Isosteviol sodium protects the cardiomyocyte response associated with the SIRT1/PGC‐1α pathway

Abstract Cardiomyocyte dysfunction is attributed to excess oxidative damage, but the molecular pathways involved in this process have not been completely elucidated. Evidence indicates that isosteviol sodium (STVNa) has cardioprotective effects. We therefore aimed to identify the effect of STVNa on cardiomyocytes, as well as the potential mechanisms involved in this process. We established two myocardial hypertrophy models by treating H9c2 cells with high glucose (HG) and isoprenaline (ISO). Our results showed that STVNa reduced H9c2 mitochondrial damage by attenuating oxidative damage and altering the morphology of mitochondria. The results also indicated that STVNa had a positive effect on HG‐ and ISO‐induced damages via mitochondrial biogenesis. The protective effects of STVNa on cardiomyocytes were associated with the regulation of the SIRT1/PGC‐1α signalling pathway. Importantly, the effects of STVNa involved different methods of regulation in the two models, which was confirmed by experiments using an inhibitor and activator of SIRT1. Together, the results provide the basis for using STVNa as a therapy for the prevention of cardiomyocyte dysfunctions.

metabolism and insulin secretion. SIRT1 also plays an important role in metabolic syndrome, cell apoptosis, cardiovascular diseases and neurodegenerative diseases, [6][7][8] and is an activator of peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α). 7 Recently, studies have reported that SIRT1 contributes to cardiac metabolism by activating PGC-1α and pyruvate dehydrogenase kinase 4 (PDK4) expressions during cardiac hypertrophy. [9][10][11] Considering the profound impact of SIRT1 on mitochondrial biogenesis and metabolism, characterizing the link between regulation of SIRT1 and HG or ISO exposure is a key issue in identifying the molecular mechanisms involved in cardiac damage induced by HG and ISO.
Isosteviol, isolated from the herb, Stevia rebaudiana, is a widely known sweetener. 12 It has been reported to have many pharmacological functions, such as anti-inflammatory, anti-tumour, and neuroprotective effects. 13,14 Isosteviol sodium (STVNa) is a sodium salt of isosteviol, and recent studies have found that it has strong effects on cardiomyocytes. 15,16 However, the underlying mechanisms of the cardiac hypertrophy effect of STVNa are still not completely clear.
The present study was therefore designed to identify the effect of STVNa on cardiomyocytes, as well as the potential mechanisms involved.

| Reagents and chemicals
Isosteviol sodium powder was provided by the Chemical

Development Laboratories of Key Biological Pharmaceutical
Company. The 2 ' ,7 ' -dichlorofluorescein diacetate (DCFH-DA), JC-1 and phalloidin were purchased from Sigma-Aldrich. The antibodies against PGC-1α were purchased from Abcam. SIRT1 and AMPK antibodies were obtained from Santa Cruz Biotechnology. Secondary antibodies were purchased from Affinity Biosciences.

| Cell culture and treatment
The H9c2 cells were purchased from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China) and cultured in Dulbecco's modified Eagle's medium (DMEM)/low glucose in an atmosphere of 5% CO 2 at 37°C. DMEM contained 10% foetal bovine serum, 100 U/mL penicillin and 100 mg/mL streptomycin.

| Cell viability assay
Cell viability was assessed using the Cell Counting Kit-8 (CCK8) assay (Beyotime). First, H9c2 cells were seeded in a 96-well plate.
When the cells reached 70% confluency, they were treated with HG or ISO with or without STVNa for 48 hours. Then, the CCK-8 diluent (0.5 mg/mL) was added at a volume of 100 μL per well. Lastly, the optical density was measured at 450 nm after incubation for 1-4 hours at 37°C.

| Assessment of intracellular reactive oxygen species (ROS)
H9c2 cells were seeded in 24-well plates. When the cells reached 70% confluency they were treated according to the experimental design. Intracellular ROS accumulation was assessed using DCFH-DA (Sigma-Aldrich). DCFH-DA (1 mmol/L) stock solution was diluted with DMEM to a concentration of 5 μmol/L. The cells were then incubated for 40 minutes at 37°C. The fluorescence intensity was measured after the cells were washed three times with phosphate buffered saline (PBS) using confocal microscopy (Carl Zeiss).

| Measurement of mitochondrial membrane potential (Δψ)
Mitochondrial membrane potential was determined using JC-1 (Sigma-Aldrich), which is a cationic dye that exhibits potentialdependent accumulation in mitochondria. First, H9c2 cells were seeded in confocal petri dishes. When the cells reached 70% confluency, they were treated according to the experimental design. JC-1 working liquid was then prepared to a final concentration of 1 μg/ mL. The JC-1 working solution was added in a volume of 500 μL per well and incubated at 37°C for 20 minutes. The dye was then removed, and the wells were washed three times with PBS. The cells were observed using a confocal microscope (Carl Zeiss), and the ratio of red-to-green fluorescence intensities were analysed to determine the Δψ.

