SAL, DMSO, trypsin, 1.5 M Tris-HCl (pH=8.8), 1.0 M Tris-HCl (pH=6.8) and penicillin-streptomycin solution were purchased from Beijing Solarbio Science & Technology Co., Ltd. Serum-free DMEM and FBS were purchased from Thermo Fisher Scientific, Inc. PBS was purchased from Lanzhou Bailing Biotech Co., Ltd. RNApure Tissue & Cell (DNase I), UltraSYBR One Step RT-qPCR, HiFiScript cDNA Synthesis and Cell Counting Kit-8 (CCK-8) were purchased from Beijing ComWin Biotech Co., Ltd. SDS-PAGE Sample Loading Buffer (5X), fluo-4/acetoxymethyl ester (Fluo-4/AM) (2 mM) and Annexin V-FITC Apoptosis Detection kit were purchased from Beyotime Institute of Biotechnology. BCA protein assay kit and 5% bovine serum albumin (BSA) were purchased from Beijing Labgic Co., Ltd. L-glutamine was purchased from Suzhou Haixing Biological Technology Co., Ltd.; 30% acrylamide (29:1) was purchased from Sinopharm Chemical Reagent Co., Ltd. PVDF membranes was purchased from MilliporeSigma. Tween-20 was purchased from Amresco Co., Ltd. Anti-EGLN1 (cat. no. #4835s) was purchased from Cell Signaling Technology, Inc. Anti-HIF-1α (cat. no. NB100-105) was purchased from Novus Biologicals, LLC. Anti-GAPDH (cat. no. 60004-1-Ig) was purchased from Proteintech Group, Inc. Horseradish peroxidase-conjugated AffiniPure Goat Anti-Rabbit IgG (cat. no. ZB2301) was purchased from OriGene Technologies, Inc. 10% SDS was purchased from China Sinopharm International (Shanghai) Co., Ltd. Enhanced chemiluminescence (cat. no. ECL-0011) was purchased from Ding Guo Prosperous Co., Ltd. Primers for HIF-1α (forward, 5'-CCGCCACCACCACTGATGAATC-3' and reverse, 5'-GTGAGTACCACTGTATGCvTGATGCC-3') and GAPDH (forward, 5'-GAAGGTCGGTGTGAACGGAT-3' and reverse, 5'-CCCATTTGATGTTAGCGGGAT-3') were purchased from Beijing Tsingke Biotech Co., Ltd. EGLN1 primers (forward, 5'-AGCTGGTCAGCCAGAAGAGT-3' and reverse, 5'-GCCCTCGATCCAGGTGATCT-3') were purchased from Sangon Biotech Co., Ltd. All other solvents and chemicals were of analytical grade. Pure water was produced using a Milli-Q purification system (MilliporeSigma).

H9C2 cell line was purchased from Suzhou Haixing Biological Technology Co., Ltd. The cells in the normoxia group were cultured in DMEM containing 10 FBS and 1% penicillin-streptomycin and incubated at 37˚C in a 5% CO₂ incubator. For the hypoxia group, cells were cultured at 37˚C with 5 CO₂ and 2% O_2.

The biocompatibility of SAL in H9C2 cells was assessed using CCK-8 assay. H9C2 cells were seeded into 96-well plates at a density of 1x10⁵ cells/well and incubated overnight at 37˚C, following which 200 µl SAL (10, 100, 250, 500, 750 and 1,000 nM) or DMEM (blank control) was added for an additional 24 h at 37 ˚C. After removing the DMEM, 110 µl fresh medium (containing 10 µl CCK-8) was added and incubated for 1.5 h. Finally, viability was determined by measuring the absorbance of each well at 450 nm using Spectrophotometer Multiskan (Thermo Fisher Scientific, Inc.).

The ability of SAL to rescue hypoxia-treated H9C2 cells was also confirmed using the CCK-8 method. H9C2 cells were cultured in 96-well plates at a density of 1x10⁵ cells/well and incubated overnight at 37˚C. H9C2 cells were transferred to a hypoxic incubator and cultured under hypoxic conditions for 48 h at 37˚C. Medium was removed and 200 µl SAL (10, 100, 250, 500, 750 and 1,000 nM) or an equal volume of fresh DMEM was added for 24 h in the hypoxic incubator at 37˚C. After removing DMEM, the cells were incubated with CCK-8 solution for 1.5 h. Finally, viability was calculated by measuring the absorbance of each well at 450 nm using Spectrophotometer Multiskan (Thermo Fisher Scientific, Inc.).

