Male C57BL/6N mice (8 weeks) were from Beijing Charles River Animal Co., Ltd. (Beijing, China). L-NAME was supplied in the drinking water for the indicated periods of time, after adjusting the pH to 7.4. Mice were fed on an HFD (D12492) and given 0.5 g/l L-NAME (in drinking water) for 10 weeks to establish the HFpEF model (Schiattarella et al., 2019). The standard controls (control group) were fed on control feed and ordinary drinking water. After 10th weeks, the HFpEF mice were given 10 mg/kg empagliflozin (oral gavage), 10 mg/kg AS-IV (intraperitoneal injection), or 20 mg/kg AS-IV (intraperitoneal injection) for 4 weeks.

The tail-cuff method and an animal non-invasive blood pressure monitor (BP98AWU, Tokyo, Japan) were used to evaluate blood pressure in conscious mice. The mice were placed in a cylindrical heating sleeve and heated at 37°C for 3 min. The non-invasive blood pressure meter was used to measure the blood pressure of the mice under conscious conditions and in a quiet environment until three stable continuous measurements, including SBP and DBP, were obtained.

A Vevo2100 ultrahigh-resolution ultrasound system (VisualSonics, Canada) was used for standard transthoracic echocardiogram analysis after 28 days of drug intervention. The left ventricular parameters, including ejection fraction (EF), fractional shortening (FS), interventricular septum thickness (IVS), LV posterior wall thickness (LVPW), and LV mass, were measured via Standard B-mode and M-mode imaging. The isovolumic relaxation time (IVRT) and isovolumic contraction time (IVCT) were evaluated using a pulse wave Doppler.

The cardiac hemodynamics were monitored using the Millar pressure-volume system. The catheter was connected to a microcomputer instrument via a pressure transducer and then inserted into a 5 mL syringe filled with room-temperature PBS buffer for calibration. The anesthetized mouse, injected with 0.1 mL of bromoethanol per 20 g, was fixed on a mouse board. A longitudinal incision of about 2 cm was made from the neck, and the muscle was bluntly separated using toothless microforceps to expose the trachea. A retention needle was used for tracheal intubation. The ventilator was connected, the respiratory rate was adjusted, the chest skin was cut, the muscle was bluntly separated, and the hemostatic forceps were used to separate the third and fourth intercostal spaces bluntly. The pericardium was torn open, and the heart was extruded. The catheter was inserted into the left ventricle from the apex. After the waveform stabilized, the left ventricular systolic pressure (Pes), left ventricular diastolic pressure (Ped), left ventricular maximum rate of rise and fall (±dp/dt max), and minimum rise/fall rate (dp/dt min) were recorded.

The heart tissues were fixed using 4% paraformaldehyde for 48 h, subsequently embedded in paraffin, and sliced (4–5 μm). Histologic sections of tissues were stained with hematoxylin/eosin (H&E), masson, sirius red, and wheat germ agglutinin (WGA).

Firstly, the mice were subjected to adaptive training on a treadmill with a slope of 0°, a speed of 9 m/min, a current of 0.25 mA, and trained for 30 min. The formal experiment was conducted on the fourth day. The experimental conditions are a slope of 20°, with a speed of 9 m/min for 5 min to adapt, and then the speed is increased to 12 m/min, with a speed increase of 1 m/min every 10 min and a current of 0.25 mA. The endpoint is determined to be that the mouse cannot maintain normal running speed and state after receiving a continuous 3-s electric shock. The mouse is removed from the track and the running distance is recorded.

For TEM, heart tissues were processed for ultrastructural analysis using the method previously described (Li et al., 2020; Kirshenbaum and Singal, 1992). Left ventricles were cut into 1 mm³ random areas of the cubes. Heart cubes were fixed in 2% glutaraldehyde. Then, they were osmicated in 2% OsO4, after which ultrathin sections were stained with uranyl acetate and lead citrate and then subjected to ultrastructural examination. Pictures were taken from three random areas from three sections per mouse.

