Hypertensive BPH/2J Mice Exhibit Sex-Specific Heart Failure Progression and Late-Life Microvascular Rarefaction

Abstract

Heart failure (HF) involves structural and functional impairments in ventricular filling or blood ejection, and is a growing health burden in the United States. Sex differences in HF with mildly-reduced ejection fraction (HFmrEF) has been observed, and this condition is exacerbated by endothelial cell (EC) microvascular rarefaction. HF with preserved ejection fraction (HFpEF) is more prevalent in women, with hypertension being the major risk factor. However, the mechanisms by which hypertension contribute to HFpEF development remain poorly understood. We hypothesized that male hypertensive BPH/2J mice develop HFmrEF later in life with cardiac EC rarefaction, while female hypertensive BPH/2J mice show HFpEF. Male and female BPN/3J (control) and BPH/2J mice were assessed for blood pressure (6 weeks and 1.5 years of age). Cardiac function was assessed by echocardiography. Cardiac EC density and stem-cell antigen-1 (SCa1)⁺ cells were evaluated by immunofluorescence. BPH/2J mice exhibited cardiac dysfunction at 6 weeks of age, prior to hypertension, compared to controls. By 1.5 years of age, BPH/2J mice were hypertensive and developed HF-like features in a sex-dependent manner. Male BPH/2J mice exhibited several characteristics of HFmrEF, while female BPH/2J mice developed some features of HFpEF. Cardiac EC rarefaction was observed in male BPH/2J mice. Female BPH/2J mice (1.5-year-old) retained a significant SCa1⁺ population in coronary arteries compared to hypertensive males. These findings establish BPH/2J mice as a novel sex-specific model of hypertension-induced features of HF, revealing distinct endothelial and progenitor cell dynamics in males and females.

NEWS & NOTEWORTHY

Male hypertensive BPH/2J mice develop characteristics of HFmrEF and cardiac microvascular rarefaction, while female hypertensive BPH/2J mice recapitulate features of HFpEF. SCa1⁺ cells in the heart might play a role in left ventricular ejection fraction (LVEF) worsening. Preventing the loss of cardiac EC can be a strategy to reduce fibrosis and stimulate angiogenesis to improve cardiac repair in HF.

Introduction

Heart failure (HF) involves structural and functional impairment in ventricular filling or blood ejection. It affects ~6.7 million U.S. adults, representing a significant health and economic burden¹. Hypertension is the major preventable risk factor for HF, accounting for 20% of total HF cases². HF is commonly classified based on the left ventricular ejection fraction (LVEF) as HF with reduced ejection fraction (<40%; HFrEF), HF with mildly-reduced ejection fraction (41-49%; HFmrEF), or HF with preserved ejection fraction (≥50%; HFpEF)¹. Male sex, previous myocardial infarction, and LV hypertrophy are associated with HFrEF². However, the HFpEF phenotype is commonly observed in females and in early menopause^3,4. Conversely, studies show no clear consensus regarding the sex-based prevalence of HFmrEF^5-8.

Although animal models of HFpEF exist⁹, there are still limitations in understanding the progressive development of it, particularly when triggered by hypertension. In this context, preclinical models for HF are often based on surgery (e.g., transverse aortic constriction), genetic manipulation (e.g., leptin receptor-deficient db/db mouse strain), or treatments (e.g., high-fat diet and others)^9,10. This challenge makes it difficult to uncover novel mechanisms underlying the spontaneous development of HF induced by essential hypertension. It is well-known that microvascular rarefaction contributes to the clinical progression of HF, while stimulating angiogenesis can improve cardiac function¹¹. We hypothesized that in an animal model of essential hypertension, male BPH/2J mice would develop HFmrEF later in life accompanied by cardiac endothelial cell (EC) microvascular rarefaction, while female BPH/2J mice would develop HFpEF. In the present study, we observed that the BPH/2J mouse strain develops several features of HF, and in a sex-dependent manner.

