Echinacoside Reverses Myocardial Remodeling And Improves Heart Function

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Yajuan Ni1 | Jie Deng1 | Xin Liu1 | Qing Li1 | Juanli Zhang1 | Hongyuan Bai1 | Jingwen Zhang2

Abstract

Myocardial remodeling is an important pathological basis of HF, mitochondrial oxidative stress is a promoter of 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 tumors, 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 hypothesize that ECH may reverse myocardial remodeling and improve the heart function of HF via regulating the SIRT1/FOXO3a/ MnSOD signaling axis and inhibiting mitochondrial oxidative stress in cardiomyocytes. Here, we firstly induce a 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 an HF rat model induced by ISO and pre-treat with ECH. Indexes of heart function, myocardial remodeling, mitochondrial oxidative stress and function, expression of SIRT1/FOXO3a/MnSOD signaling axis are measured, the data indicate that ECH improves heart function, inhibits myocardial hypertrophy, fibrosis and apoptosis increases the expression of SIRT1/FOXO3a/MnSOD signaling axis, reduces the mitochondrial oxidative damages, protects mitochondrial function. We conclude that ECH reverses myocardial remodeling and improves cardiac function via up-regulating the SIRT1/FOXO3a/MnSOD axis and inhibiting mitochondrial oxidative stress in HF rats.

KEYWORDS echinacoside, heart failure, mitochondrial oxidative stress, myocardial remodeling

fresh herbs cistanche to mitochondrial oxidative stress

1 | INTRODUCTION

Heart failure (HF) is the ultimate outcome of most cardiovascular diseases with high prevalence and poor prognosis, and with the aggravating trend of an aging 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 remodeling is an important pathological basis of HF and is characterized by myocardial hypertrophy, apoptosis, interstitial fibrosis, the myocardial cells are disorganized, and lead to a reduction of ejection fraction (LVEF), an 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 remodeling.3-7

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 to the ROS effects.9 Studies in HF identified increased ROS generation from complex I of the respiratory chain.8 Increased ROS production attacks mitochondrial components 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,11-13 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 remodeling and even HF.15-17

Echinacoside (ECH), a natural phenylethanoid glycoside, is the major active component of Cistanches Herba, which is traditional Chinese medicine. In recent years, ECH has been extensively studied in the nervous system and tumors, and it has been reported to possess a variety of pharmacological effects, such as antioxidant, anti-inflammatory, anti-apoptosis, and anti-tumor 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 plays 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 signaling axis is the most important antioxidant axis and is responsible for the suppression of mitochondrial oxidative stress, MnSOD distributes widely in mitochondria and is primarily responsible for the 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 its pharmacological effects via regulating multiple signaling pathways, such as p-AKT, mTOR/STAT3, TGF-beta 1/Smads, and SIRT1 signaling pathway.26-29 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 remodeling. However, research on the effects of ECH has focused on the nervous system or tumor, the effects and mechanisms of ECH on mitochondrial oxidative stress in cardiomyocytes and myocardial remodeling remain unknown. Here, we firstly induce a cellular model of oxidative stress by ISO and confirm the effect of ECH on mitochondrial oxidative stress and function in vitro. Then, we establish an HF rat model induced by ISO and demonstrate that ECH reverses myocardial remodeling and improves cardiac function via up-regulating the SIRT1/ FOXO3a/MnSOD signaling axis and inhibiting mitochondrial oxidative stress in HF rats. The results are intended to provide experimental evidence for the development of new drugs to prevent heart failure.

2 | MATERIALS AND METHODS

2.1 | Cell culture and treatment

AC16 cells are purchased from Bena Culture Collection. The cells are maintained in high glucose Dulbecco's modified Eagle's medium (DMEM; Hyclone, GE Healthcare, USA) supplement with 10% fetal bovine serum are (10%FBS; Gibco) in T-75 flasks (Corning Glassworks, Corning, NY, USA). Conditions are maintained at 37℃ in a humidified atmosphere containing 5% CO2. Once the cells reach 70%-80% confluence, ISO (Sigma, USA) group cells are incubated with a medium containing 10uM ISO (Sigma, USA) for 24h, ECH group cells are pre-treated with 50uM ECH (MCE, USA) for 30min prior ISO exposure. Control cells (Ctrl) are treated nothing.

