Secretome from myoblasts statically loaded at low intensity promotes tenocyte proliferation via the IGF‐1 receptor pathway

Exercise is widely recognized as beneficial for tendon healing. Recently, it has been described that muscle‐derived molecules secreted in response to static exercise influence tendon healing. In this study, the optimal static loading intensity for tendon healing and the composition of secretome released by myoblasts in response to different intensities of static strain were investigated. In an in vitro coculture model, myoblasts were mechanically loaded using a Flexcell Tension System. Tenocytes were seeded on transwell inserts that allowed communication between the tenocytes and myoblasts without direct contact. Proliferation and migration assays, together with RNA sequencing, were used to determine potential cellular signaling pathways. The secretome from myoblasts exposed to 2% static loading increased the proliferation and migration of the cocultured tenocytes. RNA‐seq analysis revealed that this loading condition upregulated the expression of numerous genes encoding secretory proteins, including insulin‐like growth factor‐1 (IGF‐1). Confirmation of IGF‐1 expression and secretion was carried out using qPCR and enzyme‐linked immunosorbt assay (ELISA), revealing a statistically significant upregulation in response to 2% static loading in comparison to both control conditions and higher loading intensities of 5% and 10%. Addition of an inhibitor of the IGF‐1 receptor (PQ401) to the tenocytes significantly reduced myoblast secretome‐induced tenocyte proliferation. In conclusion, IGF‐1 may be an important molecule in the statically loaded myoblast secretome, which is responsible for influencing tenocytes during exercise‐induced healing.


| INTRODUCTION
The natural tendon healing process is slow and often results in scar formation, which can impede tendon function and increase the risk of postoperative tendon rupture. 1 Cell migration and proliferation are critical events during tendon wound healing.3][4] Therefore, unraveling the factors that regulate tendon cell migration and proliferation is crucial for developing effective treatment strategies for tendon healing.
][7][8][9][10] It has been suggested that mechanical loading of tendons promotes tendon stem/progenitor cell proliferation and tenogenic differentiation, resulting in the generation of tenocytes that replenish the lost tenocytes at the wound site, ultimately improving tendon wound healing. 11However, this explanation only addresses the effect of mechanical loading within the tendon.It cannot account for the observation that a denervated tendon, which is subjected to passive loading exhibits degenerative features in comparison with a tendon that is loaded by muscle contraction. 12This highlights the potential role of the muscle-derived secretome in enhancing tendon healing.
4][15][16][17][18][19] For example, in bone tissue there is an evidence that exercise-induced muscle-derived secretory proteins, such as insulin-like growth factor 1 (IGF-1) and interleukin-15 (IL-15) regulate bone formation. 20n skin, exercise retards skin aging and rescues slow wound healing via a mechanism that involves exercise or exercise-mimicking IL-15 treatment. 16,21In tendon, platelet-rich fibrin matrices have been shown to accelerate the healing process of Achilles tendon tears and facilitate a faster return to sports for individuals. 22Furthermore, plasma proteomic patterns observed during exercise were found to be associated with various wound healing-related pathways, including wound healing, apoptosis, cellular stress response, as well as inflammation and immune system responses. 23ifferent types, intensities, frequencies, and durations of exercise have different functions.For example, low-intensity exercise accelerates wound healing rates, but high-intensity exercise does not. 24We believe that the different functions of diverse forms of exercise partly is due to the release of distinctive concentrations and compositions of secretome into the circulation. 16,25,26or instance, a study found that marathon runners had a significant increase in plasma interleukin-6 (IL-6), tumor necrosis factor alpha (TNF-α), and matrix metallopeptidase 9 (MMP-9) levels as compared with half-marathon runners. 27Moderate and sprint intensity training was shown to decrease the concentration of IL-6, Hepatocyte Growth Factor (HGF), and Leptin, whereas moderate intensity training, but not sprint intensity, increased monocyte chemoattractant protein-1 (MCP-1) levels. 28Multiplex assays for mouse serum cytokines show different sets of cytokines activated in response to a single bout of sprint, endurance run, and one-week voluntary wheel running. 29In humans, plasma concentrations of 184 distinct proteins were responsive to moderate-intensity exercise while 598 proteins changed with high-intensity exercise. 30Based on the aforementioned studies, it is conceivable that various profiles of muscle-derived secretory proteins have unique regulatory functions that impact tendon wound healing in different healing phases.
In our previous study, we observed that static loading of myoblasts increased tenocyte migration, collagen type I/III ratio, and the expression of tenocyte markers when compared with dynamic loading. 31Building upon these findings, the objective of the current study was to investigate the optimal intensity of static loading applied to myoblasts in order to induce the release of an effective secretome that promotes tendon healing while also elucidating the underlying signaling pathways involved.To address this aim, we used an in vitro model that incorporated an indirect coculture approach, utilizing Transwell inserts and the FlexCell tension system to load myoblasts.We assessed the impact of the myoblast-derived secretome on tenocyte proliferation and migration and employed RNA sequencing of the myoblasts to identify relevant signaling pathways and explore the potential involvement of specific molecular candidates.razor blade.The Achilles tendons were cut into 1.0 mm 3 pieces and digested with 2 mg/mL collagenase (Sigma-Aldrich, C0130, USA) dissolved in Dulbecco's modified Eagle medium (DMEM) + GlutaMAX™ -I (Gibco, 61 965-026, USA) supplemented with 10% fetal bovine serum (FBS) (Sigma-Aldrich, 0001648078, USA) in a 5% CO 2 cell culture incubator overnight.Subsequently, the digested pellets were collected by centrifugation at 500 g for 5 min, resuspended in DMEM supplemented with 10% FBS, and seeded in a 6-well plate.Primary tenocytes were passaged when they reached approximately 80% confluency and frozen at −80°C at passages 3-4.Thus, passage fourth to sixth cells were used in experiments.For all experimental conditions, the FBS concentration was reduced to 1%.

