Uncovering a Sex-Specific cAMP Landscape in Vascular Smooth Muscle

Figures

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  Figure 1:. Isolation and characterization of primary mouse aortic smooth muscle cells. A) Cartoon of ICUE and B) CUTie cAMP biosensors. C) Workflow for establishing unpassaged VSM cultures; (Ci) aorta freshly dissected from a mouse, (Cii) aorta after removal of the surrounding fat tissue, (Ciii) removal of the adventitia from the medial layer after enzyme incubation, Civ) representative image of cultured aortic smooth muscle cells (10X objective). D) Flow cytometric analyses of smooth muscle cells: (left) background fluorescence with isotype controls, (right) Smooth muscle cells identified as α-smooth muscle actin-positive, CD31-, CD45-, lineage-negative (α-SMA+/Thy-/CD31-/CD45-/Lin-). X- and Y-axes represent arbitrary units. Representative results are shown. N = 9 mice.

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  Figure 2:. Sexually dimorphic cAMP signaling domains in VSM. (n=cells/arteries; N=mice) A-C) Time courses of normalized FRET ratios in response to isoproterenol (ISO), followed by the maximal response to forskolin plus the phosphodiesterase inhibitor IBMX, and summary plots of ISO-induced FRET responses expressed as a percentage of the forskolin + IBMX response. cAMP responses were measured using targeted ICUE3 sensors at the A) plasma membrane (PM), B) cytosol (Cyt), and C) sarcoplasmic reticulum (SR) in VSMs from male and sham-operated female mice. D) Time courses and summary plots of SR-localized cAMP responses measured using the CUTie sensor in VSMs from male and sham-operated female mice. Data are presented as the mean ± SEM. Sample sizes are as follows (n=cells and N= cultures): ICUE3 experiments: male: PM (n/N= 29/5); Cyt (n/N= 22/8); SR (n/N 45/3); sham: PM (n/N = 22/7), Cyt (n/N= 21/5), SR (n/N= 51/4). CUTie experiments, male (n/N= 25/6); sham ( n/N = 17/10). P-values are shown in the figure. Statistical analysis by Kruskal-Wallis test followed by Dunn’s multiple-comparisons test.

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  Figure 3:. Role of ovarian hormone loss on compartmentalized cAMP signaling. A) Cartoon of the ovariectomy procedure. B) Representative brightfield images of vaginal cytology stained with 0.1% crystal violet from (i) a sham-operated mouse, showing nucleated and cornified epithelial cells, and (ii) an ovariectomized (ovx) mouse, showing predominantly leukocytes. Images were acquired using a 20× objective. C-F) Effects of ovariectomy on the C) uterine/body weight ratio (sham N=18; ovx N=18); and estradiol (sham N= 7; ovx N=11); D) progesterone (sham N=7; ovx N=7); and E) 20-dihydroprogesterone (20-DHP) (sham N=8; ovx N=9) (F) levels (N=mice). Data are presented as the mean ± SEM. P-values are shown in the figure; Statistical analysis: Mann-Whitney. G-J) Time courses of normalized FRET ratios in response to isoproterenol (ISO), followed by the maximal response to forskolin plus the phosphodiesterase inhibitor IBMX, and summary plots of ISO-induced FRET responses expressed as a percentage of the forskolin + IBMX response. cAMP responses were measured at the G) plasma membrane (PM), H) cytosol (Cyt), and I, J) sarcoplasmic reticulum (SR) in VSMs from male, sham-operated female, and ovx mice. The…

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  Figure 4:. Association of PDE3 and PDE4 with the SR of VSM. A) Relative expression of PDE isoforms in mesenteric arterial lysates from male (N=9), sham (N=9), and ovx (N=9) mice, presented as ΔCT values. B) Representative fluorescence images and quantification of proximity ligation assay (PLA) puncta/μm3 in acutely isolated mesenteric VSM co-labeled for C) RyR2 and PDE3 or D) RyR2 and PDE4. Representative images are shown for male (i, iv), sham-operated female (ii, v), and ovx female (iii, vi) mice. Sample size (n=cells and N= mice). RyR2-PDE3 PLA: male (n/N= 59/7); sham (n/N 28/6); ovx (n/N = 31/6). For RyR2-PDE4 PLA: male (n/N= 56/6); sham (n/N= 21/5); ovx(n/N= 57/7). E) Representative fluorescence images and quantification of PLA puncta per μm3 in unpassaged aortic vascular smooth muscle cells co-labeled for F) RyR2 and PDE3 or G) RyR2 and PDE4. Sample size: RyR2-PDE3 PLA: male (n/N=12/3); sham (n/N= 15/4); ovx ( n/N= 18/3). RyR2-PDE4 PLA: male (n/N= 18/5); sham (n/N= 19/4); ovx (n/N=18/5). Data represent mean ± SEM; P-values are shown in the figure; one-way ANOVA with Šidák’s multiple comparisons. H) The PDE4 inhibitor rolipram unmasks sarcoplasmic reticulum-localized cAMP responses in…

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  Figure 5:. Distinct SR PDE distribution is associated with altered RyR-mediated Ca2+ signaling in VSM. A-C) Representative pseudocolor images of VSM showing Ca2+ spark events and D-F) representative spark recording traces from male, sham female, and ovx mice. Summary plots show G) Ca2+ spark amplitude, H) frequency, I) rise time, J) half-width, and K) decay time in cells from male (N = 5), sham female (N = 6), and ovx (N = 6) mice. Data are presented as the mean ± SEM for each animal, with measurements for individual cells (small dots) overlaid. P-values are shown in the figure. Statistical tests were performed using the Kruskal-Wallis test, followed by Dunn’s multiple-comparisons test.

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  Figure 6:. Sexually dimorphic vasomotor response in the mesenteric arteries and thoracic aorta. A) Representative myography traces recorded in mesenteric arteries from male (top), sham female (middle), and ovx female (bottom). Summary plots of B) 60mM KCl-induced tension and C) Phe-induced tension in mesenteric arteries from male, sham female, and ovx mice. D) Concentration-response curve for ISO-induced dilation of mesenteric arteries precontracted with 2 μM Phe. Responses were normalized to the Phe-induced precontraction and are expressed as percent relaxation. Sample sizes: male 8; sham 13; ovx 23. Data are presented as the mean ± SEM. P values are indicated in the figure; one-way ANOVA with Šidák’s multiple comparisons test. For panel D, *P < 0.05, male versus sham; and #P < 0.05, sham versus ovx; Mixed-effects analysis followed by Dunnett’s multiple-comparisons test. E) Representative myography traces recorded in thoracic aorta rings from male (top), sham female (middle), and ovx female (bottom). Summary plots of F) 60 mM KCl-induced tension and G) Phe-induced tension in aortic rings from male, sham-operated female, and ovx mice. H) Concentration-response curves for ISO-induced…

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  Figure 7:. In vivo aortic pulse wave velocity (PWV) and Doppler-derived aortic flow parameters from male, sham female, and ovx females. Representative ultrasound/Doppler images of the A) distal and B) proximal aorta (top) and Doppler velocity waveforms (bottom). Bar plot of calculated C) PWV and D) heart rate. Sample size: male, 17; sham, 18; and ovx, 17. Data represent mean ± SEM. P-values are indicated in the figure. One-way ANOVA with Tukey’s multiple comparisons.