Canagliflozin Ameliorates Ventricular Remodeling through Apelin/Angiotensin-Converting Enzyme 2 Signaling in Heart Failure with Preserved Ejection Fraction Rats

Among patients with heart failure (HF), the proportion of patients with preserved ejection fraction (HFpEF) (ejection fraction, EF ≥50%) increased [1]. The hospitalization rate and mortality rate of such patients are high. Although drug treatment for HF has made progress, the current treatment methods are insufficient to improve the clinical manifestations and prognosis of HFpEF patients [2]. This means that there is an urgent need for treatment options for this type of HF. Ventricular remodeling plays an important role in the occurrence and progression of HF and is an important reason for poor prognoses. Inhibiting ventricular remodeling and finding relevant therapeutic targets are important research objectives for HF, especially HFpEF.

Sodium-glucose cotransporter-2 (SGLT2) inhibitors (SGLT2i) are a new type of hypoglycemic drug that can inhibit the activity of SGLT2. The protein participates in the glucose reabsorption of proximal tubules, thereby playing a hypoglycemic role [3]. SGLT2i improves the prognosis of patients with type 2 diabetes, especially those with high cardiovascular (CV) risk. The CV effects of SGLT2i have been evaluated in randomized clinical trials [4]. These have shown that SGLT2i can significantly reduce the HF hospitalization rate, CV and HF mortality risks, and the risk of major adverse CV events in patients with coronary artery disease. Moreover, these protective effects of SGLT2i have nothing to do with its hypoglycemic effects. Interestingly, SGLT2i has proven effective in patients with HFpEF [5]. Many studies are exploring the mechanisms of action of SGLT2i drugs on HFpEF. Canagliflozin (CANA) is the first SGLT2i approved by the US Food and Drug Administration [6], but its improvement mechanism of HFpEF is still unclear.

The renin-angiotensin-aldosterone system (RAAS) and the apelin-APJ receptor system are important endocrine systems that regulate CV physiological functions. Apelin was first discovered in 1998 as an endogenous peptide that can bind APJ (G protein-coupled receptor protein related to the angiotensin II [Ang II] protein 1). It was originally considered an orphan G protein-coupled receptor [7]. The apelin-APJ pathway is associated with HF. Several clinical and experimental studies have demonstrated that the apelin-APJ axis has cardiorenal protective effects in regulating fluid homeostasis, myocardial contractility, vasodilation, angiogenesis, cell differentiation, apoptosis, oxidative stress, cardiac and renal fibrosis, and dysfunction [8‒10]. Angiotensin-converting enzyme 2 (ACE2) is a key enzyme in the RAAS that maintains CV homeostasis by regulating Ang II. The Ang II/AT1R axis, a classical pathway in the RAAS system, leads to vasoconstriction, water and sodium retention, inflammatory responses, oxidative stress, myocardial fibrosis, and cardiac remodeling. ACE2 can specifically catalyze the conversion of Ang II to Ang (1–7). Ang (1–7) can negatively regulate the action of RAAS through its specific receptor MASR. Therefore, the ACE2/Ang (1–7)/MASR axis can exert its cardioprotective effects such as vasodilation, anti-inflammatory, anti-oxidative stress, and anti-cardiac remodeling by counteracting the Ang II/AT1R axis [11, 12]. It was found that ACE2 gene deletion promotes increased expression of pro-inflammatory factors and aggravates vascular inflammation and atherosclerotic process. In contrast, ACE2 overexpression reduces blood pressure levels and improves vascular endothelial function in hypertensive rats [13]. In patients with hypertension combined with acute HF or chronic HF, plasma levels of ACE2 were reduced with upregulation of Ang II and downregulation of Ang (1–7). After treatment with recombinant human ACE2 (rhACE2), plasma Ang II was downregulated and Ang (1–7) levels were increased in patients with hypertension combined with HF versus those with HF alone, suggesting that rhACE2 has important clinical applications in the treatment of hypertension [14]. Notably, rhACE2 effectively activates the ACE2/Ang (1–7)/Mas axis and regulates Ang II/Ang (1–7) production, with protective effects against acute lung injury, myocardial infarction-induced HF, and hypertensive kidney injury [15, 16]. In the process of HF, ACE2 expression increased, which changes the balance of the RAAS from the Ang (1–7)/MASR to the Ang II/AT1R, leading to the deleterious effects of HF [17]. Apelin is a positive regulator of ACE2, which binds to the promoter region of ACE2 and enhances its expression. Apelin is also a negative regulator of the Ang II/AT1R pathway during hypertension and HF [18]. Apelin upregulates ACE2, and the balance of RAAS shifts toward the Ang (1–7)/MASR axis, thereby inhibiting disease progression and exerting a protective effect. Thus, the apelin/ACE2 signaling may be a promising therapeutic target for HF. Pharmacological antagonism of the RAAS with ACE inhibitors or Ang II type 1 receptor blockers is the basis for the treatment of HF patients with reduced EF. However, some studies have shown that RAAS antagonists are also beneficial in HFpEF [19‒22].

