Echinacoside reverses myocardial remodeling and improves heart function via regulating SIRT1/FOXO3a/MnSOD axis in HF rats induced by isoproterenol

Abstract Myocardial remodelling is important pathological basis of HF, mitochondrial oxidative stress is a promoter to myocardial hypertrophy, fibrosis and apoptosis. ECH is the major active component of a traditional Chinese medicine Cistanches Herba, plenty of studies indicate it possesses a strong antioxidant capacity in nerve cells and tumour, it inhibits mitochondrial oxidative stress, protects mitochondrial function, but the specific mechanism is unclear. SIRT1/FOXO3a/MnSOD is an important antioxidant axis, study finds that ECH binds covalently to SIRT1 as a ligand and up‐regulates the expression of SIRT1 in brain cells. We hypothesizes that ECH may reverse myocardial remodelling and improve heart function of HF via regulating SIRT1/FOXO3a/MnSOD signalling axis and inhibit mitochondrial oxidative stress in cardiomyocytes. Here, we firstly induce cellular model of oxidative stress by ISO with AC‐16 cells and pre‐treat with ECH, the level of mitochondrial ROS, mtDNA oxidative injury, MMP, carbonylated protein, lipid peroxidation, intracellular ROS and apoptosis are detected, confirm the effect of ECH in mitochondrial oxidative stress and function in vitro. Then, we establish a HF rat model induced by ISO and pre‐treat with ECH. Indexes of heart function, myocardial remodelling, mitochondrial oxidative stress and function, expression of SIRT1/FOXO3a/MnSOD signalling axis are measured, the data indicate that ECH improves heart function, inhibits myocardial hypertrophy, fibrosis and apoptosis, increases the expression of SIRT1/FOXO3a/MnSOD signalling axis, reduces the mitochondrial oxidative damages, protects mitochondrial function. We conclude that ECH reverses myocardial remodelling and improves cardiac function via up‐regulating SIRT1/FOXO3a/MnSOD axis and inhibiting mitochondrial oxidative stress in HF rats.


| INTRODUC TI ON
Heart failure (HF) is the ultimate outcome of most cardiovascular diseases with high prevalence and poor prognosis, and with the aggravating trend of an ageing population, the hospitalization and mortality due to chronic heart failure (CHF) increased sharply all around the world. 1 During the past 20 years, drug treatments for HF have made great progress, but the effect is very limited, so new drugs are urgently needed. Myocardial remodelling is important pathological basis of HF and is characterized by myocardial hypertrophy, apoptosis, interstitial fibrosis, the myocardial cells are disorganized, and lead to reduction of ejection fraction (LVEF), increase of left ventricular end-diastolic and systolic dimensions (LVIDd and LVIDs), and decrease of left ventricular fractional shortening (LVFS). 2 But the mechanisms underlying were still not fully understood. In recent years, numerous studies have confirmed that oxidative stress is an important promoter of myocardial remodelling. [3][4][5][6][7] Oxidative stress is a condition in which reactive oxygen species (ROS) or free radicals are generated excessively and lead to oxidative damage of cell components, this in turn causes cell dysfunction. 8 Mitochondria are the main source of intracellular ROS, as a result of leakage from the respiratory electron transport chain, and mitochondria are the most sensitive position about the ROS' effects. 9 Studies in HF identified increased ROS generation from complex I of the respiratory chain. 8 Increased ROS production attacks mitochondrial component firstly and results in excess oxidative stress, manifests as carbonylation of proteins, lipid peroxidation, and DNA damage, 10 and dysfunction of mitochondria, which consequently lead to excessive formation of ROS, 8 forms a vicious circle. The sustained oxidative stress is demonstrated can cause myocardial hypertrophy and myocardial fibrosis, [11][12][13] and cause apoptosis of myocardial cells through activation of mitochondria-dependent apoptosis as a result of carbonylation of mitochondrial membrane proteins and loss of mitochondrial membrane potential. 14 So, it is considered that inhibition of mitochondrial oxidative stress may be a new strategy to prevent myocardial remodelling and even HF. [15][16][17] Echinacoside (ECH), a natural phenylethanoid glycoside, is the major active component of Cistanches Herba, which is a traditional Chinese medicine. In recent years, ECH has been extensively studied in nervous system and tumour, and it has been reported to possess a variety of pharmacological effects, such as antioxidant, anti-inflammatory, anti-apoptosis and anti-tumour properties. 18,19 In nerve cells, ECH significantly enhances antioxidant capacity, reduces ROS production, improves mitochondrial membrane potential (MMP) and increases cell viability, 20 exhibits protective effects on mitochondrial function and inhibits mitochondrial oxidative stress, it protects against oxidative injury and improves memory, eventually prevents neurodegeneration. 21,22 ECH also increases the activity of SOD and play a protective role in brain tissue and retina tissue, 19,23 but the specific mechanism for its antioxidant effect is unclear. SIRT1 is closely related to oxidative stress, and SIRT1/FOXO3a/ MnSOD signalling axis is the most important antioxidant axis and responsible for suppression of mitochondrial oxidative stress, MnSOD distributes widely in mitochondria and primarily responsible for clearing of mitochondrial ROS, thus inhibiting oxidative stress, compounds which activate this antioxidant axis can inhibit oxidative stress. 24,25 Recent studies have shown that ECH exerts it pharmacological effects via regulating multiple signalling pathways, such as p-AKT, mTOR/STAT3, TGF-beta 1/Smads and SIRT1 signalling pathway. [26][27][28][29] And a new study finds that ECH binds covalently to SIRT1 as a ligand and up-regulates the expression of SIRT1 in brain cells. 26 It suggests that ECH may inhibit mitochondrial oxidative stress in cardiomyocytes and reverse myocardial remodelling. However, researches on the effects of ECH have focused on the nervous system or tumour, the effects and mechanisms of ECH on mitochondrial oxidative stress in cardiomyocytes and myocardial remodelling remain unknown. Here, we firstly induce cellular model of oxidative