Methods

Animals

All animal procedures were approved by the University of South Carolina School of Medicine Animal Care and Use Committee and adhered to the NIH Guide for the Care and Use of Laboratory Animals and ARRIVE guidelines. Male and female BPN/3J (RRID:IMSR_JAX:003004) and BPH/2J (RRID:IMSR_JAX:003005) mice were obtained from The Jackson Laboratory and maintained as inbred colonies. Mice were studied at either 6 weeks or 13-18 months of age (here as 1.5 years old) under a 12-hour light/dark cycle with ad libitum access to water and standard chow. Only 30% of mice were used at 13 months of age, and inclusion criteria for HF^12,13 are presented in the Supplemental Material (Figshare doi: 10.6084/m9.figshare.29510060; link: https://figshare.com/s/04fed8ba231c5c8df968) Table S1.

Echocardiography

Male and female BPN/3J and BPH/2J mice underwent transthoracic echocardiography, as previously published¹⁴. Anesthesia was maintained with 1% isoflurane, ensuring normothermia and physiological heart rates. A 40-MHz probe on VisualSonics VEVO3100 system was used to acquire 2D and M-mode recordings from standard views. Measurements, averaged over five cardiac cycles, included left ventricular (LV) inner dimensions, wall thickness, mass, and ejection fraction (LVEF, by Simpson's method)¹⁴, all adhering to murine echocardiography guidelines. Diastolic function was assessed via pulse wave doppler.

Blood Pressure Measurement by Radiotelemetry System

Male and female BPN/3J and BPH/2J mice (1.5-year-old) were anesthetized with 1% isoflurane, and positioned supine on a heated pad. Meloxicam (0.5mg/kg, s.c.) was administered pre-operatively. A small cervical incision was made to expose the left carotid artery, which was catheterized with a radiotelemetry transmitter (HD-X10-DSI). The device was placed subcutaneously, and the incision was sutured. Post-operative analgesia (meloxicam, 0.5mg/kg, s.c.) was continued for two days. Six days after surgery, baseline recordings of systolic and diastolic blood pressure (SBP and DBP, respectively), mean arterial pressure (MAP), and heart rate (HR) were collected continuously over 24-h, using Ponemah Software (DSI).

Blood Pressure Measurement by Left Carotid Catheterization

Since the telemetry device is not suitable to measure blood pressure in mice with body weight <20g (i.e., 6-week-old BPH/2J body weight: 15-18g; Table 1), we also assessed blood pressure via left carotid artery catheterization in both sexes and at both ages. BPN/3J and BPH/2J mice underwent invasive hemodynamic assessment. Under isoflurane anesthesia (1%), a heparinized catheter was inserted into the left carotid artery to record pulsatile arterial pressure for 20min, from which SBP and HR were calculated. After, blood was collected via the catheter for plasma isolation (1,000xg, 15min, 4°C) and storage. Subsequently, mice were euthanized for organ harvesting. LV were dissected for subsequent analyses. LV hypertrophy and fibrosis were evaluated by hematoxylin and eosin or Mason’s trichrome staining, respectively. Lung edema was quantitatively determined by the wet-to-dry weight ratio, and heart weight was normalized to tibial length to account for body size differences.

Table 1:

Male and female BPH/2J mice showed features of HF induced by hypertension in a sex-dependent manner.

