https://orcid.org/0009-0002-0539-5104
Figures
Abstract
Heart failure with preserved ejection fraction (HFpEF) is a major cardiovascular disease that accounts for 50% of all cases of heart failure. Patients with HFpEF have limited therapeutic options because of the complex pathogenesis of this disease. Decreased nitric oxide (NO) levels and increased renin–angiotensin system (RAS) activity may be associated with HFpEF pathogenesis. However, whether soluble guanylate cyclase (sGC) stimulators and RAS inhibitors protect against HFpEF remains unclear. This study aimed to evaluate the preventive effects of RAS inhibitors captopril (Cap) and/or sacubitril/valsartan (Sac/Val) and sGC stimulator vericiguat (Ver) on HFpEF progression. HFpEF was induced in 8-week-old male Wistar rats through intake of L-arginine methyl ester and a high-fat diet. Results showed that the survival rate after 8 weeks of treatment was 100% in the normal diet (Cont group), Cap, and Sac/Val groups, whereas it was approximately 20% in the HFpEF and Ver groups. No significant differences in the left ventricular systolic function were found. In addition, histochemistry revealed that myocardial hypertrophy and interstitial fibrosis obviously increased in the HFpEF group but not in the Cap and Sac/Val groups compared with the Cont group. Furthermore, RNA sequencing analysis showed that the expression of genes related to inflammatory response, hypertrophy, and extracellular matrix–receptor interaction increased in the HFpEF group and decreased in the Cap and Sac/Val groups. In conclusion, early administration of Cap or Sac/Val may reduce the risk of developing HFpEF by inhibiting the RAS pathway rather than the NO-sGC-cGMP pathway.
Citation: Kono Y, Sonoda K, Ohtake K, Ota A, Yamamoto S, Nakayama H, et al. (2026) Early administration of renin–angiotensin system inhibitors improves survival and cardiac remodeling in heart failure with preserved ejection fraction. PLoS One 21(3): e0339600. https://doi.org/10.1371/journal.pone.0339600
Editor: Mohanad Alkhodari, University of Oxford, UNITED KINGDOM OF GREAT BRITAIN AND NORTHERN IRELAND
Received: September 15, 2025; Accepted: December 9, 2025; Published: March 12, 2026
Copyright: © 2026 Kono et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: The original source data (titled: raw data) for the Figures and Tables used in our manuscript is contained in the Supporting Information files. Regarding Figure 4, which was generated from our RNA-sequencing results, the raw data has been deposited in the Gene Expression Omnibus (GEO) at the following link: https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE297653.
Funding: This work was supported by a JSPS Grant-in-Aid for Research Activity Start-Up [grant number: 22K21220] and a Kinjo Gakuin University Research Grant. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Introduction
Heart failure with preserved ejection fraction (HFpEF), characterized by a left ventricular ejection fraction (LVEF) of ≥50%, accounts for approximately 50% of patients with heart failure [1]. Several therapeutic options are available for patients with heart failure with reduced ejection fraction (HFrEF), characterized by an LVEF of ≤40%, but most of them are ineffective for patients with HFpEF [2–8]. SGLT2 inhibitors alone may reduce the risk of cardiovascular death or hospitalization by approximately 20% in patients with HFpEF [9,10]. Currently, SGLT2 inhibitors are recommended to prevent heart failure in Europe and the United States [1,11]. However, patients with HFpEF do not always benefit from such therapeutics because of the complexity and heterogeneity of this disease [12]. Therefore, further evidence is required to establish a novel method to prevent HFpEF.
The onset of HFpEF is related to decreased nitric oxide (NO) levels and increased oxidative stress resulting from high-fat diet (HFD) intake [13]. NO lowers blood pressure by activating the soluble guanylate cyclase (sGC)-cyclic GMP (cGMP) system. Therefore, a decrease in NO levels enhances contraction and increases diastolic tone in the heart, leading to hypertension, cardiac hypertrophy, and fibrosis [14]. Additionally, low NO bioavailability increases renin production, which subsequently activates the renin–angiotensin system (RAS) [15]. Therefore, decreased NO levels and increased RAS activity may be involved in HFpEF pathogenesis. However, the preventive effects of sGC stimulators and/or RAS inhibitors on HFpEF remain obscure.
Thus, we aimed to evaluate the protective effects of RAS inhibitors captopril (Cap) and sacubitril/valsartan (Sac/Val) and sGC stimulator vericiguat (Ver) on a rat model of HFpEF induced by the ingestion of an NO synthase inhibitor, NG-Nitro-L-arginine methyl ester (L-NAME), and a HFD. The possible molecular mechanisms by which these drugs suppress L-NAME/HFD-induced HFpEF were also discussed.
