United States), and APC-conjugated/cyanine7-conjugated anti-mouse Sca1 antibody (Biolegend, CA, United States) for 30 minutes at 4 °C in the dark. The gating strategy used was CD31^-CD45^-integrinα7^-Sca1⁺. The stained cells were then subjected to FACS with a FACSAria III (BD Biosciences, NJ, United States).

Histological testing

TA muscle samples were collected and immediately fixed in 10% formalin for 24 hours before being dehydrated in a 30% sucrose solution for 48 hours. The samples were then flash frozen or embedded in paraffin embedded and cryo-sectioned at a thickness of 10 mm. For samples from Pdgfra-H2B::eGFP mice, the slices were then directly observed under a luminescence microscope.

For samples from WT mice and PdgfraCre^ERT::Rosa^DTA mice, hematoxylin and eosin (H&E) staining (Solarbio, Beijing, China), modified Masson’s trichrome (TRI) staining (Solarbio, Beijing, China) and oil red O (ORO) staining (Solarbio, Beijing, China) were performed before observation. H&E staining was used to facilitate the quantification of the cross-sectional area (CSA) of the TA muscle fibers to assess the degree of atrophy in the sample. TRI staining was used to determine the collagen content in order to evaluate fibrosis in the TA muscle. ORO staining was performed to visualize the lipid content to evaluate fatty infiltration of the TA muscle. FBXO32 (Affinity, OH, United States) and alpha-smooth muscle actin (α-SMA) (Affinity, OH, United States) immunohistochemistry (IHC) were performed to detect the pro-atrophic muscle fibers that had been labeled with FBXO32, as well as the pro-fibrotic content labeled with α-SMA within the TA muscle. Perilipin-1 (Abcam, MA, United States) immunofluorescence (IF) was performed to detect adipogenesis within the TA muscle. The procedures for staining, IHC and IF followed the corresponding product protocols.

Statistical analysis

For the histological tests, cross sections were chosen randomly from the mid-bellies of the TA muscle, and further assessments were made on 3 different sections, with 5 randomly selected areas on each section. All images were assessed by 2 blinded researchers using ImageJ, and the average value was calculated for each sample. CSA was measured for muscle fibers in H&E-stained sections. The severity of fibrosis was evaluated on the basis of the fraction of the entire sample area with aniline blue on TRI-stained sections. Similarly, the severity of fatty infiltration was evaluated by dividing the area of fat stained with ORO by the entire sample area on ORO-stained sections.

The fraction of the sample area positive for FBXO32 on IHC, α-SMA on IHC and perilipin 1 on IF were also determined by dividing the area of positive expression by the whole area to show the changes over time of muscle atrophy, fibrosis and adipogenesis, respectively. The collected FACS data were further analyzed with FlowJo v10. The change in the population of FAPs was determined by calculating the proportion of CD45^-CD31^- cells accounted for by FAPs, the proportion of live cells accounted for by FAPs, and the number of FAPs per gram of TA muscle. All the data were analyzed using two-way ANOVA with SPSS. Significance was determined at a P value < 0.05.

RESULTS

FAPs proliferated after TAT and peaked in number at 1 week post-TAT

A mouse model (pdgfra-H2B::eGFP) using platelet-derived growth factor alpha, a classical surface marker for FAPs, was constructed. The TA muscles of Pdgfra-H2B::eGFP mice that underwent TAT at 0-3 days, and 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, and 6 weeks post-operation were collected and sectioned, and the number of FAPs at each time point were then quantified via luminescence microscopy. The results (Figure 1) revealed that the FAPs were located mainly amongst the myofibers, both before and after TAT. In addition, there were only 1.3 ± 0.5 FAPs per field of view in the muscle before TAT; however, this number increased significantly to 8.3 ± 1.2 per field at 3 days post-TAT (0 day vs 3 days, P < 0.001) and further reached 11.7 ± 1.2 per field at 1 week post-TAT (0 day vs 1 week, P < 0.001). After that time point, the number of FAPs decreased, and there was no significant difference in the number of FAPs per field between 0 day and 6 weeks post-TAT (2.3 ± 0.5 vs 1.3 ± 0.5, P > 0.05).

