Estradiol deficiency and skeletal muscle apoptosis: Possible contribution of microRNAs.

BACKGROUND: Menopause leads to estradiol (E2) deficiency that is associated with decreases in muscle mass and strength. Here we studied the effect of E2 deficiency on microRNA (miR) signaling that targets apoptotic pathways. METHODS: C57BL6 mice were divided into control (normal estrous cycle, n = 8), OVX (E2 deficiency, n = 7) and OVX + E2 groups (E2-pellet, n = 4). Six weeks following the OVX surgery, mice were sacrificed and RNA isolated from gastrocnemius muscles. miR-profiles were studied with Next-Generation Sequencing (NGS) and candidate miRs verified using qPCR. The target proteins of the miRs were found using in silico analysis and measured at mRNA (qPCR) and protein levels (Western blot). RESULTS: Of the apoptosis-linked miRs present, eleven (miRs-92a-3p, 122-5p, 133a-3p, 214-3p, 337-3p, 381-3p, 483-3p, 483-5p, 491-5p, 501-5p and 652-3p) indicated differential expression between OVX and OVX + E2 mice in NGS analysis. In qPCR verification, muscle from OVX mice had lower expression of all eleven miRs compared with OVX + E2 (p < 0.050). Accordingly, OVX had higher expression of cytochrome C and caspases 6 and 9 compared with OVX + E2 at the mRNA level (p < 0.050). At the protein level, OVX also had lower anti-apoptotic BCL-W and greater pro-apoptotic cytochrome C and active caspase 9 compared with OVX + E2 (p < 0.050). CONCLUSION: E2 deficiency downregulated several miRs related to apoptotic pathways thus releasing their targets from miR-mediated suppression, which may lead to increased apoptosis and contribute to reduced skeletal muscle mass.

Tissue selective effects of bazedoxifene on the musculoskeletal system in female mice.

The actions of selective estrogen receptor modulators are tissue dependent. The primary objective of the current study was to determine the tissue selective effects of bazedoxifene (BZA) on the musculoskeletal system of ovariectomized (OVX) female mice, focusing on the strengths of muscle-bone pairs in the lower hindlimb. Treatment with BZA after ovariectomy (OVX+BZA) did not prevent body or fat mass gains (P < 0.05). In vivo plantarflexor muscle isometric torque was not affected by treatment with BZA (P = 0.522). Soleus muscle peak isometric, concentric and eccentric tetanic force production were greater in OVX+BZA mice compared to OVX+E2 mice (P ≤ 0.048) with no effect on maximal isometric specific force (P = 0.228). Tibia from OVX+BZA mice had greater cortical cross-sectional area and moment of inertia than OVX mice treated with placebo (P < 0.001), but there was no impact of BZA treatment on cortical bone mineral density, cortical thickness, tibial bone ultimate load or stiffness (P ≥ 0.086). Overall, these results indicate that BZA may be an estrogen receptor agonist in skeletal muscle, as it has previously been shown in bone, providing minor benefits to the musculoskeletal system.

Estrogen Regulates the Satellite Cell Compartment in Females.

Skeletal muscle mass, strength, and regenerative capacity decline with age, with many measures showing a greater deterioration in females around the time estrogen levels decrease at menopause. Here, we show that estrogen deficiency severely compromises the maintenance of muscle stem cells (i.e., satellite cells) as well as impairs self-renewal and differentiation into muscle fibers. Mechanistically, by hormone replacement, use of a selective estrogen-receptor modulator (bazedoxifene), and conditional estrogen receptor knockout, we implicate 17ß-estradiol and satellite cell expression of estrogen receptor α and show that estrogen signaling through this receptor is necessary to prevent apoptosis of satellite cells. Early data from a biopsy study of women who transitioned from peri- to post-menopause are consistent with the loss of satellite cells coincident with the decline in estradiol in humans. Together, these results demonstrate an important role for estrogen in satellite cell maintenance and muscle regeneration in females.

Effects of ovarian hormones and estrogen receptor α on physical activity and skeletal muscle fatigue in female mice.

