Introduction
The aging of the world's population poses serious medical, social, and economic challenges. In postmenopausal women, hormonal changes such as estrogen decline contribute to the development of sarcopenia and osteoporosis [
1,
2]. Sarcopenia describes the coexistence of reduced muscle quality or quantity and physical limitations [
3].
Direct hormone replacement therapy is essential in certain cases of hormone deficiency in men and women [
4‐
6]. Nevertheless, severe side effects such as thromboembolism have prompted the development of selective androgen and estrogen receptor modulators (SARMs and SERMs, respectively) with higher tissue selectivity [
5,
7,
8].
The biochemical hypothesis for the superior bioavailability and pharmacokinetic profile of SARMs compared to testosterone is the resisted aromatization of 5-α-reduction [
9]. However, while SARMs are not yet approved [
10], SERMs have been shown to be a safe therapeutic option for postmenopausal symptoms with fewer side effects compared to estrogen [
7].
The SARM ostarine (OST), also known as S-22, MK-2866, enobosarm or GTx-024, showed increased vascularization and citrate synthase activity in skeletal muscle in a rat model of postmenopausal osteoporosis [
11], and beneficial effects on muscle in orchiectomized rats [
12]. In addition, clinical studies have shown improved physical function and beneficial effects on body mass and muscle in elderly men and postmenopausal women, and reduced muscle wasting in cancer patients [
13‐
15].
Similarly, the SERM raloxifen (RAL) was found to increase lean body mass in postmenopausal women [
16] and improve body composition in orchiectomized rats [
12]. In mice of both sexes suffering from muscular dystrophy, skeletal muscle function and structure were improved by RAL [
17]. More recently, the combination of OST and RAL showed equivalent effects on muscle in terms of weight gain in the levator ani muscle compared to OST alone in an orchiectomized rat model, but reduced the androgenic potential of OST in the prostate [
12].
Studies on the effects of combined OST and RAL treatment on muscle structure and metabolism in the female organism are lacking. Therefore, the present study was conducted to investigate the effects of the combination of OST and RAL on skeletal muscle and metabolism in an established rat model of postmenopausal conditions and to compare it with OST and RAL treatments alone. The treatments were used as a phrophylaxis against the detrimental changes under hormone deficiency. Potential side effects were analyzed.
Discussion
In this study, we analyzed the effects of the SARM ostarine and the SERM raloxifen on muscle structure and metabolism in ovariectomized rats as a model of postmenopausal musculoskeletal system deterioration. We found that OST treatment exerted beneficial effects on muscle tissue, whereas RAL or combined OST + RAL treatments had less effect on muscle.
After OVX, rats showed increased BW as a common response to estrogen deprivation, which is consistent with previous studies [
11,
32‐
35] and could be explained by an increased food intake and other metabolic changes in rats observed after OVX [
36]. While OST + RAL treatment did not alter BW in the OVX rats, RAL administration resulted in a decrease in food intake and BW, possibly similar to the mechanisms of estrogens in blunting the increase in BW [
36]. OST administration did not change BW throughout the experiment; however, during the last four weeks of the experiment, OST rats showed increased food intake. It is possible that changes in BW and food intake are time dependent. This would be consistent with the results of Kearbey, Gao [
37], who found increased BW in OVX rats after 120 days of SARM S-4 administration, while body fat was reduced and lean mass was increased. Overall, the rats in the present study had a higher food intake than in our previous study [
11], which resulted in a higher uptake of the test compounds. However, the doses were comparable to the other rodent studies [
38,
39].
The activity of the rats was not affected by any of the treatments. Previous studies have reported a decrease in physical activity in OVX rats that promotes weight gain [
40,
41], whereas estrogen replacement therapy has been associated with a return to normal physical activity and body weight [
40]. The limitations of the physical activity assessment test used in this study are its short duration and application time at the end of the experiment. We did not perform more comprehensive tests because analysis of physical activity and behavior were not the primary objectives of the study.