In this study, we established a classical HFpEF model. Rats were treated with CANA and irbesartan (IRB) to evaluate the effects of SGLT2i on the apelin/ACE2 signaling and HFpEF ventricular remodeling. We further evaluated the effect of the combined application of CANA and IRB.

The tests including creatures were affirmed by the Creature Care and Administration Committee of Hebei General Hospital (permit number SYXK (JI) 2015-0065) and were following the International Regulations on the Management of Laboratory. This study was performed strictly following the National Institutes of Health Guide for the Care and Use of Laboratory Animals and was approved by the Ethics Committee of Hebei General Hospital (code: 2022086). A total of 39 male Dahl salt-sensitive (DSS) rats (weighing 180–250 g) were purchased at 7–8 weeks of age from Vital River, Beijing, China. They were maintained in the clinical research center of Hebei General Hospital on a 12/12-h light/dark cycle with a room temperature of 23–25°C and with free access to water and food. Low-salt diet (AIN-76A 0.3% NaCl) and high-salt diet (HSD) (AIN-76A 8% NaCl) were purchased from Beijing Keao Xieli Feed Co., Ltd. After a week of adaptation, the rats were randomly divided into five groups and fed for 12 consecutive weeks. The five groups were grouped as follows. (1) Control group (normal-salt diet [NSD] group, n = 7): DSS rats gotten a low-salt diet (AIN-76A + 0.3% NaCl with irradiation) and gotten intragastric administration of vehicle, hydroxypropyl methylcellulose (0.5%) (2 mL/kg/day). (2) HFpEF group (HSD group, n = 8): DSS rats gotten a HSD (AIN-76A + 8% NaCl with irradiation) and gotten intragastric administration of vehicle (0.5%) (2 mL/kg/day). (3) HSD+CANA group (n = 8): DSS rats received a HSD (AIN-76A + 8% NaCl with irradiation) and gavaged with CANA (20 mg/kg/day) dissolved in vehicle (0.5%). (4) HSD+IRB group (n = 8): DSS rats gotten a HSD (AIN-76A + 8% NaCl with irradiation) and gavaged with IRB (20 mg/kg/day) dissolved in vehicle (0.5%). (5) HSD+CANA+IRB group (n = 8): DSS rats were bolstered with a HSD (AIN-76A + 8% NaCl irradiation) and gavaged with CANA and IRB (20 mg/kg/day) dissolved in vehicle (0.5%). Rat body weight was measured weekly. The volume of drug to be gavaged was calculated based on the rat’s body weight. The dose of the vehicle hydroxypropyl methylcellulose is 2 mL/kg/day, and the volume of the vehicle to be gavaged was calculated for each rat. The drug was then dissolved in the vehicle and gavaged. This procedure was continued for 12 weeks. A schematic timeline of the experiments is shown in Figure 1.

Nutritional intake, water intake, and urine output were recorded for 24 h using a metabolic cage (SA104, Jiangsu SANS Natural Innovation Co., Ltd., China) at week 10 of the trial. Body weight was assessed on weekly.

After 1 week of acclimatization feeding, the blood pressure of DSS rats was checked using a tail sphygmomanometer (BP-2,000, Visitech Frameworks, Inc., USA). Blood pressure was measured once a week. Each rat was measured three times and the mean was taken.