| Relative mitochondrial (mt)DNA content measurements
Total DNA was extracted from H9c2 cells using an EZNA™ Tissue DNA Kit (Omega) according to the manufacturer's instructions. The mtDNA was measured using quantitative real-time polymerase chain reaction (qRT-PCR) analysis. Primers for cytochrome c oxidase subunit I (COXI) encoded by the heavy chain of mtDNA, NADH dehydrogenase subunit 1 (ND1), NADH dehydrogenase subunit 6 (ND6) and β-globin were designed for qRT-PCR by Generay. The primers are listed in Table 1.

| The qRT-PCR
Total RNA samples were prepared using a standard TRIzol protocol (Generay Biotech) and quantitatively measured using ultraviolet spectroscopy. Total RNA was amplified by qRT-PCR using a two-step RT-PCR kit (Vazyme, Nanjing, China) according to the manufacturer's protocol. The qRT-PCR analyses were repeated at least three times.
The primers are listed in Table 1.

| Western blot analysis
The H9c2 cells were collected in 1.5 mL microcentrifuge tubes, and 80 μL ice-cold RIPA lysis buffer with phenylmethylsulfonyl fluoride and protein kinase inhibitors was added to the tubes, which were then centrifuged at 13800 g at 4°C for 10 minutes. The concentration of acquired protein was detected using a BCA kit (Beijing Dingguo

| Immunohistochemistry
The cells were seeded in confocal petri dishes, and cultured at 37°C in 5% CO 2 for 24 hours and then fixed with cold 4% paraformaldehyde for 10 minutes. After three washes in PBS, the cells were immersed in 1% Triton X-100 for 5 minutes and then were incubated with anti-SIRT1 antibody overnight at 4°C. The cells were then washed and incubated with tetramethylrhodamine isothiocyanateconjugated secondary antibody for 1 hour. After three washes in

| Statistical analysis
Data are presented as the mean ± standard error of the mean (SEM).
Differences between groups were determined by one-way analysis of variance followed by Tukey's post hoc test. Statistical significance was indicated by a value of P < .05. All statistical analyses were performed using GraphPad Prism, version 5.0 (GraphPad software).

| STVNa promoted H9c2 cell viability following exposure to HG or ISO by decreasing the intracellular ROS accumulation
Compared with the control group, the viability of H9c2 cells treated with ISO or HG for 48 hours was significantly decreased (P < .05), as shown by the CCK-8 staining assay. However, STVNa protected cells from damage caused by ISO or HG ( Figure 1A,B). Specifically, the cell viability was increased following treatment with 5, 10 and 50 μmol/L STVNa, so we used 5, 10 and 50 μmol/L STVNa in subsequent studies. To determine whether STVNa decreased the intracellular ROS accumulation that occurred following exposure to ISO or HG, we examined the intracellular ROS levels. The results showed that H9c2 cells had a higher fluorescence intensity following treatment with ISO or HG, when compared with the control group ( Figure 1C), showing that the ISO and HG groups produced high levels of ROS.
However, the fluorescence intensity decreased significantly after 48 hours in the presence of STVNa, indicating that treatment with STVNa reduced intracellular ROS accumulation ( Figure 1C,D).

| STVNa protected H9c2 cells against HG-and ISO-induced cardiomyocyte hypertrophy
The

| STVNa restored mitochondrial membrane potential (Δψ), maintained mitochondrial morphology, and increased mitochondrial biogenesis following exposure to HG or ISO
If mitochondrial function is disturbed, the cell viability decreases, so a decrease in Δψ is an indication of failing mitochondria. We therefore used the membrane sensitive dye, JC-1, to stain cells to assess the effect of STVNa on Δψ. Figure Figure 3B shows that the morphology of mitochondria became fragmented or smaller following HG or ISO exposure. The length of mitochondria was measured using ImageJ software ( Figure 3D), indicating that STVNa significantly inhibited the effect of HG or ISO, to maintain the morphology of the mitochondria. the protein levels of SIRT1 and PGC-1α using immunofluorescent staining and Western blot assays. The results indicated that the nuclear protein levels of SIRT1 and PGC-1α were significantly increased after treatment with STVNa in cells exposed to HG or ISO ( Figure 4A,B). However, the regulatory effects of STVNa on the protein expressions of SIRT1 and PGC-1α in the HG and ISO models and the dosage effects of STVNa in these two groups were different ( Figure 4C,D). We also showed that the mRNA expressions of SIRT1, PGC-1α and AMPK were significantly increased by treatment with STVNa ( Figure 4E-G). These results suggested that the cardiomyocyte protective effects of STVNa were associated with the up-regulation of the SIRT1/PGC-1α signalling pathway.
However, the mechanism of action of STVNa may be different in these two models.