Materials and methods
Drug
L-NAME was obtained from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). The angiotensin-converting enzyme (ACE) inhibitor Cap was obtained from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan). The angiotensin receptor–neprilysin inhibitor (ARNI) Sac/Val was purchased from Novartis Pharma K.K. (Tokyo, Japan). The sGC stimulator Ver was obtained from Bayer Yakuhin, Ltd. (Osaka, Japan).
Animal models and diets
Male Wistar rats (7-week-old) were obtained from Japan SLC, Inc. (Shizuoka, Japan). The rats were fed ad libitum with water and standard rat chow (CE-2; CLEA Japan Inc., Tokyo, Japan) for the first week and allowed to acclimatize to the environment. The rats were divided into five groups at 8 weeks of age (Fig 1A): (1) Wistar rats + CE-2 (Cont group, n = 18), (2) Wistar rats + HFD + L-NAME (HFpEF group, n = 41), (3) Wistar rats + HFD + L-NAME + Cap (Cap group, n = 9), (4) Wistar rats + HFD + L-NAME + Sac/Val (Sac/Val group, n = 10), and (5) Wistar rats + HFD + L-NAME + Ver (Ver group, n = 16). CE-2 was obtained from Funabashi Farm (Chiba, Japan), and HFD was obtained from EP Trading Co., Ltd. (Tokyo, Japan). L-NAME was dissolved in drinking water to 0.5 g/L in all groups [13]. Cap was dissolved in drinking water to 0.1 g/L. Sac/Val and/or Ver were mixed into the diet at concentrations of approximately 667 and 16.7 mg/kg, respectively. The concentration of the drug was five times the usual adult dose, referring to the animal equivalent volume from humans to rats [16]. Body weight was measured once every 2 weeks from 8 to 16 weeks of age.
(A) Experimental design. (B) EF and FS: evaluation of left ventricular ejection fraction, n = 10 in the Cont group, n = 11 in the HFpEF group, n = 7 in the Cap group, and n = 8 in the Sac/Val group. Organ weights corrected by body weight (C) for the heart (D) and lung (E), n = 18 in the Cont group, n = 27 in the HFpEF group, n = 9 in the Cap group, and n = 10 in the Sac/Val group. (F) Overall view of the left ventricle myocardium stained with Sirius red. Scale bar = 5 mm. (G) Cardiac interstitium of the left ventricle stained with Sirius red. Scale bar = 300 µm. (H) Left ventricle muscle-to-lumen ratio, n = 8 in the Cont group, n = 11 in the HFpEF group, n = 8 in the Cap group, n = 9 in the Sac/Val group. (I) Fibrosis area as a percentage of left ventricular area, n = 8 in the Cont group, n = 11 in the HFpEF group, n = 8 in the Cap group, and n = 9 in the Sac/Val group. Data are shown as the mean ± SE. *p < 0.05 vs. the Cont group, †p < 0.05 vs. the HFpEF group. Cont: control; HFpEF: heart failure with preserved ejection fraction; Cap: captopril; Sac/Val: sacubitril/valsartan; EF: ejection fraction; FS: fractional shortening; SE: standard error.
Animal care and handling were performed by research staff who had completed animal experimentation training. The animals were reared for a maximum of 8 weeks or until they exhibited the following symptoms, which were used as criteria for humane endpoint: quadriplegia, reduced mobility, decreased food intake, and loss of 5% or more of body weight within 1 week. The health and behavior of the animals were monitored daily. The animals that reached the humane endpoint were immediately and humanely euthanized under anesthesia using thiobutabarbital (Inactin®, 80 mg/kg i.p.). Of the 41 animals in the HFpEF group, 9 were euthanized and 24 died unexpectedly without displaying the criteria for humane endpoint above. Of the 16 animals in the Ver group, 7 were euthanized and 5 died unexpectedly without displaying the criteria for humane endpoint above. Most animals in the Ver group were euthanized or died; hence, no further rearing or analysis was performed because of animal distress. The mortality rate was 0% in the Cont, Cap, and Sac/Val groups.
All animal experiments were performed in compliance with the guidelines of the Kinjo Gakuin University Animal Center. The study protocol was approved by the Committee on Ethics of Animal Experiments of the Kinjo Gakuin University Animal Center (approval number: 228).
Echocardiographic analysis
All rats were intraperitoneally anesthetized using thiobutabarbital (Inactin®, 80 mg/kg i.p.; St. Louis, MO, USA) and M-mode LV echograms before dissection. Echocardiography was performed with a 12 MHz transducer (SonoScape E2V, Shoei Japan Co., Ltd, Osaka, Japan). Anterior and posterior wall thicknesses and LV internal dimensions were measured at end-diastole and end-systole by using a modification of the American Society for Echocardiography leading-edge method of obtaining at least three consecutive cardiac cycles for M-mode tracings [17–20]. The basic parameters for LV ECHO, LV end-diastolic and end-systolic dimensions, interventricular septum thickness, and LV posterior wall thickness were measured. LV systolic function was evaluated with respect to LV fractional shortening (%FS) and LVEF. LVEF was calculated using the formula of Teichholz et al.
