key: cord-013567-qnp65w53
authors: Cheng, Qiao-qiao; Wan, Yu-wei; Yang, Wei-min; Tian, Meng-hua; Wang, Yu-chuan; He, Hai-yan; Zhang, Wei-dong; Liu, Xuan
title: Gastrodin protects H9c2 cardiomyocytes against oxidative injury by ameliorating imbalanced mitochondrial dynamics and mitochondrial dysfunction
date: 2020-03-12
journal: Acta Pharmacol Sin
DOI: 10.1038/s41401-020-0382-x
sha: 
doc_id: 13567
cord_uid: qnp65w53

Gastrodin (GAS) is the main bioactive component of Tianma, a traditional Chinese medicine widely used to treat neurological disorders as well as cardio- and cerebrovascular diseases. In the present study, the protective effects of GAS on H9c2 cells against ischemia–reperfusion (IR)-like injury were found to be related to decreasing of oxidative stress. Furthermore, GAS could protect H9c2 cells against oxidative injury induced by H(2)O(2). Pretreatment of GAS at 20, 50, and 100 μM for 4 h significantly ameliorated the decrease in cell viability and increase in apoptosis of H9c2 cells treated with 400 μM H(2)O(2) for 3 h. Furthermore, we showed that H(2)O(2) treatment induced fragmentation of mitochondria and significant reduction in networks, footprint, and tubular length of mitochondria; H(2)O(2) treatment strongly inhibited mitochondrial respiration; H(2)O(2) treatment induced a decrease in the expression of mitochondrial fusion factors Mfn2 and Opa1, and increase in the expression of mitochondrial fission factor Fis1. All these alterations in H(2)O(2)-treated H9c2 cells could be ameliorated by GAS pretreatment. Moreover, we revealed that GAS pretreatment enhanced the nuclear translocation of Nrf2 under H(2)O(2) treatment. Knockdown of Nrf2 expression abolished the protective effects of GAS on H(2)O(2)-treated H9c2 cells. Our results suggest that GAS may protect H9c2 cardiomycytes against oxidative injury via increasing the nuclear translocation of Nrf2, regulating mitochondrial dynamics, and maintaining the structure and functions of mitochondria.

INTRODUCTION Gastrodin (GAS) (p-hydroxymethylphenyl-b-D-glucopyranoside) is the main bioactive component of Rhizoma Gastrodiae (the dried rhizome of Gastrodia elata Blume, also known as Tianma), a popular traditional Chinese medicine. In Shennong's Classic of Materia Medica, the first herbal monograph in Chinese history, written during the Han Dynasty, Tianma was described as a "top grade medicine" that can rejuvenate the body, enhance health, and extend life without toxicity, and can be used long term without harm [1] . Although Tianma is traditionally used in the treatment of nervous diseases, Tianma has been reported to have protective activities against convulsion, oxidation, depression, epilepsy, obesity, asthma, and inflammation, in addition to the effects on analgesia, sedation, learning and memory improvement, and neuroprotection [2] . Tianma is currently used clinically to treat various cardiovascular and cerebrovascular diseases, as well as nervous diseases, including hypertension, headache, convulsion, epilepsy, and coronary heart diseases [3] [4] [5] [6] . However, the mechanisms of Tianma and GAS have not been fully clarified. Furthermore, most of the previous studies on GAS focused on its effects on neurons, and little is known about the effects of GAS on cardiomyocytes.

Previous studies showed that GAS ameliorated myocardial ischemia-reperfusion (IR) injury in rats [7] . GAS pretreatment ameliorated myocardial IR injury by decreasing calcium overload [8] . GAS also protected H9c2 cardiomyocytes against injuries induced by serum deprivation [9] or LPS treatment [10] . In the present study, we examined the protective effects of GAS on H9c2 cells that underwent ischemia-like injury (deprivation of oxygen, glucose, and serum for 9 h) or IR-like injury (deprivation of oxygen, glucose, and serum for 9 h, and then culture under normal conditions in complete medium for an additional 3 h). Based on the observation of the antioxidant activity of GAS in protecting H9c2 cells against IR-like injury, we focused on studying the effects and mechanisms of GAS on H9c2 cells that underwent oxidative injury induced by H 2 O 2 treatment.

Recent studies have suggested that imbalanced mitochondrial dynamics and related mitochondrial dysfunction play important roles in cardiac IR injury [11] [12] [13] [14] . Mitochondria are highly dynamic organelles that undergo coordinated cycles of fission (to generate discrete fragmented mitochondria) and fusion (to form an interconnected elongated phenotype), referred to as "mitochondrial dynamics", to maintain their shape, distribution, and size [15, 16] . The balance of mitochondrial fusion and fission determines mitochondrial morphology, and is necessary to support mitochondrial functions. Mitochondrial dynamics are coupled to the bioenergetics and signaling functions of mitochondria. Therefore, imbalanced mitochondrial dynamics cause mitochondrial dysfunctions such as changes in mitochondrial respiration and the induction of apoptosis [17] [18] [19] . Mitochondrial dynamics may be a therapeutic target for treating cardiac diseases [14] . In the present study, we observed the morphology and functions of mitochondria in H9c2 cells under H 2 O 2 -induced oxidative injury with or without GAS pretreatment, and attempted to clarify the mechanisms by which GAS protects H9c2 cells against oxidative injury.