Figure 1 Change in fibro-adipogenic progenitors population after tendon rupture observed in fibro-adipogenic progenitor-tracing mice. A-H: Images of frozen sections of tibialis anterior muscle at 0 day, 3 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, and 6 weeks after tibialis anterior tenotomy in Pdgfra-H2B::eGFP mice, respectively; I: The statistical analysis of the number of EGFP⁺ cells per field counted under microscopy. ^aP < 0.001, 3 days compare to 0 day; ^bP < 0.001, 1 week compare to 0 day; ^cP < 0.001, 2 weeks compare to 0 day; ^dP < 0.01, 3 weeks compare to 0 day; ^eP < 0.01, 4 weeks compare to 0 day; ^fP < 0.01, 5 weeks compare to 0 day; ^gP > 0.05, 6 weeks compare to 0 day. Pdgfra⁺ cells were labeled with enhanced green fluorescence protein. Yellow arrowheads indicate EGFP⁺ cells (i.e., fibro-adipogenic progenitors). Scale bar = 50 μm. TAT: Tibialis anterior tenotomy; FAPs: Fibro-adipogenic progenitors.

In addition, TAT was also performed in WT mice, and corresponding SSs were conducted on the contralateral side (TAT/SS model). The muscles from both sides at 0-3 days, and 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, and 6 weeks after injury were then processed and analyzed using FACS (gating strategies showed in Figure 2A). The results showed that the proportion of FAPs among CD45^-CD31^- cells in the muscle began to increase at 3 days post-TAT and subsequently remained significantly greater than that of the SS side (primary results showed in Figure 2B, further analysis shown in Figure 2C). However, this proportion demonstrated a random pattern of changes from 1 week to 6 weeks post-TAT (Figure 2C). Therefore, we further analyzed the proportion of FAPs among all live cells in the muscle (Figure 2D) and detected no obvious pattern of change. Considering that the alteration in the proportion of FAPs among CD45^-CD31^- cells and live cells demonstrated with FACS could be largely influenced by the significant change in the quantity of other cells, such as immune cells, it is reasonable to assume that the changes in those proportions could be biased, rather than reflecting the actual changes in the absolute numbers of FAPs themselves. Therefore, the results from FACS were analyzed again, and the number of FAPs per gram of muscle was calculated. The results (Figure 2E) revealed that the number of FAPs per gram of muscle began to increase at 3 days post-TAT, reached (17.1 ± 0.6) × 10⁵ per gram of muscle at 1 week post-TAT, and was significantly greater than that of the SS side (P < 0.001). The number of FAPs per gram then declined and was close to, but still greater, than that on the SS side at 6 weeks post-operation. This result demonstrated a similar trend to the change pattern of FAPs obtained in Pdgfra-H2B::eGFP mice. On the basis of the above evidence, it was determined that 1 week post-TAT is a crucial timepoint, as that is when the number of FAPs was highest after TAT.

Figure 2 Change in fibro-adipogenic progenitors population after tendon rupture observed in C57BL/6 mice. A: The gating strategy of fluorescence-activated cell sorting for fibro-adipogenic progenitors (FAPs) in the tibialis anterior muscle of C57BL/6 mice; B: The fluorescence-activated cell sorting results of FAPs in the muscle at 0 day, 3 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, and 6 weeks, respectively, after tibialis anterior tenotomy (TAT) or sham surgery (SS) in C57BL/6 mice; C: Subsequent analysis showing the proportion of FAPs among CD45^-CD31^- cells (^aP < 0.001, ^bP < 0.001, ^cP < 0.001, ^dP < 0.001, ^eP < 0.001, ^fP < 0.001, ^gP < 0.001, TAT compared to SS side at 3 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, and 6 weeks, respectively); D: The proportion of FAPs among live cells (^aP < 0.001, ^bP < 0.001, ^cP < 0.001, ^dP < 0.001, ^eP < 0.001, TAT compared to SS side at 3 days, 1 week, 2 weeks, 3 weeks, and 4 weeks, respectively); E: The number of FAPs per gram tibialis anterior muscle (^aP < 0.001, ^bP < 0.001, ^cP < 0.001, ^dP < 0.001, ^eP < 0.001, ^fP < 0.001, ^gP < 0.001, TAT compared to SS side at 3 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, and 6 weeks, respectively). TAT: Tibialis anterior tenotomy; SS: Sham surgery; FAPs: Fibro-adipogenic progenitors.