| Indirect coculture
The L6 rat myoblast cell line was purchased from American Type Culture Collection (ATCC).L6 myoblasts were seeded at a density of 5 × 10 5 cells per well on FlexCell plates (Flexcell International Corporation, BF-3001C, USA) and 3 × 10 5 tenocytes were seeded in cell culture inserts (8 μm pore size, #353093 or 1 μm pore size, #353102, Corning USA).After 24 h, both myoblasts and tenocytes were washed with Hanks' Balanced Salt Solution (HBSS) (Gibco, 14170112, UK) and the inserts with tenocytes were placed in the FlexCell plates with myoblasts.Pores in the insert enabled cell-cell communication via soluble factors without direct contact.

| Mechanical loading
The FlexCell Tension System (Flexcell International Corporation, FX5000, USA) was used to generate mechanical loading to the adherent myoblasts.FlexCell plates were placed on 25-mm-diameter round loading posts.The membranes of the FlexCell plates were stretched by vacuum suction to create equi-biaxial loading.The membrane received static mechanical loading of 2%, 5%, and 10%.The loading protocol was as follows: 1 h loading followed by a 2 h rest period; this loading and rest period was repeated once before a resting period of 6 h.The protocol was repeated for 24 h.

| Cell migration
Tenocytes were seeded on cell culture inserts (with 8.0 μm pore size) at a density of 3 × 10 5 cell per insert, and L6 myoblasts were seeded on the FlexCell plates at a density of 5 × 10 5 cell per well.Tenocytes were cultured alone or cocultured with loaded or unloaded myoblasts.After 24 h, tenocytes that had migrated from the top of the membrane to the underside of the membrane were analyzed.Tenocytes remaining on the top side of the membrane were carefully removed by cotton swabs and the migrated tenocytes on the underside of the membrane were fixed with methanol for 20 min followed by 20 min air drying.Tenocytes were stained with 0.1% crystal violet (Sigma-Aldrich, St. Louis, Missouri, USA) for 30 min at room temperature (RT) and washed 4 times with milli-Q water.The inserts were left to air dry for 2 h until no liquid remained.The crystal violet staining was dissolved with 500 μL extraction solution (30% methanol, 10% acetic acid, and 60% milli-Q water) and the absorbance was measured with a microplate reader at 590 nm wavelength.

| Cell proliferation
Tenocytes were seeded on cell culture inserts (with 1.0 μm pore size) at a density of 3 × 10 5 cells per insert, and L6 myoblasts were seeded on FlexCell plates at a density of 3 × 10 5 cells per well.Tenocytes were cultured alone or cocultured with loaded or unloaded myoblasts for 24 h.During the experiment, cells were exposed to EdU solution to allow measurement of cells synthesizing DNA using a Click-iT Plus EdU Imaging kit (Thermo Fisher, C10639, USA) according to the manufacturer's protocol.Briefly, the membranes of the inserts were cut off and fixed with 10% Formalin (3.7% formaldehyde) (Sigma-Aldrich, HT501128, USA) for 15 min before cells were washed in 3% bovine serum albumin (BSA) (Sigma-Aldrich, A7030, USA).Afterwards, membranes were incubated in 0.5% Triton X-100 (Sigma-Aldrich, 102 115 323, USA) for 20 min and again washed in 3% BSA.Subsequently, tenocytes were incubated in Click-iT Plus reaction cocktail for 30 min at RT before staining with 5 mg/mL Hoechst 33342 for 30 min.The membranes were mounted on glass slides with ProLong Gold antifade reagent (Thermo Fisher, P36930, USA).Images of EdU and Hoechst 33342 stained nucleus were captured respectively by Leica Thunder Widefield microscope (Leica Microsystems, Germany) with 200-fold magnification.Ten randomly selected field of views were chosen for each repeat.In total, five repeats were performed for each group.The number of EdUor Hoechst 33342-stained nucleus were counted by Fiji (https://fiji.sc/).In each field of view, the proliferation rate was calculated by dividing the number of Hoechst 33342-stained nucleus with the number of EdU-stained nucleus.The average proliferation rate from 10 field of views in each repeat were used for further statistical calculation.