Under the condition of feeding food and water, urine was collected from rats in metabolic cages for 24 h the week before the end of the experiment. After 12 weeks of high-salt feeding, rats fasted overnight and were anesthetized with 3% pentobarbital sodium (30 mg/kg) by intraperitoneal injection. Then, the whole blood was collected from the abdominal aorta, the serum was collected and stored at −80°C. A programmed biochemical analyzer (DS-261, SINNOWA Restorative Science and Innovation Co., Ltd., China) was utilized to identify serum protein, serum sodium, serum creatinine, urine sodium, and urine protein. A rodent brain natriuretic peptide (BNP) enzyme-linked immunosorbent assay kit (CSB-E07972r, CUSABIO, China) was used for the measurement of circulating BNP. Blood levels of Ang II and Ang (1–7) were measured using an enzyme-linked immunosorbent assay kit (Shanghai Jijie Biotechnology, Shanghai, China) according to the manufacturer’s instructions.

Before putting the animal to death, transthoracic echocardiography was performed using a Vevo^® 2100 Imaging System (FUJIFILM VisualSonics Inc., Toronto, Canada) under isoflurane-based anesthesia, and M-mode and two-dimensional pulse-wave Doppler images were obtained. Echocardiography was mainly used to detect evaluation of the ratio of peak E to peak A (E/A), left ventricular anterior wall end diastole, left ventricular anterior wall end systole, left ventricular posterior wall end diastole, left ventricular posterior wall end systole, left ventricular internal diameter end diastole (LVIDd), and left ventricular internal diameter end systole (LVIDs). The left ventricular EF was calculated by using the Teichholz method of estimated LV volumes. Left ventricular fractional shortening was to evaluate the systolic function of rats, and the calculation formula was LVFS% = (LVIDd–LVIDs)/LVIDd*100. All measurements were performed by two experienced technicians blinded to the experimental group of the subject animals, and the data are reported as the averages from three consecutive cycles per loop.

Total RNA was extracted with TRIzol total RNA reagent (TIANGEN, China), and cDNA was created with FastKing RT Kit (TIANGEN, China). By using SuperReal PreMix Plus (TIANGEN, China), quantitative real-time polymerase chain reaction was carried out with the following primers (GENERAL BIOL, China); apelin, forward: 5′-AGT​TTG​TGG​AGT​GCC​ACT​GAT​GT-3′ and reverse: 5′-TCC​TGG​TCC​AGT​CCT​CGA​AGT​T-3′; beta-actin, forward: 5′-CAC​CAT​GTA​CCC​AGG​CAT​TG-3′ and reverse: 5′-CCT​GCT​TGC​TGA​TCC​ACA​TC-3′. Reaction conditions were as follows: pre-denaturation at 95°C for 15 min, followed by 40 cycles of denaturation at 95°C for 10 s, and annealing and extension at 60°C for 32 s. Beta-actin served as an internal control.

Tissues were homogenized with cold radioimmunoprecipitation assay lysis buffer (Solarbio Science & Technology Co., Beijing, China), and the protein concentration of the supernatant was quantified by a BCA kit (Solarbio Science & Technology Co., Beijing, China). Fifty micrograms of protein from each sample were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electrophoretically transferred to polyvinylidene difluoride membranes. The membranes were blocked with 5% nonfat milk for 2 h at 22°C and then incubated with primary antibodies against ACE2 (1:8,000, Abcam, UK), MASR (1:4,000, Proteintech, USA), collagen I (1:1,000, Abcam, UK), and GAPDH (1:10,000, Zen BioScience, China) overnight at 4°C. Then, the membranes were incubated with horseradish peroxidase-conjugated goat anti-rabbit (1:5,000, Abcam, UK) secondary antibodies. Signals were detected by WB chemiluminescent gel imaging (MiniChemi 610 Plus, Beijing Saizhi Venture Technology Co., Ltd., China), and band intensities were quantified by ImageJ software (National Institutes of Health, Bethesda, USA).

The rats were anesthetized using sodium pentobarbital, and their hearts were collected and fixed in 4% paraformaldehyde for 24 h at 4°C. Heart tissue was then embedded in paraffin and sliced into 5-μm cross-sections. Using Masson’s trichrome stain, the myocardium and fibrotic tissues were stained red and blue, respectively. Images were collected using a multifunctional microscope (Nikon Instech Co., Ltd., Konan, Tokyo, Japan) under the same parameters. Cardiac tissue fibrosis from 3 sections in each group was calculated by Image-Pro Plus v6.0 software (Media Cybernetics, Inc.) [23]. The percentage of fibrotic tissue (blue) relative to the myocardial surface area (red + blue) was calculated to evaluate cardiac fibrosis.

