The Loss of Gonadal Hormones Has a Different Impact on Aging Female and Male Mice Submitted to Heart Failure-Inducing Metabolic Hypertensive Stress

Abstract

Background: Aging and the female sex are considered risk factors for the development of heart failure with preserved ejection fraction (HFpEF). Unlike other risk factors, such as hypertension, obesity, or diabetes, they do not represent therapeutic targets.

Methods: In a recently developed two-hit murine HFpEF model (angiotensin II + high-fat diet; MHS), we studied the relative contributions of the biological sex, aging, and gonadal hormones to cardiac remodeling and function. We aimed to reproduce a frequent HFpEF phenotype in mice characterized by aging, hypertension, the female sex, menopause, and metabolic alterations. Using the MHS mouse model, we studied cardiac remodeling and function in C57Bl6/J mice of both sexes, young (12 weeks) and old (20 months), that were gonadectomized (Gx) or not.

Results: We observed that in mice, aging was associated with body weight gain, cardiac hypertrophy (CH), left ventricle (LV) concentric remodeling, and left atrial (LA) enlargement. Diastolic parameters such as E and A wave velocities were modulated by aging but only in females. Submitting young and old mice to MHS for 28 days induced the expected HFpEF phenotype consisting of CH, LV wall thickening, LA enlargement, and diastolic dysfunction with a preserved EF except for old males, in which it was significantly reduced. Young mice were Gx at five weeks, and old mice at six months (over a year before MHS). Gx increased myocardial fibrosis in MHS females and helped preserve the EF in males.

Conclusions: Our results suggest that MHS has sex-specific effects on old mice, and the loss of gonadal hormones significantly impacts the observed heart failure phenotype.

Keywords: HFpEF; aging; cardiac hypertrophy; heart failure; mouse; preclinical model; sex differences.

Figure 4

MHS in young and old mice: impact of gonadectomy. (A) Schematic representation of experimental design. (Created in BioRender. Couet, J. (2025), https://BioRender.com/aqzg5bj, accessed on 1 May 2025). (B) Body weight in young and old intact and Ovx males. (C) Body weight in young and old intact and Ovx females. (D) Heart weight of males. (E) Heart weight of females. (F) Left atrial weight of males. (G) Left atrial weight of females. Data are presented as mean +SEM (n = 7–14 per group). Two-way ANOVA was performed, followed by Holm–Sidak post-test. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001 between indicated groups.

Figure 5

Effects of age (males) and age + Ovx (females) on cardiac response to MHS. (A) Indexed heart weight (iHeart; males, left and females, right). (B) iHeart gain from MHS in % (HW/TL). (C) Indexed left atrial weight (iLA). (D) Lung water weight (difference between wet and dry weight). Echocardiography data. (E) LV wall thickness in diastole (PWd + IVSd). (F) LV relative wall thickness (LV walls/EDD; RWT). (G) End-diastolic LV diameter (EDD) and (H) ejection fraction (EF). Data are presented as mean ± SEM (n = 7–14 per group). Two-way ANOVA was conducted, followed by Holm–Sidak post-test. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001 between indicated groups. (I) B-mode LV diastolic tracings of an old control male (left; EF: 52.3%) and an old MHS male (right; EF: 40.1%).

Figure 6

Cardiac myocyte hypertrophy and myocardial interstitial fibrosis after MHS. (A) Representative images of WGA-FITC staining from LV sections of various indicated groups. (B) Cross-sectional area of cardiomyocytes quantified by WGA-FITC staining control (Ctrl; left) and MHS (middle) groups. CSA gain (% over control; right) from MHS. (C) Representative images of picrosirius red staining of old female and male heart sections (Ctrl vs. MHS). (D) Myocardial fibrosis (picrosirius red staining). Data are presented as mean ± SEM (n = 7–8 per group). Two-way ANOVA was conducted, followed by Holm–Sidak post-test. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001 between indicated groups.

Figure 1

The effects of gonadectomy (Gx) on cardiac morphology and function in young (4 months) and old (20 months) mice of both sexes. (A) A schematic representation of the experimental design (created in BioRender. Couet, J. (2025), https://BioRender.com/4zt874x, accessed on 1 May 2025. Male and female C57Bl6/J/J mice were Gx at 5 weeks or 6 months and were allowed to age until 4 and 20 months. All mice were fed a standard diet as described in Section 2. Older animals had a running device installed in their cages after Gx to prevent unwanted behavior and for environmental enrichment. (B) The body weight, (C) tibial length, and (D) indexed heart weight (iHeart) of male (blue) and female (orange) mice. Gx animals (Ocx or ovx) are represented as solid dots. Echocardiography data. (E) End-diastolic LV diameter (EDD). (F) LV wall thickness in diastole (PWd + IVSd). (G) LV relative wall thickness (LV walls/EDD; RWT), (H) The ejection fraction (EF). (I) LV stroke volume (SV), (J) Cardiac output (CO). Diastolic function. (K) Pulsed-wave Doppler E wave velocity (E wave). (L) A wave velocity and (M) E/A ratio. (N) Tissue Doppler E’ wave velocity (E’ wave), (O) E/E’ ratio, and (P) left atrial weight. The results are expressed as the mean ± standard error of the mean (SEM). A two-way ANOVA was conducted, followed by the Holm–Sidak post-test. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001 between indicated groups (n = 8–14 mice/group).