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2.2 | Detection of mitochondrial ROS level using luminol chemiluminescence

Mitochondria are isolated and purified from AC-16 cells using Mitochondria Isolation Kit (Sigma, USA) according to the manufacturer's instructions. Protein concentrations are determined by the BCA protein assay kit (Biotech). Mitochondrial ROS level is detected by luminal (3-aminophthalic hydrazide 5-amino-2,3-dihydro-1,4-phthalazinedione) using an Elisa kit (GERMED SCIENTIFIC INC, USA), it reacts with ROS and causes the emission of photons, which is a band spectrum having a peak at about 460nm, and Relative Light Unit (RLU) is detected by chemiluminescence measuring instrument and converted into RLU/μg mitochondrial protein.

2.3 | 8-OHdG is used to evaluate the oxidative damage of mitochondrial DNA

Mitochondrial DNA oxidative damage is evaluated by detecting the content of 8-Hydroxy-2-deoxyguanosine (8-OHG). Purified mitochondria in cells are isolated using a mitochondria isolation kit as above (Sigma). Protein concentrations are determined by the BCA protein assay kit (Biotech). Then, they are dissociated to determine the level of 8-OHdG using an Elisa kit (GERMED SCIENTIFIC INC, USA) according to the manufacturer's instructions. The absorbance of the color change is measured in a spectrophotometer at the wavelength of 450 nm. The concentration of 8-OHdG in the samples is then determined by comparing the OD of the samples to the standard curve and converted into ng/ml.

2.4 | 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 a 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 represented as FLU/μgP.

2.5 | Detection of apoptotic cells by flow cytometry in AC-16 Cells

Flow cytometry is used to assess cellular apoptosis. An Annexin V-FITC/PI apoptosis detection kit (CWBIO, China) is used to detect apoptotic and necrotic cells according to the supplier's instructions. Early-stage apoptosis, late-stage apoptosis or necrosis, cellular debris, and viable cells are identified as annexin V (+)/PI (−), annexin V (+)/PI (+), annexin V (−)/PI (+) and annexin V (−)/PI (−), respectively. Fluorescence intensities are analyzed using a flow cytometer (BD FACSVerse™ Flow Cytometer, BD Biosciences, San Jose, CA, USA). Each experiment is repeated 3 times.

2.6 | Assessed the intracellular ROS by flow cytometer

After treatment, cells are trypsinized, washed twice with PBS. The cells are then resuspended in PBS, stained with DCFH-DA (Beyotime, China) at the final concentration of 1 μM and incubated for 1 h at 37℃ in an incubator with CO2, and then tested in a flow cytometer at 488 nm excitation. The fluorescence intensity represents the level of intracellular ROS.

2.7 | 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 sulfate to precipitate mitochondrial nucleic acids, protein concentration is determined. The carbonyl protein content is tested using an Elisa kit (GERMED SCIENTIFIC 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.

2.8 | 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 the BCA protein assay kit (Biotech), 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.

2.9 | Animals and treatment

Young male Sprague-Dawley rats weighing 120-140g are acquired from Xi'an Jiao tong University Laboratorial Animal Center (Shaanxi, China). The investigation conforms to The Guide for the Care and Use of Laboratory Animals, published by the US National Institutes of Health (NIH publication no.85-23, revised in 1996), and the experimental protocols were approved by the local authorities (Biomedical Ethics Committee of Medical Department of Xi ' a Jiao tong University). The 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 heart function.

2.10 | Echocardiographic measurements

Animals are anesthetized by an intraperitoneal injection of 300 mg/kg of chloral hydrate. The SONOS-2500 ultrasound system with an ultrasound transducer of 7.5 MHz (HP, USA) is used to make echocardiographic examinations. The left ventricular ejection fraction (LVEF), left ventricular fractional shortening (LVFS), left ventricular end-diastolic dimensions (LVIDd), left ventricular end-systolic dimensions (LVIDs), heart rate (HR), interventricular septal thickness in diastole (IVSTd), left ventricular posterior wall thickness in diastole (LVPWTd) are measured as described previously by us.32

FIGURE 1 50μmol ECH effectively inhibits mitochondrial oxidative stress of AC-16 cells induced by 10μmol/L ISO and protects mitochondrial function and reduces apoptosis.