The trend toward muscle degeneration emerged early after TAT in C57BL/6 mice

The TAT/SS model was again established in WT mice, and the muscles at 0 day, and 1 week, 2 weeks, 4 weeks, and 6 weeks post-operation were collected for further histological evaluation. The results of H&E staining (Figure 3A and B) reveal that the CSA of post-TAT muscle fibers was lower than that of post-SS muscle fibers, especially at 4 weeks and 6 weeks after TAT (both P < 0.05). Furthermore, FBXO32 IHC staining (Figure 3C and D) revealed that at 1 week after TAT, the muscle was more inclined to atrophy at all time points compared with that of the post-SS side (all P < 0.001).

Figure 3 The progression of muscle atrophy after tendon rupture vs sham surgery in C57BL/6 mice. A: The hematoxylin and eosin staining results of the tibialis anterior muscle at 0 day, 1 week, 2 weeks, 4 weeks, and 6 weeks after tibialis anterior tenotomy (TAT) or sham surgery (SS) in C57BL/6 mice; B: The statistical analysis of average myofiber cross-sectional area which represents the severity of muscle atrophy (^aP < 0.05, ^bP < 0.05, TAT compared to SS side at 4 weeks and 6 weeks, respectively); C: The FBXO32 immunohistochemistry staining results of the muscle at 0 day, 1 week, 2 weeks, 4 weeks, and 6 weeks after TAT or SS in C57BL/6 mice; D: The statistical analysis of the area fraction of positive stained area which represents the tendency towards muscle atrophy (compared to SS side, ^aP < 0.001, ^bP < 0.001, ^cP < 0.001, ^dP < 0.001, TAT compared to SS side at 1 week, 2 weeks, 4 weeks, and 6 weeks, respectively). Scale bar = 100 μm. TAT: Tibialis anterior tenotomy; SS: Sham surgery.

In addition, TRI staining (Figure 4A and B) and α-SMA IHC staining (Figure 4C and D) of the tissue sections reveal that fibrosis at 1 week after TAT was significantly more severe in the post-TAT muscle than in the post-SS side (all P < 0.01). In addition, ORO staining (Supplementary Figure 1) and perilipin 1 IF staining were also used to evaluate fatty degeneration of the muscle (Figure 5A and B). Although the results of ORO staining revealed no lipid droplets in any of the muscle samples, IF staining revealed that the expression of perilipin 1, a marker indicating the early phase of adipogenesis, was significantly greater at 1 week post-TAT, than at 1 week post-SS (all P < 0.05).

Figure 4 The progression of muscle fibrotic degeneration after tendon rupture vs sham surgery in C57BL/6 mice. A: The modified Masson trichrome staining results of the tibialis anterior muscle at 0 day, 1 week, 2 weeks, 4 weeks, and 6 weeks after tibialis anterior tenotomy (TAT) or sham surgery (SS) in C57BL/6 mice; B: The statistical analysis of area fraction of collagen which represents the severity of fibrosis (^aP < 0.05, ^bP < 0.001, TAT compared to SS side at 4 weeks and 6 weeks, respectively); C: The alpha-smooth muscle actin immunohistochemistry staining results of the muscle at 0 day, 1 week, 2 weeks, 4 weeks, and 6 weeks after TAT or SS in C57BL/6 mice; D: The statistical analysis of the area fraction of positive stained area. Area fraction of collagen (%) = area of collagen staining of the entire sample area which represents the tendency of fibrosis (^aP < 0.001, ^bP < 0.001, ^cP < 0.001, ^dP < 0.001, TAT compared to SS side at 1 week, 2 weeks, 4 weeks, and 6 weeks, respectively). Scale bar = 100 μm. TAT: Tibialis anterior tenotomy; SS: Sham surgery.

Figure 5 The progression of muscle adipogenic degeneration after tendon rupture vs sham surgery in C57BL/6 mice. A: Images of perilipin 1 immunofluorescence staining of the tibialis anterior muscle at 0 day, 1 week, 2 weeks, 4 weeks, and 6 weeks after tibialis anterior tenotomy or sham surgery in C57BL/6 mice; B: The statistical analysis of the area fraction of positive stained area which represents the tendency of adipogenesis (^aP < 0.05, ^bP < 0.05, ^cP < 0.01, ^dP < 0.001, tibialis anterior tenotomy compared to sham surgery side at 1 week, 2 weeks, 4 weeks, and 6 weeks, respectively). Scale bar = 100 μm. TAT: Tibialis anterior tenotomy; SS: Sham surgery.