| The effect of the SIRT1 activator, resveratrol, and the SIRT1 inhibitor, Ex527, on SIRT1 activity in H9c2 cells
Resveratrol (3,5,4′-trihydroxy-trans-stilbene) is a polyphenol phytoalexin present in a variety of plant species and has drawn much attention due to its beneficial effects. 15 Additionally, recent studies have shown that resveratrol could improve the expression of Experiments were performed as in (D). Quantitative analysis of SIRT1 and PGC-1α normalized to GAPDH levels and expressed as relative fold changes versus the control group. The mRNA expressions of SIRT1 and PGC-1α were measured by qRT-PCR (E, F and G). Data are presented as the mean ± SEM. # P < .05, ## P < .01, ### P < .01 vs the control group; * P < .05, ** P < .01, *** P < .001 vs the isoprenaline or highglucose group SIRT1 and activate the AMPK-SIRT1-autophagy signal pathway. 16 In the present study, resveratrol (RES) was used as an agonist of SIRT1 and Selisistat (EX-527) was an inhibitor of SIRT1 enzymatic activity. To further ascertain the involvement of the SIRT1/PGC-1α signalling pathway in the mechanism of STVNa protection against cardiomyocyte dysfunction, we determined the influence of the SIRT1 activator, resveratrol (RES), and the SIRT1 inhibitor, Ex527, on SIRT1 activity. H9c2 cells were incubated with ISO or HG in the presence or absence of STVNa (10 μmol/L). EX-527 (10 μmol/L) or RES (30 μmol/L) was added 1 hour before STVNa treatment. Figure 5 shows that pre-treatment with RES prevented ISO-and HG-induced SIRT1 down-regulation. In addition, the expression of SIRT1 was much higher when pre-treatment combined with RES and STVNa than pre-treatment with RES alone. It indicated that STVNa could further activate SIRT1 expression and the downstream signal pathway ( Figure 5A-C). SIRT1 was suppressed by EX527, and the effect of STVNa regulating on SIRT1 was significantly attenuated when H9c2 cells were treated with EX527 and STVNa ( Figure 5D,E).
Interestingly enough, the expression trend of PGC-1α was consistent with SIRT1 in the HG group rather than in the ISO group. Notably, compared with the HG group, the expression of SIRT1 in H9c2 cells did not affect the expression of PGC-1α following treatment with EX-527 or RES ( Figure 5). These results suggested that there may be different mechanisms of action of STVNa in these two models. The different mechanisms may be associated with the different mechanisms of regulation of STVNa on the expression and the post-translational modification of PGC-1α. STVNa may activate the expression levels of SIRT1 and PGC1-1α during HG treatment, whereas SIRT1 may activate PGC-1α by regulating deacetylation instead of simply up-regulating its protein levels when H9c2 cells treated with STVNa were exposed to EX-527 and RES in the ISO group.

| D ISCUSS I ON
Previous studies have demonstrated the cardioprotective effects of STVNa. For example, isosteviol has been shown to ameliorate diabetic cardiomyopathy in rats, 17,18 and STVNa has been shown to protect H9c2 cells against myocardial ischaemia reperfusion injury. 19 However, these studies have not identified the specific molecular mechanisms by which STVNa acts at the cellular level. Our study showed that STVNa exerted a cardioprotective effect in ISO-/HGinduced myocardial hypertrophy through a mitochondrial-mediated signalling pathway.
Myocardial hypertrophy is the pathological manifestation of hypertensive heart disease, which is an independent risk factor leading to an increase in cardiovascular disease morbidity and mortality. HG and ISO are traditional methods used to induce cardiac hypertrophy.
Previous studies have reported that cell models are successful when the concentration of ISO is 10 μmol/L 20 and when the glucose concentration is 30.5 nmol/L. We therefore first analysed the cytotoxic effect of STVNa with ISO/HG on H9c2 cells. Our results showed that STVNa had no toxic effect on cells when the concentration was <200 µmol/L in the presence of HG or ISO. If ROS generation exceeds its elimination, it results in cell damage that ultimately leads to death. We found that exposure to HG and ISO induced a significant increase in ROS. However, treatment with STVNa reduced intracellular ROS levels and protected the mitochondria.
Mitochondria are the major sites of ATP synthesis, involving the process of oxidative phosphorylation. Mitochondrial health must be monitored carefully because mitochondria are sensitive to the major metabolic pathways of the cell, 21  repair of defective mtDNA through mixing and redistribution of the mitochondria to sites requiring high energy production. 25,26 The results of the present study showed that STVNa protected the mtDNA from damage, showing that treatment with STVNa increased mitochondrial biogenesis in H9c2 cells. Many studies have found that SIRT1 occurs upstream of ROS.
We could not directly infer that ROS production was downstream of SIRT1, but our results indicated that STVNa increased the level of SIRT1, and activation of SIRT1 led to increased ROS damage and oxidative stress. We therefore concluded that STVNa reduced oxidative stress through the SIRT1/PGC-1α pathway. The SIRT1/ These differences may be the reason that the regulatory effects of STVNa on the protein expressions of SIRT1 and PGC-1α in HG and ISO models and the dosage effects of STVNa in these two groups were different in this study. Overall, these results suggested that the molecular mechanisms of STVNa in the ISO and HG models may differ ( Figure 6).
In the present study, we showed a role for STVNa in inhibiting HG-and ISO-induced oxidative stress and mitochondrial damage in H9c2 cells, and found that it attenuated oxidative stress and restored the mitochondrial membrane potential to restore mitochondrial morphology. Furthermore, we found that STVNa reduced

CO N FLI C T O F I NTE R E S T
The authors declare that they have no competing interests.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.