2.11 | Heart weight to body weight ratio (HW/BW) is measured

Rat is weighed before anaesthetization, the fresh heart is isolated and washes 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.

2.12 | 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).

2.13 | TUNEL staining

Myocardial tissue from the left ventricle is collected and the TUNEL assay is carried out as described previously by us32

2.14 | Transmission electron microscope

Fresh heart tissue is fixed in 2.5% glutaraldehyde at 4℃, tissue is then cut into 1mm3 in volume and fixed again in 2.5% glutaraldehyde at 4℃ for 2h, then is washed with 0.1mol/L PBS, fixed in 1% osmic acid, dehydrated with acetone, embedded with epoxy resin, ultrathin slices which thin about 800nm are made, stained with uranyl acetate and lead citrate, and then are observed under a transmission electron microscope (H-7650, Hitachi Limited, Japan). To quantify the protective effect of ECH in mitochondrial morphology, the mean mitochondrial circumference, mean mitochondrial cross-sectional area, percentage of degeneration, and percentage of vacuolization in mitochondria are analyzed statistically with Image-Pro Plus 6.0 (Media Cybernetics, USA).

2.15 | 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: 5′-GTGGAGTCGGCAATTGCA-3′. GAPDH forward 5'-GGC AAG GTC ATC CCA GAG CT-3', reverse 5'-CGC CTG CTT CAC CAC CTT CT-3'. 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.

2.16 | Western blot analysis of SIRT1, FOXO3a, MnSOD protein expression

Total protein of left ventricular tissue is extracted on ice using cold RIPA Buffer (BioRad, USA) with Protease Inhibitor Cocktail (Sigma-Aldrich). The concentration is determined by the BCA method. Procedures of Western blot are described previously.33 Briefly, the protein sample is fractionated on 15% sodium dodecyl sulfate-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 the mean ray value to β-actin. Protein bands are detected by a chemiluminescence system (ChemiDoc XRS, BioRad).

2.17 | Measurement of mitochondrial oxidative stress in rat myocardium

Purified mitochondria of rat myocardium are isolated using Mitochondria Isolation Kit (Sigma, USA) according to the manufacturer's instructions, mitochondrial ROS, MMP, oxidative damage of mtDNA, carbonylated protein content, and lipid peroxidation level in mitochondria are all detected using the same methods as cell experiments described above. Of note, ROS in myocardial tissue is detected with DCFH-DA fluorescent probes, fluorescence intensity is detected with the enzyme-labeling measuring instrument, the results are represented as RLU/μg mitochondrial protein.

2.18 | Statistical analysis

All data are presented as means ± SEM. One-way ANOVA is used to compare differences among multiple groups, followed by the 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.

3 | RESULTS

3.1 | 50 μM ECH effectively inhibits mitochondrial oxidative damage and apoptosis in AC-16 cells

Untreated cells are set as a Ctrl group. Firstly, as Figure 1A shows, ECH inhibits mitochondrial ROS induced by ISO. ISO treated cells show a significant increase in mitochondrial ROS compared with Ctrl and this increase is significantly attenuated by pre-treatment with 50 μM ECH. Secondly, ECH can inhibit oxidative damage of mtDNA induced by ISO. 8-OHdG is believed to be one of the most abundant DNA lesions resulting from oxidative stress and is a biomarker of oxidative DNA damage and repair. As Figure 1B shows, the content of 8-OHdG in purified mitochondria from AC-16 cells is detected, the results show that the level of 8-OHdG in mitochondria from ISO treated cells dramatically increased, but the change was reversed by 50 μM ECH. Thirdly, ECH is effective to protect the MMP, which is impaired by ISO. The fluorescence intensity is significantly decreased in cells treated with ISO compared with Ctrl cells, whereas pre-treatment with 50 μM ECH remarkably attenuates the reduction of MMP, as Figure 1C shows. 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, compared to the Ctrl cells, whereas pre-treatment cells with 50 μM ECH significantly reduces ISO-induced apoptosis.

