1. Introduction

Tendon tissue along the Extracellular Matrix (ECM) is a muscle that mechanically and structurally adapts to the muscle with mechanical load [2]. Although the mechanical and morphological changes that occur in tendons in response to resistance exercise are well documented, little is known about the underlying cellular and molecular mechanisms that regulate these responses.

The “Transforming Growth Factor Beta 1 (TGF-β1) family” messaging pathway appears to play a major role in tendon adaptation to resistance exercise. Myostatin is a member of the large TGF-β family, the expression of which negatively regulates skeletal muscle growth [5].

Both TGF-β1 and myostatin stimulate tendon fibroblast proliferation and type I collagen synthesis [4, 6]. Matrix Metalloproteinases (MMPs) are zinc-dependent endopeptidases that break down collagen and other structural molecules. MMPs are essential for baseline ECM homeostasis. The expression of MMPs is regulated by various cytokines and messenger molecules, including TGF-β1 [2].

However, the exact components of the signal transduction pathways that regulate MMP expression following exercise have not yet been fully elucidated. Therefore, aim of the present study was to investigate the effect of 6 weeks of aerobic exercise on the expression of TGF-β1, myostatin and MMP9 genes in the fast and slow-twitch muscle tendons of male Wistar rats.

2. Materials and Methods

Twelve male Wistar rats (8-week-old) were randomly divided into 2 groups of “exercise” (n=6) and “control” (n=6). The exercise group performed aerobic exercise for 6 weeks, which included 5 sessions of treadmill running per week for 6 weeks [13].

Forty-eight hours after the last training session, all rats were killed. Then, the Soleus Tendon (SOL) and the Extensor Digitorum Longus muscle (EDL) of their right foot were immediately and carefully extracted and kept at minus 80° C for further measurements. The mRNA expression levels of TGF-β1, myostatin and MMP9 genes were measured using Real time-PCR. Independent t-test was used to analyze the obtained data.

3. Results

The results showed that there was a significant difference between the TGF-β1 gene values ​​of EDL and soleus muscles in the aerobic exercise group compared to the control group. TGF-β1 gene expression in EDL and soleus muscles was significantly increased in the aerobic exercise group compared to the control group (EDL muscle: 0.63±0.09 compared to 0.14±0.04, P≥0.001, and soleus muscle: 0.36±0.08 compared to 0.17±0.04, P≥0.001) (Figure 1).

The results of independent t-test showed that myostatin gene expression was significantly reduced only in the EDL muscle of the aerobic exercise group compared to the control group (EDL muscle: 0.3±0.1 compared to 0.58±0.07, P≥0.001, and soleus muscle: 0.20±0.09 compared to 0.29±0.05, P≥0.001) (Figure 2).

Also, no significant difference was observed between MMP9 gene expression in EDL and soleus muscles between the aerobic exercise and control groups (EDL muscle: 0.21±0.08 compared to 0.15±0.02, P>0.05, and soleus muscle: 0.27±0.07 compared to 0.19±0.1, P>0.05) (Figure 3).

 

4. Discussion

The results of the present study showed that following a 6-week aerobic exercise program, TGF-β1 mRNA expression increased in both EDL and soleus muscles. Heniemir et al. (2007) reported increased mRNA levels of TGF-β1 genes and type I and III collagens in the gastrocnemius muscle and Achilles tendon following isometric, concentric and eccentric contractions by stimulating the sciatic nerve for 4 days [15].

Also, another study showed that long-term endurance training caused a significant increase in TGF-β1 mRNA in soleus muscle [8]. The study showed that the antioxidant status of muscle affected the expression of TGF-β1 [16]. Evidence suggests that TGF-β1 is the main mediator of inducing collagen synthesis in fibroblasts through mechanical loading [15] and a similar role for TGF-β1 in tendons has been suggested [17].

An increase of more than 453% in the expression of TGF-β1 mRNA in the EDL muscle tendon, compared with a 203% increase in its expression in the soleus tendon following aerobic exercise - observed in the present study - is more likely to indicate the involvement of fast-twitch muscles in aerobic exercise, which are less used in normal daily practices.

There is much evidence to support the idea that myostatin regulation is specific to the type of muscle fibers and is strongly associated with myosin heavy chain IIb isoform [21], and High concentrations of myostatin protein have been observed in fast-twitch muscle compared to slow-twitch fibers [22].

These reports could justify the results of the present study, which showed a more than 49% reduction in myostatin mRNA expression in the EDL muscle compared with a slight 24% reduction in the expression of this gene in the soleus muscle following aerobic exercise.

Exercise appears to damage collagen tissue in skeletal muscle and tendons, and MMP9 levels increase to remove these damaged elements from tissue. In addition, MMPs have been reported to be essential for the induction of exercise-induced angiogenesis in skeletal muscle [25].

In the present study, exercise increased MMP9 expression by 40% in EDL muscles and by 28% in soleus muscles; but these changes were not statistically significant. Consistent with the results of the present study, Rollman et al. (2009) reported a non-significant increase in MMP9 expression in the human vastus lateralis muscle following aerobic exercise [26].

The decrease in myostanin levels following aerobic exercise observed in the present study can be considered as a mechanism for the relative increase in MMP9 mRNA expression in tendon tissue. Mendias et al. (2015) reported a 141% increase in MMP9 gene expression in the tendon tissue of myostatin-free rats (rats whose myostatin was genetically inactivated) [18].

In addition, activation of the ERK1/2 and JNK-NF-κB signaling pathways via ROS has been shown to be essential for the positive regulation/activation of MMP9 and cell migration induced by TGF-β1 [27], which can be another mechanism to justify the relative increase in MMP9 mRNA expression in tendon tissue in this study.

5. Conclusion 

In general, aerobic exercise positively regulates basal levels of TGF-β1 gene and negatively regulates basal levels of myostatin gene in fast- and slow-twitch muscle tendons; and these effects are significantly greater in fast-twitch muscle tendons. It seems that aerobic exercise can modulate the expression of genes Involved in the regulation of tendon tissue collagen in a muscle-dependent manner.

Ethical Considerations

Compliance with ethical guidelines

All stages related to animals were performed in accordance with Ethical Instructions and after obtaining license (Code: IR.IAU.PS.REC.1398.296) from the Vice Chancellor for Research, Islamic Azad University Central Tehran Branch.

Funding

The present paper was extracted from the PhD. thesis of the second author, Department of Sports Physiology, Faculty of Physical Education and Sports Sciences, Islamic Azad University, Central Tehran Branch.

Authors' contributions

Conceptualization, review, subject definition, writing – review & editing: All authors; Methodology, data analysis: Ghasem Mohammadnejad.

Conflicts of interest

The authors declared no conflict of interest.

Acknowledgements

The authors would like to thank Dr. Reza Gharakhanloo, Department of Physical Education and Sports Science, Faculty of Human Sciences, Tarbiat Modares University, and Faculty of Human Sciences for their cooperation in conducting this study.

 

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