TGF-β1 response to eight weeks combined training with different orders in slow and fast twitch muscles in Wistar rats

Skeletal muscle is a highly malleable tissue capable of significant metabolic and morphological adaptations in response to disruptions in cellular homeostasis induced by exercise. Resistance and endurance training represent divergent exercise modes, with each inducing distinct responses within the muscle milieu that act to minimize cellular stress. The well-known adaptive features of chronic muscle contraction include muscle hypertrophy and increased strength. These features are induced by high-intensity muscle contraction, such as that involved in resistance exercises. Other typical adaptations are increases in oxidative capacity and glucose uptake, which are induced by low-intensity, high volume muscle contraction, such as that observed during EE. combined training, a combination of endurance exercises (EE) and resistance exercise (RE) in succession, is used widely in exercise prescription to simultaneously improve muscular strength and cardiovascular function. Interestingly, accumulating evidence indicates that this combination of the two exercise modes may hamper strength and muscle hypertrophic adaptations to RE. However, studies on the effects of physiological adaptations of EE on RE-induced muscle hypertrophy have shown mixed results. Intriguingly, a recent study in humans reported that EE performed prior to RE increased muscle mass to a greater extent than long-term RE alone, suggesting that EE may not always attenuate RE-induced muscle hypertrophy.
Transforming growth factor beta 1 (TGF-β1) is a polypeptide member of the transforming growth factor beta superfamily of cytokines. TGF-β1 plays essential roles in various biological processes, including cell growth, differentiation, apoptosis, tissue development, and inflammation. While transient TGF-β expression may contribute to muscle regeneration after injury, the chronic elevated expression of TGF-β in skeletal muscle may be detrimental. Although the role of TGF-β in muscle mass regulation and skeletal muscle fibrosis has been studied extensively, the effects on hypertrophy signaling pathways muscle cells and underlying mechanisms are not well understood. Furthermore, taking into account the functional and mechanistic similarities between TGF-β and myostatin, as well as the fact that both ligands have been implied as possible therapeutic targets for muscle wasting disorders, the purpose of the present study was to determine the effect of eight weeks of combined training with different order on TGF-β1 in fast and slow twitch muscles in male wistar rats.

In this study, 40 adult male Wistar rats (age: 10 weeks, weight: 180-200 gr) were maintained on a 12:12-h dark-light cycle in a temperature-controlled environment (22 oC) with free access to standard laboratory chow and tap water. Before the allocation to their respective groups, all animals underwent three familiarization sessions, separated by 48-h intervals, to get acquainted with the exercise protocols (i.e., endurance and strength exercises). Rats were randomly allocated into resistance training (n=8), endurance training (n=8), resistance- endurance training (n=8), endurance- resistance training (n=8) and control (n=8) groups in equal laboratorial condition. Resistance training was conducted for 8 weeks (5 sessions/week) on a special 1-meter high ladder (divided by 26 stairs) with the loading of percent of overloading test (%75 of weight body) in the first week and increased to 30gr per week. Training included 10 sets of with 2 min rest between sets. Endurance training consisted of treadmills run, that speed and duration of running gradually increased during training period, from 9 m/min and 10 minutes in the first week to 30 m/min and 60 minutes in the last week. Animals in the endurance- resistance group performed the EE protocol first. 5 min after the completion of the EE bout, they were placed in the strength-training apparatus to perform the RE protocol. In the resistance- endurance group performed RE 5 minutes before EE. Animals were killed by decapitation 24 h after the exercise protocols and the flexor hallucis longus (FHL) and soleus muscles were removed from both legs, weighed on an analytical balance, frozen in liquid nitrogen, and stored at -80oC for further analysis. The proteins levels of TGF-β1 were measured by ELISA method. After normality (Shapiro Wilk test) and variance assurance (Levene test), the groups were compared by one-way analysis of variance and the Tukey post hoc test was applied for multiple comparison purposes. The level of significance was set at P ≤ 0.05.

The results showed that weight of FHL and soleus muscles significantly increased in all experimental groups compared to control group. The TGF-β1 in fast twitch muscle decreased significantly in strength group (p=0.008). Although both resistance- endurance training and endurance- resistance training resulted in no significantly decrease in TGF-β1 for FHL and soleus, but there were no significant differences between combined training with differing order (P≥0.05).

It seems that exercise order in combined training result in no different effect on TGF-β1 in fast and slow twitch muscles in wistar rats. The majority of current molecular data suggests that endurance exercise does not compromise early anabolic responses to resistance exercise. Thus, sports scientists but mostly the coaches should be very careful about the training strategies which they are willing to follow, when a combination of endurance and resistance training is needed, by taking into consideration how different training modalities interact between them. In an effort to maximize the training adaptations from a combined and thus limit the effects, sports scientists and coaches should consider the level of fatigue from both modules and the need of inter-session time intervals to minimize the training induced overall fatigue. However, further elucidation of the molecular factors mediating the specificity of training adaptation in human skeletal muscle is warranted, which in turn may provide additional mechanistic insight into the combined interference phenomenon. Ultimately, improved understanding of the roles of individual combined training variables, including within-session exercise order in modulating the interference effect is required to guide exercise prescription for simultaneously maximizing divergent training adaptations. Future work should aim to further clarify the roles of these training variables in acute and particularly chronic interference in trained individuals to inform practical recommendations for minimizing interference between combined resistance and endurance exercise.