The mice were euthanized in the morning for metabolomics analysis. Metabolic profiling based on NMR measurements was performed using the method previously described (Jiang et al., 2019). The raw data from the mass spectrometer downstream were converted to mzML format by ProteoWizard, and peak extraction and retention time correction were performed using the XCMS program. Peak areas were corrected using the “SVR” method, and peaks with >50% missing were filtered in each sample group. The corrected and filtered peaks were searched in the laboratory’s database and integrated with public libraries, AI prediction libraries, and metDNA methods to obtain metabolite identification information.

Real-time quantitative PCR was performed as previously described (Kirshenbaum and Singal, 1992). The primer sequences of ANP, BNP, a-SKA, and b- MHC were synthesized by Sangon Biotech Co., Ltd. (Shanghai, China) as follows: for ANP, AAG​AAC​CTG​CTA​GAC​CAC​CTG​GAG and TGC​TTC​CTC​AGT​CTG​CTC​ACT​CAG; for BNP, TAA​CGC​ACT​GAA​GTT​GTT​GTA​GG and CGC​TAT​GTT​TAT​TAT​GTT​GTG​GC; for Collagen I, GAC​AGG​CGA​ACA​AGG​TGA​CAG​AG and CAG​GAG​AAC​CAG​GAG​AAC​CAG​GAG; for Collagen III, ACG​AGG​TGA​CAA​AGG​TGA​AAC​TGG and AGA​ACC​TGG​AGG​ACC​TGG​ATT​GC; for CTGF, CAA​AGC​AGC​TGC​AAA​TAC​CA and GGC​CAA​ATG​TGT​CTT​CCA​GT; for IL-1β, TCG​CAG​CAG​CAC​ATC​AAC​AAG​AG and AGG​TCC​ACG​GGA​AAG​ACA​CAG​G; for IL-6, CTT​CTT​GGG​ACT​GAT​GCT​GGT​GAC and AGG​TCT​GTT​GGG​AGT​GGT​ATC​CTC; for Caspase-1, ACA​ACC​ACT​CGT​ACA​CGT​CTT​GC and CCA​GAT​CCT​CCA​GCA​GCA​ACT​TC; and for GAPDH, CGGCCGCATCTTCTTGTG and CACC GACCTTCACCATTTTGT.

Western blot analysis was performed using the method previously described (Ni et al., 2020). The heart protein was extracted with lysis buffer (Solarbio, Beijing, China). Lysates were centrifuged, and supernatants were collected. The protein content of the lysates was quantified using a BCA assay kit (Thermo Fisher Scientific, United States). Protein samples were resolved by SDS-PAGE and transferred to Polyvinylidene fluoride membranes (Millipore, Bedford, MA, United States) in a wet transfer system (Bio-Rad, Hercules, CA, United States). Membranes were blocked with 5% defatted milk powder for 2 h at room temperature and subsequently immunoblotted with specific primary antibodies against MMP1, MMP2, MCP1, NLRP3, VCAM1, MPC1, GLUT1, GLUT4, Ndufb8, SDHB, UQCRC2, MTCO2, ATP5A1, and GAPDH at 1,000 times dulition (Proteintech or CST, United States) overnight. Then the membrane was incubated at room temperature for 2 h with horseradish peroxidase-conjugated secondary antibody diluted 10,000 times (Proteintech, China). Specific protein bands were visualized by enhanced chemiluminescence (Beyotime, Shanghai, China) and quantified by densitometry with an Amersham Imager 680 ultra sensitive chemiluminescence imaging instrument (GE Healthcare, United States). The relative values were corrected to GAPDH expression levels and normalized with respect to baseline controls.

PDH activity and LDH activity were examined with commercial kits (Solarbio, Beijing, China). ComplexⅠ activity was examined with commercial kits (Abbkine Scientific Co., Ltd., Wuhan, China). Hearts from mice fasted overnight were homogenized in PBS. PDH activity, LDH activity, and ComplexⅠ activity were determined as above.