Parameters	Male				Female
	6-week-old		1.5-year-old		6-week-old		1.5-year-old
	BPN/3J	BPH/2J	BPN/3J	BPH/2J	BPN/3J	BPH/2J	BPN/3J	BPH/2J
24h SBP (mmHg)	n.d.	n.d.	123±1.22	151±1.05#	n.d.	n.d.	115±1.51	147±2.53#
24h DBP (mmHg)	n.d.	n.d.	96±1.05	112±1.62#	n.d.	n.d.	90±1.20	118±1.73#
24h MAP (mmHg)	n.d.	n.d.	108±1.14	127±1.37#	n.d.	n.d.	103±1.35	131±1.9#
24h HR (B.P.M.)	n.d.	n.d.	535±7	583±6	n.d.	n.d.	575±7	572±6
Body weight (g)	21.5±0.9	18.7±0.5	32.9±1.2*	28.6±2.0**	17.1±0.9	15.4±0.7	34.0±1.1*	28.6±1.3**
Tibial length (cm)	1.73±0.04	1.53±0.05*	1.99±0.05*	1.78±0.04**	1.65±0.03	1.57±0.03	1.97±0.06*	1.82±0.04**
Right lung weight wet/dry (edema)	7.9±0.5	8.2±0.9	8.1±0.6	8.0±1.2	9.9±0.5	9.0±0.4	7.4±0.8*	7.4±0.5**
Heart weight/tibia length	0.07±0.001	0.10±0.005*	0.10±0.002*	0.11±0.009	0.07±0.003	0.07±0.003	0.09±0.002*	0.09±0.002**
LV mass (mg)	118±5.1	131±6.0*	214±13.6*	193±8.3**	116±10.3	127±11.8	177±21.4	178±7.1**
LV volume (d) (μL)	36.3±2.3	53.5±4.0 *	49.8±4.6	78.0±4.5#	34.7±2.0	48.2±3.1	53.3±3.2	65.7±2.5#,**
LV diameter (d) (mm)	3.0±0.1	3.5±0.2*	3.5±0.2	4.1±0.1#	3.0±0.1	3.3±0.1	3.5±0.1	3.9±0.1#,**
E/e’	37.9±2.9	43.8±4.1	39.0±1.2	54.8±4.0#,**	39.8±3.5	43.2±1.9	32.7±4.6	39.2±3.5
IVRT (ms)	22.2±1.2	21.6±1.8	21.2±1.7	26.3±2.1	24.5±1.8	24.1±2.7	23.6±2.7	26.8±2.3
ET (ms)	56.4±3.4	63.1±2.6	52.9±2.6	48.1±2.4**	60.1±2.4	64.8±4.0	56.1±3.1	49.3±1.1**
E wave (mm/s)	647.4±41.2	802.7±28.5*	765.6±38.8	623.0±42.0**	684.6±43.3	731.1±33.6	804.8±65.6	867.0±41.2**
A wave (mm/s)	355.9±27.1	459.1±33.3*	414.5±33.7	331.6±41.3	351.6±31.7	362.4±20.6	343.9±35.5	224.7±23.5
Fasting blood glucose (mg/dL)	86.3±2.1	92.9±1.6*	75.4±5.3*	123.9±13.1#,**	83.6±3.3	92.0±7.4	110.3±3.6*	114.6±4.2**
Fasting β-ketones (mM)	1.35±0.1	0.98±0.1*	1.48±0.4	0.89±0.1	1.58±0.1	1.41±0.2	0.44±0.1*	0.62±0.1**
Plasma noradrenaline (pg/mL)	122.7±16.1	92.9±18.0	80.9±11.5	38.6±9.8#	93.1±21.2	39.6±2.4	31.8±5.7*	7.5±2.3#,**
Plasma NT-proBPN (pg/mL)	136.0±4.4	125.8±2.6	122.7±1.4*	120.4±1.4	126.0±3.2	119.5±1.9	134.3±8.7	124.3±3.9

^*Different from BPN/3J 6-week-old

^#different from BPN/3J 1.5-year-old

^**different from BPH/2J 6-week-old (p<0.05). Data are presented as mean ± SEM of independent and different experimental n (n=4-14). The 24-h radiotelemetry was used only in male and female mice at 1.5 years of age to evaluate SBP, DBP, MAP and HR, and values represent the average of a 24-h measurement period (dark and light phases), in conscious (non-anesthetized) mice. Cardiac function was evaluated by transthoracic echocardiography. SBP=systolic blood pressure; DBP=diastolic blood pressure; MAP=mean arterial pressure; HR= heart rate; LV=left ventricle; d=diastole; IVRT= isovolumic relaxation time; ET=ejection time; NT-proBPN=N-terminal pro-brain natriuretic peptide; n.d.=not determined.