| Measurement of MMP by JC-1
Mitochondria are isolated and purified of AC-16 and protein concentration is determined as mentioned above. Then the mitochondria are resuspended at 1mg/ml concentration, the MMP is measured using JC-1 assay kit according to the manufacturer's instructions. The fluorescence intensity is measured at excitation wavelength: 490nm, slit 5nm, emission wavelength: 590 nm, slit 5nm with a spectrophotometer instrument, 30 the results are represent as FLU/μgP.

| Test carbonyl protein content in mitochondria of AC-16 cells
After treatment, purified mitochondria are isolated and sonicated on ice and are treated with 1% streptomycin sulphate to precipitate mitochondrial nucleic acids, protein concentration are determined.
The carbonyl protein content is tested using an Elisa kit (GENMED SCIENTIFICS INC, USA) based on derivatization of the carbonyl group with dinitrophenylhydrazine (DNPH), according to the instructions of the kit, OD is measured by a microplate reader and converted into nmol/mg protein by comparing to the standard curve.

| Determination of lipid peroxidation level in mitochondria of AC-16 cells
After treatment, cells are collected and lysed, mitochondria are isolated and purified and cracked by ultrasound, protein concentration is determined by BCA protein assay kit (Bio tech), lipid peroxidation is determined by the level of malondialdehyde (MDA) via the TBARS assay, using a kit (Nan jing jian cheng, China) and MDA level is measured following the manufacturer's protocol. OD is detected at 586 nm by a microplate reader and converted into μmol/g protein by comparing to the standard curve. tong University). Rat model of HF is established as described previously by us. 31 Briefly, Isoproterenol (10 mg/kg) is administered once daily by intraperitoneal injection for 2 weeks (defined as HF group), ECH is administered with 20 ug/g once daily by intraperitoneal injection at 30min before ISO is treated (defined as ECH group), and the administration lasts for 2 weeks. Control animals are administrated with 0.9% NaCl (defined as Ctrl group). Echocardiography measurements are performed to evaluate the heart function.

| Heart weight to body weight ratio (HW/BW) is measured
Rat is weighed before anaesthetization, fresh heart is isolated and wash out blood with 0.1mol/L PBS, cut away vessels and other redundant tissue and the heart is weighed, then HW/BW(mg/100g) is calculated.

| Histological staining
The hearts of rats are dissected and fixed in 4% paraformaldehyde, embedded in paraffin and sectioned into 5 μm-thick slices.
Haematoxylin-Eosin (HE) staining is used to observe the pathological changes and Masson's trichrome is used to evaluate collagen fibres of rats myocardium tissue. The cardiac collagen volume fraction (CVF) and cardiomyocytes cross-sectional area is measured with Image-Pro Plus 6.0 (Media Cybernetics, Bethesda, MD, USA).

