Extended Abstract

Introduction
Myosins are a family of proteins that continuously interact with actin filaments and move along them using adenosine triphosphate (ATP). Exercise and sports supplements can cause changes in myosin heavy chain isoforms, leading to alterations in muscle fiber phenotypes. Numerous studies have shown that even a single session of intense resistance exercise can increase the expression of genes encoding myosin heavy chain isoforms. Glutamine supplementation has beneficial effects on muscle growth and function, specifically preventing muscle atrophy caused by glucocorticoids. It can increase muscle fiber size and the abundance of myosin heavy chain isoforms, enhance functional recovery, help maintain the myosin profile, promote muscle growth, and reduce intramuscular fat deposition. Myofibrils of type IIX are a type of muscle fiber responsible for fast and powerful contractions, characterized by high myosin ATPase activity and the ability to generate substantial force. However, type IIX myofibrils are also highly susceptible to fatigue. This study aimed to determine the effect of a single session of intense resistance exercise combined with glutamine supplementation on the relative expression of α and IIX myosin heavy chain genes in fast-twitch muscle fibers of male rats.
Methods
This experimental study was conducted on 30 male Wistar rats (eight-week-old) with an approximate weight of 220±20 g. The rats were divided into three groups of 10: the control group, the intense resistance exercise group (experimental group 1), and the intense resistance exercise group with glutamine supplementation (experimental group 2).
The intense resistance exercise involved climbing an 85° inclined surface to a height of 1.5 m, with 4 sets of 5 repetitions, 30 s of rest between repetitions, and 2 min of rest between sets. Glutamine supplementation was administered as a powder dissolved in 100 mL of distilled water at a dose of 0.5 g per kg of body weight per day for 5 days via gavage.
Eight hours after the exercise and supplementation, the rats were fasted for 12 h with free access to water. The rats were then anesthetized using ketamine and xylazine via intraperitoneal injection, following the ethical protocols. After blood was drawn from the heart, a midline incision was made on the rat's hind leg to expose the extensor digitorum longus (EDL) muscle by carefully separating the surrounding tissues. Once the muscle was freed, it was placed in a sterile container and then transferred into 1.5 or 2 μL microtubes containing RNA later at -70°C for gene expression studies.
The expression of α and IIX myosin heavy chain genes was measured using real-time PCR. The Q-PCR reaction was performed using RealQ Plus 2x Master Mix Green on an Applied BioSystem DNA Analyzer according to the manufacturer's protocol. Primer sequences were obtained from the NCBI website, and primers for the target genes and β-actin were designed and reviewed using Genrunner and Oligo software. Primer specificity was confirmed using the BLAST program. The GAPDH gene was used as the reference gene.
Results
A single session of intense resistance exercise alone in adult male rats significantly increased the relative expression of the α myosin heavy chain gene in fast-twitch muscle fibers (1.93±0.298) compared to the control group (P<0.001). The combination of intense resistance exercise and glutamine supplementation also significantly increased the relative expression of the α myosin heavy chain gene in fast-twitch muscle fibers (1.65±0.195) compared to the control group (P<0.001). The increase in the relative expression of the α myosin heavy chain gene in the exercise-only group was significantly higher than in the exercise plus glutamine supplementation group (P<0.001). A single session of intense resistance exercise alone significantly increased the relative expression of the IIX myosin heavy chain gene in fast-twitch muscle fibers (1.42±0.239) compared to the control group (P<0.001). The combination of intense resistance exercise and glutamine supplementation also significantly increased the relative expression of the IIX myosin heavy chain gene in fast-twitch muscle fibers (1.26±0.190) compared to the control group (P<0.001).
Conclusion
According to the results of the present study, a single session of intense resistance exercise with or without glutamine supplementation significantly increased the expression of α myosin heavy chain and IIX myosin heavy chain genes in fast-twitch muscle fibers of male rats. Although the increase in both genes was more pronounced in the exercise-only group compared to the group receiving glutamine supplementation, the increase in the IIX gene was not statistically significant. It appears that the administered dose of glutamine was not effective in improving gene expression, and the intensity of the exercise was likely more influential than the supplementation in these gene changes.
Consistent and safe resistance training can lead to metabolic benefits and physiological and structural adaptations associated with improved neuromuscular function and motor performance. Additionally, resistance training induces extensive interstitial remodeling in both types of skeletal muscle. Glutamine is a key nutrient for the proliferation and differentiation of muscle stem cells, which may indirectly promote the synthesis of new muscle fibers, leading to increased expression of the β myosin heavy chain gene. Resistance exercise itself can directly activate muscle stem cells, which are dormant in skeletal muscle tissue. These activated stem cells merge with damaged muscle fibers to form new myonuclei, contributing to muscle growth and increased strength. Glutamine may enhance the activation of muscle stem cells by providing the necessary nutrients for their proliferation and differentiation. This stem cell activation may indirectly increase the expression of the β myosin heavy chain gene. Resistance training primarily causes hypertrophy of type II fast-twitch fibers in skeletal muscle. Type II muscle fibers preferentially respond to high-intensity training protocols, while type I muscles are more susceptible to high-volume training. It appears that exercise intensity is a more effective factor in changing the expression of myosin heavy chain and IIX genes in the extensor digitorum longus muscle of adult male rats than glutamine supplementation, and further research in this area is necessary.
Ethical Statement
The present study was approved by the Research Ethics Committees of the Marvdasht Branch, Islamic Azad University (IR.IAU.M.REC.1401.035).
Funding
This article was extracted from Mansur Mottahedy's Ph.D. dissertation in the field of Sports Physiology, Damghan Branch, Islamic Azad University, Damghan, Iran.
Conflicts of Interest
The authors have no conflicts of interest.
Acknowledgement
We extend our sincere gratitude to the relevant laboratories at Damghan Branch, Islamic Azad University, for their invaluable support.

Key Message
A single session of intense resistance exercise with or without glutamine supplementation significantly increases the relative expression of the α myosin heavy chain gene and the IIX motor unit gene in fast-twitch muscle fibers of the extensor digitorum longus muscle in adult male rats.