Blood and organ analysis
At 16 weeks of age, blood samples were drawn from the inferior vena cava of all rats under anesthesia with thiobutabarbital (Inactin®, 80 mg/kg i.p.). The blood samples were centrifuged at 3,000 rpm for 10 min at 4°C, and the plasma was stored at −80°C. The heart and lungs were excised and weighed, and a portion was sectioned for histopathological analysis.
Arterial blood pressure
The rats were anesthetized with thiobutabarbital (Inactin®, 80 mg/kg, i.p.), which provides a stable anesthetic effect for 3–4 h without suppressing autonomic reflexes (depth of anesthesia was monitored using respiratory rate and tail reflex). Thiobutabarbital exerts a limited effect on cardiovascular function (heart rate, blood pressure, and arterial pH) compared with other anesthetics [21]. A cannula (INTERMEDICTM PE-50 tubing; Becton Dickinson and Company, Sparks, MD, USA) was inserted into the femoral artery to measure arterial blood pressure while the animal’s body temperature was maintained on a waterbed. Heparin (30 IU/mL) diluted in physiological saline was injected to prevent blood coagulation. The cannula was then connected to a transducer (MLT0670, BP transducer, AD Instruments, Oxford, UK), and blood pressure was continuously monitored for 10 min using the PowerLab® system (AD Instruments), with the average value calculated [22].
Histopathological analysis of the heart
The hearts were fixed in 10% formalin (FUJIFILM Wako Pure Chemical Corporation) for 48 h, paraffin-embedded, and then sectioned into 4 µm slices for histological analysis. The sections were stained with Sirius red, which stains the fibers red. Sirius red-stained tissue from the entire heart was imaged using a stereomicroscope (SZ61; Olympus, Tokyo, Japan) and an optical microscope equipped with a high-resolution video camera (CX41; Olympus, Tokyo, Japan). The captured images were used to calculate heart tissue fibrosis, LV myocardial area, LV lumen area, and LV lumen-to-total LV area ratio.
B-type natriuretic peptide (BNP) and atrial natriuretic peptide (ANP) concentration measurement
The plasma levels of BNP and ANP were determined using ELISA kits (Phoenix Pharmaceuticals, Inc., California, USA) in accordance with the manufacturer’s protocols.
Nitrite and nitrate concentration measurement
Blood samples collected for dissection were immediately immersed in liquid nitrogen and stored at –80°C until use. The samples were treated with plasma-methanol (1:1, v/v) to remove proteins and then centrifuged (10,000 × g, 5 min, room temperature). The concentrations of nitrate and nitrite ions (NOx) were measured using an HPLC system (ENO-20, Eicom, Kyoto, Japan) based on the Griess method for NOx quantification [23,24].
RNA-Seq analysis
Heart tissue (n = 5 each) samples collected during dissection were immediately immersed in RNA stabilizing solution (RNA later® solution; Ambion, Carlsbad, CA, USA) and stored at –30°C until measurement. RNA was extracted from heart tissues using ISOGEN II (Nippon Gene Co., Ltd., Tokyo, Japan). Total RNA concentration was quantified using a Dw-K2800 Nucleic Acid Analyzer (Shanghai Drawell Scientific Instrument Co., Ltd., Shanghai, China). Total RNA (800 ng) from each heart sample was converted to Illumina-compatible libraries using the NEBNext Ultra II RNA Library Prep Kit for Illumina (NEB) in accordance with the manufacturer’s protocol. RNA sequencing was performed using a NovaSeq6000 sequencer (Illumina, Inc., San Diego, CA, USA) with 20 million paired-end reads per sample. All reads were checked, and low-quality reads were removed using fastp (ver0.23.2). Raw RNA-seq reads were aligned to the rat reference genome assembly mRatBN7.2 by using STAR (ver2.7.10a). FeatureCounts (ver2.0.1) was used to calculate the counts for each gene. Counts were performed on an Ensembl Gene ID basis as defined in mRatBN7.2. Expression count data were normalized for calculation. The distance between samples was calculated from transcripts per million (TPM). Distance was defined as “1 – correlation coefficient (Spearman’s rank correlation).” Hierarchical clustering was performed using Ward’s method to visualize the dendrograms.