The experimental processes and data analyses were carried out following randomization. Statistical analysis was performed by one-way ANOVA or Kruskal–Wallis test of repeated experiments followed by SPSS 22.0 statistical software. A p-value <0.05 was considered statistically significant.

To investigate the effect of AS-IV in HFpEF, either AS-IV (10 mg/kg/day or 20 mg/kg/day) or saline was administered daily via intraperitoneal injection. After 4 weeks of treatment, echocardiography was performed. The transverse echocardiographic evaluation showed that the EF was unchanged in all groups (Figures 1A, B). The content of NT-proBNP, a heart failure marker, significantly increased in the plasma of HFpEF mice. After treatment with AS-IV, the content of NT-proBNP was significantly reduced (Figure 1C). The pulse Doppler results showed that the E/A value of HFpEF mice was significantly reduced, and both IVCT and IVRT were prolonged. After intervention with astragaloside IV, the E/A value was significantly increased, and both IVCT and IVRT were shortened (Figures 1D–F). The LV compliance and hemodynamics were further evaluated via invasive cardiac catheterization. The maximum rise/fall rate (dp/dt max) and minimum rise/fall rate (dp/dt min) significantly decreased in HFpEF mice, indicating a loss of LV compliance (Figures 1G, H). Moreover, the reduced LV compliance was further confirmed by the increased LV pressure (Pes and Ped) in HFpEF mice (Figures 1I, J). It was found that AS-IV significantly increased dp/dt max and dp/dt min, and reduced Ped (Figures 1G–J). Furthermore, it was found that HFpEF mice showed significant hypertension compared to healthy controls. This was reflected by the significantly higher blood pressure (both systolic and diastolic) in HFpEF mice (Figures 1K, L). By contrast, systolic and diastolic blood pressure (SBP and DBP) significantly decreased after AS-IV treatment, indicating that AS-IV can prevent hypertension in HFpEF mice. Through exercise tolerance testing, it can also be seen that the motor function of HFpEF mice significantly declined, and the administration of AS-IV improved these conditions (Figure 1M). These results indicated that AS-IV relieved HFpEF cardiac function.

HFpEF mice showed pathological hypertrophy of the entire myocardium, as reflected by increased HW/TL, LV Mass/TL, IVS, and LVPW (Figures 2A–F). Plasma ANP levels significantly increased in HFpEF mice (Figure 2G). Furthermore, myocardial ANP and BNP mRNA levels significantly increased in HFpEF mice (Figures 2H, I). These heart function parameters were significantly reduced after 28 days of AS-IV treatment, indicating that AS-IV relieves HFpEF myocardial hypertrophy. WGA showed cardiomyocyte hypertrophy in HFpEF mice (Figures 2J, K). The differences between cardiomyocytes and their surface area were significantly increased. AS-IV treatments reduced the cardiomyocyte difference and significantly reduced the surface area of cardiomyocytes (Figures 2J, K). HE staining (Figure 2J) showed nucleus aggregation and disordered cell arrangement in HFpEF mice. AS-IV treatment group showed different relief in myocardial necrosis after 28 days of treatment. Masson staining and Sirius red staining showed significant collagen deposition and fibrosis in the myocardial tissue of HFpEF mice, and administration of astragaloside significantly reduced collagen deposition and fibrosis in HFpEF myocardium (Figure 2J). Moreover, AS-IV treatment also reduced the collagen area and the expression levels of collagen I and III (Figures 2L, M). Furthermore, cardiac matrix metalloproteinase protein levels (MMP-1 and MMP-2) were significantly restored in the AS-IV-treated mice compared with the model mice (Figures 2N–P).

The expression of inflammatory factors CRP, IL1RL1, and GDF15 increased in the heart of HFpEF mice, suggesting that HFpEF is associated with the activation of Systemic inflammation (Figures 3A–C). AS-IV treatment reduced CRP, IL1RL1, and GDF15 levels, indicating a slightly decreased systemic inflammatory status. HFpEF mice showed increased mRNA levels of IL-1, IL-6, and Caspase-1 in the myocardium. Western blot results showed that MCP-1, NLRP3, and VCAM-1 expression in HFpEF mice was significantly upregulated. These genes promote the production of pro-inflammatory cytokines. After administration of astragaloside IV, the expression of IL-1, IL-6, Caspase-1, NLRP3, and VCAM-1 in the myocardium of HFpEF mice was significantly reduced (Figures 3D–J).

