NGR1 (purity ≥98%, molecular weight = 933.14) was purchased from Aladdin Biochemical Technology Co., Ltd. (Shanghai, China). Simvastatin was purchased from Merck Sharp & Dohme Limited (U.K.). Triglyceride (TG), non-esterified fatty acids (NEFA) and 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) probe were purchased from Nanjing Jiancheng Institute of Biotechnology (Nanjing, China). CK-MB kits were obtained from Beijing Beijian Xinchuangyuan Biotechnology Co., Ltd. (Beijing, China). Cardiac troponin I (cTnI) was purchased from Wuhan Huamei Biotech Co., Ltd. (Wuhan, China). Ceramide were purchased from Ruixin Biotech Co., Ltd. (Fujian, China) and DAG were purchsaed from Mlbio Biotech Co., Ltd. (Shanghai, China). Dulbecco’s modified eagle medium (DMEM), fetal bovine serum (FBS), streptomycin and penicillin were obtained from Corning (New York, United States). Bovine serum albumin (BSA), palmitic acid (PA), LipidTOX™ neutral lipid stains, 4′,6′-Diamidino-2-phenylindole (DAPI) were purchased from Sigma (St. Louis, MO, United States). Hoechst/PI assay was obtained from Beijing Solarbio Technology Co., Ltd. (Beijing, China). The CCK-8 assay (Dojindo, Kumamoto, Japan) was obtained from Dojindo Laboratories (Kumamoto, Japan). RIPA lysis buffer, protein phosphatase inhibitor and bicinchoninic acid (BCA) assay were obtained from Beijing Pulilai Gene Technology (Beijing, China).

Forty male-ICR mice (28 ± 2 g) in SPF grade were purchased from Beijing Sibeifu Laboratory Animal Technology Co., Ltd. (Beijing, China). The model of HF was induced by coronary artery left anterior descending (LAD) artery as previously described ( Zhang et al., 2019). Briefly, the mice were weighed and anaesthetized with pentobarbital sodium (0.5%, 0.08 ml/10 g) by intraperitoneal injection, followed by tracheal intubation and ventilation with an HX-101 E constant-volume rodent ventilator (Chengdu Techman Software Co., Ltd., China). The chest cavity of mice were exposed to accomplish the ligation of LAD with 7–0 surgical line. The sham group only threaded but not ligated. Mice were randomly divided into four groups, including sham group, model group, NGR1 group (7.14 mg/kg/days) and positive drugs group (simvastatin, 2.9 mg/kg/days) after the LAD operation. In this study, the doses administered for NGR1 were converted based on rat dose equivalence (He et al., 2014), and the doses administered for the positive drug simvastatin were converted based on clinical treatment dose equivalence. All mice gavagly administered for 14 days.

Two-dimensional echocardiography (Vevo TM 2100; Visual Sonics, Toronto, ON, Canada) was used to evaluate the cardiac function. After mice were anesthetized by isoflurane, the ejection fraction (EF), fractional shortening (FS), left ventricular internal diameter at end-diastole (LVID; d) and left ventricular internal diameter at end-systole (LVID; s) of mice were detected and at least three cardiac cycles consecutively were captured.

Heart tissues were fixed in 4% paraformaldehyde, embedded in paraffin, and cut into 5 mm thick sections. The paraffin sections were stained with hematoxylin-eosin (H&E) and Masson simultaneously, then they were scanned under an optical microscope (Leica Microsystems GmbH).

The blood was extracted from the abdominal aorta under anesthesia and centrifuged at 3,000 r/min for 20 min. Serum triglyceride (TG), cholesterol (CHOL), high density lipoprotein-cholesterol (HDL-C), low density lipoprotein-cholesterol (LDL-C), non-esterified fatty acids (NEFA) and creatine kinase MB (CK-MB) were detected using automatic biochemical analyzer (HITACHI 7080, Japan). Cardiac troponin I (cTnI) was detected using Enzyme-linked immunosorbent assay (ELISA).