Chemicals and materials GAS with a purity >98% was purchased from Nature Standard Company (Shanghai, China). SRB (sulforhodamine B), TCA (trichloroacetic acid), oligomycin, FCCP (2-[2-[4-(trifluoromethoxy)phenyl]hydrazinylidene]-propanedinitrile), and rotenone were purchased from Sigma-Aldrich (St. Louis, MO, USA). MitoTracker Green FM and the Alexa Fluor 488 Annexin V/Dead Cell Apoptosis Kit were purchased from Thermo Fisher Scientific (Waltham, MA, USA). DCFH-DA (2′,7′-dichloro-fluorescin diacetate) and the bicinchoninic acid (BCA) protein assay kit were obtained from Yeasen (Shanghai, China). Antimycin A was purchased from Abcam Biotechnology (Cambridge, MA, USA). The reversetranscription kit was obtained from TaKaRa (Shiga, Japan). The FastStart Essential DNA Green Master for PCR was purchased from Roche (Basel, Switzerland). Antibodies, except where specifically noted, were purchased from Cell Signaling Technology (Danvers, MA, USA).

Cell culture and treatments H9c2 cells were obtained from the cell bank of the Institute of Biochemistry and Cell Biology (Shanghai, China), and cultured in Dulbecco's modified Eagle's medium (DMEM) containing 4.5 g/L D-glucose supplemented with 10% fetal bovine serum, 100 mg/mL streptomycin, and 100 U/mL penicillin, all bought from HyClone (Logan, Utah, USA), at 37°C in a humidified incubator with 5% CO 2 . The cells were passaged every 2 or 3 days. For treatments, H9c2 cells were seeded in 96-well plates at a density of 5 × 10 3 cells per well. After culturing for 24 h and reaching~70%-80% confluence, the cells were pretreated with the indicated concentrations of GAS (dissolved in culture medium) for 4 h before induction of ischemialike injury or IR-like injury, or treatment with H 2 O 2 at the indicated concentrations. Ischemia-like or IR-like injury was induced in H9c2 cells using methods similar to those in a previous report [20] . For ischemia-like injury, the cells were deprived of oxygen, glucose, and serum for 9 h; briefly, the culture medium was replaced with serum-and glucose-free medium, and the plates were placed in an incubator with 1% O 2 , 94% N 2 , and 5% CO 2 air for 9 h. For IR-like injury, the cells were deprived of oxygen, glucose, and serum for 9 h, and then cultured under normal conditions in complete medium for an additional 3 h. For H 2 O 2 treatment, the cells were treated with the indicated concentrations of H 2 O 2 for different time periods.

Cell viability assay Cell viability was determined using the Sulforhodamine B (SRB) assay [21] . Briefly, after treatments, the cells were fixed with cold 10% trichloroacetic acid at 4°C for 60 min. The plates were washed five times with tap water and then air dried. SRB solution (100 μL) at 0.4% (w/v) in 1% acetic acid was added to each well and incubated at room temperature for 10 min. After staining, the plates were washed five times with 1% acetic acid to remove unbound dye and then air dried. Bound dye in the cells was subsequently solubilized with 10 mM Trizma base, and the absorbance was read using an automated plate reader at a wavelength of 515 nm. Three independent experiments (each with at least triplicate samples) were conducted, and the statistical results are shown.

After treatments, the cells were incubated with 10 μM DCFH-DA in serum-free and phenol red-free medium for 30 min. Then, after two phosphate-buffered saline (PBS) washes, the fluorescence intensity of each well was detected using a Cytation 5 Cell Imaging Multi-Mode Reader (BioTek, Winooski, VT, USA) at an excitation wavelength of 488 nm and an emission wavelength of 525 nm. Three independent experiments (each with triplicate samples) were conducted, and the statistical results are shown. Images of the cells were also captured with an inverted fluorescence microscope (Leica, Wetzlar, Germany).

Cell morphology observation and flow cytometric analysis of apoptosis The morphological changes in H9c2 cells after treatments were observed with an optical microscope (DMil, Leica). For flow cytometric analysis, cells (both adherent and detached) were collected, washed with PBS, and stained by using an Alexa Fluor 488 Annexin V/Dead Cell Apoptosis Kit (Thermo Fisher Scientific) according to the manufacturer's instructions. Then, flow cytometric analysis was conducted using a FACSCalibur flow cytometer, and data analysis were performed with CellQuest software (BD Biosciences, Sparks Glencoe, MD, USA). Three independent experiments (each with triplicate samples) were conducted, and the results of one representative experiment are shown.