Either two-group comparison or multiple test was performed to identify differentially expressed genes (DEGs), which were defined as fold change (FC) > 2.0 with p-value < 0.01 (for two-group comparison) or q-value < 0.05 (for multiple test). For Gene Ontology (GO) analysis, the DEGs (defined by multiple tests) in the HFpEF group compared with those in the Cont group were further refined as FC > 1.5. GO analysis with the DEGs (FC > 1.5, q-value < 0.05 in the multiple tests; p-value < 0.01 in the two-group comparison) was performed using functional annotation in DAVID Bioinformatics (https://davidbioinformatics.nih.gov/) with the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. A heat map was constructed using HeatMapper (http://www.heatmapper.ca/). The raw RNA-seq data used in this study have been deposited in the National Center of Biotechnology Information Gene Expression Omnibus (NCBI GEO; accession no. GSE297653).
Results
Preventive administration of either Cap or Sac/Val ameliorates the HFpEF-like phenotype following HFD and L-NAME intake
Assessment of left ventricular contractile function demonstrated that the mean EF and FS in the Cont group were 87.0% and 52.2%, respectively, which were within the normal range (S1 Table). The mean EF and FS in the HFpEF group administered L-NAME and HFD were 86.8% and 52.9%, respectively, with no significant differences compared with the Cont group (Fig 1B). Similarly, the Cap and Sac/Val groups did not show significant differences in the mean EF and FS compared with either the Cont or HFpEF group (Fig 1B). The body weight in the HFpEF group was significantly lower than that in the Cont group, whereas the Cap and Sac/Val groups showed significantly suppressed weight loss induced by HFpEF (S1 Table, Fig 1C). Both heart and lung weights adjusted for body weight were significantly higher in the HFpEF group than in the Cont group (S1 Table, Fig 1D, 1E). By contrast, the increase in heart and lung weights induced by HFpEF was suppressed in the Cap and Sac/Val groups, with values comparable to those in the Cont group (S1 Table, Fig 1D, 1E). Sirius red staining of cardiac tissue showed that the left ventricular lumen ratio significantly increased in the L-NAME- and HFD-treated groups compared with the Cont group (Fig 1F, 1H). Fibrosis in the myocardial interstitial tissue, which contributes to diastolic dysfunction, also significantly increased in the L-NAME- and HFD-treated groups compared with the Cont group (Fig 1G, 1I). By contrast, the lumen ratio and fibrosis area significantly decreased in the Cap and Sac/Val groups compared with the L-NAME- and HFD-treated groups, with no significant difference compared with the Cont group (Fig 1F–1I).
Preventive administration of either Cap or Sac/Val improves survival following HFD and L-NAME intake
Survival analysis indicated that mortality began at 10 weeks of age (2 weeks after administration) in the HFpEF group. Ultimately, at 8 weeks of administration, the survival rate of the HFpEF group was approximately 20%, which was significantly lower than that of the Cont group (p < 0.001, S1 Table). Similarly, the survival rate of the Ver group 8 weeks after administration was approximately 25%, which was comparable to that of the HFpEF group (S1 Fig). By contrast, the survival rates of the Cap and Sac/Val groups were maintained at 100% (p < 0.001), which was significantly higher than that of the HFpEF group (Fig 2).
n = 18 in the Cont group, n = 41 in the HFpEF group, n = 9 in the Cap group, and n = 10 in the Sac/Val group.
Preventive administration of either Cap or Sac/Val improves increase in blood pressure following HFD and L-NAME intake
Systolic, mean, and diastolic blood pressures were significantly higher in the HFpEF group than in the Cont group (S1 Table, Fig 3). A similar significant increase in blood pressure was observed in the Cap group compared with the Cont group. By contrast, the blood pressure in the Sac/Val group was significantly lower than that in the HFpEF group and reached levels comparable to that in the Cont group (S1 Table, Fig 3).
(A) SBP. (B) MBP. (C) DBP. Data are shown as the mean ± SE; n = 14 in the Cont group, n = 10 in the HFpEF group, n = 9 in the Cap group, n = 10 in the Sac/Val group. *p < 0.05 vs. the Cont group, †p < 0.05 vs. the HFpEF group. *p < 0.05 vs. the Cont group, †p < 0.05 vs. the HFpEF group. SBP: systolic blood pressure; MBP: mean blood pressure; DBP: diastolic blood pressure; SE: standard error.
Preventive administration of either Cap or Sac/Val improves increase in plasma BNP and ANP levels following HFD and L-NAME intake
The BNP and ANP levels were significantly higher in the HFpEF group than in the Cont group (S2 Fig). By contrast, the BNP and ANP levels in the Cap group were significantly lower than those in the HFpEF group (S2 Fig). The BNP and ANP levels in the Sac/Val group were lower than those in the HFpEF group, but the difference was not statistically significant (S2 Fig).
Preventive administration of either Cap or Sac/Val improves increase in plasma NOx levels following HFD and L-NAME intake
In HFpEF, the biological availability of NO and the levels of NOx in plasma fluctuate [14]. Therefore, we decided to investigate the plasma NOx levels. HPLC analysis showed that plasma NOx levels did not significantly differ between the Cont and HFpEF groups but significantly decreased in the Cap and Sac/Val groups compared with the Cont and HFpEF groups (S3 Fig).