The clipped myocardial tissue was added to a glass homogenizer with the corresponding volume of phosphate-buffered saline (PBS) (generally at a weight-to-volume ratio of 1:9) and ground thoroughly on ice. The mixture was centrifuged at 2000 r/min for 20 min and the supernatant was carefully collected. After processing the samples, ceramide, DAG and NEFA were performed in strict accordance with the kit instructions.

H9C2 cells were purchased from China Infrastructure of Cell Line Resources (Chinese Academy of Medical Sciences, China). The cells were cultured in dulbecco’s modified eagle medium (DMEM) with 10% fetal bovine serum (FBS) and 100 mg/ml streptomycin and 100 U/mL penicillin at 37°C in a humidified incubator (Thermo, NYC, United States).

The PA solution was prepared by saponifying PA with sodium hydroxide (NAOH) and mixing it with fatty acid-free bovine serum albumin (BSA) (Yang et al., 2019). As follows, PA was added to NAOH solution and placed in thermostat water bath for 30 min at 70°C. Next, the saponification solution was mixed with 1% fatty acid-free BSA to form a 1 mM stock solution, which was diluted with DMEM containing 5% FBS for use.

PA was used to stimulate H9C2 cells, which is the most commonly used method to induce lipotoxicity in vitro (Moscoso et al., 2019; Chang et al., 2021; Li et al., 2021). When H9C2 cell density reached 70–80%, the medium was removed and they were stimulated with 50–800 μmol/L PA for 24 h. In subsequent experiments, H9C2 cells were divided into four groups: normal group, PA (100 μmol/L) group, NGR1 (40 μmol/L) group and NGR1+Compound C (10 μmol/L) group.

The CCK-8 assay was used to determine cell viability. Briefly, after different treatments, the original medium was discarded and CCK-8 solution was added to each well and incubated for 2 h at 37°C in the dark. Then cell viability was measured at 450 nm using a microplate reader (Thermo, NYC, United States).

After the cells were treated, cells were fixed with 4% paraformaldehyde for 15 min at room temperature. Then, the LipidTOX™ neutral lipid stains was performed at a dilution ratio of 1:1,000 for at least 30 min, followed by 3 times washes in PBS, then nuclei were stained with 4′,6′- Diamidino-2-phenylindole (DAPI) for 15 min and photographed after 3 times washes in PBS. Images were collected using a laser confocal microscopy (Leica Microsystems GmbH) and analyzed with ImageJ software (National Institutes of Health).

Cells were collected, adequately lysed and fragmented after treatment. NEFA and TG assay were performed in strict accordance with the manufacturer’s instructions.

Hoechst/PI staining was used to assess cell apoptosis. After treatment, cells were co-stained with Hoechst 33,342 and PI for 20 min at 4°C in the dark and subsequently photographed by a laser confocal microscopy. ImageJ software was used to analyze fluorescent images.

According to the manufacturer’s instructions, the intracellular ROS levels were measured by the ROS assay. After different stimuli, cells were incubated with 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) probe for 30 min at 37°C in the dark, and finally washed 3 times with PBS and photographed. The fluorescence images of intracellular ROS obtained by confocal microscopyImages were acquired by a laser confocal microscopy and analyzed with ImageJ software.

Total protein was obtained from myocardial tissue and H9C2 cells using pre-cold RIPA lysis buffer and 1% protein phosphatase inhibitor and protein concentration was measured by bicinchoninic acid (BCA) assay. Subsequently, protein samples were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes. After that, PVDF membranes were blocked in 5% milk, incubated with primary antibodies at 4°C overnight, incubated with secondary antibody for 1 h at room temperature and exposed to the ECL in gel imager. Image-Lab software was used to analyze data. The antibodies were used as follows: rabbit anti-AMPKα (5,831, Cell Signaling Technology, Germany), rabbit anti-AMPK-phosphor-T172 (CY5608, Abways, China), rabbit anti-SPTLC1 (DF12752, Affinity, China), rabbit anti-SPTLC2 (DF12231, Affinity, China), mouse anti-GPAT (sc-398135, Santa cruz, China), rabbit anti-CPT-1A (DF12004, Affinity, China), rabbit anti-phosphor-AKT (CY6569, Abways, China), rabbit anti-AKT (CY5551, Abways, China), mouse anti-Bcl-2 (AB3359, Abways, China), rabbit anti-Bax (00089105, proteinch, China), rabbit anti-GAPDH (AB0037, Abways, China).