| TUNEL staining
Myocardial tissue from the left ventricle is collected and the TUNEL assay is carried out as described previously by us 32

| Transmission electron microscope
Fresh heart tissue is fixed in 2.5% glutaraldehyde at 4℃, tissue is then cutted into 1mm 3 in volume and fixed again in 2.5% glutaraldehyde at

| Quantitative PCR
Total RNA is prepared from left ventricular tissue with Trizol reagent (Invitrogen, Carlsbad, USA). cDNA is synthesized using the TUREscript 1st Strand cDNA Synthesis Kit (Takara, Japan).
Specific primer sequences of collagen I used for real-time PCR is as follows: forward: 5′-GTCGTATCCAGTGCGTGTC-3′, revers: Real-Time quantitative PCR (qPCR) is performed with SYBRPremix Ex Taq (Perfect Real Time) (Takara, Japan). The relative level of mRNA is calculated by normalizing to GAPDH, according to the 2 -ΔΔCT method.

| Western blot analysis of SIRT1, FOXO3a, MnSOD protein expression
Total protein of left ventricular tissue are extracted on ice using cold RIPA Buffer (BioRad, USA) with Protease Inhibitor Cocktail (Sigma-Aldrich). The concentration is determined by BCA method.
Procedures of Western blot are described previously. 33 Briefly, protein sample is fractionated on 15% sodium dodecylsulphate-polyacrylamide gels, transferred to PVDF membranes (BioRad, USA), blocked by 5% skim milk and incubated with MYH antibody from mouse (diluted 1:200, Santa Cruz, USA). Then, incubated with a horseradish peroxidase-conjugated goat mouse antibody (diluted 1:2,000, Santa Cruz, USA). The relative level of protein is calculated by normalizing mean ray value to β-actin. Protein bands are detected by a chemiluminescence system (ChemiDoc XRS, BioRad).

| Statistical analysis
All data are presented as means ± SEM. One-way ANOVA is used to compare differences among multiple groups, followed by Tukey post hoc test for significance. All statistics are determined using SPSS15.0 software (SPSS Inc, IL, USA). A probability value of P < .05 is considered significant.  Figure 1C shows.

| 50 μM ECH effectively inhibits mitochondrial oxidative damage and apoptosis in AC-16 cells
Fourthly, ECH protects against oxidative damage of mitochondrial protein in AC-16 cells. As Figure 1D shows, the carbonyl protein content in mitochondria of ISO treatment cells is significantly increased and ECH attenuates the effect. Fifthly, ECH also inhibits mitochondrial lipid peroxidation in AC-16 cells induced by ISO. As Figure 1E shows, the level of lipid peroxides in mitochondria of ISO cells is obviously higher than that of in Ctrl cells, while this increase is significantly reduced by 50μM ECH pre-treatment. Ultimately, ECH inhibits the accumulation of intracellular ROS. As Figure 1F shows, the level of intracellular ROS in ISO cells is markedly increased and is prominently decreased in ECH pre-treated cells. Figure 1G shows the apoptosis rate of AC-16 cells is significantly increased following 24h incubations with 10μM ISO, comparing to the Ctrl cells, whereas pre-treatment cells with 50 μM ECH significantly reduces ISO-induced apoptosis.

| ECH reverses myocardial remodelling and improves cardiac function of ISO-induced HF rats
As shown in Figure 2A, hearts in HF rats are obvious hypertrophy and is reversed by treatment with ECH. As Figure 2B-D and L shows, LVPWTd is increased in HF, but no difference between ECH and Ctrl, or between ECH and HF, all as Figure 2E-K shows.
Results of HE staining indicates that the myocardial hypertrophy, arranged disorder or degeneration necrosis, inflammatory cell infiltration and interstitial hyperaemia and oedema in HF rats, and ECH significantly alleviates these damages, myocardial hypertrophy and arranged disorder are relieved, the degeneration necrosis and inflammatory infiltrate are reduced, as shown in Figure 3A.
The cardiomyocytes cross-sectional area is measured, it is significantly increased after ISO and is decreased in ECH treated group, as Figure 3E shows. Assessments of collagen deposition by Masson staining reveals that left ventricular collagen content in HF rat is increased obviously and apparently decreased after treatment with ECH, which obviously decreases CVF. Real-time PCR analysis reveals that procollagen type Ⅰ α transcripts are up-regulated in ISO rat and down-regulated in ECH rat, these results are consistent with each other, as Figure 3B and F-G shows, the data indicate that ECH effectively reduces collagen deposition. The number of TUNEL-positive cells in cardiomyocytes is significantly increased in HF rats compared with Ctrl, ECH markedly reduces myocardial cell apoptosis, and Figure 3C-D shows the representative image of TUNEL-positive cells in low and high power field respective, and Figure 3H shows the apoptosis rate of myocardial cells. These results indicate that ECH effectively reverses myocardial hypertrophy, myocardial interstitial fibrosis and apoptosis, and improves heart function in HF rat induced by ISO.