Mitochondrial morphology observation and network quantification Mitochondria in living H9c2 cells were stained with 100 nM MitoTracker Green FM (Invitrogen, Carlsbad, CA, USA) for 30 min. After washing twice with PBS and transfer to phenol red-free DMEM, mitochondrial morphology was observed using a DeltaVision OMX SR imaging system with super resolution (GE Healthcare Bio-Sciences, Pittsburgh, PA, USA). The mitochondrial morphology in individual cells was quantified as described in previous reports [22, 23] . Three independent experiments were conducted, and the results of one representative experiment are shown. Briefly, random fields of cells (100 cells per condition) in each group were evaluated. The mitochondrial morphology types included long tubular, short tubular, and fragmented. The percentage of different mitochondrial morphology types in 100 cells was quantified. Furthermore, the ImageJ macro tool MiNA was used to analyze mitochondrial network morphology.

Mitochondrial respiration assay A mitochondrial respiration assay in living cells was conducted [24] using a Seahorse XFe96 Extracellular Flux Analyzer (Seahorse Bioscience, North Billerica, MA, USA). The real-time oxygen consumption rate (OCR), which indicated the mitochondrial respiration potential of the cells, was measured. Briefly, H9c2 cells were seeded into XFe96 cell culture microplates (Seahorse Bioscience) at a density of 8000-10000 cells/well. After treatments, the culture medium was changed to unbuffered DMEM (pH 7.4) supplemented with 1 mM pyruvate, 2 mM glutamine, and 10 mM D-glucose at 1 h before the assay. After baseline rate measurement, oligomycin (1 μM), FCCP (1 μM), and rotenone (0.5 μM) combined with antimycin A (0.5 μM) were injected sequentially through ports in the Seahorse Flux Park cartridges. The OCR was recorded and normalized to the number of 1000 cells per well, and then the data were analyzed by using Wave Desktop Software provided by Seahorse Bioscience. Three independent experiments (each with at least triplicate samples) were conducted, and the results of one representative experiment are shown.

Transfection of siRNAs H9c2 cells were transfected with siRNA for Nrf2 (5′-CAAACA GAATGGACCTAAA-3′) or a nontargeting siRNA, both purchased from GenePharma (Shanghai, China) by using Lipofectamine 3000 (Invitrogen) according to the manufacturer's instructions. Briefly, H9c2 cells were plated in 24-or 6-well plates and incubated until they reached 70% confluence. Lipofectamine 3000 and siRNA were mixed and incubated for 15 min at room temperature before being added to the cells. The cells were incubated with siRNA at a final concentration of 50 nM for 24 h. The expression of Nrf2 in negative control cells (cells treated with nontargeting siRNA) and Nrf2-siRNA-treated cells was evaluated using both RT-PCR and Western blot analysis.

RNA extraction and RT-PCR Total cellular RNA was extracted using RNAiso Plus reagent (Takara, Shiga, Japan) dissolved in RNase-free water. The quantification of total RNA was conducted by detecting ultraviolet absorption (optical density, 260/280 nm) using a NanoDrop 2000 (Thermo Scientific, USA). An equal amount of total RNA (500 ng) was reverse transcribed to cDNA using PrimeScript TM RT Master Mix (Takara) according to the manufacturer's instructions. RT-PCR amplifications were performed using the FastStart Essential DNA Green Master and LightCycler® 96 Instrument (Roche, Basel, Switzerland). The thermal profile was 95°C for 10 min, followed by 40 cycles at 95°C for 10 s and 60°C for 30 s. Each sample was analyzed in triplicate, and the mean cycle threshold (Ct) value was calculated. The relative expression level was calculated using the ΔΔCt method. The mRNA expression of actin was used as an internal control. Primer sequences for RT-PCR analysis are listed in Supplementary Table S1 . Three independent experiments (each with at least triplicate samples) were conducted, and the statistical results are shown.

Immunofluorescence staining After treatments, the cells were fixed in 4% paraformaldehyde for 20 min at room temperature, and then washed three times with PBS. The cells were permeabilized in 0.1% Triton X-100 (Sigma-Aldrich) for 10 min, and then blocked in 2% bovine serum albumin for 1 h at room temperature. After an overnight incubation with mouse monoclonal anti-Nrf2 antibody (#ab89443, 1:100 dilution, Abcam, Cambridge, Cambridgeshire, UK) at 4°C, the cells were washed three times with PBS, and then incubated with secondary antibody (#A0568, FITC-labeled goat anti-mouse IgG, 1:1000 dilution, Beyotime Biotechnology) for 1 h at room temperature. Afterward, the cells were washed three times with PBS, and stained with DAPI for 10 min at room temperature. The cells were then observed using a DeltaVision OMX SR imaging system. Three independent experiments were conducted, and the results of one representative experiment are shown.

