Sex Hormones Promote Opposite Effects on ACE and ACE2 Activity, Hypertrophy and Cardiac Contractility in Spontaneously Hypertensive Rats

Abstract

Background: There is growing interest in sex differences and RAS components. However, whether gender influences cardiac angiotensin I-converting enzyme (ACE) and angiotensin-converting enzyme 2 (ACE2) activity is still unknown. In the present work, we determined the relationship between ACE and ACE2 activity, left ventricular function and gender in spontaneously hypertensive rats (SHRs).

Methodology/principal findings: Twelve-week-old female (F) and male (M) SHRs were divided into 2 experimental groups (n = 7 in each group): sham (S) and gonadectomized (G). Fifty days after gonadectomy, we measured positive and negative first derivatives (dP/dt maximum left ventricle (LV) and dP/dt minimum LV, respectively), hypertrophy (morphometric analysis) and ACE and ACE2 catalytic activity (fluorimetrically). Expression of calcium handling proteins was measured by western blot. Male rats exhibited higher cardiac ACE and ACE2 activity as well as hypertrophy compared to female rats. Orchiectomy decreased the activity of these enzymes and hypertrophy, while ovariectomy increased hypertrophy and ACE2, but did not change ACE activity. For cardiac function, the male sham group had a lower +dP/dt than the female sham group. After gonadectomy, the +dP/dt increased in males and reduced in females. The male sham group had a lower -dP/dt than the female group. After gonadectomy, the -dP/dt increased in the male and decreased in the female groups when compared to the sham group. No difference was observed among the groups in SERCA2a protein expression. Gonadectomy increased protein expression of PLB (phospholamban) and the PLB to SERCA2a ratio in female rats, but did not change in male rats.

Conclusion: Ovariectomy leads to increased cardiac hypertrophy, ACE2 activity, PLB expression and PLB to SERCA2a ratio, and worsening of hemodynamic variables, whereas in males the removal of testosterone has the opposite effects on RAS components.

Fig 1. Initial and final systolic blood pressure.

Male sham, male orchiectomized (OQX), female sham and female ovariectomized (OVX). The results are the mean ± S.E.M. (n = 7 per group). *P < 0.05 vs. females of the same group; (two-way ANOVA and Fischer test).

Fig 2. Histological analysis of the left ventricle in SHR.

Sham and gonadectomized animals. A: number of myocyte nuclei/ high power field, B: cardiac fiber diameter. Representative images of histological analyses in C: sham male SHR, D: sham female groups, E: orchiectomized male groups and F: ovariectomized female groups. Samples were stained with hematoxylin/eosin (H&E): 400x. The results are the mean ± S.E.M. (n = 7 per group). Statistical significance is indicated by *P < 0.05 vs. females of the same group; ⁺P < 0.05 vs. sham animals of the same sex; (two-way ANOVA and Fischer test).

Fig 3. Left ventricle ACE and ACE2 activity.

(A) ACE and (B) ACE2 activity values were obtained as fluorescence arbitrary units (AFU/μg.prot.) in male and female SHR, sham and gonadectomized groups. The results are the mean ± S.E.M. (n = 7 per group). Statistical significance is indicated by*P < 0.05 vs. females of the same group; ⁺P < 0.05 vs. sham animals of the same sex; (two-way ANOVA and Fischer test).

Fig 4. Hemodynamic variables.

(A) positive first derivatives dP/dt maximum Left Ventricle (mmHg/sec) and (B) negative first derivatives dP/dt minimum (mmHg/sec) in male and female SHR, sham and gonadectomized groups. The results are the mean ± S.E.M. (n = 7 per group). *P < 0.05 vs. females of the same group; ⁺P < 0.05 vs. sham animals of the same sex; (two-way ANOVA and Fischer test).

Fig 5. Densitometric analysis of Western blots.

(A) SERCA2a; (B) phospholamban (PLB), (C) PLB to SERCA2a ratio, in hearts from male and female sham and gonadectomized rats. (SM: sham male; SF: sham female; GM: gonadectomized male; GF: gonadectomized female). The values are expressed as the means ±S.E.M (n = 6 per group). *P < 0.05 vs. females of the same group; ⁺P < 0.05 vs. sham animals of the same sex (two-way ANOVA and Fischer test).