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Exercise is well known to promote tendon healing, an effect traditionally attributed to mechanical loading-induced responses within the tendon itself. However, skeletal muscle also functions as a secretory organ, releasing bioactive factors (secretome) during exercise that influence various tissues. We hypothesized that muscle-derived secretome released during exercise may also contribute to tendon healing. To test this, we applied mechanical loading to cultured muscle cells (myoblasts) using the FlexCell tension system to simulate exercise in vitro. Our previous studies, using 2D-cultured tendon cells (tenocytes), have demonstrated that secretome from statically loaded myoblasts, particularly under 2% loading, enhanced tendon healing-related responses. Building upon these findings, we employed a 3D tendon construct model to more closely mimic in vivo healing conditions. We found that secretome derived from statically loaded myoblasts, especially at 2% loading, promoted tendon healing-related processes as compared with the control group, which received no secretome treatment (no conditioned media). These included increased cell-covered area, expression of the tenocyte marker scleraxis (SCX), and elevated production of Type I and III collagens at an early stage (Day 7). Additionally, a reduction in type III collagen production was found at a later stage (Day 14), suggesting a potentially accelerated healing process. These findings highlight the therapeutic potential of the muscle-derived secretome in promoting tendon healing and may inform future strategies for rehabilitation and regenerative medicine.

Exercise has been shown to promote wound healing, including tendon repair. Myokines released from the exercised muscles are believed to play a significant role in this process. In our previous study, we used an in vitro coculture and loading model to demonstrate that 2% static loading of myoblasts increased the migration and proliferation of cocultured tenocytes─two crucial aspects of wound healing. IGF-1, released from myoblasts in response to 2% static loading, was identified as a contributor to the increased proliferation. However, the factors responsible for the enhanced migration remained unknown. In the current study, we subjected myoblasts in single culture conditions to 2, 5, and 10% static loading and performed proteomic analysis of the cell supernatants. Gene Ontology (GO) analysis revealed that 2% static loading induced the secretion of NBL1, C5, and EFEMP1, which is associated with cell migration and motility. Further investigation by adding exogenous recombinant proteins to human tenocytes showed that NBL1 increased tenocyte migration but not proliferation. This effect was not observed with treatments using C5 and EFEMP1.

Background: Exercise is widely recognized for its health benefits, including the release of bioactive proteins into the bloodstream, which exert systemic effects on various organs. Previous research has demonstrated that certain types of exercise can promote tendon healing, however, the specific exercise modalities that yield the most beneficial effects, as well as the underlying mechanisms, remain poorly understood. This thesis project aimed to address these knowledge gaps by utilizing an in vitro cell loading model to simulate exercise and investigate how different types and intensities of mechanical loading on myoblasts (muscle cells) influence the secretion (i.e. production and release) of bioactive proteins that may enhance tendon healing.

Aim: This thesis comprised four studies. The first study aimed to determine whether the secretome, derived from statically or dynamically loaded myoblasts, has a greater impact on tendon wound healing. This was assessed through measuring key processes, such as tenocyte (tendon cell) migration, proliferation, healing phenotype, and collagen production. The second and third studies sought to identify the optimal intensity of static loading that induces the secretion of proteins with potential roles in tendon healing. The fourth study employed a three-dimensional (3D) tendon formation model to elucidate how factors secreted by mechanically loaded myoblasts influence tendon cell phenotype, extracellular matrix (ECM) protein production, tendon structure, and the underlying molecular mechanisms.

Results: The Paper I demonstrated that secretory factors from statically loaded myoblasts significantly enhanced tenocyte migration, increased the type I/III collagen ratio and induced a myofibroblast-like phenotype in tenocytes compared with both dynamically loaded myoblasts and unloaded controls. These results suggest that molecules secreted from statically loaded myoblasts play a crucial role in tendon healing. In the Papers II and III, RNA sequencing and proteomic analyses, followed by validation experiments, identified insulin-like growth factor 1 (IGF-1) and neuroblastoma suppressor of tumorigenicity 1 (NBL1) as key factors secreted from myoblasts subjected to low-intensity (2%) static loading as compared with mild (5%) and high (10%) intensity loading and unloaded control. IGF-1 was found to enhance tenocyte proliferation, while NBL1 promoted tenocyte migration. The Paper IV revealed that secretome derived from myoblasts under 2% static loading increased the expression of key ECM proteins in tenocytes, including type I and III collagen, while also upregulated the expression of tenocyte-specific markers using an in vitro 3D tendon formation model.

Conclusion: This thesis work showed that the secretome derived from myoblasts subjected to low-intensity static loading improved tendon healing related parameters in tenocytes. This presents a potential novel strategy to support tendon healing during the critical immobilization phase following tendon injury. By stimulating the secretion of bioactive proteins into the circulation through targeted muscle loading—without directly subjecting the injured tendon to mechanical stress—this approach presents a promising method for promoting tendon healing. Furthermore, IGF-1 and NBL1 may serve as potential therapeutic targets for enhancing tendon healing.

