Cordycepin ameliorates cardiac hypertrophy via activating the AMPKα pathway

Abstract Increase of myocardial oxidative stress is closely related to the occurrence and development of cardiac hypertrophy. Cordycepin, also known as 3'‐deoxyadenosine, is a natural bioactive substance extracted from Cordyceps militaris (which is widely cultivated for commercial use in functional foods and medicine). Since cordycepin suppresses oxidative stress both in vitro and in vivo, we hypothesized that cordycepin would inhibit cardiac hypertrophy by blocking oxidative stress‐dependent related signalling. In our study, a mouse model of cardiac hypertrophy was induced by aortic banding (AB) surgery. Mice were intraperitoneally injected with cordycepin (20 mg/kg/d) or the same volume of vehicle 3 days after‐surgery for 4 weeks. Our data demonstrated that cordycepin prevented cardiac hypertrophy induced by AB, as assessed by haemodynamic parameters analysis and echocardiographic, histological and molecular analyses. Oxidative stress was estimated by detecting superoxide generation, superoxide dismutase (SOD) activity and malondialdehyde levels, and by detecting the protein levels of gp91phox and SOD. Mechanistically, we found that cordycepin activated activated protein kinase α (AMPKα) signalling and attenuated oxidative stress both in vivo in cordycepin‐treated mice and in vitro in cordycepin treated cardiomyocytes. Taken together, the results suggest that cordycepin protects against post‐AB cardiac hypertrophy through activation of the AMPKα pathway, which subsequently attenuates oxidative stress.

effectively intervened, eventually develops into HF eventually develops. 4 Undoubtedly, cardiac hypertrophy plays an important role in the occurrence and development of HF. Pharmacological inhibition of cardiac hypertrophy may be a good way to prevent and treat HF. 5 Although the exact mechanisms of the cellular responses and pathways associated with cardiac hypertrophy remain unclear, excessive production of reactive oxygen species (ROS) and elevated levels of oxidative stress have recently been identified as conditions that accelerate the hypertrophic process. 6 Myocardial oxidative stress can be triggered by many factors, including mechanical stretching by pressure overload and various humoral factors, such as angiotensin II (Ang II) and phenylephrine. The signalling pathways closely related to oxidative stress involve oxidative stress included nuclear factor κB (NF-κB), adenosine 5'-monophosphate (AMP)-activated protein kinase α (AMPKα), mitogen activated protein kinases (MAPKs) and other proteins. 7,8 Among these molecules, AMPKα is important in the regulation of bioenergetic metabolism. 9 Numerous studies have confirmed that activation of AMPKα can inhibit cardiac hypertrophy and oxidative stress. [9][10][11] Therefore, searching for a drug that can effectively activate the AMPKα signalling pathway effectively may play a role in inhibiting cardiac hypertrophy.
Cordycepin, also known as 3'-deoxyadenosine (3'-dA), is a major natural bioactive substance extracted from C. militaris. 12 C. militaris is a valuable medicinal material commonly used in China and other East Asian countries to maintain health and for treatment of diseases involving circulation, respiration, immunity, and the glandular system. 12 Studies have shown that cordycepin can inhibit airway remodelling in chronically asthmatic rats and inhibit lung fibrosis in cellular and rat models. 13,14 In addition, cordycepin can activate AMPKα and inhibit oxidative stress. [15][16][17] However, no research has been performed to elucidate the effect of cordycepin on cardiac hypertrophy. The purpose of this study was to determine whether cordycepin can attenuate cardiac hypertrophy, induced by 1 μmol/L Ang II in cultured neonatal rat cardiac myocytes in vitro and by pressure overload in mice. We also sought to elucidate the molecular mechanism underlying the presumptive effect of cordycepin.

| Animals
Adult male C57/BL6 mice (8- Institutes of Health. After 1 week of adaptation, the mice were randomly divided into four groups: a sham operation group (sham, n = 15), a sham + cordycepin treatment group (sham + Cor, n = 15), an aortic banding (AB) group (AB + vehicle, n = 15), and an AB + cordycepin treatment group (AB + Cor, n = 15). Mouse cardiac hypertrophy models were induced by AB. In short, after anaesthetization with pentobarbital sodium by intraperitoneal injection, the left chest of each mouse was opened to identify the thoracic aorta atinin the second intercostal space. We subsequently performed AB operation using 7-0 silk sutures to band the thoracic aorta against a 27-gauge needle. The needle was removed and the air was drawn out of the chest before the thoracic cavity was closed. In the sham operation group, similar operations were performed without constricting the aorta. Beginning 7 days after surgery, we administered cordycepin (20 mg/kg/d) by oral gavage for 4 weeks. At the endpoint of the treatment, heart weight/ body weight (HW/BW, mg/g), and the heart weight/tibia length (HW/ TL, mg/mm) ratios were calculated after the mice were euthanized for both cordycepin and vehicle-treated mice.