Figure 2

Effects of aging on cardiac morphology and development of myocardial interstitial fibrosis. (A) Representative M-mode echo LV tracings of young and old mice, Gx or not. (B) Representative images of picrosirius red staining of male and female heart long-axis sections for each indicated group. (C) Myocardial fibrosis (picrosirius red staining) in males (left) and females (right). (E) Left graph represents females (red), and right graph represents males (blue). (D) Col1a1, Collagen 1 α1; (E) Col3a1, Collagen 3 α1; (F) Postn, periostin; and (G) Tbsp4, thrombospondin 4. Data are represented as mean ± SEM (n = 6–8 per group). Two-way ANOVA was conducted, followed by Holm–Sidak post-test. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001 between indicated groups.

Figure 3

Plasma protein concentration of 9 immunity-related molecules in male and female mice exposed or not exposed to MHS. Levels of these molecules were evaluated as described in Section 2. CCL2: C-C Motif Chemokine Ligand 2, Csf3: Colony-Stimulating Factor 3, Ctla4: Cytotoxic T-lymphocyte associated protein 4, Cxcl9: C-X-C Motif Chemokine Ligand 9, IL-17: interleukin 17, Il6: interleukin-6, Tnfα: tumor necrosis factor alpha, Pdcd1lg2: Programmed Cell Death 1 Ligand 2. Data are represented as mean ± SEM (n = 4). F: females; M: males. Student’s t-test was performed for young and old animals. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001 between indicated groups.

Figure 7

Modulation of LV gene expression after MHS in young and old mice. (A) Nppa, atrial natriuretic peptide. (B) Nppb, brain natriuretic peptide. (C) Col1a1, Collagen 1 α1; (D) Col3a1, Collagen 3 α1; (E) Postn, periostin; and (F) Tbsp4, thrombospondin 4. Data are presented as mean ± SEM (n = 7–8 per group). Two-way ANOVA was conducted, followed by Holm–Sidak post-test. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001 between indicated groups.

Figure 8

MHS inhibits myocardial energy metabolism via inhibition of glucose utilization. A–D. Expression levels of various genes implicated in myocardial energy production. (A) CD36/FAT, fatty acid transporter; (B) Cpt1b, carnitine palmitoyl transferase 1b; (C) Glut4, glucose transporter 4; (D) Pdk4, pyruvate dehydrogenase kinase 4; (E) Bdh1, 3-Hydroxybutyrate Dehydrogenase 1; and (F). Ampkb1, AMP kinase b1. Protein content estimated by immunoblotting. (G) PDK4, (H) phospho-PDH (pyruvate dehydrogenase), and (I) PDH. (J) Representative blots of PDK4, p-PDH, PDH, and GAPDH (Glyceraldehyde 3-phosphate dehydrogenase) contents in young controls (CtrlY), old controls (CtrlO), young MHS mice (MHSY), and old MHS (MSHO) mice. Two-way ANOVA was conducted, followed by Holm–Sidak post-test. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001 between indicated groups.

Figure 9

Loss of gonadal hormones modulates cardiac response to MHS in old male mice. (A) Indexed heart weight and lung water weight (difference between wet and dry weights); (B) ejection fraction and end-diastolic LV volume (EDV). (C) Nppa and Nppb LV mRNA levels. (D) Col1a, Col3a, Postn, and Thbs4 LV mRNA levels. Two-way ANOVA was conducted, followed by Holm–Sidak post-test. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001 between indicated groups.

Figure 10

Summary of observations made in this study. In young animals, Gx reduced cardiac growth more in males than in females, and hearts of Ocx males were smaller after MHS or in old animals. Gx in females reduced cardiac growth (hypertrophy) in old animals. MHS resulted in all groups in HFpEF-like phenotype characterized by cardiac hypertrophy, LV concentric remodeling, and left atrial enlargement except in old ocx males, which evolved towards LV eccentric remodeling and lower ejection fraction (HFrEF). Created in BioRender. Couet, J. (2025) https://BioRender.com/g0dpcwr, accessed on 1 May 2025.