Skeletal muscle adaptation to exercise involves various phenotypic changes that enhance the metabolic and contractile functions. One key regulator of these adaptive responses is the activation of AMPK, which is influenced by exercise intensity. However, the mechanistic understanding of AMPK activation during exercise remains incomplete. In this study, we utilized an in vitro model to investigate the effects of mechanical loading on AMPK activation and its interaction with the mTOR signaling pathway. Proteomic analysis of muscle cells subjected to static loading (SL) revealed distinct quantitative protein alterations associated with RNA metabolism, with 10% SL inducing the most pronounced response compared to lower intensities of 5% and 2% as well as the control. Additionally, 10% SL suppressed RNA and protein synthesis while activating AMPK and inhibiting the mTOR pathway. We also found that SRSF2, necessary for pre-mRNA splicing, is regulated by AMPK and mTOR signaling, which, in turn, is regulated in an intensity-dependent manner by SL with the highest expression in 2% SL. Further examination showed that the ADP/ATP ratio was increased after 10% SL compared to the control and that SL induced changes in mitochondrial biogenesis. Furthermore, Seahorse assay results indicate that 10% SL enhances mitochondrial respiration. These findings provide novel insights into the cellular responses to mechanical loading and shed light on the intricate AMPK-mTOR regulatory network in muscle cells.

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.

Interleukin-1 (IL-1) and transforming growth factor-beta (TGFβ) are important cytokines involved in corneal wound healing. Here, we studied the effect of these cytokines on corneal stromal cell (keratocyte) differentiation. IL-1β treatment resulted in reduced keratocyte phenotype, as evident by morphological changes and decreased expression of keratocyte markers, including keratocan, lumican, ALDH3A1, and CD34. TGFβ1 treatment induced keratocyte differentiation towards the myofibroblast phenotype. This was inhibited by simultaneous treatment with IL-1β, as seen by inhibition of α-SMA expression, morphological changes, and reduced contractibility. We found that the mechanism of crosstalk between IL-1β and TGFβ1 occurred via regulation of the NF-κB signaling pathway, since the IL-1β induced inhibition of TGFβ1 stimulated keratocyte-myofibroblast differentiation was abolished by a specific NF-κB inhibitor, TPCA-1. We further found that Smad7 participated in the downstream signaling. Smad7 expression level was negatively regulated by IL-1β and positively regulated by TGFβ1. TPCA-1 treatment led to an overall upregulation of Smad7 at mRNA and protein level, suggesting that NF-κB signaling downregulates Smad7 expression levels in keratocytes. All in all, we propose that regulation of cell differentiation from keratocyte to fibroblast, and eventually myofibroblast, is closely related to the opposing effects of IL-1β and TGFβ1, and that the mechanism of this is governed by the crosstalk of NF-κB signaling.

It is known that mechanical loading of muscles increases the strength of healing tendon tissue, but the mechanism involved remains elusive. We hypothesized that the secretome from myoblasts in co-culture with tenocytes affects tenocyte migration, cell phenotype, and collagen (Col) production and that the effect is dependent on different types of mechanical loading of myoblasts. To test this, we used an in vitro indirect transwell co-culture system. Myoblasts were mechanically loaded using the FlexCell® Tension system. Tenocyte cell migration, proliferation, apoptosis, collagen production, and several tenocyte markers were measured. The secretome from myoblasts decreased the Col I/III ratio and increased the expression of tenocyte specific markers as compared with tenocytes cultured alone. The secretome from statically loaded myoblasts significantly enhanced tenocyte migration and Col I/III ratio as compared with dynamic loading and controls. In addition, the secretome from statically loaded myoblasts induced tenocytes towards a myofibroblast-like phenotype. Taken together, these results demonstrate that the secretome from statically loaded myoblasts has a profound influence on tenocytes, affecting parameters that are related to the tendon healing process.

Exercise is well known to promote tendon healing, an effect traditionally attributed to mechanical loading-induced responses within the tendon itself. However, skeletal muscle also functions as a secretory organ, releasing bioactive factors (secretome) during exercise that influence various tissues. We hypothesized that muscle-derived secretome released during exercise may also contributes to tendon healing. To test this, we applied mechanical loading to cultured muscle cells (myoblasts) using the FlexCell® tension system to simulate exercise in vitro. Our previous studies demonstrated that secretome from statically loaded myoblasts- particularly under 2% loading- enhanced tendon healing-related responses in 2D-cultured tendon cells (tenocytes). Building upon these findings, we employed a 3D tendon construct model to more closely mimic in vivo healing conditions. We found that secretome derived from statically loaded myoblasts, especially at 2% loading, promoted tendon healing-related processes. These included increased expression of the tenocyte marker scleraxis (SCX), elevated production of collagen I and III at an early stage (day 7), and a reduction in collagen III production at a later stage (day 14), suggesting a potentially accelerated healing process. These findings highlight the therapeutic potential of the muscle-derived secretome in promoting tendon healing and may inform future strategies for rehabilitation and regenerative medicine.