FIGURE 2 Cardiac hypertrophy of HF rats induced by ISO is relieved and heart function is significantly improved by ECH treatment.

3.2 | ECH reverses myocardial remodeling and improves the cardiac function of ISO-induced HF rats

As shown in Figure 2A, hearts in HF rats are obvious hypertrophy and are reversed by treatment with ECH. As Figures 2B-D and L show, ECH reduces the HW/BW. Echocardiography results show that LVEF and LVFS are significantly reduced in the HF group, LVIDd, LVIDs, and IVSTd are significantly increased in the HF group. However, ECH significantly decreases LVIDd, LVIDs, IVSTd and improves LVEF and LVFS, there is no significant difference in HR among the three groups, 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 hyperemia and edema 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 cardiomyocyte's 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 reveal that left ventricular collagen content in HF rats 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 rats and down-regulated in ECH rats, these results are consistent with each other, as Figures 3B and F-G show, 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 fields 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 rats induced by ISO.

FIGURE 3 Results of histological staining demonstrate that ECH reverses left ventricular remodeling of HF rats induced by ISO.

3.3 | ECH protects myocardial ultrastructure in HF rats

Transmission electron micrographs results show that the damage to myocardial ultrastructure could be attenuated by ECH, as Figure 4A-B shows. Myofibrils are vertical arrangement, the sarcomere structure is obvious, the myofilaments are arranged neatly, and the Z line is clear. Mitochondria are neatly arranged and the structure is clear in Ctrl rats. However, myofibrils are disorganized, ruptured, and dissolved, the sarcomere structure is fuzzy, absence of the Z line, the myocardial contraction zone is visible, the number and volume of mitochondria are increased, mitochondria are different shapes and sizes, arranged in disorder, the structure is unclear, swelling and crista fragmentation, collapse or clumping and dissolved into vacuole in HF rats. ECH obviously attenuates these changes, the majority of the cardiac muscle fibers are neatly arranged and myocardial filaments are clear, Z line is existence, although some dissolution of filaments is apparent, the sarcomere structure is clear, the density of mitochondrial is decreased, the shapes and sizes are relieved, arranged more neatly, mitochondria swelling and crista fragmentation, collapse or clumping, and vacuolization are evidently reduced by ECH compared with HF. Mitochondria swelling and crista fragmentation, collapse or clumping, and partial vacuolization are identified as mitochondrial degeneration. The results demonstrate that ECH effectively protects myocardial ultrastructure, and especially protects mitochondrial morphology in HF rats, as Figure 4C-F shows.

FIGURE 4 ECH protects the ultrastructure of cardiomyocytes and mitochondrial morphology of HF rats induced by ISO.

3.4 | 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 rats, however, ECH obviously up-regulates the decreased protein expression. The representative protein bands of immunoblotting and statistical analysis results are shown in Figure 5. The part of the data indicates that ECH increases the protein expression of SIRT1, FOXO3a, and MnSOD.

3.5 | 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.

FIGURE 5 ECH up-regulates the protein expression level of SIRT1, FOXO3a, and MnSOD which is down-regulated in HF rats.

4 | DISCUSSION

This study demonstrates that ECH exerts effective roles in the prevention of myocardial remodeling and improvement of heart function via up-regulating the SIRT1/FOXO3a/MnSOD signaling axis and inhibiting mitochondrial oxidative stress.