Menopause is associated with declines in physical activity and skeletal muscle strength. Physical activity is also reduced in rodents after ovariectomy (OVX) and whole-body estrogen receptor α (ERα) knockout. However, it is unclear if the effects are estradiol (E2) specific. Thus, the overall purpose of this study was to investigate the effects of the ovarian hormones, E2 and progesterone (P4), and skeletal muscle ERα (skmERα) on physical activity and skeletal muscle contractility in female mice. METHODS: Study 1: Forty female C57Bl/6J mice were given free access to running wheels for 2 weeks to assess baseline running and randomized into 4 treatment groups: OVX, OVX + E2, OVX + P4, OVX + E2 + P4. All mice underwent OVX, returned to wheels for 2 weeks, received hormone pellet implants and returned to running wheels for 6 weeks, after which soleus muscle contractility testing was completed. Study 2: Thirty-two skeletal muscle specific ERα knock-out (skmERαKO) mice and wildtype (WT) littermates were randomized into 4 groups: skmERαKO-Run, skmERαWT-Run, skmERαKO-Sed, and skmERαWT-Sed. Run mice were given free access to wheels for 20 wk and sedentary (Sed) mice maintained normal cage activities. At the end point, muscle contractility was tested. RESULTS: Study 1: OVX + E2 + P4 group ran greater distances than both the OVX and OVX + P4 groups (p ≤ 0.009). After fatiguing contractions, soleus muscles of the OVX + E2 + P4 group maintained greater submaximal force than those of other groups (p = 0.023). Immediately after the fatiguing contractions, OVX + E2 + P4 muscles had greater maximal force production than the OVX + E2 group (p = 0.027). Study 2: There were no differences in running distance between skmERαWT and skmERαKO mice (p = 0.240). Soleus muscles of skmERαKO mice were more fatigable (p < 0.001) and did not recover force as well as skmERαWT mice (p < 0.001). In vivo isometric, concentric and eccentric torque was decreased in skmERαKO mice compared to skmERαWT mice (p ≤ 0.029). CONCLUSIONS: Combined treatment of E2 + P4 in OVX mice restored physical activity, predominantly driven by E2, and protected soleus muscles against fatigue. Muscle of skmERαKO mice was weak regardless of physical activity. Although 20 wk of wheel running partially prevented force loss during fatigue in skmERαKO mice, force production during recovery remained low, indicating that estradiol functions through ERα in skeletal muscle.

Deletion of estrogen receptor α in skeletal muscle results in impaired contractility in female mice.

Estradiol deficiency in females can result in skeletal muscle strength loss, and treatment with estradiol mitigates the loss. There are three primary estrogen receptors (ERs), and estradiol elicits effects through these receptors in various tissues. Ubiquitous ERα-knockout mice exhibit numerous biological disorders, but little is known regarding the specific role of ERα in skeletal muscle contractile function. The purpose of this study was to determine the impact of skeletal muscle-specific ERα deletion on contractile function, hypothesizing that ERα is a main receptor through which estradiol affects muscle strength in females. Deletion of ERα specifically in skeletal muscle (skmERαKO) did not affect body mass compared with wild-type littermates (skmERαWT) until 26 wk of age, at which time body mass of skmERαKO mice began to increase disproportionally. Overall, skmERαKO mice had low strength demonstrated in multiple muscles and by several contractile parameters. Isolated extensor digitorum longus muscles from skmERαKO mice produced 16% less eccentric and 16-26% less submaximal and maximal isometric force, and isolated soleus muscles were more fatigable, with impaired force recovery relative to skmERαWT mice. In vivo maximal torque productions by plantarflexors and dorsiflexors were 16% and 12% lower in skmERαKO than skmERαWT mice, and skmERαKO muscles had low phosphorylation of myosin regulatory light chain. Plantarflexors also generated 21-32% less power, submaximal isometric and peak concentric torques. Data support the hypothesis that ablation of ERα in skeletal muscle results in muscle weakness, suggesting that the beneficial effects of estradiol on muscle strength are receptor mediated through ERα. NEW & NOTEWORTHY We comprehensively measured in vitro and in vivo skeletal muscle contractility in female estrogen receptor α (ERα) skeletal muscle-specific knockout mice and report that force generation is impaired across multiple parameters. These results support the hypothesis that a primary mechanism through which estradiol elicits its effects on strength is mediated by ERα. Evidence is presented that estradiol signaling through ERα appears to modulate force at the molecular level via posttranslational modifications of myosin regulatory light chain.