Melatonin attenuates doxorubicin‐induced cardiotoxicity through preservation of YAP expression

Abstract There are increasing concerns related to the cardiotoxicity of doxorubicin in the clinical setting. Recently, melatonin has been shown to exert a cardioprotective effect in various cardiovascular diseases, including cardiotoxic conditions. In this study, we examined the possible protective effects of melatonin on doxorubicin‐induced cardiotoxicity and explored the underlying mechanisms related to this process. We found that in vitro doxorubicin treatment significantly decreased H9c2 cell viability and induced apoptosis as manifested by increased TUNEL‐positive cells, down‐regulation of anti‐apoptotic protein Bcl‐2, as well as up‐regulation of pro‐apoptotic protein Bax. This was associated with increased reactive oxygen species (ROS) levels and decreased mitochondrial membrane potentials (MMP). In vivo, five weeks of doxorubicin treatment significantly decreased cardiac function, as evaluated by echocardiography. TUNEL staining results confirmed the increased apoptosis caused by doxorubicin. On the other hand, combinational treatment of doxorubicin with melatonin decreased cardiomyocyte ROS and apoptosis levels, along with increasing MMP. Such doxorubicin‐melatonin co‐treatment alleviated in vivo doxorubicin‐induced cardiac injury. Western Blots, along with in vitro immunofluorescence and in vivo immunohistochemical staining confirmed that doxorubicin treatment significantly down‐regulated Yes‐associated protein (YAP) expression, while YAP levels were maintained under co‐treatment of doxorubicin and melatonin. YAP inhibition by siRNA abolished the protective effects of melatonin on doxorubicin‐treated cardiomyocytes, with reversed ROS level and apoptosis. Our findings suggested that melatonin treatment attenuated doxorubicin‐induced cardiotoxicity through preserving YAP levels, which in turn decreases oxidative stress and apoptosis.


| INTRODUC TI ON
Doxorubicin (Dox) is an effective anti-neoplastic medication, widely used in the treatment of solid cancers and haematological malignancies. 1 However, it has limited clinical use owing to its acute and chronic cardiotoxicity, which mainly manifests in the form of left ventricular dysfunction and ultimate heart failure. In fact, a study conducted by Tan et al showed that in a cohort of women treated with anthracyclines and trastuzumab, left ventricular end-diastolic and end-systolic volumes increased, while ejection fraction, strain and strain rate decreased at the end of treatment compared with baseline. There, no recovery was present during >2 years follow-up. 2,3 It has been demonstrated that Dox induces cardiomyocyte toxicity and cell death through a variety of mechanisms, the most prominent being the production of excess reactive oxygen species (ROS). 4 Cardiac tissue contains abundant mitochondria, which are essential for cardiomyocytes to sustain sufficient ATP production for contractile function and cell survival. 5,6 Dox specifically targets mitochondria, where its accumulation there results in the destruction of mitochondrial membrane structure, interference with oxidative respiration, and reduction of mitochondrial membrane potential (MMP), all of which eventually leads to cardiomyocyte apoptosis. 7 The apoptotic effects of Dox were further proved by studies showing that Dox treatment is capable of significantly increasing the expression of pro-apoptotic protein Bax, as well as decreasing the expression of anti-apoptosis protein Bcl-2. [8][9][10] Melatonin (Mel), endogenously-produced by the pineal gland of mammals, has recently been implicated in the modulation of various cardiovascular diseases. 11 Studies have shown that Mel alleviates post-infarct cardiac remodelling and dysfunction through up-regulating autophagy, decreasing apoptosis and modulating mitochondrial integrity and biogenesis. 11 Furthermore, there is evidence suggesting that Mel is able to reduce the infarct area, sustain myocardial function and suppress cardiomyocyte death during cardiac ischaemia-reperfusion injury. 12,13 Mel also abrogates diabetic cardiomyopathy, by reducing ROS level and rescuing impaired mitophagy activity. 14,15 Additional studies also showed Mel being involved in alleviating mitochondrial oxidative damage and apoptosis caused by Dox in cardiomyocytes. 2,16 However, the exact mechanism mediating this protective effect of Mel on Dox-induced cardiotoxicity remains unclear.
YAP (Yes-associated protein, also known as YAP1) is the downstream effector of the Hippo signalling pathway, where it participates in diverse physiological and pathological processes related to heart development, apoptosis, hypertrophy, autophagy, angiogenesis and basal homoeostasis. 17 Inactivation of YAP increases cardiomyocyte apoptosis and fibrosis, as well as aggravating cardiac dysfunction after a myocardial infarction (MI). 18 Conversely, cardiac-specific YAP activation after MI has been demonstrated to mitigate myocardial injury and improve cardiac function, the latter being associated with enhanced cardiomyocyte survival via encouraging a less mature cardiac gene expression profile. This profile entails the stimulation of cell cycle genes. 19 Human ischaemic and non-ischaemic heart failure activates the Hippo pathway, while its inactivation reverses systolic heart failure after MI. 20 Another YAP-related activity is the modulation of antioxidant capacity, where YAP inactivation suppresses FoxO1 activity and decreases antioxidant gene expression, thus aggravating ischaemia-reperfusion induced heart injury. 21 The present study is aimed to investigate whether Mel can protect cardiomyocytes from Dox-induced oxidative stress injury and apoptosis, as well as underlying mechanism and mediators involved if such protection actually took place. We showed that Mel treatment attenuated Dox-induced cardiotoxicity through preserving YAP levels, which decreased oxidative stress and apoptosis.