| ECH protects myocardial ultrastructure in HF rats
Transmission electron micrographs results show that the damage of myocardial ultrastructure could be attenuated by ECH, as

| ECH up-regulates protein expression of SIRT1, FOXO3a and MnSOD
Western Blot analysis demonstrates that the protein expression level of SIRT1, FOXO3a and MnSOD are significantly down-regulated in HF rat, however, ECH obviously up-regulates the decreased protein expression. The representative protein bands of immunoblotting and statistical analysis results are showed in Figure 5. The part of the data indicates that ECH increases the protein expression of SIRT1, FOXO3a and MnSOD.

| ECH inhibits mitochondrial oxidative stress in left ventricular myocardial tissue
In this part, mitochondrial ROS, MMP, oxidative damage of mitochondrial DNA, carbonyl protein content and lipid peroxidation level in mitochondria in left ventricular myocardial tissue of rats are all measured, and the results are consistent with cell experiments, that is to say, ECH decreases mitochondrial ROS, protects MMP, relieves the damage degree of mtDNA, reduces carbonyl protein content and lipid peroxidation in mitochondrial, as Figure 6 shows.

| D ISCUSS I ON
This study demonstrates that ECH exerts effective roles in the prevention of myocardial remodelling and improvement of heart function via up-regulating SIRT1/FOXO3a/MnSOD signalling axis and inhibiting mitochondrial oxidative stress.  36 In HF, the activity of MnSOD is significantly decreased and the generation of mitochondrial ROS is elevated. 37,38 In cardiac specific MnSOD gene knockout mice, mitochondrial ROS could not be cleared and ROS is increased in cardiomyocytes, this led to mitochondrial dysfunction and progressive cardiac enlargement, eventually lead to heart failure. 39 In present study, we confirm that the expression of MnSOD is significantly decreased, which is attenuated by ECH treatment, and thus, mitochondrial ROS is reduced and mitochondrial oxidative stress is inhibited. It means that ECH protects cardiomyocytes against oxidative damage through up-regulation of the expression of MnSOD, we further explore the molecular mechanisms underlying these effects.
The expression of MnSOD is regulated by FOXO3a, which is a transcription factor and binds in the gene promoter region of MnSOD and leads to the promotion of gene transcription. 40  also, there are abnormalities in mitochondrial size and shape, reactive myocardial hypertrophy and interstitial fibrosis, which secondary to myocyte loss. 44 We also observed the above changes  45 the myocardial glycogen level is also measured, the results indicates that there is no significant difference among the three groups, as shown in Figure 7J.
The previous study also detected myocardial hibernation in a non-ischaemic heart failure pig model by pacing the LV free wall with high-frequency, 46 because microvascular obstruction plays important roles in myocardial hibernation and the resulting contractile dysfunction, and that are marked improved when the microvascular obstruction is reversed by drug. 47 At present, some study conclude that revascularization therapy is effective to hibernating myocardium, 43  cardioprotective effects by increasing expression of MnSOD, 48,49 and rising of MnSOD is related to increased myocardial capillary density and VEGF expression. We performed RT-PCR to detected the expression of VEGF, the data are consistent with the finding of histological staining, it means that ECH may promote angiogenesis and increase myocardial capillary density via regulating VEGF expression, as shown in Figure 7I. The present study finds that ECH can protect and increase myocardial capillary and arteriolar via up-regulating SIRTI/FOXO3a/MnSOD pathway and the expression of VEGF, thereby suppressing hibernating myocardium and contributing to improve heart function.
Of note, cardiomyocyte apoptosis is well known to be an important pathologic changes in heart failure, and the present study indeed confirms that large amount of apoptotic cells in HF rats and ECH significantly reduces the apoptosis via regulating SIRTI/FOXO3a/MnSOD pathway and inhibiting mitochondrial oxidative stress, as mentioned above.
Above all, ECH reverses myocardial remodelling and improves heart function via up-regulating SIRT1/FoxO3a/MnSOD signalling axis, and thus inhibits mitochondrial oxidative stress and reduces hibernating myocardium. Thus, it is suggested that ECH is a potential drug for prevention or treatment of myocardial remodelling and HF. And we thanks for the financial assistance.

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

AUTH O R CO NTR I B UTI O N S
JW-Z and YJ-N designed the study, planned and performed experiments, analysed data and drafted and revised the manuscript. JD, XL and QL helped to perform experiments, administrated experiments, collected and analysed data. JL-Z validated data, supported software and revised the manuscript.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data used to support the findings of this study are available from the corresponding author upon request.