| RNA isolation and real-time PCR
Total cellular RNA in myoblasts was extracted using RNeasy mini kits (Qiagen, 74140, Germany) according to the manufacturer's protocol.Subsequently, a high-capacity cDNA reverse transcription kit (Thermo Fisher, 4368814, Lithuania) was used to reverse transcribe 1000 ng of RNA into cDNA.TaqMan™ Fast Advanced Master Mix (Thermo Fisher, 4444963) was used with Igf1 probe (Thermo Fisher, Rn00710306_m1) and Actb probe (Thermo Fisher, Rn00667869_m1).The amplification was performed on a ViiA7 Real-Time PCR system and analyzed with associated software (Applied Biosystems, Carlsbad, CA) using the 2 −∆∆Ct method.

| RNA sequencing and bioinformatics
Three biological repeats of total RNA from unloaded or loaded myoblasts were collected.RNA Sequencing was performed at Novogene (Cambridge Genomic Sequencing Centre, UK) using an Illumina NovaSeq 6000 instrument.The sequencing workflow began with an initial step of conducting a sample quality check (sample QC), which showed that the samples met the criteria of RNA sequencing technique.Next, the library was prepared and its quality was tested (Library QC).Thereafter, a paired-end 150 bp sequencing strategy was used to sequence the lncRNA and circRNA library, and singleend 50 bp was used to sequence the small RNA library.The sequenced raw reads were filtered to remove the adapters containing reads, N > 10% (N represents base that could not be determined) containing reads and lowquality reads [The Qscore (Quality value) of over 50% bases of the read is ≤ 5].HISAT2 software was used to map the clean reads to the rat reference genome.Mapping information from all samples was combined and placed as input into the regular StringTie assembler.The assembled transfrags were compared with the reference transcripts to determine if they were sufficiently different to be considered novel.Gene expression level was estimated by the abundance of transcripts (count of sequencing) that mapped to exon.Fragments Per Kilobase of transcript sequence per Millions base pairs sequenced (FPKM) were used to estimate gene expression levels.The read count was normalized, and the p-value and FDR (false discovery rate) value were estimated based on multiple hypothesis testing.The Gene Ontology(GO) analysis was done with the upregulated and downregulated genes, respectively.Seven, six, and three enriched functions in biology process, cell components, and molecular functions of upregulated and downregulated genes were shown, respectively.

| Predicted secretome analysis
Genes encoding secretory proteins were analyzed by comparing our whole gene lists obtained from RNA-seq result with the 1903 genes encoding secretory proteins derived from the Human Protein Atlas (HPA).There were 787 genes out of the 1903 that had average FPKM values at least in one group.Genes in 2%, 5%, and 10% group that exhibited at least a 1.3-fold increase in their average FPKM values than it in the coculture control were selected and designated as upregulated.The average FPKM of these genes were shown in the heatmap made by using R pheatmap package.
Protein-protein physical interaction enrichment analysis of upregulated genes in each group was analyzed with STRING (physical score > 0.132) and BioGrid in Metascape (https://metas cape.org/gp/index.html#/main/step1).The resulting network contained the subset of proteins that formed physical interactions with at least one other member in the gene list.Molecular Complex Detection (MCODE) algorithm10 was applied to identify densely connected network components.Each color on the node graph within the same group represents the components of the same MCODE network.For each component of MCODE network, pathway and process enrichment were analyzed, and the three highest scoring functional descriptions of the corresponding components by p-value were shown with a same color bar chart.The function was significantly enriched when p-value was <.05.

| ELISA
L6 myoblasts were seeded at a density of 5 × 10 5 cells per well on FlexCell plates and either unloaded or loaded for 24 h.At the end of the experiment, the supernatant was collected and centrifuged at 1000 g for 5 min at 4°C to remove cell debris.The supernatant was stored at −80°C until all groups were collected.IGF-1 concentration was measured by using a Rat IGF-I enzyme-linked immunosorbent assay (ELISA) Kit (Thermo, ERIGF1, USA) according to the manufacturer's protocol.Absorbance was measured at 450 nm by a microplate reader.