Fasting Blood Glucose, β-Ketones and Lipid Panel

BPN/3J and BPH/2J mice were fasted for 10h prior to measurement. Blood glucose, β-ketone levels and the lipid panel as total cholesterol, high-density lipoprotein cholesterol (HDL), triglycerides and calculated low-density lipoprotein cholesterol (LDL) were assessed using whole blood collected from the tail tip. Glucose was measured with Accu-Chek Guide strips (Roche, #07453744001), β-ketone with Precision-Xtra Blood β-Ketone strips (Abbott, #70745-65), and the lipid panel with CardioCheck Plus pts Panels (pts Diagnostics, #1710).

Confocal Microscopy and Immunofluorescence

LV sections from BPN/3J and BPH/2J mice were fixed with 4% paraformaldehyde for 20min and permeabilized with 0.1% Triton X-100 and 0.01M glycine for 1h at 37°C. After blocking, sections were incubated overnight at 4°C with either AlexaFluor 488-goat anti-mouse cluster of differentiation-31 antibody (CD31; 1:300, R&D Systems, AF3628) or rabbit anti-stem cell antigen-1 antibody (Sca1/Ly6A/E; 1:300, Boster, A30403). Subsequently, secondary antibody AlexaFluor 555-conjugated donkey anti-rabbit (1:500, BioLegend, #406412) was used for 1h at room temperature. Slides were mounted, and negative controls were incubated with secondary antibody. Images were acquired with the Leica Stellaris 5 LIAchroic confocal microscope, and analyzed using ImageJ. Fluorescence intensity was quantified (area=184.52 μm²) and reported in arbitrary units (U).

Western Blotting

Diluted plasma proteins (1:10, v/v) or 50μg of LV protein were separated on 12% SDS-PAGE gels, transferred to 0.45μm nitrocellulose membranes, and blocked for 2h. The primary antibodies were incubated, overnight, at 4°C: rabbit anti-phosphorylated-vascular endothelial (VE)-cadherin (Y685, 1:750, Abcam, ab119785), mouse anti-cardiac troponin I (1:500, Invitrogen, MA1-20112), and rabbit anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH; 1:1,000, Invitrogen, MA5-35235). After, membranes were incubated with HRP-conjugated secondary antibodies, donkey anti-rabbit (Jackson ImmunoResearch, #711-035-152) or goat anti-mouse (Jackson ImmunoResearch, #115-035-062), at 1:1,000, for 1h at room temperature. Immunoreactivity was detected using Pierce ECL Western Blotting Substrate (ThermoFisher, #32106). Ponceau S staining was used to verify transfer efficiency and normalize circulating troponin I. GAPDH served as loading control for LV proteins. Images were analyzed by ImageJ.

Circulating Noradrenaline Levels

Circulating levels of noradrenaline from BPN/3J and BPH/2J mice were measured using the Mouse Norepinephrine ELISA-Kit (MyBioSource, MBS2600834), according to the manufacturer’s instructions.

Circulating N-Terminal-Pro-Brain Natriuretic Peptide

Circulating levels of the N-terminal-Pro-Brain Natriuretic Peptide (NT-pro-BNP) from BPN/3J and BPH/2J mice were measured using the Mouse N-Terminal-Pro-Brain Natriuretic Peptide ELISA-kit (MyBiosource, MBS761753), according to the manufacturer’s instructions.

Statistical Analysis

Data analysis was performed in a blinded manner. Statistical analyses were conducted using GraphPad Prism 10 (GraphPad Software). Data are presented as mean±S.E.M. (p<0.05). Bar graphs show individual value dispersion (n). For normally distributed data (Shapiro-Wilk test), Two-way ANOVA with Tukey's post-hoc identified interaction factors and compared groups parametrically. Nonparametric data used the Kruskal-Wallis test with Dunn's correction. Significance was set at 0.05. Sample size (n) per experiment indicates independent samples.