All data were presented as the mean ± standard deviation (SD) and all statistical analyses were performed using Graphpad Prism 8 software. The data were determined by One-way analysis of variance (ANOVA) and t-test. Significance was accepted at p < 0.05.

After 14 days of treatment, echocardiography indicated that the HF model was successfully established, as evidenced by a significant decrease in EF and FS in the mice of the model group as compared with the sham group (p < 0.001) (Figures 1A,B). After NGR1 intervention, the EF and FS values were up-regulated (p < 0.001) and the LVID; d and LVID; s values were down-regulated (p < 0.001) (Figures 1A,B). H&E staining showed that the myocardial cells in the sham group were well-arranged, with intact nuclei and no inflammatory cell infiltration. However, rupture and disorder of myocardial fibers and edema of myocardial tissue were observed in the model group. The pathological changes were reversed by NGR1 treatment (Figure 1C). Results of masson staining indicated that the degree of fibrosis was increased in the model group, which could be attenuated after NGR1 treatment (Figures 1D,E). CK-MB and cTnI are known as specific markers of myocardial injury (Xie et al., 2019). Our results showed that compared with the sham group, the levels of serum CK-MB and cTnI were elevated in the model group (p < 0.05, p < 0.001), while NGR1 treatment reversed these changes (p < 0.01, p < 0.001) (Figure 1F). These results suggested that NGR1 treatment exerted a cardio-protective effect in the HF mice model. Simvatatin had similar effects as NGR1 (Figures 1A,F).

Ceramide, DAG and NEFA are considered as lipotoxicity mediators due to their effect on heart dysfunction (D'Souza et al., 2016; Kretzschmar et al., 2021). The levels of ceramide, DAG and NEFA in the model group were up-regulated compared with those in the sham group (p < 0.001, p < 0.01). After NGR1 intervention, ceramide, DAG and NEFA levels were reduced (p < 0.001) (Figures 2A,C). Cardiac lipotoxicity is a manifestation of disorders of cardiac lipid metabolism. Therefore, we used an automated biochemical analyzer to measure serum lipid levels. The results showed that levels of TG, CHOL, LDL-C and NEFA were increased (p < 0.05) and HDL-C level was reduced in the model group (p < 0.001), as compared with sham group. These results suggest that lipid metabolism disorders occurred in HF mice. NGR1 treatment effectively reversed the changes of the serum lipid levels, demonstrating that NGR1 could regulate lipid metabolism (Figures 2D,H).

AMPK is an important regulator of energy homeostasis in cardiomyocyte. The α subunit was the main catalytic subunit of AMPK and it could be activated when phosphorylated at Thr172 site (Feng et al., 2018). Activated AMPK can effectively reduce myocardial dysfunction, inhibit myocardial cell apoptosis and attenuated lipotoxicity by regulating downstream targets, including SPT and GPAT. We investigated whether NGR1 attenuated cardiac lipotoxicity in HF mice by regulating the AMPK pathway. We found that the expressions of p-AMPK were significantly decreased (p < 0.001) and the expressions of GPAT, SPTLC1 and SPTLC2 were all increased in the model group (p < 0.05, p < 0.001) compared with sham group, suggesting that production of toxic lipids were increased in HF mice heart. In addition, the expression of CPT-1A was significantly down-regulated in the model group (p < 0.05), suggesting that FAO was impaired in the hearts of model mice. NGR1 treatment could regulate the expressions of p-AMPK and key enzymes involved in toxic lipid production and lipid metabolic pathway (p < 0.05, p < 0.01) ( Figures 3A,B ). We further examined the expression of anti-apoptotic protein Bcl-2 and pro-apoptotic protein Bcl-2 Associated X Protein (Bax). The results showed that NGR1 treatment could regulate expressions of Bcl-2 and Bax (p < 0.05, p < 0.01), thereby attenuating the lipotoxicity-induced apoptosis (Figure 3C).