Western blotting assay H9c2 cells were harvested and lysed in RIPA buffer containing the protease inhibitor phenylmethane-sulfonyl fluoride. The total protein concentrations were determined by using a BCA protein assay kit. Equal amounts of total proteins (20 μg) from each treatment group were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis at 100 V for 2 h, and then electrophoretically transferred to polyvinylidene fluoride membranes. The membranes were blocked using TBST (1× TBS and 0.05% Tween-20) containing 5% skim milk for 2 h, and then incubated with rabbit polyclonal anti-Nrf2 (c-20) (#sc722, 1:200 dilution, Santa Cruz Biotechnology, Dallas, TX, USA) or rabbit polyclonal anti-actin (#4967S, 1:10000 dilution, Cell Signaling Technology, Danvers, MA, USA) overnight at 4°C. After washing for 10 min in TBST (1× TBS and 0.05% Tween-20) three times, the membranes were incubated with secondary polyclonal antibody (goat anti-rabbit HRP-conjugated IgG, 1:10000 dilution, Absin, Shanghai, China) for 1 h at room temperature. After the membranes were washed again in TBST, the immune complexes were detected by using ECL-Plus TM chemiluminescent detection reagent (P0018A, Beyotime Biotechnology), and visualized with a Molecular ImagerR ChemiDoc TM XRS + System (Bio-Rad Laboratories, Hercules, CA, USA). Three independent experiments were conducted, and the results of one representative experiment are shown.

Values are expressed as the mean ± standard deviation. All statistical analyses were performed using GraphPad Prism 5.0 software, and comparisons between two groups were analyzed using two-tailed Student's t test. A P value less than 0.05 was considered statistically significant.

GAS protected H9c2 cells against ischemia-like injury, IR-like injury, and H 2 O 2 -induced injury As shown in Fig. 1a , treatment with GAS at concentrations ranging from 5 to 100 μM exhibited no significant influence on the viability of H9c2 cells. As shown in Supplementary Fig S1, pretreatment with GAS significantly protected H9c2 cells against ischemia-like injury (Fig S1a) , as well as IR-like injury (Fig S1b) . Intracellular reactive oxygen species (ROS)-level measurements indicated that the increase in intracellular ROS induced by IR-like injury was significantly ameliorated by GAS pretreatment (Fig S1c) . These results suggest that GAS-mediated protection of H9c2 cells against IR-like injury is related to a decrease in oxidative stress. Therefore, the present study focused on studying the effects of GAS on H9c2 cells that underwent oxidative injury induced by

First, the viability of H9c2 cells treated with different concentrations of H 2 O 2 for 24 h (Fig. 1b) was observed. As shown in Fig. 1b , H 2 O 2 at 200, 300, 400, and 500 μM induced~10%, 30%, 60%, and 80% decreases in the viability of H9c2 cells, respectively. To determine the conditions in which cell viability would decrease by~40% or 50% in less than 24 h, the effect of 400 μM GAS treatment for different times (3, 6, 9, and 24 h) on cell viability was further examined. As shown in Fig. 1c , treatment with 400 μM H 2 O 2 for 3 h induced an approximately 40% decrease in cell viability, and thus was used as a condition for H 2 O 2 treatment in the subsequent experiments. As shown in Fig. 1d , pretreatment with GAS at 20, 50, and 100 μM significantly ameliorated the decrease in viability in H 2 O 2 -treated H9c2 cells. Intracellular ROSlevel measurements indicated that the H 2 O 2 -induced increase in ROS was significantly ameliorated by GAS pretreatment (Fig. 1e) . Representative photographs of the intracellular ROS signal in H9c2 cells treated with 400 μM H 2 O 2 for 3 h with or without GAS pretreatment (Fig. 1f ) also show that GAS pretreatment inhibited the H 2 O 2 -induced increase in intracellular ROS. These results suggest that GAS protects H9c2 cells against H 2 O 2 -induced oxidative injury.

GAS attenuated H 2 O 2 -induced apoptosis of H9c2 cells Representative photographs of cultured H9c2 cells treated with 400 μM H 2 O 2 with or without GAS pretreatment are shown in Fig. 2a . Treatment with H 2 O 2 induced considerable cell death in H9c2 cells, while GAS pretreatment ameliorated H 2 O 2 -induced cell death (Fig. 2a) . Flow cytometry results showed that H 2 O 2 -induced cell death included both early and late apoptosis (Fig. 2b) . The cells that were Annexin V-FITC + /PI − were defined as early apoptotic cells, while the cells that were Annexin V-FITC + /PI + were defined as late apoptotic cells. As shown in the quantification of the flow cytometry results, GAS significantly decreased the percentage of apoptotic cells (Fig. 2c) . These results suggest that GAS attenuates H 2 O 2 -induced apoptosis in H9c2 cells. Notably, no obvious dose-response effect of GAS was observed in either the apoptosis or cell viability analysis (as shown in Fig. 1d ). The reason for this is unclear. It is possible that the protective effects of GAS on H9c2 cells were mainly due to decreasing ROS levels, and the response to ROS elimination might plateau. (Fig. 3a) . Quantification of 100 random cells in each group confirmed that GAS significantly ameliorated the decrease in the percentage of cells with long tubular mitochondria, and the increase in the percentage of cells with short tubular mitochondria or fragmented mitochondria induced by H 2 O 2 (Fig. 3b) . Mitochondrial network morphology characteristic analyses using the ImageJ macro tool MiNA are shown in Fig. 3c -g. GAS significantly attenuated the H 2 O 2 -induced change in networks (Fig. 3c) , mitochondrial footprint (Fig. 3e) , and mean branch length (Fig. 3f) . These results suggest that H 2 O 2 treatment induces considerable imbalance in mitochondrial dynamics in H9c2 cells, while GAS pretreatment partly attenuates the H 2 O 2 -induced change in mitochondrial morphology, and might be helpful in maintaining mitochondrial function.