Results

Early Cardiac Dysfunction Precedes Hypertension and Progresses to Sex-Dependent HF in BPH/2J Mice

BPH/2J mice are commonly described as a genetic model of spontaneous hypertension^15-17. Typically, SBP elevation in BPH/2J mice begins after 7 weeks of age^15-17. However, there is a lack of literature describing the cardiovascular impact of the BPH/2J genetic background before the onset of hypertension. To address this, we characterized the cardiac function of male and female BPH/2J mice prior to the onset of hypertension (6-week-old). We compared these results to older mice (1.5-year-old), when the hypertension is established, to assess this strain's potential as a model of spontaneous HF due to hypertension and aging.

At 6 weeks of age, BPH/2J had similar SBP to the BPN/3J mice (1% isoflurane), confirming they were not yet hypertensive (Figure 1). Male but not female BPH/2J mice at 6 weeks showed decreased LVEF (63.66±3.49%, n=12) compared to age-matched BPN/3J mice (81.05±1.27%, n=19). Figures 1 and 2, and Table 1 summarize the cardiac function and morphological parameters observed in mice.

Figure 1: Cardiac dysfunction in BPH/2J mice prior to hypertension is followed by sex-dependent HF-like development in later life.

The SBP and HR values of male (A and B) and female (C and D), respectively, in BPN/3J and BPH/2J mice were analyzed by left carotid artery catheterization (1% isoflurane) at 6 weeks and 1.5 years of age. Representative short-axis view on M-mode of the LV (E) from male and female mice analyzed by echocardiography (1% isoflurane). LVEF in male (F) and female (G) mice. LVFS in male (H) and female (I) mice. Data are presented as mean ± SEM. *different from BPN/3J 6-week-old; ^#different from BPN/3J 1.5-year-old; **different from BPH/2J 6-week-old (p<0.05).

Figure 2: Diastolic dysfunction in male and female BPH/2J mice.

Representative images of pulse wave doppler in the LV mitral valve (A) in male and female BPN/3J and BPH2J mice. Bar graphs in B and C represent E/A ratio in male and female mice, respectively. Isovolumic contraction time is displayed in D and E. The LV myocardial performance index values are presented in F and G. Representative Western blotting membranes (H, upper: males; bottom: females) and bar graph quantification of cardiac troponin I normalized by GAPDH (I and J). Representative membranes (upper) and bar graphs of circulating levels of troponin I normalized by the Ponceau red staining in male (K) and female (L) mice. Data are presented as mean ± SEM. *different from BPN/3J 6-week-old; ^#different from BPN/3J 1.5-year-old; **different from BPH/2J 6-week-old (p<0.05).

At 1.5 years of age, BPH/2J mice were hypertensive (Table 1; non-anesthetized 24-h radiotelemetry) and recapitulated features of HF. As observed in Figure 1E-G, male BPH/2J mice developed several features of HFmrEF (LVEF BPH/2J: 43.3±1.21%, n=13; BPN/3J: 69.4±3.56%, n=11). In contrast, female BPH/2J mice exhibited some characteristics of HFpEF (LVEF BPH/2J: 51.4±3.13%, n=12; BPN/3J: 63.4±5.60%, n=5). This study, to the best of our knowledge, represents the first report in the literature which not only describes male and female BPH/2J mice as a model of hypertension-induced features of HF, but also, highlights the impact of mouse sex on different outcomes. HR increased in male but not in female BPH/2J mice at 1.5 years of age compared to controls when assessed by carotid artery catheterization (Figure 1B and D) or echocardiography (Figure S1A and B), but not by 24-h radiotelemetry (Table 1). The male BPH/2J group also showed a decrease in LV fractional shortening (LVFS) compared to controls, while only aging affected this measurement in BPH/2J females (Figure 1H-I). Changes in the LV anterior wall (LVAW) or posterior wall (LVPW) thickness, LVID, cardiac output and stroke volume, and cardiomyocyte area are displayed in Figures S1 and S2.

