Myostatin and Follistatin mRNA Expression in Castrated Rats Submitted to Resistance Training.
Myostatin (MSTN) is a negative regulator of skeletal muscle mass. It is a member of the transforming growth factor-[beta] (TGF-[beta]) and a growth-differentiating factor-8 (GDF-8). Knock-out rats for the MSTN gene have increased muscle mass from 100 to 200% in adult animals engaged in muscle hypertrophy protocols (11,18). MSTN is the target of therapies in patients with diabetes, cancer, and AIDS. MSTN has been the target of commercial supplements that promise to inhibit MSTN that allows for muscle hypertrophy (21). The increase in skeletal muscle from knock-out animals to MSTN is attributed not only to the proliferation of satellites of the muscle fiber, but also according to Amthor et al. (2) have shown that the absence of MSTN involves hypertrophy and little or no satellite cell.
MSTN acts on hypertrophy in two ways. It does so by the inhibition of PDK1 (phosphoinositide-1-dependent protein kinase) and PI3K (phosphatidylinositol-3 kinase), which stimulates Akt in which it stimulates mTOR (rapamycin target protein) and promotes protein synthesis, and also by stimulating Smads 2 and 3 that bind to Smad 4 to form a complex that translocates to the nucleus and increases the transcriptional activity of the apoptosis enzymes (3,14,18).
There are several ways to inhibit MSTN, such as Folistatin (FS) that is responsible for inhibition by extracellular binding (8,14,18), or by other inhibitors such as follistatin-related gene (FLRG), differentiation and growth factor associated with serum protein 1 (GASP-1) and associated adeno virus (AAV) (3,8,24). Little is known about FS and its action or relationship with other inhibitors of MSTN action. Lee and McPherron (18), working with MSTN knock-out animals and with FS superexpression, had a four-fold increase in muscle mass compared to the control animals.
Kovacheva et al. (15) demonstrated that testosterone stimulates cell growth by inhibiting apoptosis and atrophy. It also stimulates proliferation and differentiation of satellite cells. Testosterone is an anabolic hormone. It causes hypertrophy when it is administered exogenously, and its action is a dependent dose (4,28).
The effect of MSTN, FS, and testosterone is not fully known. However, MSTN is a mediator of sarcopenia, and FS and testosterone among other hormones that are mediators of muscle hypertrophy. Morisette et al. (23) demonstrated that aged animals have lower mass muscle than adults and that knock-out animals for MSTN have greater muscle mass only in adult animals.
In adult skeletal muscle, chronic overload attenuates sarcopenia by inducing hypertrophy and increasing strength (12). Jones et al. (11) related to sarcopenia with two factors: decreased protein synthesis due to a decrease in production and secretion of hormones such as GH and testosterone in aging, as well as an increase in protein degradation through sedentarism and increased MSTN. Goto et al. (7), on the other hand, demonstrated that resistance training increases muscle mass.
Therefore, the castration performed in the present study and the resistance training was to relate the effects of the absence of testosterone and training in Wistar rats on the gene expression of MSTN and FS. Thus, we hypothesized that the absence of testosterone will increase MSTN and that the training will reduce the deleterious effects of this increase even with castration. We also hypothesized that FS will help the MSTN regulation process in the absence of testosterone.
Wistar rats (N = 32) from the University of Sao Paulo (USP), Ribeirao Preto-SP, Brazil, with the initial weight of 180 [+ or -] 1.2 g were used in this study. The animals had free access to water and feed plus a controlled environment with a temperature of 22 [+ or -] 20[degrees]C, humidity, and a 12 hr light/dark cycle.
The research was approved by the ethics committee of animal experiments of the Universidade Federal de Sao Carlos (protocol no. 034/2010), and consistent with the rules of the National Council of Control of Animal Experimentation (CONCEA).
The trained groups underwent 8 wks of resistance training. They were divided into four groups (8 animals per group): intact sedentary (IS), castrated sedentary (CS), intact trained (IT) and castrated trained (CT). All animals had 1 wk of adaptation before castration surgery.
Castration was performed when the animals were at a body mass of 229.3 [+ or -] 4.1 g (precastration) in accordance with the work of Leal and Moreira (17) with 10 d of recovery after surgery. The training protocol performed after the 3-d adaptation (pre-training) started with a test and retest of maximum load with 75% of the animals' mass of which 30 g were added for subsequent climbs until the animals could not voluntarily climb. Fatigue of the animals was determined with three successive stimuli in the unresponsive syringe. The training sessions consisted of 4 climbs, 65%, 85%, 95%, and 100% of the previous loading capacity, which was determined in the previous session, plus the subsequent climbs with an additional 30 g with a maximum of 9 climbs per session and then the determination of a new maximum load (6).