The mitochondrial respiration potency of H9c2 cells treated with H 2 O 2 with or without GAS pretreatment was measured. As shown in Fig. 4a , H 2 O 2 treatment induced a considerable decrease in mitochondrial respiration potency. Treatment with GAS alone exhibited no influence on mitochondrial respiration, while pretreatment with GAS partly ameliorated the H 2 O 2 -induced inhibition of mitochondrial respiration in H9c2 cells (Fig. 4a) . Analysis of the mitochondrial respiration parameters, such as basal respiration, maximal respiration, and ATP production, are shown in Fig. 4b-d , respectively. GAS pretreatment significantly ameliorated the H 2 O 2 -induced decrease in basal respiration (Fig. 4b) , maximal respiration (Fig. 4c) , and ATP production (Fig. 4d) in H9c2 cells. These results suggest that H 2 O 2 -induced mitochondrial dysfunction in H9c2 cells was partly attenuated by GAS.

GAS induced nuclear translocation of the Nrf2 protein in H9c2 cells As shown in Fig. 5a , the Western blotting results showed that H 2 O 2 treatment with or without GAS pretreatment did not cause a significant change in the protein level of Nrf2. The Nrf2 protein only showed a slight increase in cells that were treated with GAS. Interestingly, as shown in Fig. 5b , the distribution of Nrf2 protein in H9c2 cells exhibited great differences in the different groups of cells. In control cells, Nrf2 protein was almost exclusively observed in the cytoplasm. Treatment with H 2 O 2 induced translocation of Nrf2 protein from the cytoplasm into the nucleus, which indicated activation of Nrf2; therefore, the yellow signal (merge of the red signal of Nrf2 and the green signal of nuclear DAPI staining) Quantitative results of the percentage of cells with long tubular mitochondria, short tubular mitochondria, or fragmented mitochondria. c-g Quantitative results of mitochondrial network characteristics, including networks (c), individuals (d), mitochondrial footprint (e), mean branch length (f), and median branch length (g). The data presented are the mean ± SD and # P < 0.05 vs. the control group, *P < 0.05 vs. the H 2 O 2 -induced group increased in these cells. Importantly, pretreatment with GAS enhanced the nuclear translocation of Nrf2, and the cells exhibited a strong yellow signal, which suggested that GAS induced activation of Nrf2 in H9c2 cells (Fig. 5b) .

Nrf2 was involved in the protective effects of GAS on H 2 O 2 -treated H9c2 cells To examine whether Nrf2 was involved in the protective effects of GAS on H9c2 cells, Nrf2 expression in H9c2 cells was knocked down using siRNA transfection, and then the effects of GAS on these Nrf2-siRNA-treated cells were compared with the effects on negative control cells (cells treated with nontargeting siRNA). As shown in Fig. 6a (RT-PCR results) and Fig. 6b (Western blotting results), Nrf2-siRNA transfection successfully decreased both the mRNA and protein expression of Nrf2 in H9c2 cells. In negative control cells, H 2 O 2 treatment induced a significant decrease in cell viability, while GAS pretreatment significantly ameliorated the H 2 O 2 -induced decrease (Fig. 6c) . In contrast, in Nrf2-siRNA-treated cells, H 2 O 2 treatment also induced a significant decrease in cell viability, but GAS pretreatment did not ameliorate the H 2 O 2induced decrease in cell viability (Fig. 6c) . Analysis of intracellular ROS levels showed similar results: in Nrf2-siRNA-treated cells, GAS pretreatment did not ameliorate the H 2 O 2 -induced increase in ROS levels (Fig. 6d) . These results suggest that Nrf2 knockdown attenuated the protective effects of GAS against oxidative stress, and that Nrf2 is involved in the mechanisms of GAS.

Knockdown of Nrf2 expression attenuated the effects of GAS on H 2 O 2 -induced imbalanced mitochondrial dynamics As shown in Fig. 7a , in negative control cells, H 2 O 2 treatment induced imbalanced mitochondrial dynamics, while GAS pretreatment ameliorated the imbalanced mitochondrial dynamics induced by H 2 O 2 . In contrast, in Nrf2-siRNA-treated cells, GAS pretreatment did not successfully ameliorate the imbalanced mitochondrial dynamics induced by H 2 O 2 (Fig. 7b) . Quantitative analysis of mitochondrial network characteristics showed similar results: in Nrf2-siRNA-treated cells, GAS pretreatment did not ameliorate the decrease in mitochondrial footprint, mean branch length, or median branch length induced by H 2 O 2 (Fig. 7c) . These results suggest that Nrf2 is involved in the effects of GAS in maintaining the mitochondrial morphology of H9c2 cells that underwent H 2 O 2 treatment.