The rescue effect of SAL on hypoxic cells was quantitatively analysed using flow cytometry. H9C2 cells were co-inoculated into 3-cm dishes at a density of 2.5x10⁵ cells/ml. A total of three groups was used: Normoxia, hypoxia and hypoxia + SAL. Cells in the normoxia group were cultured under normoxic conditions for 48 h and incubated in fresh DMEM for an additional 24 h at 37˚C. Cells in the hypoxia group were incubated under hypoxic conditions for 48 h at 37˚C, followed by incubation in DMEM for an additional 24 h. In the hypoxia + SAL group, cells were incubated under hypoxic conditions for 48 h at 37˚C, followed by incubation with 200 µl SAL (100 nM) for an additional 24 h at 37˚C. Cells were collected via centrifugation (500 x g for 5 min at 4˚C) and washed twice with PBS. Cells were suspended in 100 µl binding buffer mixed with 5 µl Annexin-V/FITC and incubated for 5 min in the dark at room temperature. The cells were mixed with 10 PI stain and 400 µl PBS. The stained cells were analyzed by flow cytometry (BD Biosciences) and analysed using WinCyte software (CompuCyte).

The ability of SAL to maintain intracellular calcium homeostasis was evaluated using Fluo-4/AM. H9C2 cells were cultured in 12-well plates with cell slides at a density of 1.5x10⁵ cells/well. Normoxia group was cultured at 37˚C with 5% CO₂ and 2% O₂. Hypoxia group was cultured at 37˚C with 5% CO₂. The culture medium was removed following incubation, the cells were washed with PBS twice and 200 µl Fluo-4/AM (2.5 µM) was added. The cells were then incubated at 37˚C for 15 min in the dark. The stained H9C2 cells were washed with PBS three times, 300 µl 4% paraformaldehyde was added to fix the cells for 30 min at 37˚C, and cells were observed under a fluorescence microscope at 100x magnification (Leica GmbH).

A total of 1x10⁷ cells/tube (three tubes from each group) were subjected to TMT proteomic analysis to screen differentially expressed proteins, which were analysed at the functional level as reported (32). Gene Ontology (GO; david.ncifcrf.gov/tools.jsp) and Kyoto Encyclopaedia of Genes and Genomes (KEGG) enrichment (http://www.kegg.jp) analyses were performed to determine whether differentially expressed proteins were significantly enriched in hypoxia-related pathways. For each protein, the fold-change (FC) was calculated as the ratio of mean values of all biological measurements in each two groups.

RNA was extracted from cells using an RNApure Tissue and Cell kit (DNase I) according to the manufacturer's protocol. cDNA was synthesised using HiFiScript cDNA Synthesis kit according to the manufacturer's protocol. UltraSYBR One Step RT-qPCR Kit was used to detect the expression of HIF-1α and EGLN1. Thermocycling conditions were as follows: Initial denaturation at 95˚C for 30 sec, followed by 45 cycles of 95˚C for 5 sec and 60˚C for 30 sec. The relative expression levels of HIF-1α and EGLN1 were evaluated by the 2^-∆∆Cq method (33).

Cell lysate was extracted using RIPA assay buffer and protein lysates were obtained following centrifugation (12,000 x g for 10 min at 4˚C). Proteins were quantified using a BCA assay kit. The protein extracts were isolated and transferred onto a PVDF membrane, which was blocked with 5% BSA at 37˚C for 2 h. Subsequently, the membrane was incubated overnight with primary antibody at 4˚C, followed by incubation with secondary antibody at 37˚C for 1 h. Chemiluminescent imaging system (Clinx Science) was used to detect the signals. The bands were quantified using Image J software 6.3 (imagej.net/software/).

All data were analysed by SPSS 16.0 software (SPSS, Inc.). Continuous variables are expressed as the mean ± standard deviation of three independent replicate experiments. One-way analysis of variance followed by Tukey multiple comparisons test were employed to determine significance between different groups. Fisher's exact test was used to find enriched GO and KEGG terms. The corresponding P-value was calculated as the significance index. Benjamini-Hochberg False Discovery Rate was used to correct the P-value. P<0.05 was considered to indicate a statistically significant difference.

Hypoxic incubator models create a hypoxic environment and induce cell apoptosis (34,35). Morphology of the adherent H9C2 cells was fusiform; however, following hypoxic culture, the cell morphology became round and some cells fell off the bottom of the culture dish (Fig. 1A). These morphological changes were not apparent in SAL treatment group. Compared with the hypoxia group, cells of the hypoxia + SAL group adhered to culture dishes and exhibited spindle-shaped shape, which was notably improved.