Euthanasia occurred 48 hrs after the last training session (post-training). All samples were weighed, immediately frozen in liquid nitrogen, and then stored in a -80[degrees]C freezer for further analysis.
Isolation of RNA and Real-Time Quantitative (RT) Polymerase Chain Reaction (PCR)
Total RNA was extracted from the white gastrocnemius with Trizol (Invitrogen Corporation ([R]), California, USA) according to the manufacturer's specifications. The integrity and quality of the purified RNA were determined by agarose electrophoresis gel and measured at [A.sub.260]/[A.sub.280] frequency with Nanodrop ([R]). For the removal of the genomic DNA, 1 [micro]g of total RNA from each sample was treated with Deoxyribonuclease I (DNase I, Invitrogen Corporation, California, USA) following manufacturer's specifications. The treated RNA was transcribed with cDNA using reverse M-MLV transcriptase (Promega Corporation ([R]), Madison, WI, USA). For the qRT-PCR procedure, 20 ng of cDNA and 0.5 [micro]M of each primer was used in 25 [micro]L containing SYBR Green PCR Master Mix (Applied Biosystems ([R]), Fosters City, CA). Samples were analyzed in duplicates. The temperature cycles were 95[degrees]C for 10 min, followed by 40 cycles of 94[degrees]C for 15 sec, 57 to 61[degrees]C for 30 sec, and 72[degrees]C for 60 sec. The melting curve demonstrated that only one PCR product was amplified. [beta]-Actin was used as endogenous control. The relative expression of the qRT-PCR products was determined by the [DELTA][DELTA]Ct method.
The data are presented as mean [+ or -] standard error of the mean (SEM). A homoscedasticity and normality test by Kolmogorov-Smirnov was performed, and analysis of one-way variance (ANOVA) used to compare resistance training and castration variables. Tukey's post hoc test was used in the event of statistical significance, which was considered at an alpha level of P[less than or equal to]0.05. All the analysis was performed using SPSS ([R]) version 22 (IBM ([R]) corporation). Sample power of 0.80 was determined and sample size was calculated by GPower ([R]) software after pilot study.
No statistical difference was found between the body mass of the animals between groups (data not shown). Only difference between the time of the experiment (expected data) was found. There was no statistical difference between the TC and TI groups in the first or last training session. There was a difference between the first and last training session within their respective CT or IT group, as expected (P<0.0001) and shown in Figure 1.
There was a statistical difference in the expression of MSTN (P<0.05) between the IS, IT, and CT groups in relation to the CS group. However, no difference was found between the trained groups or between the trained groups and the IS group as shown in Figure 2. We found a statistical difference in FS expression (P<0.05) between the CT and IS groups, and also between the CT and IT groups shown in Figure 3, and there was no difference between the sedentary groups.
The difference in relative loading of the load found between the first and last training session was expected because of the adaptation to loads during training. However, there was no difference between the groups trained (6). In this sense, other anabolic hormones may have been responsible for acting in the absence of testosterone and increasing muscle strength. One limitation of this study was not to have evaluated other anabolic hormones, such as IGF, GH, and insulin that may be associated with the training adaptation.
The increase in MSTN mRNA expression in the CS group compared to the other groups demonstrates the effect of testosterone on the expression of MSTN. This result goes against the study of Mendler et al. (23) who evaluated MSTN expression after 7, 11, and 14 d of castration of prepubertal rats with and without testosterone treatment, and there was no difference between the control group and the castrated group. The absence of testosterone causes increased expression of MSTN, which may contribute to the atrophic process and consequent loss of muscle mass (11). In the present study, castrated animals were exposed to the effects of the absence of testosterone for slightly more than 10 wks that is likely the reason for the increase of MSTN in the CS group. We did not have a group using testosterone, but we demonstrated the influence of testosterone on MSTN in playing an important role in the regulation of hypertrophy and cell cycle that are inhibited by MSTN.
Resistance training did not decrease the MSTN expression between the trained groups and the IS group, although there are other studies that have demonstrated the decrease in MSTN after resistance training (13,20,25,26). However, it is clear that our findings confirm the results of several studies (1,9,16,27) that have demonstrated resistance training being unable to decrease MSTN expression. Nevertheless, of these studies, only Adams et al. (1) worked with rats, and used a different training protocol from the present study. So, the present study demonstrated new results on testosterone, MSTN, and FS in rat resistance training. Although we did not find a statistically significant difference in the MSTN of the CT group in relation to IT, the findings indicate that even in the absence of testosterone resistance training, the animals were able to regulate MSTN expression.