| Protein extraction and western blot
Tenocytes cultured in inserts were washed twice with PBS before the cell suspension was collected in PBS and centrifuged at 14 000 g for 1 min.Cells from four repeats were combined into one sample.The cell pellet was lysed in PierceTM RIPA buffer (Thermo scientific, 89901, USA) supplemented with protease and phosphatase inhibitor cocktail (Thermo scientific, 1861282, USA) on ice for 40 min.The cell lysate was centrifuged at 14 000 g for 15 min at 4°C to collect proteins.Total protein concentration was determined with the PierceTM BCA Protein Assay Kit (Thermo scientific, 23225, USA).Samples containing 10 μg of proteins were separated by SDS-polyacrylamide gels (Bio-Rad, 4561044, USA) and transferred to PVDF membrane (Cytiva, 10600023, Germany).The membranes were blocked with 5% BSA in 0.1% TBS-T(tween-20) (VWR, 9005-64-5, France) for 1 h before staining with IGF-I receptor β (Cell signaling, 3027, 1:1000) and βactin (Cell signaling, 4967, 1:1000) antibodies overnight at 4°C.The PVDF membranes were washed and stained with Anti-rabbit IgG, HRP-linked antibody (Cell signaling, 7074, 1:2000) for 1 h at RT. Finally, the membranes were washed before incubation with enhanced chemiluminescent (ECL) horseradish peroxidase (HRP) substrate (Thermo, 34577, USA) and analyzed in an Odyssey Fc Dual-Mode Imaging System (LI-COR Biotechnology, Nebraska, USA).
2.12 | Statistics 2.12.1 | RNA-seq statistics Bulk RNA-seq differential analysis was performed using DESeq2 R package, which is an empirical Bayes approach to integrate the dispersion and fold change estimates and uses the Wald test tests the gene differential expression.The adjusted p-values were calculated using Benjamini and Hochberg's approach for controlling the False Discovery Rate (FDR).Genes with an adjusted p-value <.05 found by DESeq2 were assigned as differentially expressed.GO enrichment analysis was carried out using GOseq R package, the p-values were calculated by Wallenius noncentral hyper-geometric distribution.
For other experiments, statistical analysis was performed using One-way ANOVA with Tukey's multiple comparison post hoc test.All experiments were performed in at least triplicates for each experiment, and exact number is specified in the results section.All experiments were repeated at least twice.Differences were considered statistically significant at a p-value of <.05.

| Secretome from 2% statically loaded myoblasts increases tenocyte migration and proliferation
We first investigated whether secretome released from statically loaded myoblasts affects parameters of tendon healing in an intensity-dependent manner.Myoblasts were indirectly cocultured with tenocytes and subjected to 2%, 5%, and 10% static loading for 24 h (Figure 1A).Tenocyte migration and proliferation, which are important features of tendon healing, were examined.Tenocytes cultured alone or cocultured with unloaded myoblasts served as control groups.Tenocyte migration was significantly increased by secretome from 2% statically loaded myoblast as compared with all other groups (Figure 1B,C).Secretome from 2% statically loaded myoblasts also significantly increased tenocyte proliferation as compared with all groups.The proliferation was significantly decreased in 5% and 10% static loading as compared with 2% and the coculture control group (Figure 1D,E).
These results showed that the secretome from 2% statically loaded myoblast increased tenocyte proliferation and migration when compared with the higher intensities of static loading.

2% and 5% intensities show enriched function in protein synthesis
To gain a comprehensive view of statically loaded myoblasts, we performed RNA sequencing (RNA-seq).Myoblasts were indirectly cocultured with tenocytes and subjected to 2%, 5%, and 10% static loading for 24 h.Unloaded myoblasts cultured alone or cocultured with tenocytes were used as control groups.The differential gene expression in 2%, 5%, and 10% static loading groups was analyzed by comparing with the unloaded coculture control.
The predicted functions of upregulated genes in statically loaded myoblasts showed that both 2% and 5% intensities upregulated genes related to protein synthesis, such as translation, peptide biosynthetic process, and amide biosynthetic process in the biological processes (BP) section, which was not seen in 10% static loading (Figure 2A).Although not significantly different, the predicted functions of downregulated genes showed that 5% static loading downregulated genes related to protein secretion, including exocytosis, secretion by cell, and secretion in the BP section (Figure 2B).We speculated that in both 2% and 5% statically loaded myoblasts, the protein synthesis is increased, but that in 5% loading, an inhibition of protein secretion may occur.