| siRNA transfection and in vitro experimental groups
Commercially available rat YAP and control siRNA were purchased from RiboBio Co. LTD. H9c2 cells were plated on 6-well plates, at 2 × 10 5 cells per well, in 2 mL of antibiotic-free normal growth medium supplemented with 10% FBS. After the cells reached 30%-50% confluence, they were transfected with either control or targeted siRNA duplex, according to the kit instructions. Cells were harvested for further experiments after 48 hours transfection.
Cells were assigned into four groups in the initial experiment:

| Analysis of cell apoptosis by TUNEL assay
Apoptosis of H9c2 cells and mice heart were detected by the TUNEL assay, according to the manufacturer's instructions. H9c2 cells were inoculated in 48 well plates, at 2.5 × 10 4 per well. In vivo paraffin sections were cut to 5 μm thickness, and images obtained under fluorescence microscopy (Olympus). Apoptotic index was expressed as the ratio of TUNEL-positive (green) to DAPI-positive myocytes (blue).

| Determination of mitochondrial membrane potential
A mitochondrial membrane potential assay kit utilizing JC-1 (Beyotime) was applied to measure mitochondrial membrane potential (MMP). The experimental procedure was operated according to the manufacturer's instructions. At high MMP, JC-1 accumulated in the mitochondrial matrix, forming polymers (J-aggregates), which yielded a red fluorescent light. At low MMP, JC-1 dissociated into monomers, which were unable to attach to the mitochondrial matrix. These monomers emitted a green fluorescent light. Fluorescent intensity was immediately measured under fluorescence microscopy (Olympus). MMP was quantified as the ratio of mean green fluorescent intensity (monomer)/mean red fluorescent intensity (polymer).

| Real-time quantitative polymerase chain reaction (RT-qPCR)
Total RNA was extracted with TRIzol reagent, which was then reverse

| Western blot
Total cell protein was exacted using RIPA lysis buffer, containing protease and phosphatase inhibitors. BCA protein assay was used to determine protein concentration (Beyotime Institute of Biotechnology). The protein samples (20μg) of each group were fractionated by SDS-PAGE, and then transferred to PVDF membranes, which were blocked with 5% non-fat milk in TBS-T (Tris-buffered saline with 0.1% Tween-20) for 1-2 hours at room temperature.
The membranes were incubated with primary antibodies against YAP, Bax, Bcl-2 and β-actin overnight at 4°C, after which they were washed in TBS-T and exposed to the corresponding secondary antibodies (1:10 000) at room temperature for 1-2 hours. Fluorescent signal was detected with a Tanon 5100 imaging system, and quantified using Image J.

| Immunofluorescence staining
Immunofluorescence staining for YAP in cells was performed using anti-YAP antibody in a humidified box overnight at 4°C, followed by incubation with a fluorescein-labelled secondary antibody for 1 hour at 37°C. Cell nuclei were stained with DAPI (5.0 μg/mL) for 5 minutes. The resulting immune-stained samples were imaged by fluorescence microscopy (Olympus) at 200× magnification.

| Echocardiography
One week before initiation, as well as 7 days after the final injec-

| Haematoxylin-Eosin staining (H&E staining)
Hearts were excised from anaesthetized mice, and the left ventricle was bisected along the long axis. One of the two parts was fixed overnight in 4% paraformaldehyde for paraffin embedding, while the other half was frozen in liquid nitrogen, followed by storage in −80°C. Formalin-fixed, paraffin-embedded myocardial tissue sections (5 μm thick) were deparaffinized, rehydrated and stained with haematoxylin and eosin (H&E). Morphological changes in myocardial tissues were observed under a light microscope.

| Masson's trichrome staining
Masson's trichrome staining (Solarbio) was performed to measure cardiac collagen fraction. Briefly, paraffin-embedded heart tissues were cut into 5 μm serial sections and placed on slides. Following deparaffinization and rehydration, sections were stained with standard Masson trichrome to analyse myocardial fibrosis and collagen fibre density. After staining, the normal myofibre was stained red, and the collagen was stained blue. The collagen fraction was defined as the blue-stained area divided by the total field.

| Immunohistochemical staining
Formalin-fixed, paraffin-embedded myocardial tissue sections (5 μm thick) were deparaffinized, rehydrated, washed in PBS and primary antibody applied overnight at 4°C. Thereafter, slides were incubated with peroxidase-conjugated second antibody, diluted 1:100 in PBS, for 30 minutes. After washing in PBS, colouring reaction was carried out.

