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Natural aging and ovariectomy induces parallel phosphoproteomic alterations in skeletal muscle of female mice

ABSTRACT: The loss of skeletal muscle strength mid-life in females is associated with the decline of estrogen. Here, we questioned how estrogen deficiency might impact the overall skeletal muscle phosphoproteome after contraction, as force production induces phosphorylation of several muscle proteins. Phosphoproteomic analyses of the tibialis anterior muscle after contraction in two mouse models of estrogen deficiency, ovariectomy (Ovariectomized [Ovx] vs Sham) and natural aging-induced ovarian senescence (Older Adult [OA] vs Young Adult [YA]), identified a total of 2,593 and 3,507 phosphopeptides in Ovx/Sham and OA/YA datasets, respectively. Further analysis of estrogen deficiency-associated proteins and phosphosites identified 66 proteins and 21 phosphosites from both datasets. Of these, 4 estrogen deficiency-associated proteins and 4 estrogen deficiency-associated phosphosites were significant and differentially phosphorylated or regulated, respectively. Comparative analyses between Ovx/Sham and OA/YA using Ingenuity Pathway Analysis (IPA) found parallel patterns of inhibition and activation across IPA-defined canonical signaling pathways and physiological functional analysis, which were similarly observed in downstream GO, KEGG, and Reactome pathway overrepresentation analysis pertaining to muscle structural integrity and contraction, including AMPK and calcium signaling. IPA Upstream regulator analysis identified MAPK1 and PRKACA as candidate kinases and calcineurin as a candidate phosphatase sensitive to estrogen. Our findings highlight key molecular signatures and pathways in contracted muscle suggesting that the similarities identified across both datasets could elucidate molecular mechanisms that may contribute to skeletal muscle strength loss due to estrogen deficiency.

INSTRUMENT(S): Orbitrap Fusion

ORGANISM(S): Mus Musculus (mouse)

SUBMITTER: Mina Peyton

LAB HEAD: Dawn Lowe

PROVIDER: PXD035171 | Pride | 2023-09-01

REPOSITORIES: Pride

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Project description:Despite an abundance of evidence to the contrary from animal studies, large clinical trials on humans have shown that estrogen administered to post-menopausal women increases the risk of cardiovascular disease. However, timing may be everything, as estrogen is often administered immediately after ovariectomy (ovx) in animal studies, while estrogen administration in human studies occurred many years post-menopause. This study investigates the discrepancy by administering 17ß-estradiol (E2) in a slow-release capsule to Norway Brown rats both immediately following ovx and 9 weeks post-ovx (Late), and studying differences in gene expression between these 2 groups as compared to age-matched ovx and sham operated animals. Two different types of microarray were used to analyze the left ventricles from these groups: an Affymetrix array (2 samples/group, each sample contained total RNAs pooled from 3 rats) and an Inflammatory Cytokines and Receptors PCR array (N=4 /group). Key genes were analyzed by western blotting. Ovx without replacement led to an increase in caspase 3, caspase 9, calpain 2, MMP9, and TNFα. Caspase 6, STAT3, and CD11b increased in the Late group, while TIMP2, MMP14, and collagen I α1 were decreased. MADD and fibronectin were increased in both Ovx and Late. TNFα protein levels increased with Late replacement. Many of these changes were prevented by early E2 replacement. These findings suggest that increased TNFα may be involved in some of the deleterious effects of delayed E2 administration seen in human studies. The rats were randomly assigned to four groups: Sham, Ovx, Early estrogen group, and Late estrogen group. The Sham rats were anesthetized, had their body cavities opened and then immediately closed with no tissue removal; the Ovx group underwent ovariectomy. The Early estrogen group after ovariectomy had a 0.5 mg 17-β estradiol 90-day slow release pellet (Innovative Research of America, Sarasota, FL) implanted subcutaneously in the back of the neck at the time of surgery. The Late estrogen group underwent ovariectomy and had a subcutaneous estrogen pellet placed, as above, at 9 weeks post-ovx. RNA target was prepared according to the instructions of the manufacturer (Affymetrix). For each RNA sample, good-quality total RNA from three rat hearts were pooled together in equal amounts and used to synthesize double-strand cDNA using a one-cycle cDNA synthesis kit (Affymetrix, P/N 900431). This approach allows repetitive analysis of all groups by the array in a group representative and cost effective manner, and has been used effectively by other investigators. Two pooled RNA samples were prepared for each group, and all the RNA samples were proceeded with Affymetrix expression chip analysis.

Project description:Purpose: Using RNA-sequencing technology to screen the effect of moderate-intensity treadmill exercise on the key genes that affect bone mass in the peripheral blood mononuclear cells (PBMCs) of ovariectomized (OVX) rats. Methods: Three-month-old female Sprague–Dawley rats of Specific Pathogen Free (SPF) grade were randomly divided into the sham operation (SHAM) group, OVX group, and OVX combined exercise (OVX+EX) group. The OVX+EX group performed moderate-intensity treadmill exercise for 17 weeks. Upon completion of these exercises, the body composition and bone mineral density (BMD) were measured using dual-energy X-ray absorptiometry, and the bone microstructure of the femur was observed using micro-computed tomography scanning. PBMCs were collected from the abdominal aorta, and the differential genes were analyzed by transcriptome sequencing. The Metascape software was used for gene ontology and pathway enrichment analysis to further screen key genes. Results: 1. In the OVX group, the body weight and body fat content were significantly higher than in the SHAM group and the body muscle content and BMD were significantly lower. 2. The trabecular bone parameters in the OVX group were significantly lower than in the SHAM group, and they were significantly higher in the OVX+EX group than in the OVX group. When compared with the SHAM group, the microstructure of the distal femur trabecular in the OVX group was severely damaged, the trabecular bones were sparse, and there was a large gap between the trabecular bones. The number and continuity of the trabecular bones were higher in OVX+EX group than in the OVX group. 3. A Venn diagram showed that there were 58 common differential genes, with a fold change ≥2 and p value <0.05. and the differential genes were mainly enriched in the PI3K-Akt signaling pathway. Five key genes were screened including CCL2, Nos3, Tgfb3, ITGb4, and LpL. Conclusion: Moderate-intensity treadmill exercise may improve the body composition and bone mass of the OVX group by upregulating CCL2 and other genes of the PBMC. The results also showed that the PBMCs in the peripheral blood can be a useful tool for monitoring the effect of exercise on bone health in postmenopausal osteoporosis.

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Natural aging and ovariectomy induces parallel phosphoproteomic alterations in skeletal muscle of female mice

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