Increased Cardiac and Circulating Troponin I Levels in BPH/2J Mice

Cardiac troponin is a significant prognostic indicator in patients with HF¹⁸. BPH/2J mice showed elevated cardiac troponin I levels prior to the onset of hypertension (Figures 2H-J). These cardiac markers further increased with HF-like phenotype. However, only age predicted an increase in circulating troponin I in BPH/2J mice (Figures 2K and L).

Blood glucose levels were higher in BPH/2J males compared to controls at an early age, and remained elevated even after developing characteristics of HFmrEF. However, BPH/2J females exhibited high blood glucose levels only after HFpEF-like characteristics. There were reduced values of β-ketone in young BPH/2J males, with pronounced decreases in normotensive and hypertensive females. Circulating noradrenaline decreased in both 1.5-year-old BPH/2J males and females. However, circulating NT-proBNP did not increase in either male or female BPH/2J mice (Table 1). The 1.5-year-old BPH/2J mice showed attenuation of triglycerides compared to controls (Figure S3), and only the female BPH/2J group (1.5-year-old) presented lower total cholesterol and HDL compared to controls. No differences in LDL levels were observed.

Interstitial LV and Perivascular Fibrosis in BPH/2J Mice with HFmrEF- or HFpEF-Like Characteristics

Cardiac fibrosis is prevalent in patients with HF and plays a pivotal role in disease progression¹⁹. Studies have demonstrated that cardiac fibroblasts derive from activated fibroblasts^20,21 or ECs through endothelial-to-mesenchymal transition (EndMT)²². We have previously detected EndMT in arteries from BPH/2J mice¹⁴. We observed that BPH/2J mice exhibited high fibrosis in the LV and in the perivascular region of coronary arteries (Figures 3A-E). Additionally, 6-week-old BPH/2J males but not females (Figure 3F-G) showed increased VE-cadherin phosphorylation compared to controls, suggesting its internalization and EndMT in cardiac ECs²³.

Figure 3: Cardiac fibrosis is followed by microvascular rarefaction in male but not female BPH/2J mice with HF-like characteristics.

In A, representative images of collagen staining (Mason’s Trichrome) of LV and coronary arteries from male and female BPN/3J and BPH/2J mice, at 6 weeks and 1.5 years of age. Fibrotic area quantification (%) in male (B and C) and female (D and E) BPN/3J and BPH/2J mice. Western blotting representative membranes (top) and bar graphs (bottom) of phosphorylated-VE-cadherin in male (F) and female (G) mice. GAPDH was used as loading control. Representative images (H) of cardiac ECs (CD31⁺, in green), and bar graphs show its density (% area) in male (I) and female (J) in BPN/3J and BPH/2J mice. Bar represents 50μm, and DAPI stained cell nuclei (blue). Representative images (K) of cardiac Sca1⁺ progenitor cells (gray), and bar graphs show its fluorescence intensity in male (L) and female (M) BPN/3J and BPH/2J mice. Bar represents 50μm. Comparison of the cardiac Sca1⁺ cells (N) shows higher levels in female BPH/2J with HFpEF than in male BPH/2J mice with HFmrEF. Negative control images were incubated with secondary antibodies. Data are presented as mean ± SEM. *different from BPN/3J 6-week-old; ^#different from BPN/3J 1.5-year-old; **different from BPH/2J 6-week-old (p<0.05).

Cardiac Microvascular Rarefaction Is Present in Male but not Female BPH/2J Mice

Microvascular rarefaction contributes to the clinical progression of HF, while stimulating angiogenesis can improve cardiac function¹¹. We noticed that the cardiac EC density (CD31⁺ cells) was reduced with aging, a change that was more evident in BPH/2J males (Figure 3H-J), suggesting a process of EC rarefaction. Female BPH/2J mice did not show loss of EC density, suggesting a protective mechanism in their hearts.