For immunohistochemistry, all sections were deparaffinized at 60°C. Antigen retrieval was performed using citrate buffer (pH 6.0) for 7 min at 100°C followed by blocking with normal goat serum for 1 h. Slides were then incubated with appropriate concentrations of primary antibodies against Apelin (1:100, Abcam, UK), ACE2 (1:32,000, Abcam, UK), and MASR (1:500, Proteintech, USA) followed by incubation with corresponding secondary antibodies conjugated to horseradish peroxidase. All slides were counterstained with hematoxylin, the integral absorbance was examined under a light microscope, and the results were quantified by Image-Pro Plus 6.0 system software.

Results are presented as the mean ± standard deviation (SD) of at least three independent replicates and were analyzed by SPSS 25.0 software (SPSS, Inc., Chicago, IL, USA). One-way ANOVA and Welch variance analysis were used for comparisons of multiple groups. p < 0.05 was considered statistically significant. GraphPad Prism 8 software was used to make figures.

Table 1 lists the water and food intake, urine volume, body weight, heart weight, and heart weight to body weight ratio (HW/BW) of rats in the NSD, HSD, HSD+CANA, HSD+IRB, and HSD+CANA+IRB groups after 12 weeks of CANA and IRB drug intervention. After 12 weeks of a HSD, rats in the HSD group had increased water intake, increased heart weight, decreased body weight, and elevated HW/BW. Compared with the HSD group, rats in the HSD+CANA group had increased water intake, food intake, urine output, and decreased heart weight and body weight. Food intake was reduced and HW/BW was decreased in the HSD+IRB group compared to the HSD group. The HSD+CANA+IRB group had increased water and food intake, decreased heart weight, and decreased HW/BW compared to the HSD group. The HSD+CANA+IRB group had the lowest heart weight and HW/BW compared to the HSD+CANA+IRB group and was statistically significant. The above results were consistent with the results of the heart images of rats in each group (Fig. 2a). Compared with the normal rat heart, the heart of the HSD model group was significantly larger, and the heart size of the HSD+CANA group and the HSD+CANA+IRB group returned to the normal level. These results indicate that CANA can reduce body weight, increase urine volume, and prevent the occurrence of heart hypertrophy in HFpEF rats.

We analyzed the trend of blood pressure changes within 12 weeks (Fig. 2b–e). After 4 weeks of drug treatment, systolic and diastolic blood pressure in the HSD+CANA, HSD+IRB, and HSD+CANA+IRB groups were significantly lower than those in the HSD group. Systolic blood pressure in the HSD+CANA+IRB group was significantly lower than that in the HSD+CANA group. These trends remained until the end of the study. There was no significant difference in heart rate among the groups. The results indicate that CANA can prevent hypertension in HFpEF rats, and the combination of CANA and IRB has the best antihypertensive effect, especially on systolic blood pressure.

As shown in Figure 3, urine sodium, urine albumin, urine creatinine, and serum creatinine were significantly higher in rats in the HSD group after a HSD compared with the NSD group, and there were no significant differences in serum sodium and serum albumin. Compared with the HSD group, the three dosing groups, HSD+CANA, HSD+IRB, and HSD+CANA+IRB, had lower urine sodium, urine albumin, serum creatinine, and urine creatinine, and no significant difference in serum albumin. Serum sodium was elevated in the HSD+IRB group compared to the HSD group. In conclusion, CANA and IRB can improve renal function by reducing creatinine and urine albumin. CANA is a hypoglycemic agent. Therefore, we evaluated its effect on blood glucose in nondiabetic rats. There was no statistically significant difference in blood glucose between the five groups.

