Mapping the relative frequencies of stromal cell populations across menopause in skeletal muscle
Authors: Hettinger Zachary , Kopchak Rylee , Gilmer Gabrielle , Miller Amanda , Mkandawire Tabo , Sinkar Suyash , Ambrosio Fabrisia 
Published: May 2023

Intro: Nearly three-quarters of postmenopausal women report muscle pain and present with sarcopenia, the age-related decline in muscle function. Sarcopenia is mediated by both muscle cell dysfunction and shifts in surrounding stromal cells, such as muscle stem cells (MuSC), fibro-adipogenic progenitor cells (FAPs), and immune cells. Because rodents do not display a menopausal phenotype, ovariectomies have been performed to simulate menopause. However, chemically-induced models better mimic natural human menopause due to the inclusion of perimenopause and preservation of ovarian tissue. As such, the purpose of this research was to employ a chemically-induced model to analyze shifts in stromal cell abundances within skeletal muscle from perimenopause through menopause. We hypothesized that the abundance of FAPs and pro-inflammatory markers (cytotoxic T cells) would increase over time at the detriment of MuSCs and anti-inflammatory markers (M2 macrophages) in menopausal mice when compared to non-menopausal mice. Methods: Using a well-established model, we injected an ovarian toxin, 4-vinylcyclohexene diepoxide (VCD), into middle-aged (14-16 months) C57/BL6 female mice. Age-matched, control mice received vehicle (sesame oil) injections (SO). We then isolated and sorted stromal cells from VCD and SO mice using Fluorescent-Activated Cell Sorting (FACS) at timepoints across perimenopause and menopause (start of perimenopause, mid-perimenopause, start of menopause, mid-menopause, late menopause). Specifically, CD45+Sca1-α7integrin- cells were defined as immune cells, CD45-Sca1+α7integrin- cells were defined as FAPs, and CD45-Sca1-α7integrin+ cells were defined as MuSC. To analyze the relative abundance of immune cell subpopulations, we implemented imaging flow cytometry to characterize cells with conjugated fluorescent antibodies specific to cytotoxic T cells (Cd8a+) and M2 macrophages (Cd163+). A repeated-measures ANOVA was utilized to evaluate time x group effects. Results: Muscle from VCD mice contained a higher proportion of FAPs (+30.1%, p=0.001), a lower proportion of immune cells (-46.7%, p=0.032), and no changes in MuSC (-23.4%, p=0.623) compared to SO mice at all time points. At the start of menopause, we observed a decline in the percentage of Cd163+ macrophages in VCD mice but not in SO mice (-47.3%, p=0.047), with no changes in Cd8+ T cells between groups (+16.8%, p=0.056). Discussion: M2 macrophages aid in resolving inflammation and tissue repair by monitoring FAP activity. Thus, it is possible the decline in M2 macrophages results in FAP dysregulation, leading to increased fat and collagen deposition in muscle and, ultimately, menopause-associated sarcopenia. Our next steps are to evaluate muscle size, fat infiltration, and fibrosis across menopause and perimenopause. By implementing this model to study menopause, we can gain critical physiologically relevant insights into sarcopenia in the setting of female aging. NIA R01AG052978 R01AG061005, NIH T32GM008208, University of Pittsburgh Hillman Academy This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.