The degeneration of the muscle after TAT was aggravated by FAP-depletion

To explore the role of FAPs in muscle degeneration after TAT, PdgfraCre^ERT::Rosa^DTA mice were injected with tamoxifen (dissolved in corn oil) to deplete FAPs (FAP-depleted mice), and the control group was injected with an equal volume of corn oil only. The efficacy of depletion was validated using FACS (Supplementary Figure 2). Then, both the FAP-depleted mice and their control group underwent TAT, and the muscles at 0 day, and 1-6 weeks post-TAT was collected. The aforementioned histological tests were performed to compare the severity of muscle degeneration between the two groups.

The results of H&E staining (Figure 6A and B) showed that the CSA of myofibers in the muscle of the FAP-depleted mice was lower than that of the control group at 0 day, and 1-6 weeks post-TAT, although the difference was not significant (all P > 0.05). The results of FBXO32 IHC staining (Figure 6C and D) revealed that, compared with those in the control group, the muscle in the experimental group was significantly more prone to atrophy at 1 week post-TAT (1.96 ± 0.16 times vs 1.72 ± 0.17 times, P < 0.05), whereas the difference was not significant at 0 day and 6 weeks post-TAT (P > 0.05).

Figure 6 The progression of muscle atrophy after tendon rupture in fibro-adipogenic progenitor-depleted mice. A: The hematoxylin and eosin staining results of the tibialis anterior muscle at 0 day, 1 week, and 6 weeks after tibialis anterior tenotomy (TAT) in PdgfraCre^ERT::Rosa^DTA mice induced with Tamoxifen or corn oil; B: The statistical analysis of average myofiber cross-sectional area which represents the severity of muscle atrophy; C: The FBXO32 immunohistochemistry staining results of the tibialis anterior muscle at 0 day, 1 week, and 6 weeks after TAT in PdgfraCre^ERT::Rosa^DTA mice induced with Tamoxifen or corn oil; D: The statistical analysis of the area fraction of positive stained area which represents the tendency of muscle atrophy (^aP < 0.05, compared to control group). Scale bar = 100 μm. TAT: Tibialis anterior tenotomy.

TRI staining revealed that neither the FAP-depleted mice nor the control mice formed obvious fibrotic scars in the muscle before TAT, but the muscle of the FAP-depleted mice demonstrated significantly more severe fibrosis than did that of the control mice at 1-6 weeks post-TAT (Figure 7A and B). Moreover, the results of α-SMA IHC staining (Figure 7C and D) reveal that, compared with that in the control group, the trend toward fibrosis in the muscle was significantly greater at 1 week post-TAT in FAP-depleted mice (1.26 ± 0.04 times vs 1.16 ± 0.03 times, P < 0.01), but the difference was not significant at 0-6 weeks after TAT (P > 0.05).

Figure 7 The progression of muscle fibrotic degeneration after tendon rupture in fibro-adipogenic progenitor-depleted mice. A: The modified Masson trichrome staining results of the tibialis anterior muscle at 0 day, 1 week, and 6 weeks after tibialis anterior tenotomy (TAT) in PdgfraCre^ERT::Rosa^DTA mice induced with Tamoxifen or corn oil; B: The statistical analysis of area fraction of collagen which represents the severity of fibrosis (^aP < 0.05, ^bP < 0.001, compared to control group); C: The alpha-smooth muscle actin immunohistochemistry staining results of the tibialis anterior muscle at 0 day, 1 week, and 6 weeks after TAT in PdgfraCre^ERT::Rosa^DTA mice induced with Tamoxifen or corn oil; D: The statistical analysis of the area fraction of positive stained area which represents the tendency of fibrosis. Area fraction of collagen (%) = area of collagen staining of the entire sample area (^aP < 0.01, compared to control group). Scale bar = 100 μm. TAT: Tibialis anterior tenotomy.

The results of ORO staining (Supplementary Figure 3) showed that no muscle among the two groups exhibited interfibrous lipid accumulation. However, the results of perilipin 1 IHC staining (Figure 8A and B) showed that the expression of perilipin 1 was significantly greater at 6 weeks post-TAT in FAP-depleted mice than in control mice (49.16 ± 12.72 times vs 17.06 ± 4.04 times, P < 0.05). However, no significant difference was found between the groups at 0-1 week after TAT.