| Antibodies and reagents
Cordycepin specified to be over 99.

| Echocardiography and haemodynamics
Mice were anaesthetized by inhalation of 1.5% isoflurane, and echocardiography was performed to evaluate the structure and function of the left ventricle using a MyLab system (Esaote SpA, Genoa, Italy) equipped with a 10-MHz probe. Parasternal short axis images at the mid-papillary muscle level were recorded in M-mode. The left ventricular (LV) dimensions, including LV end-systolic diameter (LVEDs), LV end-diastolic diameter (LVEDd) and posterior wall thickness were measured and averaged from ten consecutive cardiac cycles. Based on these data, the LV ejection fraction (EF) and fractional shortening (FS) were calculated.
After echocardiography, haemodynamic parameters were measured. The mice were anaesthetized (1.5% isoflurane) sequentially. A 1.4-French microtip catheter transducer (SPR-839; Millar Instruments, TX) was inserted into the right carotid artery and advanced into the left ventricle. The heart rate and pressure signals were recorded continuously with a Millar Pressure-Volume System (Millar Instruments, USA). We selected a stable section of the pressure volume curve and analysed it with PVAN data analysis software.

| Morphological analysis
Cardiac tissue samples were washed in 10% potassium chloride solution, and fixed in 10% buffered formalin immediately. After transversely cutting the bottom, the hearts were embedded with paraffin. Several sections of the heart (5 μm thick) were prepared, stained with haematoxylin and eosin (HE) for histopathology or with picrosirius red (PSR) for assessment of interstitial fibrosis, and then visualized by light microscopy. Single myocytes (between 150 and 200 LV myocytes were outlined in each sample, a total of 6 sample) and the LV collagen volume fraction (calculated from the PSRstained sections as the area stained by PSR divided by the total area) were measured using a quantitative digital image analysis system (Image-Pro Plus, IPP, version 6.0).

| Cell viability
Cell viability was assessed with a Cell Counting Kit-8 (CCK8) assay (Dojindo, GB707, Japan). After the cells were treated with graded concentrations of cordycepin for 24,48, and 72 hours, 10 μL of CCK8 solution was added to each well of a 96-well plate and the absorbance was measured at 450 nm by an ELISA reader (SynergyHT, Bio Tek) after

| Immunofluorescence staining
Neonatal rat ventricular myocytes were analysed for cardiac α-actinin expression by immunofluorescence to assess cardiomyocyte hypertrophy. The cells were washed three times with PBS, fixed with 4% paraformaldehyde, permeabilized in 0.2% Triton™ X-100 in PBS, and blocked with 8% goat serum for 1 hour at room temperature.
Then, the cells were stained with a monoclonal anti-α-actinin antibody at a dilution of 1:100 in 1% goat serum overnight. The cells were then incubated with an Alexa Fluor 488-conjugated goat anti-rabbit immunoglobulin G (IgG) (Invitrogen, USA) secondary antibody for 1 hour at 37°C. DAPI was used for visualization of nuclei. Images were obtained with a fluorescence microscope (Olympus DX51, Japan). Single myocytes was measured using IPP 6.0. Quantitative data for myocyte size were obtained from >150 randomly selected myocytes in three independent experiments.

| Determination of oxidative stress
Superoxide generation and the levels of malondialdehyde (MDA) and SOD, were measured in homogenized cardiac tissue samples using commercially available kits (Nanjing Jiancheng Bioengineering Institute, China) according to the manufacturer's protocol. 21 The leavel of glutathione peroxidase (GSH-Px) was detected using an assay kit (Beyotime, China) according to the manufacturer's protocol. The levels of superoxide generation rate was detected using an assay kit (Solarbio, China). The general steps are as follows: the samples were homogenized and the supernatant was centrifuged.
According to the instructions, the standard products were prepared and the corresponding reagents were added. Measuring 530 nm absorbance, drawing standard curve and calculating rate.

| Quantitative real-time polymerase chain reaction (RT-PCR)
Total RNA was extracted from pulverized and homogenized LV  Table 1 and the results were normalized against glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression.