We used several key parameters measured by echocardiography to explore the structural changes in the heart. The echocardiographic correlates were as follows (Fig. 4). Compared with the control group, serum BNP was significantly elevated in the HSD group, suggesting the presence of cardiac insufficiency in the rats. The E/A ratio was decreased and the EF value was unchanged in the HSD group, suggesting left ventricular diastolic insufficiency in the rats. After drug treatment, BNP decreased and the E/A ratio increased in the HSD+CANA group, HSD+IRB group, and combined drug group, with the most significant changes in the combined drug group. The above results suggest that drug treatment can improve cardiac insufficiency and left ventricular diastolic function in rats, and the effect is more obvious after the combination of CANA and IRB. Compared with the control group, the HSD group showed signs of left ventricular diastolic dysfunction and myocardial hypertrophy with thickened ventricular walls, especially with significantly higher left ventricular anterior wall end diastole, left ventricular posterior wall end diastole, HW/BW, and left ventricular mass. These changes were reversed in the HSD+CANA group and the HSD+CANA+IRB group. There were no significant changes in LV EF and short-axis shortening in the groups. Taken together, these data suggest that CANA may improve myocardial hypertrophy and left ventricular diastolic insufficiency.

As shown in Figure 5, compared with the animals in the normal group, the rats in the HSD group showed a significant increase in collagen and myocardial fibrosis in the myocardial tissue. This phenomenon improved in all three groups of rats treated with drugs, with reduced collagen and fibrosis in myocardial tissue. There was no significant difference between the three dosing groups. In conclusion, these data indicate that both CANA and IRB can alleviate myocardial interstitial fibrosis in HFpEF rats.

Compared with the NSD group, Ang II was elevated, Ang (1–7) was decreased (Fig. 6a, b), and apelin mRNA was decreased (Fig. 6c) in the HSD group. Immunohistochemical analysis showed that the expression of apelin, ACE2, and MASR were decreased in the hearts of rats in the HSD group (Fig. 7). Western blot further confirmed that both ACE2 and MASR protein expression was decreased and collagen I protein expression was significantly increased in the hearts of rats in the HSD group (Fig. 6d–g), leading to increased myocardial fibrosis in HFpEF rats. After CANA intervention, Ang (1–7) levels were increased in the HSD+CANA group and HSD+CANA+IRB group (Fig. 6b), apelin, ACE2, and MASR were increased (Fig. 6, 7), and collagen I was decreased (Fig. 6d, g). CANA ameliorated myocardial fibrosis in HFpEF rats by increasing apelin and ACE2 levels. Ang II levels were significantly decreased in the HSD+IRB group compared with the HSD group (Fig. 6a), while apelin, ACE2, and MASR levels did not change significantly (Fig. 6, 7). Taken together, our results suggest that CANA exerts cardioprotective effects through upregulation of the apelin/ACE2 signaling pathway, which may be a therapeutic target for SGLT2i to inhibit the development of ventricular remodeling in HFpEF rats. IRB inhibits the formation of ventricular remodeling by reducing Ang II levels and inhibiting Ang II receptors. Therefore, in the present study, both CANA and IRB improved ventricular remodeling in HFpEF rats.

HF is estimated to occur in 1–12% of the adult population and is expected to increase as the population ages [24]. HF is a major public health problem related to a significant risk of death, with a 10-year survival rate of 27% [25]. In cases of HF, the proportion of patients with HFpEF continues to increase. Moreover, the hospitalization rate and mortality rate of such patients are high. The current treatment methods for HF are not good enough to improve the clinical manifestations and prognosis of HFpEF patients, so further research and exploration are still needed.

Ventricular remodeling is characterized by myocardial hypertrophy and fibrosis. Most CV diseases involve pathological myocardial remodeling characterized by cardiac fibrosis. Myocardial remodeling is not only the main cause of HF but is also directly related to the increased risk of CV death [26]. Therefore, it is necessary to explore anti-myocardial remodeling drugs and identify relevant therapeutic targets to treat HFpEF.

Current treatment methods for HF have not seen success in improving the clinical manifestations and prognosis of HFpEF patients. The EMPEROR-Reserve trial for HFpEF patients clearly showed that empagliflozin could reduce the overall risk of CV death, HF-related hospitalization, and HF emergency treatment in HFpEF patients, whether the patient had diabetes or not [27]. The American Heart Failure Management Guidelines jointly updated by ACC/AHA/HFSA issued during the 71st American College of Cardiology Annual Meeting (ACC2022) also pointed out that SGLT2i are effective at treating HFpEF. There have been many studies on the exact potential mechanism and the overall effect of SGLT2i in treating HFpEF, but they are still not fully understood.