| Static loading at 2% intensity upregulates IGF-1 gene expression in myoblasts
After assessing the whole genome in myoblasts following static loading, we further explored the gene expression related to secretory proteins from myoblasts in response to the different intensities.RNA-seq identified 787 genes related to secretory proteins.Upregulated genes in 2%, 5%, and 10% static loading were analyzed by comparing them with the unloaded coculture group.
The FPKM of upregulated genes from 2%, 5%, and 10% static loading are shown in the heatmap (Figure 3A).Two percent and 5% statically loaded myoblasts shared similarities in the gene expression profiles according to the color pattern while 10% static loading induced a clearly different gene expression pattern as illustrated by the heatmap.We speculated that some of the unique genes upregulated in 2% static loading potentiate tenocyte migration and proliferation.Thus, we performed an analysis of the predicted functions of the upregulated genes in the different intensities of static loading.In the 2% static loading group, the gene expressions of IGF-1 transport and uptake were upregulated as well as genes related to MAPK and PI3K signaling pathways (Figure 3B), which are classic downstream pathways of IGF-1 receptor signaling.In 5% and 10% loading, similar effects were not seen (Figure 3C,D).The isolated gene expression of IGF-1 by FPKM showed that the expression of IGF-1 was intensity-dependent with the highest expression in 2% statically loaded myoblasts (Figure 3E).
These results suggest that IGF-1 might be an important effective secretory protein in 2% statically loaded myoblasts that regulates tenocyte migration and proliferation.

IGF-1 in statically loaded myoblasts
To validate IGF-1 expression in myoblasts in response to the different intensities of static loading, we measured IGF-1 gene expression and protein secretion after 24 h.The gene expression of IGF-1 showed that static loading significantly increased IGF-1 expression in a dosedependent manner, and that 2% static loading induced the highest expression.In 10% static loading, the expression of IGF-1 was significantly reduced as compared with the coculture control (Figure 4A).The protein concentration of secreted IGF-1 was measured in myoblast-derived supernatants from the conditioned medium (Figure 4B).Consistent with the gene expression results, static loading induced a dose-dependent IGF-1 secretion, and 2% static loading induced the highest secretion.
These results suggest a putative role of secreted IGF-1 from 2% statically loaded myoblasts to increase tenocyte migration and proliferation.

IGF-1 on tenocyte proliferation
To examine whether the increased IGF-1 in the secretome from 2% statically loaded myoblast is involved in the regulation of tenocyte migration and proliferation, we cultured tenocytes for 24 h with unloaded myoblast-derived conditioned medium (MCM) supplemented with IGF-1 at different concentrations for 24 h.
Tenocyte migration was not affected by the different concentrations of supplemented IGF-1 (Figure 5A,B).However, IGF-1 did significantly increase tenocyte proliferation in a dose-dependent manner, with the highest effect seen in 500 ng/mL IGF-1 (Figure 5C,D).
As exogenous IGF-1 increased tenocyte proliferation, we speculated that the secretome from 2% statically loaded myoblast activates the IGF-1 receptor pathway in tenocytes and plays a role in the increased proliferation.

| Secretome from statically loaded myoblasts induces tenocyte proliferation via IGF-1 receptor signaling pathway
To unravel whether IGF-1 receptor signaling is involved in tenocyte proliferation in response to the secretome from 2% statically loaded myoblast, we measured the activation of the IGF-1 receptor in tenocytes cocultured with 2% statically loaded myoblast after 1 h.We found that the expression of the monomer of IGF-1 receptor β (95KD) in tenocytes did not have a significant difference among groups.However, there was also a specific band at around 180KD corresponding to the size of dimerized β subunits, which was increased in tenocytes cocultured with 2% statically loaded myoblasts as compared with unloaded control and tenocytes alone (Figure 6A left panel).It has been shown that IGF-1 receptor activation requires dimerization of β subunits. 36,37As a positive control, we exogenously added 500 ng/mL IGF-1 to tenocytes and a similar pattern of bands were detected, with a peak at 30 min and 1 h after exogenous addition (Figure 6A right panel).Taken together, we speculate that the formation of the specific band at around 180KD in tenocytes when cocultured with 2% statically loaded myoblasts for 1 h indicates activation of the IGF-1 receptor.
To confirm whether the activated IGF-1 receptor pathway is involved with the increased proliferation rate in tenocytes, we pretreated tenocytes with IGF-1 receptor inhibitor PQ401 at concentrations of 5 and 10 μM for 1.5 h before coculturing with 2% statically loaded myoblasts for 24 h.The results showed that the increased tenocyte proliferation rate in coculture with 2% statically loaded myoblasts was completely blocked if tenocytes were pretreated with PQ401 (Figure 6B,C).This result indicates that the IGF-1 receptor pathway is involved in the increased proliferation of tenocytes cocultured with 2% statically loaded myoblasts.