OST treatment alone and in combination resulted in increased uterine weight, which has been previously observed and considered as a negative side effect [
11,
34,
42]. One reason for this uterotrophic effect may be that different scaffolds interact with the N-/C-terminal domains of the androgen receptor, leading to reduced tissue selectivity [
43]. In addition, OST was shown to increase the number of Ki67-positive cells in the mouse uterine stroma and epithelial cell proliferation [
39]. We did not observe any effects of RAL on uterine weight, which could be seen in line with its more estrogen-antagonistic potential on the uterus [
44].
OST treatment resulted in muscle weight gain in the GM and MS, highlighting the anabolic effect of SARM. A metabolic explanation for the ostarine-induced muscle weight gain in rats could be the stimulation of muscle cell differentiation by increasing the expression of myogenin, myoblast determination protein 1, and myosin heavy chain [
45]. Dalton, Barnette [
14] showed an increase in lean body mass and a decrease in total fat mass in postmenopausal women after OST treatment, supporting our findings. In contrast to OST, RAL treatment alone or in combination with OST maintained muscle weight at the level of intact Non-OVX rats, thereby reducing the effect of OVX. The changes in muscle weight could be due to the differences in BW of these groups, since the effect was not seen in data expressed relative to BW (data not shown) and a high positive correlation was found between muscle and body weight. Indeed, SERMs have been shown to improve muscle function and structure in mice with muscular dystrophy, possibly due to reduced fibrosis, oxidative stress, and mitochondria-mediated cell death [
17,
46]. Shen [
47] observed reduced BW in OVX rats treated with RAL and postulated regulation of the Wnt signaling pathway and a subsequent inhibition of adipogenesis.
OST administration did not affect muscle fiber size. The RAL group or RAL + OST group partially showed decreased muscle fiber sizes (e.g. STO + FTO in LM). A similar effect of RAL treatment alone or in combination was observed in previous experiments in male rats [
12]. A possible explanation could be a decrease in BW under RAL treatment, as muscle weight and muscle fiber size correlate with BW in rats in the present and previous study [
48].
Consistent with previous findings in OVX rats [
11], OST administration in the present study showed beneficial effects on capillary ratio in the LM and SM. Thus, we report an increased capillary ratio after OST administration in female OVX rats, which may indicate that female muscles are more sensitive to OST than male muscles studied by Roch, Wolgast [
12]. Better vascularization due to increased capillary ratio influences the recovery of muscle contractility and may subsequently improve muscle function [
49]. In contrast to OST, RAL treatment and the combined treatment of RAL and OST resulted in a decreased capillary ratio in the LM. The expression of Vegf-B, which influences muscle vascularization [
50] in our study, was least expressed in the combined treatment. Capillary ratio was correlated with BW and correspondingly with muscle weight, which may explain the lower blood supply in these treatment groups. In other studies, when RAL or estrogen was used as a therapeutic treatment 8 weeks after OVX, no changes in capillary ratio in skeletal muscles were reported [
51,
52].
In regard to the nucleus ratio, it was increased only in the GM by the combined treatment compared to the Non-OVX group. An increase in the number of satellite cells and myonuclei is associated with testosterone-induced muscle fiber hypertrophy [
53]. In our study, neither OST nor RAL was shown to affect the amount of myonuclei, although in general all OVX groups had a slightly non-significant higher ratio of myonuclei in muscle than Non-OVX rats.