Upregulation of the RAAS has been identified as a key pathological process of fibrosis, cardiomyocyte abnormalities, inflammation, and endothelial dysfunction, all of which are related to the deterioration of HFpEF. Animal models and clinical trials have shown that inhibition of the RAAS can improve cardiac diastolic function and downregulate these harmful processes [27], but the clinical benefits of neurohormone antagonists in HFpEF patients are uncertain. In HFpEF patients, the use of ACE inhibitors/Ang II type 1 receptor blockers is related to the improvement of prognosis, especially in terms of readmission due to HF [28]. In this study, we used DSS rats to induce an HFpEF model, intervened with CANA and IRB administration, and studied the mechanism of action of CANA. This study also explored the effects of CANA and IRB on ventricular remodeling in HFpEF rats.

The DSS rat is the first-choice model for the treatment of hypertension, salt sensitivity, and renal insufficiency. In this model, a HSD (8%) induces a significant increase in systemic blood pressure, followed by renal insufficiency, myocardial hypertrophy, and HF [29]. Cardiac diastolic dysfunction occurs in rats after 12 weeks of the HSD [30]. In this study, DSS rats were fed high salt for 12 weeks, establishing the HFpEF model. The left ventricular mass of rats increased, BNP increased, E/A was <1, and EF was >60%. This was similar to the formation of HFpEF after 12 weeks of high-salt feeding in DSS rats observed in other trials. We found that in the HFpEF model group, blood pressure, serum creatinine, and BNP increased, the ventricular wall thickened, the left ventricular mass increased significantly, fibrosis increased, and left ventricular diastolic dysfunction and cardiac remodeling occurred, leading to the formation of HFpEF. The use of CANA in rats can reduce body weight, increase urine output, reduce blood pressure, improve ventricular remodeling and function, improve myocardial fibrosis, and improve renal function. IRB can also reduce blood pressure, improve heart and kidney function, and improve myocardial fibrosis. It is worth noting that the combination of CANA and IRB has a stronger effect on lowering blood pressure and protecting the heart. SGLT2i selectively inhibits SGLT2 in renal proximal tubules, which significantly reduces the reabsorption of glucose and sodium in the urine, resulting in increased urine glucose and enhanced sodium excretion. The excretion of sodium in turn leads to an increase in osmotic diuresis [31]. Therefore, in our study, urine sodium increased significantly after the CANA intervention. However, CANA does not affect serum glucose levels, so CANA will not cause hypoglycemia in nondiabetic rats. Although urine sodium increased significantly after CANA intervention, it is interesting that CANA did not cause hyponatremia, even significantly reduced urine protein and urine creatinine, and improved renal function. We also evaluated the degree of cardio protection with the combination of CANA and IRB. The results showed that the combination of CANA and IRB was superior to drug treatment alone in reducing blood pressure and improving cardiac insufficiency and left ventricular diastolic dysfunction in rats. We further investigated the mechanism of action of CANA.

SGLT2i were originally used to treat type 2 diabetes, but growing evidence confirms that they can protect heart and kidney functions, so SGLT2i has become a focus of scientific research in recent years. SGLT2i are effective in patients with HFpEF [27]. The significant reduction in the incidence of major adverse CV events after SGLT2i intervention cannot be attributed solely to their hypoglycemic effect. Research shows that in the nondiabetic model of CV dysfunction, SGLT2i can also show benefits without hypoglycemic effects. The benefits of SGLT2i on HFpEF include weight loss, natriuretic effect, blood pressure reduction, uric acid level reduction, and extracellular fluid volume reduction [32]. SGLT2i can also protect the heart through negative sodium balance, redistribution of sodium ions [33], and influence the secretion of glucagon [5]. They can prevent left ventricular hypertrophy and fibrosis [34] and improve hemodynamics [35]. CANA can regulate energy metabolism and oxidative stress by activating the AMPK/SIRT1/PGC-1α pathway and improve cardiac hypertrophy, fibrosis, and left ventricular diastolic dysfunction caused by hypertension in DSS rats [36]. CANA can reduce iron death and improve HF in rats with preserved EF. However, there had been no experimental study on the effect of SGLT2i on apelin/ACE2 signaling in an HFpEF model [37].