| Western blotting
Total protein was extracted from heart tissue and H9c2 cells were lysed using a RIPA buffer. The protein concentrations were determined using a BCA kit (Thermo Fisher Scientific, USA). The protein concentration of each sample was normalized according to the concentration of each sample detected by the enzyme labelling apparatus. Fifty micrograms of protein lysate was subjected to SDS-PAGE, and the separated proteins were transferred to polyvinylidene fluoride (PVDF) membranes (EMD Millipore, FL00010, USA) as previously described. After blocking for 1-2 hours with 5% non-fat milk at room temperature, the PVDF membranes were incubated with the corresponding primary antibodies overnight at 4°C before being incubated with goat anti-mouse IgG (LI-COR, C11026-03) secondary antibodies for 1 hour at room temperature. The western blot bands were scanned by a two-colour infrared imaging system (Odyssey, LI-COR, USA). For quantification, the specific protein expression levels were normalized to GAPDH levels.

| Statistical analysis
The data are expressed as the means ± SEM. GraphPad Prism 5.0 software (GraphPad Software, USA) for Windows was used for the analysis. Differences among groups were tested by one way ANOVA followed by the post hoc Tukey test, whereas differences between two groups were compared using unpaired Student's t tests.
Statistical significance was defined as P < 0.05.

| Cordycepin attenuated cardiac hypertrophy induced by AB in vivo
To determine whether cordycepin antagonized the hypertrophic response to pressure overload, mice were subjected to either AB or sham surgery. AB surgery resulted in a significant increase in the hypertrophic response in vehicle-treated mice, as estimated by the heart weight to body weight (HW/BW, mg/g) and heart weight to tibia length (HW/TL, mg/mm) ratios, cross-sectional area (CSA) and foetal gene (atrial natriuretic peptide, ANP; B-type natriuretic peptide, BNP; and β-myosin heavy chain, β-MHC) expression levels, shown in Figure 1F-H), without affecting body weight or heart rate ( Table 2). These changes were significantly attenuated in cordycepin-treated AB mice ( Figure 1A-H). Furthermore, cordycepin-treated mice exhibited markedly increased LV EF and LV FS (Table 2), with representative pictures shown in Figure 1I; In addition, analysis of haemodynamic parameters showed that cordycepin treatment improved systolic (assessed by +dP/dt) and diastolic function (assessed by −dP/dt) after AB surgery (Table 2).
Collectively, these data suggest that cordycepin ameliorates cardiac hypertrophy and improves cardiac function after pressure overload.

| Cordycepin attenuated cardiac fibrosis in vivo
Cardiac interstitial fibrosis is one of the main features of cardiac hy-

| Cordycepin inhibits oxidative stress in vivo
Increasing numbers of studies have suggested that oxidative stress promotes cardiac hypertrophy; therefore, to further explore the potential mechanisms contributing to the protective effect of cordycepin, we subsequently measured the levels of protein markers associated with oxidative stress. Figure 3 demonstrates that cardiac superoxide generation, and MDA, and gp91 phox (also known as NOX2; a key component of the enzyme NADPH oxidase, which plays a critical role in ROS generation) levels were significantly higher in the AB + vehicle group than in the sham group; however, such elevation was significantly attenuated by treatment with cordycepin ( Figure 3A-D). Conversely, the levels and activity of SOD (an antioxidative enzyme), and the levels of glutathione peroxidase (GSH-Px) were notably decreased in the AB model-vehicle group than in the sham group, an effect that was significantly reversed by cordycepin ( Figure 3E-H).

| Cordycepin promoted AMPKα phosphorylation in hypertrophic hearts
To investigate the molecular mechanism by which cordycepin ameliorated cardiac hypertrophy, we examined the effects of cordycepin on AMPKα and its downstream signalling pathway. Phosphorylated ACC is a substrate of AMPKα, and its levels can thus reflect the activity of AMPKα. Our research confirm that phosphorylated AMPKα and ACC were elevated in AB + Cor mice compared to AB model mice. In addition, phosphorylation of mTOR and ERK1/2 was induced by AB. The mTOR and ERK1/2 signalling pathway is an important mediator of cardiac hypertrophy, and the observed changes in this pathway were alleviated by cordycepin treatment (Figure 4).