| DISCUSSION
In this study, we applied different intensities of static loading to myoblasts and examined how the released secretome affects the proliferation and migration of cocultured tenocytes.Our results showed that 2% static loading of myoblasts induced proliferation and migration of cocultured tenocytes while 5% and 10% static loading did not have these effects.Additionally, the effect of 2% static loading of myoblasts on increased tenocyte proliferation was regulated by the IGF-1 receptor pathway.
Exercise has been shown to improve tendon wound healing by regulating tendon cell migration and proliferation.For instance, in a rat model, it was shown that aquatic exercise increased the proliferation of tenocytes in the calcaneal tendon following partial transection. 38Similarly, treadmill running is known to increase tendon cell proliferation. 39Mobilized tendons during the process of healing exhibit greater cellular activity and collagen production than immobilized tendons. 40The beneficial effects of exercise on wound healing, such as proliferation and migration, are proposed to be partially mediated by the secretome derived from mechanically loaded muscle.Different in vitro mechanical loading regimes have been used to study the skeletal muscle secretome and its function. 41The loading intensities applied to myoblasts or myotubes are most commonly between 10%-30%.Some studies used relatively lower loading intensities around 2%-6%.However, most of the studies used dynamic loading with frequencies between 0.05 and 1.5 Hz and different rest intervals. 41In our study, we used a static loading regime with 1-h static loading alternating with rest at different intervals.By adding rest and applying mechanical strain for intervals, stimulus adaptation is avoided and the mechanical sensitivity of cells maintained.We chose static loading based on our previous study, which showed that static loading could significantly increase tenocyte migration, collagen I/III ratio, and the expression of tenocyte markers when compared with dynamic loading (5%, 0.5 Hz). 31 Most in vitro studies regarding the function of myoblastderived secretome from mechanical loading have focused on the autocrine effects on the myoblasts themselves.One study showed that the proliferation of L6 myoblasts is variably affected by different levels of dynamic loading (0.5 Hz). 424][45] Previous studies suggest that relatively higher intensities of dynamic loading (10%-17%) are needed to induce myoblast proliferation.However, our study found that when using static loading, a lower intensity of 2% was sufficient for the myoblast secretome to significantly induce tenocyte migration and proliferation.In contrast, higher intensities of 5% and 10% did not have these effects.No noticeable differences in myoblast proliferation were observed at the utilized intensities.
Our RNA-seq analysis revealed that 2% and 5% statically loaded myoblasts had increased expression of genes related to translation, peptide biosynthetic processes, amide biosynthetic processes, and peptide metabolic processes.In contrast, 10% static loading did not have these effects, which suggests that relatively low intensities of static loading (2% and 5%) increased myoblast anabolic functions, as compared with the high intensity (10%).These findings are consistent with a study that showed 5% static loading of C2C12 cells induced the expression of markers related to translation and elongation. 46It has also been found that 15% dynamic loading had similar effects as 5% static loading, while another study showed that 15% dynamic loading resulted in increased muscle hypertrophy. 47These studies suggest that a higher intensity of dynamic loading (e.g., 15%) is necessary to achieve a similar effect observed with lower intensities of static loading (2% and 5%).Of particular interest was the examination of the IGF-1 pathway, which emerged as a key player in myoblast static loading-induced effects on tenocytes.Both the mRNA expression and protein secretion of IGF-1 displayed a distinct dose-dependent relationship with the intensity of static loading.Interestingly, our study demonstrated that tenocyte proliferation following exogenous IGF-1 supplementation resulted in enhanced proliferation, mirroring the outcome observed with the secretome from 2% statically loaded myoblasts.This implies the participation of secreted IGF-1 in mediating tenocyte proliferation in response to static loading, which additionally was confirmed by including the IGF-1 receptor inhibitor PQ401.Furthermore, our results revealed that the secretome from myoblasts exposed to 2% static loading exhibited the most significant influence on tenocyte migration and proliferation.This suggests the potential advantageous impact of lower intensities of static loading with plausible clinical implication.Implementing early tensile loading following tendon rupture is also supported by others. 48Notably, divergent outcomes observed at higher loading intensities could symbolize a tipping point wherein the cellular response transitions from proliferation/migration to differentiation, considering the significant upregulation of secretory factors associated with WNT signaling observed at higher intensities.
Analysis of upregulated genes encoding secretory proteins from the RNA-seq results revealed that different intensities of static loading result in different gene expression profiles.Two percent and five percent static loading shared some common upregulated genes encoding secretory proteins while 10% static loading had a different profile of upregulated genes.Gene ontology analysis revealed that IGF-1 expression was dose-dependent with the highest expression in 2%.3][54] Increased plasma IGF-1 is beneficial for health 55,56 ; in contrast, decreased IGF-1 is seen in aging human plasma and is related to sarcopenia 57,58 by reducing DNA synthesis and altering the dynamics of mitochondria. 59,60Like other myokines, the serum levels of IGF-I can increase, [61][62][63][64] decrease, [65][66][67] or remain unchanged 68 as a result of various types and intensities of exercise.We confirmed that IGF-1 expression and secretion was the highest in 2% static loading of myoblasts as compared with 5% and 10%.Others have shown similar results, that is, that 2% rather than higher intensities of 15% dynamic loading (0.25 Hz) leads to upregulated IGF-1 and IGF-1Ea expression in C2C12 cells. 69Exogenous | 13 of 16 LI et al.
addition of IGF-1 to tenocytes increased cell proliferation in a dose-dependent manner but did not affect the cell migration.Other studies also showed that IGF-1 can induce tenocyte proliferation. 70,71When introducing exogenous IGF-1, we employed a relatively higher concentration (up to 500 ng/mL) compared to the endogenously secreted IGF-1 levels (up to 15 pg/mL) within the myoblast conditioned medium.It is important to note that exogenous IGF-1 was administered solely at the onset of the experiment, whereas in the coculture arrangement, myoblasts exhibited a continuous production and secretion of IGF-1.To our knowledge, no studies have shown that tenocyte migration is induced by IGF-1 alone.However, tenocytes exposed to conditioned medium derived from rat bone marrow mesenchymal stem cells, which is known to contain soluble IGF-1, promotes tenocyte migration. 72Thus, these findings suggest that IGF-1 plays a role in promoting tenocyte proliferation in coculture with 2% static loading of myoblasts, whereas other factors alone or in combination with IGF-1 may be responsible for the increased migration.
IGF-1 regulates cell proliferation by binding to and activating the IGF-1 receptor. 73,74We found that 2% static loading of myoblasts activated the IGF-1 receptor (IGF-1R) in cocultured tenocytes in a similar manner as exogenous addition of IGF-1 to tenocytes alone.These results confirm that IGF-1 secreted by myoblasts activates the IGF-1R in the cocultured tenocytes.By inhibiting the activation of IGF-1R, the induced tenocyte proliferation by myoblast-derived secretome was blocked, supporting that IGF-1 released by 2% static loading of myoblast contributes to the increased proliferation of the cocultured tenocyte by IGF-1R pathway.Knock-down of the expression of IGF-1R in tenocytes in an in vivo mouse model results in reduced cell proliferation and diminished tendon size upon exposure to mechanical loading. 49Disser et al. solely knocked down the IGF-1R receptor expression, leaving the IGF concentration unaltered. 49Nevertheless, the outcomes proved to be significant, indicating that mechanical loading modulates the IGF-1 concentration, derived from either the surrounding tissue or the tendon tissue itself.Our results show that the secretion of IGF-1 by myoblasts is reliant upon the type and intensity of loading, thereby implying that precise timing of loading holds a crucial role in the regulation of tendon healing and growth.
Given the experimental conditions in our current study, it is not technically feasible to directly measure the myoblast-derived secretome due to the coculturing of myoblasts and tenocytes.Hence, RNA-seq methodology represents the sole viable approach to identify potential secretory genes that may exert an influence on the process of tendon healing.Thus, it is crucial to acknowledge that the implications drawn solely from RNA expression analysis cannot be directly extrapolated to fully elucidate the actual impact of the muscle secretome.This limitation arises from the presence of various intermediate steps involved in the translation process, encompassing RNA expression, protein synthesis, and subsequent secretion.Consequently, to gain a more comprehensive understanding of this phenomenon, future investigations should employ proteomic approaches capable of directly quantifying the constituents of the muscle secretome.Such proteomic analyses should be conducted without utilizing the coculturing system, thus providing more accurate insights into the role of secretory proteins in tendon healing.
A methodological limitation of our study concerns to the potential heterogeneity in the intensity of exposure experienced by individual cells.Cells located in the peripheral region, where membrane deformation occurs within the Flexcell system, might encounter hyper-stretching.Moreover, cell orientation and cytoskeletal organization could influence cellular strain. 75Nonetheless, these conditions were consistent across all three intensity groups utilized.Given that distinct results were observed among these intensities, we can suppose that these confounding factors did not compromise the interpretation of the intensity variations.
In conclusion, our study has deepened the understanding of molecular mechanisms underlying the interaction between mechanically loaded myoblasts and tenocytes.Static loading of myoblasts at low intensity increased the proliferation and migration of cocultured tenocytes, and this effect was partially regulated by IGF-1R pathway.Identifying myokines secreted from loaded muscles that promote tendon repair provides promising insights for future treatment strategies for tendon injuries.Moreover, unraveling the involved signaling pathways is a prerequisite for the rational development of drugs.