Gene expression analysis showed increased Ar gene expression in the GM in the RAL group compared to the OVX controls. In contrast, decreased Ar expression has been reported in orchiectomized males after RAL administration [
12]. Sex differences may contribute to the differential expression of Ar in muscle. Igf-1 gene expression was increased in the RAL and RAL + OST groups. Igf-1 activates the calcium-dependent calcineurin signaling pathway in skeletal muscle, thereby promoting muscle growth [
54]. Thus, a similar effect of the combination treatment on Igf-1 expression as previously reported [
12] was confirmed. Tsai, McCormick [
55] observed that reduced estrogen levels after OVX in rats resulted in higher Igf-1 expression, possibly indicating its role in mediating the effects of estrogen deprivation. Furthermore, they showed that Igf-1 protein level decreased and Myostatin protein level increased after estrogen replacement [
55]. In our study, Myostatin expression was also increased after treatment of OVX rats with RAL, whereas OST and OST + RAL treatments did not change its expression. In male orchiectomized rats, OST administration resulted in a decreased Myostatin expression, whereas RAL treatment did not affect Myostatin expression [
12]. Myostatin controls muscle growth by inhibiting muscle differentiation and growth [
56,
57], which may explain the inhibition of muscle weight gain after ovariectomy in the RAL and OST + RAL groups.
Serum analysis showed that Ca and Mg levels were lower in all OVX groups than in the healthy Non-OVX group, with the combined treatment reducing Ca levels to a greater extent. In contrast, P levels were generally higher after OVX and increased significantly after OST and OST + RAL treatments. One explanation could be an effect of ovariectomy on the thyroid and the hypothalamic-pituitary-thyroid axis [
58]. While the changes in serum after OVX have been reported previously [
34,
59], the combination treatment failed to restore P and Ca levels as it was observed in male orchiectomized rats [
12]. Shahida [
60] found low Ca levels in osteoporotic patients, which may be due to the decrease in estrogen levels during menopause [
61]. Electrolyte imbalance should be avoided to prevent serious complications [
62] and this side effect of combined treatment should be considered. None of the treatments showed an effect on CK levels, indicating a lack of muscle damage [
63].
Serum FSH and LH levels were generally higher in all OVX rats compared to the Non-OVX rats, reaching the highest levels after the combination treatment. Both hormones, FSH and LH are elevated after the menopause in women and OVX rats due to the decreased estrogen and inhibin levels [
64]. Estrogen and RAL decrease LH levels by suppressing gonadotropin-releasing hormone (GnRH) release [
65], whereas FSH levels may not be decreased [
66]. Androgenic steroid hormones and SARMs also have the potential to suppress of LH and FSH levels [
67] and it is unclear why the combination of RAL and OST caused an increase in the levels of these hormones.
The study has several limitations. We examined metabolism, structure and gene expression and did not include a functional examination of the muscles. For clinical application, the effects of substances on muscle function should be addressed. In addition, the effects of anabolic substances on muscle metabolism and size may be influenced by concomitant exercise [
68,
69], which was not assessed in the present study. Furthermore, the Non-OVX rats did not experience surgical stress and postoperative pain, and therefore the sham-operated group would have been a more appropriate control group for this study.
Summarizing, OST administration resulted in favorable effects on muscle weight and capillary ratio, emphasizing the anabolic effect of SARMs, while RAL or combination therapy failed to do so. Neither treatment showed anabolic effects on muscle fiber size. The combination treatment increased the nucleus ratio in the GM compared to the Non-OVX group. OST administration did not change BW, whereas RAL administration reduced food intake and BW, consistent with the literature. In contrast to RAL, OST administration and the combination treatment increased uterine weight and had altered serum electrolyte concentration, suggesting possible side effects due to the limited tissue selectivity. Gene expression analysis revealed beneficial effects on muscle growth as indicated by increased Igf-1 expression after OST and RAL administration. However, RAL treatment also increased Myostatin expression, which likely slowed muscle growth and prevented the increase in muscle weight observed after OVX.
In conclusion, the effect of OST on muscle was favorable and superior to the effect of RAL alone or combined treatment in estrogen-deficient rats. However, side effects of OST on uterus and serum electrolytes should be considered before using it for therapeutic purposes. RAL and RAL + OST had less effect on muscle and showed some endocrinological side effects on pituitary–gonadal axis.
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.