Sex-Dependent Cardiac Responses to β3-Adrenergic Receptor Activation in a Murine Model of Heart Failure with Preserved Ejection Fraction

Figures

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  Figure 1. Effects of mirabegron on the mouse body and cardiac morphology with or without MHS. (A) Schematic of the experimental design (created in BioRender. Couet, J. (2025) https://BioRender.com/wmcm34z (accessed on 30 September 2025)). Eight-week-old C57BL/6J male and female mice were divided into four groups for 28 days: control mice on a standard diet receiving the vehicle; MHS mice treated with the vehicle; control mice treated with mirabegron (Mira); and MHS mice receiving Mira. (B) Indexed heart weight for tibial length (iHeart). Males: blue and left panel. Females: orange and right panel. (C) Body weight. Males: blue and upper panel. Females: orange and lower panel. (D) Tibial length. (E) Left atrial weight. (F) Cardiac hypertrophy, expressed as the ratio of heart weight in MHS mice to the average heart weight of the corresponding control group. Results are shown as mean ± standard error of the mean (SEM)—two-way ANOVA followed by Fisher’s LSD test. Two-way ANOVA results are shown below each graph, including the interaction (MHS × Mira). * p < 0.05, *** p < 0.001, and **** p < 0.0001 between indicated groups (n = 7–8 mice/group).

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  Figure 2. Mirabegron (Mira) reduces left ventricular volumes and increases EF in males but not females. (A) Diastolic LV wall thickness (posterior + septal). Males: blue and upper panel. Females: orange and the bottom panel. (B) End-diastolic LV diameter (EDD). (C) Relative wall thickness (RWT; LV walls/EDD). (D) End-diastolic volume (EDV). (E) End-systolic volume (ESV) and (F) LV ejection fraction (EF; (EDV − ESV)/EDV ∗ 100). Results are expressed as mean ± standard error of the mean (SEM)—two-way ANOVA followed by Fisher’s LSD test. The results of the two-way ANOVA are presented below the graph for each variable and the interaction (MHS × Mira). * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001 between indicated groups (n = 7–8 mice/group).

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  Figure 3. Representative M-mode echo LV tracings of control (Ctrl) and MHS male and female mice treated or not treated (Veh) with mirabegron (Mira).

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  Figure 4. Mirabegron sex-dependent pattern on lungs, liver and pathological LV hypertrophy gene expression. (A) Lung water content (wet lungs weight − dry lungs weight). (B) Liver weight. Males: blue and left panel. Females: orange and right panel. LV gene expression in control and MHS treated with or without Mira. Males: blue and upper panel. Females: orange and the bottom panel. (C) Atrial natriuretic peptide (Nppa). (D) Brain natriuretic peptide (Nppb). (E) Collagen 1 alpha (Col1a1). (F) Collagen 3 alpha (Col3a1). (G) Periostin (Postn). (H) Thrombospondin 4 (Thbs4). Results are expressed as mean ± SEM, with two-way ANOVA followed by Fisher’s LSD test. The results of the two-way ANOVA are presented below the graph for each variable and the interaction. * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001 between indicated groups (n = 7–8 mice/group).

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  Figure 5. Effects of MHS and Mira on brown fat. (A) Brown adipose tissue (BAT) scapular depot weight. Right panel: increase in BAT weight in control mice treated with Mira. Males: blue and left panel. Females: orange and right panel. (B) Representative pictures of the BAT depot in two female Mira-treated control mice (top) and two MHS mice. Scale bar: 10 mm. (C) Hematoxylin/eosin-stained BAT sections from a mouse of each group. Scale bar: 50 µm. (D) Number of nuclei per microscope field. (E) Ucp1 mRNA levels in the BAT and UCP1 protein content in the BAT in males. (F) Adrb3 gene expression. Results are expressed as mean ± SEM. The results of the two-way ANOVA are presented below the graph for each variable and the interaction (MHS × Mira). * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001 between indicated groups (n = 7–8 mice/group; 6 mice/group for the protein content).

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  Figure 6. UCP1 immunohistological detection in female BAT sections. UCP1 protein is evenly distributed in the BAT (orange-brown coloration) and is more abundant in the section from mice receiving Mira and/or MHS. Bar scale: 50 µm.

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  Figure 7. β-adrenergic receptor gene expression in the heart and myocardial morphological changes related to mirabegron treatment. (A) Beta-adrenergic receptor subtypes mRNA levels in control and MHS mice. Using bulk RNA sequencing data from the LV of young control and MHS mice (three males and three females per group; GEO accession number: GSE240171), we extracted the normalized counts of the three adrenergic receptors (Adrb1 or β1, Adrb2 or β2, and Adrb3 or β3) in control and MHS mice. (B) Cardiomyocyte cross-sectional area. (C) Representative images of wheat germ agglutinin-FITC staining from LV sections of the various indicated groups. (D) Myocardial interstitial fibrosis. (E) Representative images of picrosirius red staining of male and female LV sections. Results are expressed as mean ± SEM. The results of the two-way ANOVA are shown below each graph for each variable and the interaction (MHS × Mira). ** p < 0.01, and **** p < 0.0001 between the indicated groups (n = 7–8 mice per group).