F I G U R E 1
Secretome from low intensity statically loaded myoblasts increases tenocyte migration and proliferation.(A) Schematic diagram of experimental design.Tenocytes and myoblasts were seeded separately and after 24 h they were cocultured.In the coculture setup, myoblasts were subjected to mechanical loading for 24 h.The loading regime was 1 h of loading, followed by 2 h of rest, repeated twice before 1 h loading followed by 6 h of rest.The regime was repeated for 24 h.(B) Effect of secretome from different intensities of statically loaded myoblast on tenocyte migration after 24 h.Migrated tenocytes stained with crystal violet, resolved, and measured the absorbance at 590 nm.(C) Representative images of migrated cells (100-fold magnification).(D) Effect of secretome from different intensities of statically loaded myoblast on tenocyte proliferation after 24 h.Proliferation rate was measured by EdU and normalized to Hoechst 33342 staining.(E) Representative images of Hoechst 33342 stained cells and EdU incorporating cells (200-fold magnification).Control: cultured tenocytes alone.Co-cul control: myoblasts indirectly cocultured with tenocytes.SL = statically loaded myoblasts indirectly cocultured with tenocytes.Results were mean ± SD (n = 6 (migration), n = 5 (proliferation)).**p < .01;***p < .001;****p < .0001.F I G U R E 2 2% and 5% statically loaded myoblasts induced enriched function in protein synthesis.Red, green, and blue are GO annotation of genes to biological processes (BP), cell components (CC), and molecular functions (MF).Function descriptions of Gene Ontology were shown in the X-axis.The logarithm of the adjusted probability value (pajd) was set as the Y-axis value.The function is significantly enriched when padj <.05.(A) Enriched functions of upregulated genes and (B) downregulated genes in response to 2%, 5%, 10% statically loaded myoblasts.SL, statically loaded myoblasts indirectly cocultured with tenocytes.Results were from three biological repeats.