Female BPH/2J Mice Show High Levels of Cardiac SCa1 Progenitor Cells Compared to Male BPH/2J Mice

Evidence suggests that cardiac ECs expressing SCa1 can generate new ECs following cardiac injury^24-26. To investigate a potential mechanism preventing the loss of cardiac ECs and involving SCa1⁺ cells in BPH/2J females, we evaluated SCa1⁺ cells in the mouse heart. While BPH/2J males showed a negligible population of SCa1⁺ cells, BPH/2J females, at an early age, had higher values compared to control (Figure 3K-N). Moreover, these SCa1⁺ cells were found in the intramyocardial coronary arteries and remained elevated in BPH/2J females with features of HFpEF compared to controls.

Discussion

Nearly 6 million Americans are affected by HF, and about half are categorized as HFrEF. Despite advances in HFrEF treatments, morbidity and mortality remain high, underscoring the need for a deeper understanding of its pathophysiology²⁷. Conversely, HFpEF is a growing concern with similar mortality rates, but with limited therapeutic options. It is more common in postmenopausal women, but the role of reproductive aging and the underlying biological processes in its development are unclear²⁸. Further, the sex-specific prevalence of HFmrEF also remains unclear. Reliable animal models are crucial for unraveling the mechanisms behind sex-related differences in HF progression. To the best of our knowledge, this is the first study to demonstrate that both male and female BPH/2J mice can serve as a model of HF-like cardiac dysfunction induced by spontaneous hypertension (Figure 1). This finding represents a significant contribution to the field of cardiovascular research, offering novel insights into sex-specific HF-like phenotypes. Notably, hypertensive BPH/2J males developed several features that recapitulated HFmrEF, whereas hypertensive BPH/2J females exhibited some characteristics of HFpEF. In contrast, it is shown that hypertension-induced HF is observed in only 46% of aged spontaneously hypertensive rats, typically appearing after two years of age, and are often costly and less suited for investigating sex-dependent mechanisms²⁹.

We previously demonstrated that middle-aged hypertensive BPH/2J males and females exhibited reduced LVEF without clear evidence of LV dilation¹⁴. These impairments were accompanied by resistance artery dysfunction and evident EndMT, while estrogen deficiency did not seem to be a major contributor¹⁴. Here, we also observed that BPH/2J mice exhibit cardiac dysfunction prior to the onset of hypertension. These findings suggest a genetic predisposition in this strain that affects the cardiovascular system independently of blood pressure elevation.

Regarding the diastolic dysfunction, 1.5-year-old BPH/2J females showed elevated E/A ratio (Figure 2C), consistent with a restrictive filling pattern³⁰, while BPH/2J males exhibited elevated E/e’ ratios (Table 1), which may correspond to the severe LV dysfunction. Previous study demonstrated that patients with HF exhibit prolonged IVCT and shortened ET³¹, as observed in BPH/2J mice. Although cardiac hypertrophy is a common feature in hypertension³², we did not observe cardiomyocyte hypertrophy in BPH/2J mice (Figure S2). Conversely, Sharma and collaborators³³ observed that 18-week-old BPH/2J males had cardiomyocyte hypertrophy with no differences in fibrosis, suggesting that BPH/2J cardiac hypertrophy occurs at an early-stage of hypertension. Additionally, we did not observe changes in NT-proBNP levels, nor did the mice develop pulmonary edema (Table 1). On the other hand, blood glucose and β-ketones levels changed between the groups. Interestingly, circulating lipids did not point to dyslipidemia (Figure S2), and BPH/2J mice do not seem to be a model of metabolic syndrome, as it does not have changes in metabolic rate compared to BPN/3J or C57Bl6 mice³⁴.