Preventive administration of either Cap or Sac/Val suppresses gene expression changes following HFD and L-NAME intake
A comparison between the Cont and HFpEF groups revealed 503 upregulated and 147 downregulated genes (Fig 4A, 4D). A comparison between the Cont and Cap groups showed 69 upregulated and 88 downregulated genes (Fig 4B, 4D). The Cont vs. Sac/Val group comparison revealed 165 upregulated and 212 downregulated genes, with the number of DEGs being higher than that in the Cap group (Fig 4C, 4D). Furthermore, 58 and 94 genes were upregulated in the Cap and Sac/Val groups, respectively, compared with the HFpEF group (Fig 4D). Similarly, 394 and 402 genes were downregulated in the Cap and Sac/Val groups, respectively, compared with the HFpEF group (Fig 4D).
Volcano plots of (A) Cont vs. HFpEF, (B) Cont vs. Cap, and (C) Cont vs. Sac/Val. (D) Number of differentially expressed genes (DEGs). (E) Clustering analysis between samples. Heatmap of differentially expressed genes for ECM–receptor interaction (F), hypertrophic cardiomyopathy (G), and inflammatory response (H). Of the 1,354 genes that showed the highest expression levels in the HFpEF group compared with the other groups, 1,303 genes with FC > 1.5 were used for analysis. n = 5, per group. DEGs: differentially expressed genes; ECM: extracellular matrix; FC: fold change.
Cluster analysis revealed that the Cont and HFpEF groups belonged to distinct clusters (Fig 4E). By contrast, the Cap and Sac/Val groups clustered together, with some samples from the Cap group clustering with those from the Cont group (Fig 4E).
KEGG pathway analysis using DAVID identified 62 pathways from genes that were significantly upregulated in the Cont vs. HFpEF comparison (S2 Table). Further analysis was performed by selecting genes that were (i) significantly upregulated in the HFpEF group compared with the other groups and (ii) had a count ratio (HFpEF/Cont) of ≥1.5. A total of 1,303 genes were identified, and KEGG pathway analysis revealed 104 pathways (S3 Table). Heatmaps were generated using genes related to extracellular matrix (ECM)–receptor interaction, hypertrophic cardiomyopathy, and inflammatory response (Fig 4F–4H). Treatment with Cap and Sac/Val suppressed the upregulation of these genes (e.g., IL-6, Myh7, TGF-β, and TNF-α) in the HFpEF groups to levels comparable to those in the Cont group (Fig 4F–4H). These results suggest that Cap and Sac/Val administration prevents HFpEF development by suppressing HFD-induced gene expression and L-NAME intake in rats.
Discussion
SGLT2 inhibitors are the first pharmacotherapy to improve the clinical outcomes of patients with HFpEF [9,10]. However, patients with extensive myocardial fibrosis may be less responsive to SGLT2 inhibitors [25]. Therefore, in addition to SGLT2 inhibitors, novel preventive and therapeutic strategies are necessary to improve the quality of life of patients with HFpEF. In this study, we examined the effects of RAS inhibitors Cap and Sac/Val on the pathogenesis of HFpEF in a rat model of HFpEF. We found that early administration of Cap or Sac/Val significantly improved the pathogenic conditions (survival rate, weight loss, pulmonary congestion, myocardial hypertrophy, and cardiac fibrosis) observed in the HFpEF group. Furthermore, early administration of Cap reduced the plasma levels of BNP and ANP, biomarkers of cardiac function. Sac/Val decreased plasma BNP and ANP levels, but the difference was not significant. In addition, Cap or Sac/Val suppressed the expression of genes related to inflammation, fibrosis, and/or hypertrophy, all of which were upregulated in the HFpEF group.
Concentric LV remodeling and diastolic LV dysfunction are hallmarks of the pathophysiological changes observed in patients with HFpEF, in which comorbidities are frequently accompanied by chronic inflammation before the onset of HFpEF [14]. Salah, et al. (2018) showed that administering Cap to HFpEF model animals (obese ZSF1 rats) with metabolic syndrome and chronic kidney disease suppresses blood pressure elevation, cardiac hypertrophy, and vasculitis [26]. In addition, Nordén, et al. (2021) showed that administering Sac/Val to HFpEF model rats with aortic banding improves cardiac hypertrophy to a certain extent without affecting the formation of interstitial fibrosis [27]. Administering Sac/Val to HFpEF model animals created by feeding improves both cardiac hypertrophy and fibrosis [28,29]. In the present study, early administration of Cap or Sac/Val suppressed cardiac hypertrophy and fibrosis in HFpEF model rats. This result suggests that the RAS is closely involved in the pathogenesis of HFpEF induced by NO deficiency and HFD. Thus, administration of Cap or Sac/Val partially suppresses the progression of HF, at least in the HFpEF model animals.