GAS inhibited H 2 O 2 -induced changes in expression levels of genes related to mitochondrial fusion and fission As shown in Fig. 8a , in negative control cells, H 2 O 2 treatment induced significant changes in the expression levels of genes related to mitochondrial fusion (Mfn1, Mfn2, and Opa1) and mitochondrial fission (Fis1 and Drp1). GAS pretreatment significantly ameliorated the changes in the expression levels of Mfn2, Opa1, and Fis1, but did not influence those of Mfn1 and Drp1 (Fig. 8a) . These results suggest that Mfn2, Opa1, and Fis1 play roles in the protective effects of GAS on the mitochondria of H9c2 cells. Furthermore, as shown in Fig. 8b , Nrf2 knockdown attenuated the regulatory effects of GAS on the expression levels of Mfn2, Opa1, and Fis1. These results confirmed the involvement of Nrf2 in the mechanisms of GAS. To determine whether Nrf2 knockdown directly affects the expression levels of these genes related to mitochondrial fusion and fission, the expression levels of the genes in negative control cells and Nrf2-siRNA-treated cells were also compared. As shown in Fig. 8c , Nrf2 knockdown significantly decreased the expression level of Mfn2 but not that of Opa1 or Fis1. These results suggest that Nrf2 has direct regulatory effects on mitochondrial fusion.

Knockdown of Nrf2 expression attenuated the effects of GAS on H 2 O 2 -induced mitochondrial dysfunction As shown in Fig. 9a , in negative control cells, H 2 O 2 treatment induced a considerable decrease in mitochondrial respiration potency, while GAS pretreatment partly ameliorates the H 2 O 2induced mitochondrial dysfunction. Treatment with Nrf2-siRNA induced a slight decrease in mitochondrial respiration potency. Importantly, in Nrf2-siRNA-treated cells, H 2 O 2 treatment also induced a considerable decrease in mitochondrial respiration potency, but GAS pretreatment did not ameliorate the H 2 O 2induced mitochondrial dysfunction (Fig. 9a) . Analysis of the parameters of mitochondrial respiration, such as basal respiration, maximal respiration, and ATP production, are shown in Fig. 9b-d,   Fig. 4 GAS attenuated mitochondrial dysfunction in H9c2 cells induced by H 2 O 2 . a Mitochondrial respiration potency of control cells, 50 μM GAS-treated cells, and 400 μM H 2 O 2 -treated cells, pretreated with 50 μM GAS, was measured using a Seahorse metabolic analyzer. b Results of quantification analysis of basal respiration. c Results of quantification analysis of maximal respiration. d Results of quantification analysis of ATP production. The data presented are the mean ± SD and # P < 0.05 vs. the control group, *P < 0.05 vs. the H 2 O 2 -induced group respectively. In Nrf2-siRNA-treated cells, GAS pretreatment did not ameliorate the decrease in basal respiration (Fig. 9b) , maximal respiration (Fig. 9c) , or ATP production (Fig. 9d) induced by H 2 O 2 . These results suggest that Nrf2 is involved in the effects of GAS in maintaining the mitochondrial functions of H9c2 cells that underwent H 2 O 2 treatment.

In the present study, GAS protected H9c2 cells against ischemialike and IR-like injury. The results were consistent with previous reports about the cardioprotective effects of GAS [7] [8] [9] 25] . Importantly, the protective effects of GAS against IR-like injury were found to be related to its ROS-decreasing activity, and GAS directly protected H9c2 cells against oxidative injury (H 2 O 2 treatment). The antioxidant effects of GAS have also been observed in other types of cells, such as neurons [26, 27] , astrocytes [28] , osteoblasts [29] , bone marrow mesenchymal stem cells, and macrophages [30] . These results suggest that antioxidant activity plays important roles in the various effects of GAS.