Studies show that hypertension-induced eccentric cardiac remodeling leads to troponin I degradation³⁵ and cardiac overload also impact in capillary density in the LV¹¹. In contrast with male BPH/2J, the hypertensive females with HFpEF-like features did not exhibit EC rarefaction or EndMT, although they presented high cardiac troponin I levels (Figures 2 and 3). Interestingly, the preservation of the cardiac ECs in females coincided with a sustained high population of SCa1⁺ progenitors in the heart. These cells have been implicated in contributing to tissue repair through angiogenesis, and differentiation into EC or smooth muscle cells^24-26,36. In addition, a human LY6S was reported as the ortholog of the murine Ly6a gene³⁷, which is expressed in the human heart³⁸, emphasizing the potential for utilizing SCa1⁺ cells for regenerative medicine. Furthermore, VE-cadherin internalization is related to EndMT, EC dysfunction and increased permeability²³, which are common in HF^39,40. Neovascularization following myocardial injury is essential for supporting cardiomyocyte survival^41,42. Therefore, it is plausible that these SCa1⁺ cells play a protective role in maintaining vascular integrity and modulating the balance between injury and repair⁴³ in BPH/2J females, warranting further investigation.

EndMT-ECs of the coronary vasculature respond to profibrotic stimuli similarly to resident cardiac fibroblasts, after injury^19,44. A profibrotic signature of cardiac ECs isolated from adult BPH/2J mice corroborates with our findings of cardiac EC dysfunction and rarefaction⁴⁵, although 18-week-old BPH/2J mice do not show loss of cardiac ECs³³. ECs are the most abundant cell type in the heart⁴⁶, and they interact with cardiomyocytes through endothelial-derived factors that influence cardiomyocyte function and survival⁴⁷. We showed that microvascular rarefaction was a determinant on the development of HFmrEF-like features in males. Studies have demonstrated that inhibition of EndMT reduces cardiac fibrosis, hypertension¹⁴ and improves cardiac function²². However, whether fibrosis in BPH/2J hearts derives from EndMT-ECs or activated fibroblasts needs further investigation.

Some limitations to the present study included: cardiac function was assessed solely through echocardiography, and confirmation of intracardiac pressures requires further investigation; data reflecting the severity of HF, such as neurohormonal activation, myocardial ischemia, cardiopulmonary exercise testing, or cardiac magnetic resonance imaging, were not evaluated and could provide valuable insights in future studies; cardiac SCa1⁺ cells were assessed only by immunofluorescence, and further investigation is needed to clarify the role of these cells in cardiac repair.

Conclusion

Our findings establish the BPH/2J mouse as a novel model of spontaneous hypertension-induced HF-like symptoms that displays sex-dependent phenotypes reflective of the clinical HF spectrum. Male BPH/2J mice developed features of HFmrEF, while age-matched hypertensive females recapitulated some characteristics of HFpEF. Moreover, sex-specific differences in cardiac EC rarefaction and EndMT in males but not females, may underlie the divergent HF-like phenotypes observed in this model. The identification of a cardiac SCa1⁺ population in female BPH/2J mice raises intriguing questions about compensatory or pathogenic mechanisms in HFpEF-like characteristics. These data position the BPH/2J mouse as a physiologically relevant model for studying the mechanistic underpinnings of sex-specific HF-like development. This model will be instrumental in advancing our understanding of HF pathophysiology and in guiding the development of more targeted, sex-specific therapeutic strategies.

Supplementary Material

Supplemental Table S1, Supplemental Figures S1-S3: 10.6084/m9.figshare.29510060.v2

Glossary

BPH/2J

blood pressure high

BPN/3J

blood pressure normal

DBP

diastolic blood pressure

ECs

endothelial cells

ET

ejection time

IVCT

isovolumic contraction time

IVRT

isovolumic relaxation time

HF

heart failure

HFmrEF

heart failure with midly reduced ejection fraction

HFpEF

heart failure with preserved ejection fraction

HR

heart rate

LV

left ventricle/ventricular

LVEF

left ventricular ejection fraction

LVID

left ventricular inner dimension

LVAW

left ventricular anterior wall

LVPW

left ventricular posterior wall

MAP

mean arterial pressure

NT-proBPN

N-terminal pro-brain natriuretic peptide

SBP

systolic blood pressure

SCa1

stem-cell antigen-1

Data Availability

Data will be made available upon reasonable request to the corresponding authors.