Natriuretic peptides are biomarkers used in the diagnosis of heart failure. Among them, BNP is released in response to increased left ventricular end-diastolic volume due to left ventricular diastolic dysfunction [30]. ANP is released when the atria are subjected to increased left ventricular end-diastolic pressure [31]. In HFpEF, left ventricular hypertrophy and fibrosis lead to diastolic dysfunction, which places stress on the atria and consequently increases ANP levels [32]. Recent studies have reported that ANP is a strong predictor of early rehospitalization in patients with HFpEF [32]. Taken together, these results indicate that elevated levels of ANP and BNP reflect impaired cardiac function. In the HFpEF group, the plasma levels of BNP and ANP were elevated, suggesting reduced cardiac function. The Cap group had significantly lower plasma ANP and BNP levels than the HFpEF group. These levels also decreased in the Sac/Val group, but the difference was not significant. These results suggest that early administration of Cap or Sac/Val improves cardiac function by suppressing HFpEF-related pathologies, such as cardiac hypertrophy and fibrosis. Furthermore, because Sac/Val inhibits neprilysin, an enzyme responsible for degrading natriuretic peptides, the lack of a significant difference between the Sac/Val and HFpEF groups may be attributable to this mechanism.
Gene expression changes in heart tissues with HFpEF are distinct from those in heart tissues with HFrEF in humans and animals [33–35]. In the present study, comprehensive RNA-seq analysis revealed distinct gene expression patterns in the heart tissues of control and HFpEF rats after 8 weeks of L-NAME/HFD ingestion. GO analysis using DAVID with KEGG pathways showed that the genes related to cellular processes including the cytoskeleton in muscle cells and inflammation including the TNF, TGF-beta, NF-kappa B, and IL-17 signaling pathways were significantly enriched in the HFpEF group compared with the Cont group. This result suggests that the activation of these pathways contributes to the pathophysiology in the HFpEF model.
Type I collagen abnormally accumulates in the heart, where constitutive fibrillar collagen formation causes ECM remodeling and subsequent fibrosis [36]. In addition, the diastolic dysfunction observed in patients with HFpEF is correlated with the amount of collagen crosslinks and expression of lysyl oxidase-like 2 (LOXL2), an enzyme that catalyzes collagen crosslinks [36]. The gene expression levels of Col1a1, Col1a2, and Loxl2 were significantly higher in the HFpEF group than in the Cont group. In addition, RNA-seq analysis revealed that the expression levels of TGF-β1 and TGF-β2, both of which positively regulate ECM synthesis and promote fibrosis [37], were upregulated in the HFpEF group.
Early administration of either Cap or Sac/Val significantly suppressed the activation of ECM–receptor interaction, hypertrophic cardiomyopathy, and inflammatory response. Clustering analysis showed that the gene expression pattern in the Cap group was more similar to that in the Cont group; therefore, Cap might more effectively suppress the gene expression observed in the HFpEF group. Ye et al. (2023) investigated the gene expression patterns in patients with HFpEF through RNA sequencing of patient-derived epicardial left ventricular biopsies. They showed an overabundance of ECM gene expression, which might be related to the basis of myocardial fibrosis, whereas no activated inflammatory signature was observed in the HFpEF group [34]. Hahn et al. (2021) independently investigated the gene expression patterns in patients with HFpEF and/or HFrEF by using patient-derived right ventricular septal endomyocardial biopsies [33]. They showed that the expression levels of genes related to fibrosis, hypertrophy, oxidative stress, and inflammation are increased in HFpEF and/or HFrEF, whereas the pathways involving protein hemostasis, endoplasmic reticulum stress, and angiogenesis are uniquely upregulated in the HFpEF group [33]. This result suggests the existence of distinct and/or common pathogenesis between HFpEF and HFrEF. Interestingly, our RNA-seq analysis showed that coagulation cascades and protein processing in the endoplasmic reticulum were significantly upregulated in the HFpEF group, in addition to the inflammation-related pathway (S3 Table). This result indicates that our HFpEF model may partly reflect the pathogenesis observed in patients with HFpEF. Since a part of gene expression was not suppressed by Cap and/or Sac/Val, combination therapy targeting signaling pathways that were not inhibited by Cap or Sac/Val may further attenuate the pathogenesis of HFpEF.
