Blunting of cardioprotective actions of estrogen in female rodent heart linked to altered expression of cardiac tissue chymase and ACE2

Figures

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  Figure 1.. Agarose gel electrophoresis of PCR products for chymase 1 gene showing a 66 base-pair (bp) fragment from representative WKY and SHR heart tissue. M = DNA marker. PCR: polymerase chain reaction; SHR: spontaneously hypertensive rat; WKY: Wistar-Kyoto rat.

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  Figure 2.. Early mitral annular velocity (e′) and early transmitral filling velocity-to-mitral annular velocity ratio (E/e′) in SHRs and WKY rats. Data indicate differences in (a) septal e′ and (b) E/e′ with respect to strain and estrogen status (intact versus OVX). Values are reported as means ± SEM; SHRs, n = 6/group. WKY rats, n = 4–5/group. Black bars represent gonadal-intact rats; open bars represent OVX rats. Data were analyzed using two-way analysis of variance for strain and estrogen status (intact versus OVX) effects. Overall, SHRs had reduced ventricular lusitropy (e′) and higher filling pressures (E/e′) compared with WKY rats (strain effect on e′: F (1,17) = 17.14, p = 0.0008; and E/e′: F (1,17) = 5.24, p =0.035, respectively) *p < 0.05 and ***p < 0.001 indicate significance between strains. Even though estrogen loss reduced e′ and increased E/e′ in both strains (estrogen effect on e′: F (1,17) = 26.66, p < 0.0001; and E/e′: F (1,17) = 14.37, p = 0.002, respectively), the effects were most pronounced in the WKY rats, particularly e′ (interaction effect on e′: F (1,17) = 5.071, p = 0.039; and E/e′: F (1,17) = 1.622, not significant). Post-hoc analysis using Tukey’s multiple…

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  Figure 3.. Cardiac interstitial collagen deposition in SHRs and WKY female rats. (a) Quantification of cardiac interstitial collagen from histological analysis. Data indicate differences in % interstitial collagen deposition with respect to strain (SHR versus WKY) and estrogen status (intact versus OVX). Values are reported as means ± SEM; SHR, n = 4 intact and 5 OVX; WKY, n = 5 intact and 4 OVX. Black bars represent gonadal-intact rats; grey bars represent OVX rats. SHR hearts exhibited more collagen deposition than WKY hearts, irrespective of estrogen status (strain effect). There were no estrogen or interaction effects. *p < 0.05 (strain effect). (b) Representative images of LV free wall interstitial collagen deposition were taken at 20× magnification. The picrosirius red-stained slides show modest increases in collagen deposition in hypertensive (SHR) hearts when compared with normotensive (WKY) hearts. OVX: ovariectomy; SEM: standard error of the mean; SHR: spontaneously hypertensive rat; WKY: Wistar-Kyoto rat.

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  Figure 4.. Myocardial chymase, ACE, and ACE2 activities in SHR and WKY rats. Data indicate differences in (a) Chymase, (b) ACE, and (c) ACE2 enzymatic activities with respect to strain and estrogen status (intact versus OVX). Values are reported as means ± SEM; SHR, n = 5–6/group. WKY rats, n = 4–5/group. Black bars represent gonadal-intact rats; open bars represent OVX rats. Data were analyzed using two-way analysis of variance for strain and estrogen status (intact versus OVX). Main effects for estrogen status consist of: chymase activity F(1,15) = 15.41, p = 0.0013; ACE activity (F(1,16) = 4.204, p = 0.057; ACE2 activity (F(1,17) = 9.100, p = 0.0078. *p < 0.05 indicates significance between strains for ACE2 activity. Post-hoc analysis using Tukey’s multiple comparisons indicate significant difference within-strain for estrogen status #p < 0.05. ACE: angiotensin converting enzyme; OVX: ovariectomy; RAS: renin–angiotensin system; SEM: standard error of the mean; SHR: spontaneously hypertensive rat; WKY: Wistar-Kyoto rat.

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  Figure 5.. Relationship between cardiac RAS enzymatic activities and diastolic function, as defined by E/e′, in SHR and WKY rat hearts. Linear relationships (solid line) and 95% confidence intervals (dotted line) between (a) chymase activity and E/e′, (b) ACE activity and E/e′, and (c) ACE2 activity and E/e′ among SHRs (left panel) and WKY rats (right panel) are shown with regression equations and correlation coefficients, r. In SHRs, worsening diastolic function, indicated by elevations in E/e′, was inversely related to ACE2 activity (left panel (c)), while ACE and chymase activities were not significantly linked to diastolic dysfunction in this strain. In WKY rats, worsening diastolic function, or increases in E/e′, were positively linked to increasing cardiac chymase and ACE activities, respectively (right panel (a) and (b), and inversely related to ACE2 activity (right panel (c)). ACE: angiotensin converting enzyme; RAS: renin–angiotensin system; SHR: spontaneously hypertensive rat; WKY: Wistar-Kyoto rat.