Figure 8 The progression of muscle adipogenic degeneration after tendon rupture in fibro-adipogenic progenitor-depleted mice. A: The perilipin 1 immunofluorescence staining results of the tibialis anterior muscle at 0 day, 1 week, and 6 weeks after tibialis anterior tenotomy in PdgfraCre^ERT::Rosa^DTA mice induced with Tamoxifen or corn oil; B: The statistical analysis of the area fraction of positive stained area which represents the tendency of adipogenesis. ^aP < 0.05, compared to control group. Scale bar = 100 μm. TA: Tibialis anterior; TAT: Tibialis anterior tenotomy.

DISCUSSION

In this study, we confirmed the change in the number of FAPs after tendon rupture, indicating that the number of FAPs primarily expands shortly after injury, before gradually declining to the preinjury level. The expanded population of FAPs at the acute stage after tendon rupture might alleviate muscle degeneration, as FAP-depletion was found to result in a more severe trend toward postinjury muscle degeneration.

Tendon ruptures, such as rotator cuff and Achilles tendon tears, are among the most common diseases presented in orthopedic sports medicine clinics. Muscle degeneration after tendon rupture was found to be the key factor hindering functional muscle recovery, even if the torn tendon was repaired successfully. Inspired by the difficulties encountered in clinical practice, we chose to study the changes in and function of FAPs in skeletal muscle degeneration after tendon rupture. The mouse model of TAT is onesome of the most classical models to study tendon tearing in animals[15]. In addition, numerous studies focusing on FAPs and muscle degeneration were performed on the TA[16-19], including the first studies that identified FAPs[4,5]. Therefore, we also chose to utilize a mouse model of TAT.

PDGFRa-H2B::eGFP mice were used to characterize FAP distribution[20-22], and TA sections from PDGFRa-H2B::eGFP mice revealed that the number of FAPs increased significantly after TAT, peaking at 1 week, and then gradually declined. To further validate these results, we established a TAT/SS model in WT mice, and subsequent FACS demonstrated results with a similar trend. One previous study also tried to clarify the change pattern of FAP quantity after multi-regional clinical trial[23]. However, their FACS results revealed that the number of FAPs plus endothelial cells was greater at 28 days than at 5 days post trauma, which was neither precise nor direct enough to address this important question. Our study, however, provided evidence at multiple timepoints obtained from both histological tests in PDGFRa-H2B::eGFP mice and FACS data from WT mice, and this data focused exclusively on FAPs.

FAPs cannot exert their function without massive proliferation, as there are only a few FAPs in situ among the myofibers. Therefore, the number of FAPs could be an important factor affecting their function. Our results indicated that FAPs proliferate in the early stage of tendon rupture, before returning to quiescence at a later stage. This pattern is similar to that of FAPs in various musculotendinous injury models reported in the literature[4,5,18,22]. Therefore, our results suggest that FAPs might also act as a ‘double-edged sword’ toward the muscle in the tendon rupture model. In addition, these results could facilitate a better understanding of the alteration of FAPs in the muscle upon tendon rupture.

Next, we conducted histological tests to visualize the progression of muscle degeneration in WT mice after TAT. Compared with that in the SS group, there was obvious fibrotic deposition within the muscle at 1 week, 4 weeks, and 6 weeks post-TAT, and the CSA at 4-6 weeks post-TAT was significantly smaller than that in the SS group. This finding is not surprising, as previous studies have reported that skeletal muscle fibro-fatty degeneration occurs at 6-8 weeks post-tendon rupture[12,13]. However, interestingly, at 1 week, 2 weeks, 4 weeks, and 6 weeks after TAT, the FBXO32⁺, α-SMA⁺ or perilipin 1⁺ area fractions within the muscle, respectively representing the tendency toward skeletal muscle atrophy and fibro-fatty degeneration, were significantly greater than those in the SS group. These results confirmed that a trend toward muscle degeneration could be observed even at 1 week after TAT. Considering that the peak population of FAPs also occurred at 1 week post-TAT, these results suggest that FAPs could have a potential impact on muscle degeneration in the early phase after tendon rupture.

To verify this hypothesis, we utilized FAPs-depleted mice, as in previous studies[11,20], and performed histological tests to determine whether the absence of FAPs would affect skeletal muscle degeneration after TAT. The results demonstrated that the muscle of FAPs-depleted mice presented significantly aggravated atrophy and fibrosis at 1 week after TAT and a significantly aggravated trend toward adipogenesis at 6 week after TAT. These results showed that FAP-depletion could exacerbate the degeneration of skeletal muscle after TAT, indicating that FAPs could alleviate the trend of skeletal muscle degeneration early after tendon rupture.