In the present study, early administration of Cap and/or Sac/Val dramatically improved the mortality rate in HFpEF model rats. In addition, administration of Cap and/or Sac/Val suppressed the weight loss and increased the heart and lung weights (compensated per body weight) in the HFpEF group. This result suggests that Cap and/or Sac/Val reduce mortality by suppressing cardiac hypertrophy and pulmonary congestion. Moreover, the blood pressure in the Cap group, but not in the Sac/Val group, was significantly higher than that in the Cont group. Furthermore, cardiac fibrosis was significantly suppressed in the Cap and Sac/Val groups compared with the HFpEF group. Sonoda et al. reported that administering Cap to L-NAME-induced hypertensive rats suppresses cardiac hypertrophy and fibrosis [22]. Stanko et al. independently reported that administering ARNI to L-NAME-induced hypertensive rats suppresses left ventricular hypertrophy and fibrosis [38]. These reports strongly suggest that treatment with Cap and Sac/Val inhibits cardiac hypertrophy and fibrosis in animal models. A recent clinical trial has shown that Sac/Val and/or ACE inhibitors/ARBs are less effective in reducing total mortality and cardiovascular mortality in patients with HFpEF [39]. They also showed that Sac/Val treatment slightly reduces hospitalization events compared with ACE inhibitor/ARB treatment [39]. A meta-analysis reported that treatment with mineralocorticoid receptor antagonists and possibly ARNI slightly reduces the risk of hospitalization due to heart failure, whereas treatment with ACEi and ARB exerts little or no benefit in patients with HFpEF [6]. This finding indicates that the RAS system is involved in the early phase of pathogenesis in the HFpEF model. Further studies (e.g., treatment set after the onset of HFpEF) are needed to clarify the effects of Cap and Sac/Val on clinical outcomes in the L-NAME/HFD-induced HFpEF model. A previous study showed that administering Cap to L-NAME-induced hypertensive rats suppresses blood pressure [22]. However, in the present study, treatment with Cap did not lower the blood pressure in HFpEF rats. Cap reportedly exerts antioxidant effects in mouse hearts [40,41], suggesting that Cap treatment attenuates the pathogenesis of HFpEF by mediating antioxidant activity in the HFpEF model. The Sac/Val may cause hypotension [39]. Therefore, treatment with Cap may be more suitable for patients with low blood pressure.
In the present study, treatment with Ver did not significantly improve the survival rate during the 8-week feeding period compared with that in the HFpEF group. Similarly, an intervention study reported that Ver does not alter the incidence of cardiovascular death compared with the placebo [42]. Typically, Ver can generate cGMP, which protects heart function by activating PKG via direct stimulation of sGC even in the absence of NO [43]. Considering the low bioavailability of NO in HFpEF, we hypothesized that Ver suppresses the pathogenesis of HFpEF by rescuing the NO-cGMP-PKG axis. Given that Ver did not improve survival rate in the HFpEF model, the NO-cGMP-PKG axis may not be directly involved in HFpEF progression. LaPenna et al. reported that administering nitrite, an NO donor, to L-NAME/HFD-induced HFpEF model mice cannot suppress oxidative stress and inflammation, whereas administering nitrite with the antihypertensive drug hydralazine suppresses increased oxidative and nitrosative stress, diastolic dysfunction, and myocardial interstitial fibrosis [44]. Although the underlying mechanism remains unclear, this report suggests that rescuing vasodilation can improve the pathogenesis of HFpEF.
The blood NOx concentration in the HFpEF group was higher than that in the Cont group, despite the administration of the NOS inhibitor L-NAME. Administering Cap and/or Sac/Val partly suppressed the increase in NO levels (S3 Fig). A previous report showed an increase in inducible NOS (iNOS) transcripts in cardiomyocytes, and iNOS expression significantly increases NO production in neonatal rat ventricular myocytes in L-NAME/HFD HFpEF model mice; thus, inhibiting iNOS improves the pathology of HFpEF [13]. This result suggests that the increased NOx levels in the HFpEF group may have mediated iNOS expression. However, our RNA-seq analysis revealed no increase in iNOS expression in the HFpEF group. Therefore, further detailed single-cell analyses are necessary. iNOS expression and subsequent NO production are induced by inflammation; thus, Cap and Sac/Val may decrease NO concentration by reducing the inflammation-related NO levels in the HFpEF group.
A possible molecular mechanism by which Cap and Sac/Val prevent the progression of HFpEF is shown in Fig 5. In the HFpEF model rats, L-NAME reduced NO bioavailability and activated RAS in the body. The expression of ACE, a key gene related to RAS, increased in the HFpEF group. Subsequently, ACE-generated Ang II increases the expression of TGF-β [45,46]. This signaling cascade further activates the proliferation of fibroblasts and cardiomyocytes by mediating intracellular signaling pathways, including MAPK, in addition to promoting excessive deposition of ECM and fibrosis [45,46]. Ang II also activates the PI3K-Akt and JAK-STAT signaling pathways [47], both of which cause inflammation, hypertrophy, and apoptosis. The administration of Cap and Sac/Val can inhibit RAS and suppress downstream signaling, thereby suppressing inflammation and fibrosis.