F I G U R E 3
Myoblasts statically loaded at 2% upregulated IGF-1 gene expression.(A) Heatmap of upregulated genes related to secretory proteins in response to different intensities of statically loaded myoblasts.Groups are shown in columns; gene symbols are shown in rows.Average FPKM of genes were standardized within rows with Z-score by pheatmap package of R and displayed as colors ranging from blue, white to red indicating values from low to high.Rows are clustered by pheatmap using k means clustering.(B-D) Enriched protein-protein physical interactions calculated by Metascape.Each color on the node graph within the same group represents the components of the same network as shown in the right panel.The three highest scoring functional descriptions of the corresponding components by p-value were shown in the right panel by the bar chart.The function is significantly enriched when p-value <.05.(E) The average FPKM of IGF-1 from the RNA-seq result.Control: myoblasts cultured alone without mechanical loading.Co-cul control: myoblasts indirectly cocultured with tenocytes without mechanical loading.SL, statically loaded myoblasts indirectly co-cultured with tenocytes.Results were from three biological repeats.F I G U R E 4 Intensity-dependent secretion of IGF-1 in statically loaded myoblasts.(A) Gene expression of IGF-1 and (B) IGF-1 secretion from statically loaded myoblasts.Blank: medium only.Myo control: myoblasts cultured alone without mechanical loading.Co-cul control: myoblasts indirectly cocultured with tenocytes SL, statically loaded myoblasts indirectly co-cultured with tenocytes.Results were mean ± SD (n = 3).*p < .05;**p < .01;****p < .0001.F I G U R E 5 Dose-dependent effect of exogenous IGF-1 on tenocyte proliferation.(A) Tenocytes cultured in conditioned medium derived from unloaded myoblasts (MCM) supplemented with IGF-1 for 24 h.Migrated tenocytes were stained with crystal violet, resolved, and measured the absorbance at 590 nm (B) Representative images of migrated cells (100-fold magnification).(C) Tenocytes cultured in MCM supplemented with IGF-1 for 24 h.Proliferation rate was measured by EdU and normalized to Hoechst 33342 staining.(D) Representative images of Hoechst 33342 stained cells and EdU (100-fold magnification).Results were mean ± SD (n = 4).*p < .05;***p < .001.

2.1 | Isolation and culture of rat primary tenocytes
Primary tendon cells (tenocytes) were isolated from healthy female Sprague-Dawley rats (7-8 weeks old).The animal care and experimental procedures were carried out in accordance with Directive 2010/63/EU of the European Parliament and of the Council on the Protection of Animals used for Scientific Purposes, and were approved by the Animal Review Board at the Court of Appeal of Northern Norrland in Umeå (ethical permits DNR #A31-19).Surgically removed Achilles tendons were washed with phosphate-buffered saline (PBS) and the connected muscle was removed with a surgical