To our knowledge, no prior studies have reported the positive effects of FAPs on skeletal muscle after tendon rupture. Our findings support the notion that FAPs promote skeletal muscle regeneration and reduce skeletal muscle degeneration in other musculotendinous disease models[5,7,10,16,24-26]. Circulating factors derived from FAPs have been demonstrated to directly facilitate muscle regeneration in healthy individuals through the modulation of muscle stem cells expansion and asymmetric commitment, mediated by the Wnt-induced signaling protein 1/protein kinase B signaling pathway[27]. Additionally, FAP-derived cytokines such as interleukin-33, contribute to muscle regeneration through an indirect mechanism by regulating the homeostasis of muscle regulatory T cells, whose recruitment was found to be crucial for effective muscle regeneration following injury[24]. Few studies have investigated the alteration and function of FAPs under conditions of tendon rupture. According to the limited evidence, FAPs at the chronic stage of tendon rupture were found to be the cellular origin of fatty infiltration and fibrosis[12]. Together with our results, these findings further confirm that FAPs also function as ‘double-edged swords’ toward the muscle after tendon rupture. These findings could provide a more comprehensive understanding of FAPs, and provide a theoretical basis for future studies focusing on FAPs, with the aim of preventing or treating muscle degeneration after tendon injury.

There were certain limitations to the present study. First, in the current study, FAP-depletion was achieved prior to TAT, so the mitigating effect of degeneration after FAPs depletion may only apply to the early phase after TAT. It still remains unknown whether FAPs would exert the same function if they were depleted after TAT. In our study design, we intended to add a group in which the FAPs were depleted at 6 weeks post-TAT; however, the number of FAPs at 6 weeks post-TAT was already low, making depletion impossible. This may be because the surface markers of FAPs can change during their differentiation process in the late stages after TAT, making it difficult to detect FAPs with the same gating strategy.

In addition, the results of ORO staining in the present study did not agree with the results of prior studies. In our study, typical fat droplets could not be observed, even at late stages after TAT, whereas skeletal muscle fatty infiltration was observed at 6-8 weeks after tendon rupture in previous studies[12,14]. It is possible that this could be due to the content of FAPs, as well as their tendency toward adipogenesis differentiation in the skeletal muscle of mice hind limbs, like the TA, being lower than that of the upper limbs, such as the supraspinatus and infraspinatus muscles[28]. However, the TAT model is more mature and more convenient for subsequent intramuscular administration, which might be needed for further research; furthermore, with the use of the TAT model, fewer mice would need to be sacrificed, as the muscle mass of the TA is greater than that of the supraspinatus and infraspinatus muscles. Thus, we used the TAT model in favor of other tendon tear models.

Based on our findings demonstrating the beneficial role of FAPs in muscle preservation during the acute phase following tendon rupture, therapeutic strategies aimed at delaying or preventing muscle degeneration progression warrant further investigation. Preclinical studies in various pathological models, including age-related muscle atrophy and massive rotator cuff injuries, have explored the transplantation of genetically modified FAPs as a potential intervention for muscle degeneration in murine models[27,29,30]. However, the translation of FAP-based stem cell therapies into clinical applications remains challenging and requires substantial additional research. An alternative therapeutic approach involves pharmacological modulation of FAP-related pathways. Previous investigations have identified promising candidates, including the transforming growth factor-beta inhibitor decorin and the histone deacetylase inhibitor Trichostatin A, which have demonstrated efficacy in mitigating muscle degeneration through FAP modulation[31,32]. Further mechanistic studies elucidating the anti-degenerative properties of FAPs are expected to reveal novel molecular targets for pharmacological intervention. The current findings provide a foundation for future research directions and may contribute to the development of therapeutic strategies for post-tendon rupture muscle degeneration (Figure 9).

Figure 9 The main findings of the study. FAPs: Fibro-adipogenic progenitors.

CONCLUSION

FAP population expansion at the acute stage after tendon rupture can potentially alleviate the degeneration of skeletal muscle.

ACKNOWLEDGEMENTS

We thank Prof. Kang-Lai Tang (MD, PhD) from Department of Sports Medicine, Southwest Hospital, Army Medical University for his kind support to this research.