Effect of exercise on the mRNA expression of growth factors, metabolic genes and myosin heavy chain isoforms in skeletal muscles of the rat

Dissertation / Doktorarbeit von Antonios Matsakas

Abstract:

Skeletal muscle is a complex and heterogeneous tissue capable of remarkable adaptation in response to various stimuli such as exercise training. Molecular biology approaches have made a large contribution to our current understanding of how mechanical loading can alter gene expression and muscle protein synthesis rates in skeletal muscle. The steady-state level of a given mRNA is a function of both its rate of synthesis and its rate of degradation, implying that the rate of change in response to a cellular stimulus is dependent on the dynamics of mRNA turnover. Changes in muscle mRNA steady-state levels can be taken as an index of changes in gene expression, constituting currently one of the best described and understood molecular events that underlie muscle plasticity.

The discovery of new growth factors involved in the regulation of muscle development provides a better understanding of the molecular mechanisms involved in the adaptation of skeletal muscle to exercise training. Since it has been shown that changes in contractile function can be brought about by switching on one subset and repressing another subset of genes, it was hypothesized that the steady state level of multiple mRNAs (growth factors and metabolic genes) involved in regulatory functions in a muscle –which is frequently recruited during exercise– is different between endurance trained versus untrained rats. Recent scientific data indicate that myostatin constitutes a limiting factor in normal muscle development playing a crucial role in skeletal muscle hypertrophy. Nevertheless, there are only scarce data so far about the role of myostatin in the exercise-induced skeletal muscle adaptation. The transient changes in regulatory and structural gene expression provide the molecular basis of the adaptability of the skeletal muscle to exercise stimulus.

To get an insight into the molecular mechanisms of skeletal muscle adaptation, the main objective of this study was to examine the effect of both the short- and long- term effect of exercise (five day of swimming vs. chronic wheel running) on IGF-I and MSTN (positive and negative skeletal muscle regulators respectively) mRNA contents in male Wistar rats. Potential increase of positive and/or decrease of negative regulators of muscle growth lead to enhanced muscle-progenitor proliferation providing a new perspective in the mechanism of muscle adaptability. It was hypothesized that exercise training could be accompanied by an up-regulation of IGF-I and/or down-regulation of the MSTN gene (positive and negative skeletal muscle regulators respectively). Because MSTN expression has been reported to be more prominent in fast-twitch muscle, diverse skeletal muscles with different fiber type composition and recruitment patterns during movement were analyzed (gastrocnemius, vastus lateralis and soleus). In addition, MSTN was analyzed in combination with the mRNA contents of glucose transporter 4 (GLUT4), hexokinase II (HK II), and hydroxyacyl-CoA dehydrogenase (HAD), a set of well characterized metabolic genes. On the level of structural genes, myosin heavy chain (MHC) I, IIa and IIb mRNA were analyzed in gastrocnemius and vastus lateralis muscles of trained and untrained animals.

Rats were studied in two experiments by using two different protocols. Initially, the effect of 5-day endurance swim training on mRNA contents of MSTN and IGF-I (growth factors), HK II, GLUT4 and HAD (metabolic genes) and MHC isofors was analyzed in gastrocnemius, vastus lateralis and soleus muscles of male Wistar rats. In a second phase, the effect of a 12-week wheel running on the mRNA contents of the above mentioned genes was analyzed in a study with male Wistar rats. By using real time reverse transcription polymerase chain reaction, a method possessing the possibility to both identify and quantify slight alterations in the expression of a short number of genes. Significant differences in muscle mRNA levels between trained and untrained animals were determined by performing the Student’s t test for independent samples and the level of statistical significance was set at P < 0.05 for all analyses.

A potent decrease of the MSTN mRNA content by 65% (P < 0.05) was observed in the gastrocnemius muscle of the trained rats, followed by a medium decrease of 49% (P < 0.05) in vastus lateralis in response to short-term swim training. On the contrary, no significant differences were found in the MSTN mRNA levels of the soleus muscle between trained and untrained rats. No significant IGF-I and GLUT4 mRNA alterations were noticed in any of the three skeletal muscles analyzed. In contrast, significant higher HK II mRNA levels were seen in both gastrocnemius and vastus lateralis muscles (by 100% and 166% respectively) but not in the soleus. HAD mRNA levels revealed a similar pattern as HK II, showing a significant increase (P < 0.05) in both gastrocnemius and vastus lateralis muscle (by 139% and 158% respectively).

Running activity of the rats averaged 9.5 ± 1.8 km/day respectively during the 12-week training period. MSTN mRNA contents were significantly reduced in gastrocnemius and vastus lateralis, but not in the soleus of the trained group, exhibiting an expression pattern similar to the short-term swim study. No significant mRNA alterations were observed in IGF-I, GLUT4, HK II, and HAD in any of the muscles analyzed. With regard to the MHC isoform profile, increased MHC-I mRNA contents were found only in gastrocnemius of the trained rats without any other significant alterations neither in vastus lateralis nor in soleus. A similar pattern was observed in the male rats, which showed an additional middle increase of MHC I in vastus lateralis.

In conclusion, the present work provides evidence that endurance exercise training may directly modulate MSTN and, in part, HK II and HAD mRNA contents in rat skeletal muscle in a muscle-specific manner, supporting the notion that the molecular diversity of skeletal muscle types is also reflected in a differential global gene expression between red and white skeletal muscle. The same expression pattern of MSTN mRNA observed in both studies, indicates that MSTN may play an elementary role in the training-induced skeletal muscle adaptation and homeostasis. Future studies are warranted in order to examine whether the observed changes in mRNA levels are extended to the protein level in response to exercise training.

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