To further verify the potential regulatory mechanisms of NGR1 on lipotoxicity, we established a PA-stimulated H9C2 cell model. When stimulated cells for 24 h, we found that cell viability significantly decreased in a dose-dependent manner (p < 0.001) (Figure 4B). BSA was used to solubilize PA, so we compared BSA with the normal group and found that it had no effect on cell viability (Figure 4B). Based on CCK8 results, 100 μmol/L PA was chosen as working concentration to induce lipotoxicity in H9C2 cells for subsequent experiments (Figure 4B). The effect of NGR1 on PA-stimulated H9C2 lipotoxicity cell model were investigated. The CCK-8 assay results showed that NGR1 was not toxic to cells at concentrations ranging from 2.5 μmol/L to 80 μmol/L. NGR1 increased cell viability in PA-stimulated cells in a dose-dependent manner and 40 μmol/L was chosen as working concentration for subsequent investigations (Figures 4C,D).

The effects of NGR1 in a PA-stimulated cell model were further investigated. As shown in Figure 5A, PA treatment significantly down-regulated the expression of p-AMPK (p < 0.05). Moreover, the expressions of GPAT, SPTLC1 and SPTLC2 were significantly up-regulated (p < 0.05, p < 0.01) and the expression of CPT-1A (p < 0.05) was down-regulated by PA challenge (Figure 5B). Impressively, NGR1 treatment could reverse these changes and regulate expressions of p-AMPK and enzymes involved in lipid metabolism (Figures 5A,B). Moreover, when cells were co-treated with NGR1 and Compound C, an inhibitor of AMPK, the regulative effects of NGR1 on lipid metabolic enzymes were abrogated, demonstrating that the effects of NGR1 on lipid metabolism are mediated by AMPK (Figure 5B).

We also observed lipid accumulation in PA-stimulated cells, as shown by LipidTOX™ staining (Figure 5C). Lipid accumulation might be caused by reduced levels of FAO. NGR1 treatment could reduce lipid accumulation in H9C2 cells, whereas AMPK inhibitor could abrogate the effects of NGR1 (Figure 5C). We further assessed the levels of TG and NEFA. NGR1 could significantly reduce the levels of TG and NEFA in PA stimulated H9C2 cells (p < 0.01, p < 0.001), whereas AMPK inhibitor ameliorated the regulative effects of NGR1. These results demonstrated that NGR1 could attenuate lipid accumulation in PA-stimulated cells and this effect was mediated via AMPK pathway.

Imbalance of lipid metabolism induces the accumulation of ceramide and DAG, which in turn leads to oxidative stress and apoptosis. The effect of NGR1 on apoptosis was assessed by Hoechst/PI staining in a PA-induced lipotoxicity model. As shown in Figure 6A, fragmented, brighter fluorescence was observed in H9C2 cells after 24 h of PA stimulation, indicating a high level of intracellular apoptosis. NGR1 significantly attenuated the apoptotic rate (Figure 6A). The DCFH-DA probe was used to detect intracellular ROS levels, and the results showed that NGR1 significantly attenuated the increase in ROS levels induced by PA (Figure 6B). Ceramide and DAG have been reported to reduce Bcl-2 expression by inhibiting protein kinase B (AKT) phosphorylation, thereby exacerbating the apoptotic response. In this study, we found that the expression of p-AKT was significantly down-regulated in the model group compared to the normal group (p < 0.01). These alterations were reversed after NGR1 treatment (Figure 6C). These results indicated that lipotoxicity-induced apoptosis and oxidative stress could be inhibited by NGR1. Moreover, the effects of NGR1 on lipotoxicity-induced apoptosis and ROS production were abrogated by AMPK inhibitor, further demonstrating that the protective effects of NGR1 were mediated by AMPK pathway.