CCK-8 assay was used to evaluate the effect of SAL on viability of H9C2 cells under a normoxic environment (Fig. 1B). Cell viability was >90%, which confirmed good biocompatibility of SAL. The different incubation times had no significant effect on proliferation of H9C2 cells under normoxic conditions; therefore, 24 h was selected as the incubation time for SAL in subsequent experiments.

A hypoxia model was created to explore the ability of SAL to rescue H9C2 cells following hypoxia. With higher SAL concentrations, the survival rates of hypoxic cells were significantly higher than those of the control group (Fig. 1C). SAL concentrations of 100, 250, 500, 750 and 1,000 nM showed good rescue ability; therefore, 100 nM was selected for subsequent experiments.

To investigate changes of intracellular Ca²⁺ in the three groups, the cells were stained with Fluo-4/AM and observed using a fluorescence microscope. Compared with the control group, H9C2 cells in the hypoxia group showed brighter green fluorescence, indicating that the hypoxia induced intracellular calcium overload (Fig. 2A). Following SAL treatment in hypoxic cells, green fluorescence intensity of Ca²⁺ was notably weakened compared with the hypoxia group. These results indicated that SAL treatment reduced intracellular calcium overload in hypoxic cells, thereby inhibiting cell apoptosis.

Flow cytometric analysis of cells stained with Annexin V-FITC/PI was used to assess the degree of apoptosis. Survival rates of H9C2 cells in the normoxia and normoxia + SAL groups were >90%, which was consistent with the cell viability results obtained by CCK-8 assay (Fig. 2B). These results further confirmed that SAL has good biocompatibility. After H9C2 cells were cultured under hypoxic conditions, ~18.45% cells were apoptotic and the apoptosis rate increased by 13.5% compared with the normoxia group, indicating that apoptosis was induced via hypoxia-related pathways following hypoxia. In hypoxic cells treated with SAL, the apoptosis rate was 8.76% (early and late apoptosis, 3.24 and 5.52%, respectively), which was 9.69% lower than that in the hypoxia group. Flow cytometry showed that SAL effectively improved survival rate of hypoxic cells and attenuated the effects of hypoxia.

According to proteomic analysis, 63 proteins were up- and 42 were downregulated in the normoxia group compared with the hypoxia group. The expression of 78 proteins was up-while that of 48 proteins was downregulated in the normoxia group compared with the hypoxia + SAL group. The expression of four proteins was up- and that of 12 was downregulated in the hypoxia group compared with the hypoxia + SAL group (Fig. S1). Subsequently, differentially expressed proteins were screened for those associated with the hypoxia pathway.

Relative protein abundance of EGLN1(974) in the hypoxia group was significantly higher than that in the control group (461) following hypoxia treatment; however, the protein abundance decreased to 737 following treatment with SAL (P<0.05; Fig. 3A). EGLN1 expression was negatively associated with oxygen concentration under hypoxic conditions.

GO enriched terms were ‘response to glucose’, ‘response to hypoxia’ ‘glycolysis/gluconeogenesis’ (Fig. 3B). Therefore, cells responded to hypoxia by expressing hypoxia-related proteins; hypoxia treatment affected cellular glucose metabolism pathways. KEGG enrichment analysis showed that HIF-1 signalling pathway was enriched in the hypoxia vs. hypoxia + SAL group; seven proteins were down- and one was upregulated (Fig. 4A and B). Hypoxia affected the glycolysis/gluconeogenesis pathway, a central pathway in energy metabolism (Fig. 3C and D).

Expression of HIF-1α and EGLN1 in the hypoxia group was significantly higher than those in the normoxia group (Fig. 5A). For HIF-1α, mRNA expression under hypoxia was 4.98-fold that of the control group, whereas treatment with SAL decreased its expression. The mRNA expression levels of EGLN1 showed a similar trend. mRNA expression of EGLN1 under hypoxia was 2.40-fold higher than that in the normoxia group and the expression of EGLN1 was reduced following co-incubation with SAL. Non-hydroxylated HIF-1α is more stable than hydroxylated HIF-1α, resulting in increased mRNA expression. EGLN1 mRNA expression increased under hypoxia.

Compared to the normoxia group, expression of HIF-1α protein significantly increased following hypoxia treatment, which was consistent with mRNA levels (Fig. 5B and C). EGLN1 protein expression increased following hypoxia treatment. However, following treatment with SAL for 24 h, expression of HIF-1α and EGLN1 protein decreased.