The decrease in NO levels in vivo following the ingestion of HFD and L-NAME decreased the activity of the NO-sGC-cGMP pathway. cGMP-activated PKG has hypotensive, anti-inflammatory, and anti-fibrotic effects; therefore, decreased activity of the NO-sGC-cGMP pathway may lead to inflammation, hypertrophy, and apoptosis, leading to the development of HFpEF. Furthermore, the decrease in NO levels in vivo converts Ang I to Ang II; hence, a decrease in NO activates RAS. Ang II increased by RAS activity increases TGF-β expression, which consequently activates the MAPK signaling pathway, leading to fibroblast proliferation, ECM overdeposition, and fibrosis. Ang II also activates the PI3K-Akt and JAK-STAT signaling pathways, which may trigger inflammation, hypertrophy, and apoptosis, leading to HFpEF development. Early administration of the RAS inhibitors Cap and Sac/Val may suppress downstream signaling by inhibiting RAS, thereby reducing inflammation and fibrosis and preventing the onset of HFpEF. NO: nitric oxide; HFD: high-fat diet; L-NAME: NG-Nitro-L-arginine methyl ester; sGC: soluble guanylate cyclase; cGMP: cyclic guanosine monophosphate; PKG: protein kinase G; Ang I: angiotensin I; Ang II: angiotensin II; RAS: renin-angiotensin system; TGF-β: transforming growth factor-β; MAPK: mitogen-activated protein kinase; ECM: extracellular matrix; PI3K: phosphatidylinositol-3 kinase; Akt: serine/threonine protein kinase; JAK: janus kinase; STAT: signal transducer and activator of transcription.
This study is limited by the small number of HFpEF model animals. Thus, caution is required when extrapolating the findings to human HFpEF pathophysiology. Furthermore, although early administration of drugs successfully prevented the development of HFpEF in rats, the best period for drug administration to treat patients with HFpEF in humans is difficult to determine. Clinical studies have shown that patients with hypertension treated with antihypertensive drugs have a lower incidence of HF than those without treatment. Therefore, further studies in HFpEF model animals and human patients are required to determine whether Cap and Sac/Val treatments can suppress the onset of HFpEF.
Conclusion
RAS inhibitors Cap and Sac/Val significantly improved the survival rate and suppressed cardiac hypertrophy and fibrosis in L-NAME/HFD-induced HFpEF rats. Cap and/or Sac/Val suppressed the upregulation of genes related to ECM homeostasis, inflammation, and hypertrophy. This study provides new treatment options for preventing HFpEF progression.
Supporting information
S1 Fig. Survival rates. n = 18 in the Cont group, n = 41 in the HFpEF group, and n = 16 in the Ver group.
Cont: control; HFpEF: heart failure with preserved ejection fraction; Ver: vericiguat.
https://doi.org/10.1371/journal.pone.0339600.s001
(PDF)
S2 Fig. Plasma levels of BNP and ANP.
Data are shown as the mean ± SE. Plasma levels of BNP; n = 4 in the Cont group, n = 8 in the HFpEF group, n = 6 in the Cap group, and n = 6 in the Sac/Val group. Plasma levels of ANP; n = 6 in the Cont group, n = 8 in the HFpEF group, n = 6 in the Cap group, and n = 5 in the Sac/Val group. *p < 0.05 vs. the Cont group, †p < 0.05 vs. the HFpEF group. Cap: captopril; Sac/Val: sacubitril/valsartan; SE: standard error.
https://doi.org/10.1371/journal.pone.0339600.s002
(PDF)
S3 Fig. Plasma levels of NOx.
Data are shown as the mean ± SE; n = 10 in the Cont group, n = 11 in the HFpEF group, n = 9 in the Cap group, and n = 10 in the Sac/Val group. *p < 0.05 vs. the Cont group, †p < 0.05 vs. the HFpEF group. Cap: captopril; Sac/Val: sacubitril/valsartan; SE: standard error.
https://doi.org/10.1371/journal.pone.0339600.s003
(PDF)
S1 Table. Metabolic parameters in HFpEF and effects of drug administration.
Data are shown as the mean ± SE. *p < 0.05 vs. the Cont group, †p < 0.05 vs. the HFpEF group. EF: ejection fraction; FS: fractional shortening; SBP: systolic blood pressure; MBP: mean blood pressure; DBP: diastolic blood pressure; SE: standard error.
https://doi.org/10.1371/journal.pone.0339600.s004
(PDF)
S2 Table. Parameter information in the KEGG pathways (analysis of upregulated genes in the Cont and HFpEF groups compared with the two groups).
https://doi.org/10.1371/journal.pone.0339600.s005
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S3 Table. Parameter information in the KEGG pathways (analysis of genes upregulated only in the HFpEF group in a multigroup analysis).
https://doi.org/10.1371/journal.pone.0339600.s006
(PDF)
Acknowledgments
The authors would like to thank Editage (www.editage.com) for the English language editing.
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