According to Hulmi et al. (9), there was a decrease in the MSTN expression 1 hr after the training session, and 48 hrs after training the concentration was in the normal resting level. These findings are due to the signaling of the mTOR gene being active after resistance training causing a decrease in MSTN activity and its gene expression. This observation by mTOR demonstrates that resistance exercise is an instrument capable of modulating the expression of key molecules important for hypertrophy and to contribute for regulation of the MSTN gene. In the present study, the animals were euthanized after 48 hrs of the last training session, and we did not evaluate after 1 hr of the last training session to verify if the MSTN would have decreased as described by Hulmi et al. (9). However, the present study confirmed that after 48 hrs MSTN expression returned to resting levels that is in agreement with Hulmi et al. (9).
Mascher et al. (20) showed a significant increase in mTOR after 15 min of resistance training and 2 hrs of rest without Akt activation, which suggests an independent exercise pathway to promote hypertrophy. One limitation of our study was not evaluated as an expression of hypertrophic related proteins such as Akt and mTOR and another time of MSTN evaluation.
In addition to the fact that we did not have euthanasia in the same periods of the studies cited above, it is likely that it may have contributed to having no difference between the trained and sedentary groups for resistance training. Hence, this consideration may have led to the synthesis and secretion of glucocoticoids that have an atrophic effect and is influenced by the gene expression of MSTN. According to Ma et al. (19), dexamethasone applications in rats lead to increased expression and protective concentration of MSTN in relation to control, and when applied MSTN antagonist the increase was not observed.
The lower expression of FS mRNA in the CT group in relation to the IT and IS groups of this study goes against Diel et al. (5) that before and after resistance training there was no statistical difference in FS in humans. There was also no difference in the present study between IT and IS but Diel et al. (5) demonstrated that bodybulders have higher levels of MSTN and FS and lower relative levels of MSTN/FS. They also compared the effect on rats and demonstrated a lower FS level in castrated rats and dihydrotestosterone application. In the present study, the CT group did not receive any type of anabolic to influence the expression of the FS and still the group decreased in relation to the intact group. This result is in agreement with the findings of Jensky et al. (10), since they did not find statistical difference with resistance training in women in concentric or eccentric exercise in the expression of FS. They reported that other anabolic hormones may affect FS expression, since they were performed with healthy people.
According to Rodino-Kaplac et al. (24), FS inhibits the synthesis and secretion of the follicle-stimulating hormone (FSH) of the Hypothalamus-Hypophysis-Gonadal axis. FS binds to the actin receptor, promoting negative feedback and inhibition of FSH. According to the authors, the FS is synthesized by the ovaries, testicles, and pituitary, so castration caused the animals to have only small production of FS by the pituitary. Therefore, in the present study, the gonadal axis was altered with the absence of testosterone, losing the negative feedback of the axis, strengthening the stimulation of FSH, and FS in the hypothalamic-pituitary glands. Also, training with castration promotes the stimulation of other anabolic hormones to replace FS and testosterone. These hormones are stimulated by the active gonadal axis and, therefore, without negative feedback.
Resistance training was not able to inhibit or decrease MSTN expression in the white gastrocnemius rat muscle of this study, but resistance training may have regulated the expression of the MSTN in the CT group in relation to the SI. It may have influenced FS regulation also in the CT group preserving other axes of anabolic hormone. It is also reasonable that castration promoted the increase in MSTN expression that highlights the influence of testosterone on its expression.
There was no inverse relationship between the expression of MSTN and FS because other anabolic hormones can act in the absence of testosterone, and the training helps in this stimulation. Therefore, our study with resistance training is not the best protocol to verify this inverse relationship between MTSN and FS. Other studies will be needed to better answer these questions, and still understand the signaling pathway of MSTN and FS.
We thank Capes, CNPQ and UFSCar for the financial and technological support to complete this research.
Address for correspondence: Diego A. Marine, Departamento de Educacao Fisica, Universidade Federal de Sao Carlos, Sao Carlos, Sao Paulo, Brasil, rod. Washington Luis Km 235, CEP 13562-905. Email: firstname.lastname@example.org
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Diego Adorna Marine (1), Fernando Fabrizzi (1), Keico Okino Nonaka (1), Ana Claudia Garcia de Oliveira Duarte (2), Angela Merice de Oliveira Leal (3)
Departamento de Ciencias Fisiologicas (1), Departamento de Educacao Fisica e Motricidade Humana (2) e Departamento de Medicina (3). Universidade Federal de Sao Carlos. Sao Carlos/SP, Brasil