| Cordycepin attenuated Ang II induced cardiomyocyte hypertrophy
To determine the possible cytotoxicity of cordycepin towards cardiomyocytes, we evaluated cell viability using a CCK8 assay. NRVMs

| CpC counteracted the protective effects of cordycepin in vitro
To elucidate the anti-hypertrophic mechanism of cordycepin, NRVMs were treated with 1 μmol/L Ang II in vitro. We next investigated whether NRVMs subjected to CpC treatment showed inhibited the anti-hypertrophic effect of cordycepin. It was found that in NRVMs stimulated by Ang II, the surface area of NRVMs cells treated with CpC increased significantly and showed an aggravated hypertrophic response, as illustrated by the increased mRNA levels of ANP, BNP and β-MHC ( Figure 5). In addition, we also examined the effects of CpC on AMPKα and downstream signalling pathways.  Heart failure, the ultimate outcome of various cardiovascular diseases, remains a major cause of morbidity and mortality worldwide. At present, the main strategy for HF treatment involves intervention in HF pathogenesis and subsequent neurohumoral disorders. Despite the increasing number of treatments for HF, patient prognosis remains poor. 5,22 Cardiac hypertrophy has been identified as a key process contributing to the initiation and progression of HF; this condition mainly involves increased heart weight, altered heart geometry, interstitial fibrosis, increased myocardial cell CSA, and decreased heart function. 23,24 Over the past decades, clinical and experimental studies have confirmed that oxidative stress (defined as excessive production of ROS relative to antioxidant defence) is increased in HF. 25 When we studied the Cordycepin, the major component isolated from Cordyceps sinensis (a well-known and prized traditional Chinese medicine that is also sold as a health food in many countries) 26 has been shown to have many pharmacological actions such as antitumour, antibacterial, antioxidant, and anti-inflammatory effects. 12,26 Cordycepin is effective for attenuating age-related oxidative stress and decreasing lipid peroxidation in aged rats. 12 Cordycepin has also been shown to prevent ischaemia/reperfusion injury in rat hearts via upregulation of haem oxygenase (HO-1) expression and activation of the Akt/GSK-3β/ p70S6K signalling pathway. 27 However, our study demonstrated that cordycepin exerted a protective effect against cardiac hypertrophy that was independent of the aforementioned signalling pathway.

| D ISCUSS I ON
The underlying mechanisms by which cordycepin mediates its antihypertrophic effect still remain elusive. Cardiac hypertrophy is a pathophysiological process involving multiple signal transduction pathways and transcription factors. 25 It has been reported that cordycepin can activate AMPKα (a serine/threonine protein kinase) signalling. 28,29 AMPKα is an important sensor of cellular energy status, and that regulates this status in various pathophysiological processes. 30 One of the key characteristics of cardiac hypertrophy is increased protein synthesis and decreased protein degradation in cardiomyocytes, which leads to intracellular protein accumula- F I G U R E 5 The effects of cordycepin on hypertrophy induced by Ang II were blocked by Compound C (CpC). (A,B) Immunofluorescence staining of a-actinin and the cell surface area of NRVMs in the indicated groups (n = 6 samples, with 150+ cells per group). (C-E) The mRNA levels of ANP, BNP, and b-MHC in NRVMs in each group (n = 6). # P < 0.05 vs the control group; *P < 0.05 vs the Ang II group.
Although we have proven that cordycepin can ameliorate cardiac hypertrophy and have discussed the primary mechanism, our research has some limitations. First, we used CpC, an inhibitor of AMPKα, to conduct cell experiments in vitro, but we have not yet used this compound in animal experiments in vivo. Second, we have not clarified how cordycepin acts on intracellular AMPKα and its downstream signalling pathways. Third, we have not fully elucidated the role of fibrosis. Therefore, in subsequent experiments, we will seek to clarify the detailed protective mechanism of cordycepin.
In conclusion, we have demonstrated that cordycepin prevents cardiac hypertrophy induced by pressure-overload in vivo and by Ang II in in vitro via by AMPKα. We have also shown that inhibition of AMPKα by CpC abolishes the protective effects of cordycepin against Ang II-mediated myocyte hypertrophy. Our study provides evidence supporting the use of cordycepin for cardiac hypertrophy treatment. Future research will aim to elucidate the specific mechanism by which cordycepin protects against cardiac hypertrophy, and the potential clinical application of cordycepin will be of great interest.

CO N FLI C T O F I NTE R E S T
The authors declare no conflicts of interest.
F I G U R E 6 The effects of cordycepin on AMPKα and pro-hypertrophic pathways were blocked by Compound C (CpC). (A-E) The protein levels of phosphorylated AMPKα and related targets in indicated groups (n = 6). *P < 0.05 vs the corresponding group

DATA AVA I L A B I L I T Y S TAT E M E N T
The data will be made available after been required upon request from the corresponding author.

R E FE R E N C E S
F I G U R E 7 The effects of cordycepin on oxidative stress were blocked by Compound C (CpC) and a mechanistic simulation diagram. (A-D) The protein levels of gp91 phox , SOD1, SOD2 in the indicated groups (n = 6). *P < 0.05 vs the corresponding group