Mitochondria are the major site of generation of ROS as a by-product of oxidative phosphorylation, by their proximity to ROS, mitochondrial proteins, lipids, and mtDNA are believed to be primary targets of oxidative damage after excessive emission of mitochondrial ROS,34,35 this damage stimulates further production of ROS, the vicious circle continues, ROS and oxidative stress could lead to myocardial hypertrophy, apoptosis, interstitial fibrosis, and HF through activation of multiple signaling pathways.35 So, it seems that drugs that targeted inhibition of mitochondrial oxidative stress are effective for the prevention of myocardial remodeling. ECH is a natural phenylethanoid glycoside, a component of the traditional Chinese herb Cistanche tubulosa. it is widely studied and has been proved to possess kinds of pharmacological activities in recent years, such as anti-tumor, anti-apoptosis, anti-aging, neuroprotective action, and so on. Studies have proved that the pharmacological actions of ECH mainly depend on its property of antioxidant and anti-inflammatory activity,18,21 and ECH decreases mitochondrial ROS and protects mitochondrial function against oxidative damage in nerve cells.20,22 Here, we firstly demonstrated that ECH decreases mitochondrial ROS of cardiomyocytes, hence, improving MMP, reducing oxidative damage of mtDNA, protein, and lipids, effectively inhibiting mitochondrial oxidative damage and reducing apoptosis in vitro and in vivo, so, it reverses myocardial remodeling and improves heart function. It means that ECH is a potential new drug for the prevention of myocardial remodeling and HF.

In normal conditions, mitochondrial ROS is mainly cleared by MnSOD which distributes in mitochondria and protects against mitochondrial oxidative damage. Ang II significantly decreases MnSOD expression of cardiomyocytes and induces myocardial hypertrophy, however, resveratrol could increase the expression of MnSOD and inhibit myocardial hypertrophy induced by Ang II.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 leading to heart failure.39 In the 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 SIRT1 is an upstream molecular of FOXO3a and it can activate FOXO3a by deacetylation. SIRT1/FOXO3a/MnSOD signaling axis plays pivotal roles in protection against oxidative stress in kinds of cell types, activation of the signaling axis by estradiol inhibits oxidative stress and exerts the cardiac protection24; Activation of the signaling axis by resveratrol inhibits oxidative stress-induced cell death40; Otherwise, angiotensin II inhibits SIRT1/FoxO3a/MnSOD pathway and induces mitochondrial oxidative stress, mtDNA damage in osteoblasts,41 types 5 adenylyl cyclase increases oxidative stress by inhibition on the expression of MnSOD via the Sirt1/FoxO3a Pathway.42 The present study finds that the expression of SIRT1, FoxO3a, and MnSOD all are significantly reduced in HF rats and mitochondrial ROS is elevated and oxidative stress is enhanced, however, the changes are reversed by ECH. New research indicates that ECH directly binds SIRT1 as a ligand and forms a complex with 9 covalent bonds and up-regulates the expression of SIRT1 in nerve cells.26 Consistent with this study, the present study demonstrates that ECH also up-regulates the expression of SIRT1 in cardiomyocytes, it then up-regulates gene expression of MnSOD through FoxO3a and inhibits mitochondrial ROS and oxidative stress.

Long-term chronic ischemia can cause myocardial hibernation, these hibernating myocardial cells typically show a loss of contractile filaments, there are excess glycogen granules in the cytosol, sarcoplasmic reticulum, and transverse tubules are lost,43 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 in HF rats in our study, whereas ECH significantly relieves these changes, as the latest study reported recovery of cardiac function after ischaemic stress may be a feature of myocardial hibernation,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 role in myocardial hibernation and the resulting contractile dysfunction, and that is markedly improved when the microvascular obstruction is reversed by a drug.47 At present, some studies conclude that revascularization therapy is effective in hibernating myocardium,43 by using HE and Masson staining, we observe that myocardial capillary and arteriolar density are reduced obviously in HF rats and are relieved significantly by ECH, as shown in Figure 7A-H. Previous studies have demonstrated that other natural compounds, such as barley beta-glucan exert cardioprotective effects by increasing expression of MnSOD,48,49, and rising of MnSOD is related to the increased myocardial capillary density and VEGF expression. We performed RT-PCR to detect the expression of VEGF, the data are consistent with the finding of histological staining, which 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 improving heart function.

Cistanche contributes to improving heart function.

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Of note, cardiomyocyte apoptosis is well known to be an important pathologic change in heart failure, and the present study indeed confirms that a 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 remodeling and improves heart function via up-regulating the SIRT1/FoxO3a/MnSOD signaling axis, thus inhibiting mitochondrial oxidative stress and reducing hibernating myocardium. Thus, it is suggested that ECH is a potential drug for the prevention or treatment of myocardial remodeling and HF.