Apelin, an endogenous peptide that binds to APJ, is a heart-protecting peptide in HF. Therefore, exogenous administration of apelin can improve cardiac function by increasing cardiac output and contractility in failing hearts [38]. A decreased plasma level of apelin is associated with more severe left ventricular systolic and diastolic dysfunction in hypertensive patients [39]. In the process of HF, Ang II signal transduction is activated, and ACE2 is downregulated, leading to an imbalance in the ACE2/Ang (1–7)/MASR axis and the Ang II/AT1R axis. The balance is skewed toward the Ang II/AT1R axis, bringing a series of adverse consequences, such as inflammation, oxidative stress, and fibrosis [12]. The apelin-APJ system can counteract the effect of the ACE-Ang-II-AT1R axis, and exogenous apelin negatively regulates the RAAS [40]. Interestingly, apelin has been shown to increase the activity of the ACE2 promoter in vitro and upregulate the expression of ACE2 during HF in vivo [10]. Therefore, upregulation of the apelin/ACE2 pathway can improve HF.

In this study, we found that in HFpEF rats, Ang II levels increased, Ang (1–7) levels decreased, apelin, ACE2, and MASR levels decreased, collagen levels increased, myocardial fibrosis was significant, and ventricular remodeling has been established. This shows that after activation of the RAAS in HFpEF rats, ACE2, Ang (1–7), and MASR are decreased, the ACE2/Ang (1–7)/MASR axis is out of balance with the Ang II-AT1R axis, and ventricular remodeling is formed. After CANA intervention, Ang (1–7) levels increased, apelin, ACE2, and MASR were increased, collagen was reduced, myocardial fibrosis was significantly reduced, and cardiac function was significantly improved. CANA can effectively inhibit the development of fibrosis in HFpEF rats by upregulating apelin and ACE2, upregulating ACE2/Ang (1–7)/MASR signal, and reducing the production of collagen, thus improving cardiac function. In addition, in the IRB treatment group, the changes in Apelin, ACE2, and MASR were not significant, which shows that IRB did not affect apelin/ACE2 signaling. However, IRB downregulated Ang II levels, hindered the binding of Ang II and AT1R, and inhibited the adverse consequences of the Ang II/AT1R axis, such as increased blood pressure, left ventricular diastolic dysfunction, renal dysfunction, myocardial fibrosis, and ventricular remodeling. The above also explains why the combined treatment of CANA and IRB was superior to the single-drug treatment.

In this study, we found that in the HFpEF rat model, both apelin and ACE2 were decreased, leading to the aggravation of ventricular remodeling. More importantly, we found for the first time that CANA increases the expression of apelin and ACE2, which helps reduce ventricular remodeling and injury. However, all our experimental data are limited to an animal model, so our research results need to be further confirmed in cell tests and with human patients.

In summary, CANA treatment improved hypertension in the HFpEF rodent model, alleviated left ventricular diastolic dysfunction, and inhibited the development of ventricular remodeling. The CANA upregulated apelin/ACE2 pathway may play a crucial role in this process. Therefore, CANA is a promising drug to prevent and treat HFpEF. Moreover, more clinical data are needed to verify the safety and effectiveness of CANA, and its potential mechanism also needs to be further explored.

This study was performed strictly following the National Institutes of Health Guide for the Care and Use of Laboratory Animals. This study protocol was reviewed and approved by the Ethics Committee of Hebei General Hospital, approval number [2022086].

The authors have no conflicts of interest to declare.

This work was supported by the 2019 Hebei Science and Technology Project (No. 19277787D) and the 2019 Hebei Innovation Capability Promotion Project (No. 199776249D).

Yifang Guo designed the experiments and revised the manuscript; Tingting Zhang and Xinyu Wang performed experiments, analyzed data, and wrote the manuscript; Jianlong Zhai and Zhongli Wang performed experiments; Yan Wang, Lili He, Qingjuan Zuo, Sai Ma, and Guorui Zhang were involved in supervision and data analysis. All authors critically revised the manuscript and gave final approval of the version to be submitted for publication.

Tingting Zhang and Xinyu Wang contributed equally to this work.

All data generated or analyzed during this study are included in this article. Further inquiries can be directed to the corresponding author.