A total of 1,598 metabolites were identified as differentially expressed metabolites (DEMs) at the metabolome level, out of which 930 were altered in HFpEF mice relative to WT mice, while 668 DEMs were restored through AS-IV treatment (Figures 4A–E). Cluster analysis showed significant changes in metabolites between different groups. The volcano and VENN plots showed that 410 differential metabolites were significantly downregulated, and 520 differential metabolites were significantly upregulated in HFpEF mice. After treatment with astragaloside IV, 351 differential metabolites were significantly downregulated, and 317 differential metabolites were significantly upregulated (Figures 4A–D). Pathway enrichment analysis based on the KEGG database was performed to evaluate the potential function of DEMs. As shown in Figure 4E, DEMs were significantly enriched in pathways related to energy metabolism, such as Arachidonic acid metabolism, Biosynthesis of unsaturated fatty acids, Inositol phosphate metabolism, TCA cycle, fatty acid biosynthesis, fatty acid elongation, and pyruvate metabolism. This suggested that AS-IV had a significant regulatory effect on energy metabolism in HFpEF mice.

The vital metabolic enzymes related to glucose and fatty acid metabolism were measured to establish the potential effects of AS-IV in the energy remodeling of HFpEF. As shown in Figures 5A–D, the expression of glucose transporter 1 (GLUT1), glucose transporter 4 (GLUT4), and mitochondrial pyruvate carrier protein 1 (MPC1) remained unchanged in HFpEF mice. The content of pyruvate and lactate, critical metabolites in the glycolysis process, was detected (Figures 5E, F). The results showed that HFpEF mice had lactate accumulation in the myocardium, and there was no significant change in lactate dehydrogenase activity, which mediates lactate production from pyruvate (Figure 5G). However, pyruvate dehydrogenase activity was significantly reduced (Figure 5H). The results showed that in HFpEF mice, the decreased pyruvate dehydrogenase activity resulted in lactate accumulation in myocardial tissue. The content of citric acid during the TCA cycle was detected. However, the model group showed a decrease compared to the normal group and an increase in citric acid content after administration; there was no significant difference (Figure 5I). After administration of AS-IV, it significantly improved the myocardial PDH activity in HFpEF mice, thereby improving the accumulation of pyruvate and lactate in myocardial tissue. Metabolites related to lipid metabolism are detected. The results showed that the levels of total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C) in the plasma of HFpEF mice were significantly increased (Figures 5J–M). After administration of AS-IV, the content of TC and HDL-C in the myocardium of HFpEF mice was significantly reduced (Figures 5J–M).

Ultrastructural aspects of the mitochondria in myocardium cells revealed structural disorders with vacuolar degeneration and edematous myocardial fibers in HFpEF mice (Figures 6A, B). Upon treatment with AS-IV, the mitochondria were neatly arranged, there was a reduction in the mitochondrial edema, and the mitochondria structure became relatively clear and intact. HFpEF mice showed a significant decline in NAD+ and ATP contents compared to control mice (Figures 6C, D); these abnormalities were restored by AS-IV treatment. To further investigate the effect of AS-IV on mitochondrial function, the expression level and activity of the complex protein of the mitochondrial electron transfer chain were detected (Figures 6E–J), and the results showed that there was no significant difference in the protein expression of the mitochondrial electron transfer chain complex among the experimental groups (Figures 6E–I). However, the activity of Complex I was significantly reduced in the model group and improved after administration of AS-IV (Figure 6K). These results indicated that AS-IV’s anti-HF effect regulated mitochondrial function and structure and alleviated mitochondrial energy metabolism dysfunction.