Mitochondria are especially important in cardiomyocytes, since the heart is an organ with high bioenergetic demands, and more than 90% of ATP is supplied by mitochondria [31] . Interestingly, mitochondria are highly dynamic organelles with constant movement and morphological changes. Mitochondrial dynamics are involved in fundamental biological processes such as cell metabolism, cell survival, and death [31] . Our results in observing the morphology of mitochondria by using super resolution microscopy showed that H 2 O 2 treatment induced fragmentation of mitochondria, and a significant decrease in networks, footprints, and tubular lengths of mitochondria in H9c2 cells. Disruption of mitochondrial dynamics has been implicated in various human diseases, including developmental defects, neurodegenerative diseases, metabolic diseases, and cardiovascular diseases [31] . Previous reports have shown that mitochondrial morphological defects, such as increased mitochondrial fission and decreased fusion, are important events in cardiac ischemia and reperfusion [32] [33] [34] [35] . For example, fragmented mitochondria and a decrease in mitochondrial fusion were detected in cardiomyocytes from both heart failure patients and cardiac ischemia animal models [35] . The results of the present study clearly showed imbalanced mitochondrial dynamics in H 2 O 2 -treated H9c2 cells, as indicated by a decrease in mitochondrial fusion and an increase in mitochondrial fission. Furthermore, GAS ameliorated the imbalanced mitochondrial dynamics induced by H 2 O 2 . The effects of GAS in maintaining the normal structure of mitochondria might be the basis for protecting H9c2 cells against oxidative injury. Cell apoptosis is closely related to mitochondrial dynamics. During apoptosis, mitochondria undergo extensive fragmentation, which precedes caspase activation, to release proapoptotic factors [36] . Excessive fission or decreased fusion contributes to cell apoptosis, while inhibition of mitochondrial fission blocks or delays cell death [37] [38] [39] [40] . The GAS-mediated protection of mitochondria from fragmentation might contribute to the decrease in apoptosis in H 2 O 2 -treated H9c2 cells that were pretreated with GAS.

Mitochondrial dynamics and bioenergetics are reciprocally coupled [17] ; thus, imbalanced mitochondrial dynamics might result in dysfunctional mitochondrial respiration and ATP production. Measurement of real-time mitochondrial respiration in living H9c2 cells using a Seahorse XFe96 Extracellular Flux Analyzer showed that mitochondrial respiration in H9c2 cells, including basal respiration, maximal respiration, and ATP production, was strongly inhibited by H 2 O 2 treatment. GAS treatment alone did not affect mitochondrial respiration, while pretreatment with GAS partly ameliorated the H 2 O 2 -induced inhibition of mitochondrial respiration. Previous reports have shown that elongated mitochondria are linked to more efficient ATP production and distribution, and better sustained stress-induced cell damage, while fragmented (through fission) mitochondria are associated with decreased ATP production and increased susceptibility to injury [41, 42] . The protective effects of GAS on mitochondrial a Representative photographs of mitochondrial morphology in negative control H9c2 cells treated with 400 μM H 2 O 2 for 3 h with or without GAS pretreatment. Bar = 20 or 2 µm. b Representative photographs of mitochondrial morphology in Nrf2-siRNA-treated H9c2 cells treated with 400 μM H 2 O 2 for 3 h with or without GAS pretreatment. Bar = 20 or 2 µm. c Quantitative results of the mitochondrial network characteristics, including networks, mitochondrial footprint, mean branch length, and median branch length. The data presented are the mean ± SD and # P < 0.05 vs. the control group, *P < 0.05 vs. the H 2 O 2 -induced group respiration and ATP production might be related to its regulatory effects on mitochondrial dynamics and mitochondrial fragmentation. Notably, accumulating evidence supports that mitochondrial dynamics and bioenergetics may reciprocally influence each other, depending on the experimental condition or initial stimulation [17] . The interaction and feedback regulation between mitochondrial dynamics and mitochondrial respiration might be involved in the mechanisms of GAS.

Mitochondrial dynamics are controlled by a group of proteins, such as the fusion proteins mitofusin 1 and 2 (Mfn1/2) and optic atrophy 1 (Opa1), and the fission proteins fission protein 1 (Fis1) and dynamin-related protein 1 (Drp1). Mitochondrial fusion is an essential mechanism by which damaged mitochondria mitigate stresses by exchanging proteins, lipids, and mitochondrial DNA with healthy mitochondria [17] . Mfn1 and Mfn2 are the primary regulators of outer mitochondrial membrane fusion, while Opa1 mediates inner mitochondrial membrane fusion [43, 44] . The present study showed that H 2 O 2 treatment induced a significant decrease in the expression of Mfn1, Mfn2, and Opa1. These results suggest that both outer and inner mitochondrial membrane fusion were inhibited by H 2 O 2 treatment. GAS pretreatment significantly ameliorated the decrease in the expression levels of Mfn2 and Opa1, but not Mfn1 in H 2 O 2 -treated H9c2 cells. Mitochondrial fission is also critical in mitochondrial quality control by separating damaged mitochondria and subsequently removing them through mitophagy [17] . In yeast, mitochondrial fission is regulated by cytosolic dynamin-like GTPase (Dnm1p) and a C-tail-anchored outer membrane protein, Fis1p. In mammals, Drp1 and Fis1 are involved in mitochondrial fission as Dnm1 and Fis1 orthologs, respectively [45] . Fis1, located on the mitochondrial outer membrane, recruits Drp1 from the cytosol to the outer mitochondrial membrane, and then the GTPase activity of Drp1 provides a driving force through hydrolyzing GTP to mediate outer and inner mitochondrial membrane fission [46, 47] . The present study showed that H 2 O 2 treatment induced a significant increase in the expression of Fis1 and a decrease in Drp1. The increase in Fis1, a mitochondrial fission-stimulating protein [45] , indicates activation of the mitochondrial fission process in H 2 O 2treated H9c2 cells. The decrease in Drp1 is interesting, although the mechanism for its decrease is unknown. A previous report , and Fis1 in Nrf2-siRNA-treated cells that underwent H 2 O 2 treatment with or without GAS pretreatment. c Comparison of the expression levels of Mfn2, Opa1, and Fis1 in negative control cells and Nrf2-siRNA-treated cells. The data presented are the mean ± SD of at least three independent experiments. *P < 0.05 between the indicated two groups examining the expression of Drp1 in H9c2 cells and 143B osteosarcoma cells also showed that under H 2 O 2 treatment, Drp1 expression significantly decreases in H9c2 cells and slightly decreased in 143B cells [48] . The roles of Drp1 in oxidative stressinduced cells need further study. GAS pretreatment ameliorated the increase in Fis1, but exhibited no influence on the expression of Drp1. These results suggest that GAS ameliorated the activation of mitochondrial fission induced by H 2 O 2 .