| Statistical analyses
Data are presented as mean ± SD. SPSS 19.0 (SPSS Inc, Chicago, USA) software was used for data analysis. Student's t-test was used for two-group comparisons, while one-way analysis of variance (ANOVA) was used for comparison among 3 or more groups, followed by Bonferroni post hoc tests. Differences were considered statistically significant at P < .05.

| Mel treatment attenuated DOX-induced mitochondrial oxidative stress and cytotoxicity
To

| Mel treatment reversed the downregulation of YAP caused by Dox
To explore the possible mediators responsible for Dox-induced cytotoxicity, we investigated YAP expression in treated H9c2 cells.
Western blot analysis showed that YAP expression levels were significantly decreased after Dox treatment, which was restored back to the control level with treatment of Mel ( Figure 3A,B).
Immunofluorescence staining showed that YAP was mostly localized All the above evidence suggested that Dox treatment caused down-regulation of YAP and its target genes, which Mel could partially rescue.

| Mel protected H9c2 cells from Dox-induced cytotoxicity through restoration of YAP expression
To confirm the causal relationship between YAP and Mel in treating Dox-induced cardiotoxicity, we down-regulated YAP level with siRNA.
Western blot analysis showed that YAP expression in H9c2 cells was efficiently knocked down by YAP-targeted siRNA ( Figure S1A,B).

| Mel alleviated Dox-induced myocardial injury by restoring YAP expression
To investigate whether the in vitro finding also holds true in the in vivo cardiac injury model, we treated C57BL/6 mice with Dox and/  Figure 6F) and HR ( Figure 6G)were found to be significantly decreased in Dox-treated mice compared to controls. In addition, left ventricular remodelling was noted in the Dox group, as evidenced by dilated LVEDd ( Figure 6D) and LVESd ( Figure 6E).
However, the Mel and Dox co-treated group yielded increased LVEF,

Histological examination revealed visible myocardial damage in
Dox-treated animals. H&E staining showed that the Dox-only mice had cardiomyocyte dysplasia, vacuolar degeneration and interstitial oedema ( Figure 6H). All those conditions were found to have lower occurrences in the Mel and Dox co-treatment group ( Figure 6H). Masson's Trichrome staining revealed that the Dox significantly increased cardiac collagen fraction, which was decreased by Mel treatment (Figure 6I,J).
Wheat germ agglutinin (WGA) staining was used to visualize the myocyte membranes and measure the cardiomyocyte area. The results

| D ISCUSS I ON
Dox is an effective anticancer drug, which has been widely used for   Myc protein. 43 Additionally, a new study has shown that Mel treatment effectively down-regulated miR-24, an important oncogenic miRNA which reduces the activity of the p38-p53 axis components. Those components, in turn, are involved in DNA repair and inhibition of cell proliferation in breast cancers. 44 It is plausible to reason that apart from its cardioprotective effect on Dox, Mel may act in synergy with Dox to counteract tumour growth, making it an ideal adjuvant for antitumour medication.
To conclude, our results revealed that Mel protected cardiomyocyte viability in vitro, and cardiac tissue against Dox-induced cardiotoxicity in vivo, through reduced oxidative stress injury and apoptosis. YAP is a key signalling molecule involved in this process, whose preservation by Mel treatment reduced oxidative stress injury and maintained cardiac tissue morphology. To our knowledge, F I G U R E 7 Mel co-treatment with Dox decreased apoptosis and preserved YAP level in mouse hearts. Representative images of TUNEL staining (A) were obtained, and apoptosis index (B) was calculated from control, doxorubicin (Dox), melatonin (Mel) and Mel+Dox treated mouse hearts. (C) Representative immunohistochemical staining of Yes-associated protein 1 (YAP) in the sectioned left ventricle from the 4 groups of animals. n = 6/group, *P < .05 compared with the control group, **P < .01 compared with the control group, # P < .05 compared with the Dox-treated group, ## P < .05 compared with the Dox-treated group this is the first study to report the relationship between Mel treatment and Hippo/YAP pathway in Dox-induced cardiotoxicity. These findings will provide valuable information for mechanism behind, as well as the prevention of, Dox-induced cardiotoxicity.

ACK N OWLED G EM ENTS
This study was supported by grants from the National Natural Science Foundation of China (81671762 and 81371632) and the Postdoctoral Starting Foundation of Heilongjiang Province (LBH-Q16144).

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

AUTH O R CO NTR I B UTI O N S
Hai-ru Li performed the cytological experiments, analysed the data and wrote the paper. Chao Wang was responsible for doxorubicin and melatonin administration and echocardiography examination.
Chao Wang, Ping Sun and Dan-dan Liu performed the molecular biology experiments. Experiment design, calibration and supervision, as well as final approval, were done by Jia-wei Tian and Guo-qing Du.

DATA AVA I L A B I L I T Y S TAT E M E N T
The datasets analysed during the current study are available from the corresponding author upon reasonable request.