Sex-specific cardiovascular adaptations to simulated microgravity in Sprague-Dawley rats

Figures

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  Fig. 1. The effect of 14-days of HLU on body and organ weights in 20-week-old female and male Sprague-Dawley rats. Clear bars indicate control unexposed animals; checkered bars indicate rats exposed to hindlimb unloading (HLU). Data are mean ± SEM, *p < 0.05 versus Control Female; #p < 0.05 versus HLU Female; ^p < 0.05 versus Control Male; n = 3–9 per group.

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  Fig. 2. Aortic arch pulse wave velocity (PWV) in female and male rats in response to HLU. Clear bars indicate control unexposed animals; checkered bars indicate rats exposed to hindlimb unloading (HLU) (A). Data are mean ± SEM, *p < 0.05 versus Control Female; n = 5–8 per group. PWV was calculated as PWV = D/pulse transit time Δt, where D is the distance between two points in mm (t2 and t1) in the aortic arch, and t is the signal transit time estimated between R on electrocardiogram and the beginning of the Doppler wave in ms (B–D).

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  Fig. 3. Smooth muscle (SM) contractile protein staining of aortic media. Representative images and analysis of relative intensity of α-smooth muscle alpha actin (A, C) and smooth muscle myosin (B, D) in female and male SD rats with or without HLU exposure are shown. Data are mean ± SEM, *p < 0.05, versus Female, n = 3–4 per group. Magnification: ×20.

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  Fig. 4. Effect of HLU on total collagen or elastin area, and collagen-to-elastin ratio in aortic media of SD rats. Aortas were stained for collagen by Picrosirius Red stain (A, C) and elastin by Verhoeff’s stain (B, D). Collagen-to-elastin ratio is shown on panel E. Data are mean ± SEM, n = 4–5. Magnification: ×20.

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  Fig. 5. Collagen fiber bundling in the aorta of female and male SD rats in response to HLU. Clear bars indicate control animals; checkered bars indicate rats exposed to hindlimb unloading (HLU). Picrosirius red stained slides were imaged with polarized light, and the pixel values of representative regions were binned into red, orange, yellow, and green hues, corresponding to a respective decrease in collagen bundling. The data are presented as a ratio of the number of pixels of each color category over the total across all color categories per group. Data are mean ± SEM, n = 4–7; *p < 0.05 versus Control Female.

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  Fig. 6. Collagen fiber characterization in female and male SD rats. Female and male rat aortas in both control and HLU groups were collected after 14 days of either control or HLU exposure. Picrosirius red stained slides were imaged with polarized light, with representative images from each group shown (A). ECM characteristics were analyzed using CT-FIRE software to assess the straightness, length, and width of individual fibers (B), with n = 4–7 animals per group. Fiber alignment was quantified using CurveAlign software (B). Magnification: ×20.

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  Fig. 7. GPER levels in female and male SD rats in response to HLU. Clear bars indicate control unexposed animals; checkered bars indicate rats exposed to hindlimb unloading (HLU). Representative images of GPER immunostaining (A) and data analysis (B) are shown. Data are mean ± SEM, *p < 0.05 versus Control Female, n = 4–6. Magnification: ×20.

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  Fig. 8. Aortic injury-relevant markers in female and male SD rats in response to HLU. The analysis of immunostaining for various markers relevant for aortic injury is shown (A–G). Data are mean ± SEM, *p < 0.05 versus Control Female; #p < 0.05 versus HLU Female.

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  Fig. 9. The response to GPER agonist in the female SD rats following HLU. The analysis of aortic arch PWV (A), heart rate (B), and aortic immunostaining for the markers of oxidative stress and inflammation (C–E) are shown. Data are mean ± SEM, #p < 0.05 versus HLU Female.

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  Fig. 10. Graphical representation of discrete correlate summation (DCS) analysis of data. 8-OHdG and p47phox were combined into an oxidative stress node# (box), VSMC actin, TC, VVG, CF length and CF straightness were combined into an extracellular matrix composition node# and Heart/TL, Kidney/TL, BW, % Fat AVE and % Lean mass AVE were combined into a body composition node#. Edge (line) colors represent the log correlation ratios of male (cyan) versus female (magenta) [for control and HLU groups, log(malep/femalep)] and the log correlation ratios of HLU (purple) versus control (orange) [for female and male groups, log(HLUp/controlp)]. #Multi-feature nodes can have many edges with single feature nodes.