The present study confirmed that nuclear factor erythroid 2related factor 2 (Nrf2) is an important target of GAS. Nrf2 is a transcription factor that is activated by increased ROS production, and reduces ROS levels by upregulating antioxidant/detoxification genes [49] . Activation of Nrf2, including an increase in expression and/or nuclear translocation, has been observed in various types of cells treated with H 2 O 2 in time-and dose-dependent manners [50, 51] . In the present study, treatment with H 2 O 2 at 400 µM for 3 h induced the nuclear translocation of Nrf2 without a significant increase in the Nrf2 expression level. Importantly, GAS pretreatment resulted in a slight increase in Nrf2 expression, and strongly enhanced the nuclear translocation of Nrf2 under H 2 O 2 treatment. The roles of Nrf2 in the mechanisms of GAS have been reported in neurons [27, 28, [52] [53] [54] [55] [56] [57] , osteoblasts [29, 58] , liver HL-7702 cells [59] , and endothelial cells [60] . In the present study, knockdown of Nrf2 expression attenuated the protective effects of GAS on H9c2 cells, as indicated by increasing cell viability, decreasing ROS levels, ameliorating mitochondrial fragmentation, and maintaining mitochondrial respiration potency. These results suggest that Nrf2 is also an important target of GAS in H9c2 cardiomyocytes, and plays critical roles in the protective effects of GAS against oxidative injury. Furthermore, Nrf2 knockdown also attenuated the effects of GAS on the expression of mitochondrial fusion-and fission-related genes such as Mfn2, Opa1, and Fis1. Nrf2 knockdown also directly influenced the expression of Mfn2 in H9c2 cells. These results suggest that Nrf2 contributes to the control of mitochondrial dynamics.

In summary, GAS induces the activation of Nrf2, regulates mitochondrial dynamics, maintains the structure and functions of mitochondria, and thus protects H9c2 cells against oxidative Fig. 9 Knockdown of Nrf2 expression attenuated the effects of GAS on H 2 O 2 -induced mitochondrial dysfunction in H9c2 cells. a Mitochondrial respiration potency in negative control cells or Nrf2-siRNA-treated cells that underwent H 2 O 2 treatment with or without pretreatment with 50 μM GAS. b Results of quantification analysis of basal respiration. c Results of quantification analysis of maximal respiration. d Results of quantification analysis of ATP production. The data presented are the mean ± SD and *P < 0.05 between the indicated two groups injury. There has been a renaissance in mitochondrial research in recent years, featuring exciting developments in understanding the structure and organization of mitochondria, and the roles of mitochondria in cellular-signaling mechanisms, in addition to their traditional role as a cell powerhouse in eukaryotes [17] . The canonical and noncanonical roles of mitochondrial dynamics factors, such as Mfn1/2, Opa1, Fis1, and Drp1, suggested by recent discoveries, have added another dimension to the integration of mitochondrial functions [17] . In addition to the canonical roles of these factors in controlling the fusion/fission of mitochondria, they might also bear noncanonical roles, such as regulating mitochondrial bioenergetics and cell death, including apoptosis and autophagy [35, 37, 61] . Through dynamic changes in structure, mitochondria play important and pleiotropic roles in cells by regulating ATP production, ROS levels, ion channels, cell survival, and death under physiological conditions, as well as under injury. The present study suggests that the regulation of mitochondria is involved in GAS-mediated protection of H9c2 cardiomyocytes, and confirmed the critical role of mitochondria in cell regulation under oxidative injury. However, the mechanisms of mitochondrial dynamics regulation have not been fully clarified, and further study in this area is necessary and important.

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QQC conducted the experiments, analyzed the data, and drafted the paper. YWW participated in the experiments. WMY, MHT, YCW, and HYH helped to design the experiments. WDZ and XL supervised the research and revised the paper. All authors reviewed and approved the paper.

The online version of this article (https://doi.org/10.1038/s41401-020-0382-x) contains supplementary material, which is available to authorized users.Competing interests: The authors declare no competing interests.