Erythropoietin activates SIRT1 to protect human cardiomyocytes against doxorubicin-induced mitochondrial dysfunction and toxicity

Accepted Manuscript Title: Erythropoietin Activates SIRT1 to Protect Human Cardiomyocytes against Doxorubicin-Induced Mitochondrial Dysfunction and Toxicity Authors: Lan Cui, Jiabin Guo, Qiang Zhang, Jian Yin, Jin Li, Wei Zhou, Tingfen Zhang, Haitao Yuan, Jun Zhao, Li Zhang, Paul L. Carmichael, Shuangqing Peng PII: DOI: Reference:

S0378-4274(17)30168-6 http://dx.doi.org/doi:10.1016/j.toxlet.2017.04.018 TOXLET 9757

To appear in:

Toxicology Letters

Received date: Revised date: Accepted date:

3-3-2017 21-4-2017 25-4-2017

Please cite this article as: Cui, Lan, Guo, Jiabin, Zhang, Qiang, Yin, Jian, Li, Jin, Zhou, Wei, Zhang, Tingfen, Yuan, Haitao, Zhao, Jun, Zhang, Li, Carmichael, Paul L., Peng, Shuangqing, Erythropoietin Activates SIRT1 to Protect Human Cardiomyocytes against Doxorubicin-Induced Mitochondrial Dysfunction and Toxicity.Toxicology Letters http://dx.doi.org/10.1016/j.toxlet.2017.04.018 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Erythropoietin Activates SIRT1 to Protect Human Cardiomyocytes against Doxorubicin-Induced Mitochondrial Dysfunction and Toxicity Author: Lan Cui1, Jiabin Guo1, *, Qiang Zhang3, Jian Yin1, Jin Li2, Wei Zhou1, Tingfen Zhang1, Haitao Yuan1, Jun Zhao1, Li Zhang1, Paul L. Carmichael2, and ShuangqingPeng1, * 1Evaluation

and Research Centre for Toxicology, Institute of Disease Control

and Prevention, Academy of Military Medical Sciences, Beijing 100071, China; 2Unilever

Safety and Environmental Assurance Center, Colworth Science

Park, Sharnbrook, Bedfordshire MK44 1LQ, UK; and 3Department

of Environmental Health, Rollins School of Public Health, Emory

University, Atlanta, GA 30322, USA. *Corresponding Jiabin Guo, e-mail: [email protected]; Shuangqing Peng, e-mail:[email protected]. The authors declare no potential conflicts of interest with the data presented as a result of this article.

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Graphical Abstract



Highlights 

DOX induced mitochondrial biogenesis dysfunction in AC16 cells.

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EPO attenuated DOX mitotoxicity by improving mitochondrial function and biogenesis.

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Suppression of SIRT1 caused by DOX was mitigated by EPO.

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SIRT1 deficiency partly diminished EPO’s benefits and enhanced DOX mitotoxicity.

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EPO’s protection against DOX was mediated by SIRT1.

Abstract The hormone erythropoietin (EPO) has been demonstrated to protect against chemotherapy drug doxorubicin (DOX)-induced cardiotoxicity, but the underlying mechanism remains obscure. We hypothesized that silent mating type information regulation 2 homolog 1 (SIRT1), an NAD+-dependent protein 2

deacetylase that activates peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α), plays a crucial role in regulating mitochondrial function and mediating the beneficial effect of EPO. Our study in human cardiomyocyte AC16 cells showed that DOX-induced cytotoxicity and mitochondrial dysfunction, as manifested by decreased mitochondrial DNA (mtDNA) copy number, mitochondrial membrane potential, and increased mitochondrial superoxide accumulation, can be mitigated by EPO pretreatment. EPO was found to upregulate SIRT1 activity and protein expression to reverse DOX-induced acetylation of PGC-1α and suppression of a suite of PGC-1α-activated genes involved in mitochondrial function and biogenesis, such as nuclear respiratory factor-1 (NRF1), mitochondrial transcription factor A (TFAM), citrate synthase (CS), superoxide dismutase 2 (SOD2), cytochrome c oxidase IV (COXIV), and voltage-dependent anion channel (VDAC). Silencing of SIRT1 via small RNA interference sensitized AC16 cells to DOX-induced cytotoxicity and reduction in mtDNA copy number. Although with SIRT1 silenced, EPO could reverse to some extent DOXinduced mitochondrial superoxide accumulation, loss of mitochondrial membrane potential and ATP depletion, it failed to normalize protein expression of PGC-1α and its downstream genes. Taken together, our results indicated that EPO may activate SIRT1 to enhance mitochondrial function and protect against DOX-induced cardiotoxicity.

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Keywords: Erythropoietin; ; ; ; , SIRT1, Doxorubicin, mitochondrial dysfunction, cardiotoxicity

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1. Introduction Doxorubicin (DOX) is a chemotherapeutic anthracycline antibiotic isolated from Streptomyces peucetius in 1967. As one of the most frequently used antitumor agents, DOX is highly effective against a broad spectrum of neoplasms. However, its clinical application is compromised by cardiovascular side effects, which occur either as an acute syndrome or as a cumulative and dose-dependent toxicity leading to progressive heart failure and irreversible cardiac dysfunction (Von Hoff et al., 1977). Multiple mechanisms have been proposed to account for DOX-induced cardiac toxicity, including oxidative stress (Guo et al., 2015), nitrosative and nitrative stress (Pacher et al., 2003; Mukhopadhyay et al., 2009), DNA damage (Hixon et al., 1981; Ashley and Poulton, 2009), dysregulation of metabolites (Tokarska-Schlattner et al., 2006), inflammation (Bai et al., 2004), and apoptosis of cardiomyocytes (Arola et al., 2000). Among them, oxidative stress-mediated mitochondrial impairment and mitochondrial biogenesis dysfunction have been implicated as one of the major causes of DOX-induced cardiotoxicity (Wallace, 2003; Berthiaume and Wallace, 2007; Carvalho et al., 2014). Erythropoietin (EPO) is generally acknowledged as a hematopoietic cytokine that is required for the regulation of mammalian erythropoiesis through binding to its specific cellular surface receptor EPOR. Besides hematopoietic cells, EPOR is also widely found in many other tissues and organs including the heart (Cai et al., 2003; Tramontano et al., 2003). Accumulating evidence 5

has proved that EPO exhibits remarkable beneficial effects in the cardiovascular system, providing protection against myocardial infarction (Moon et al., 2003; Parsa et al., 2003; Hanlon et al., 2005), cardiac ischemiareperfusion injury (Gobe et al., 2013; Watson et al., 2013; Jun et al., 2014), heart failure (Yamada et al., 2013), and DOX-induced cardiomyopathy (Longhu Li et al., 2006; Kim et al., 2007). It was reported that EPO attenuated DOX-induced myocardial dysfunction such as cardiomyocyte apoptosis (Fu and Arcasoy, 2007) and cardiomyopathy (Li et al., 2006; Saher Hamed et al., 2006) in vivo and in vitro. Recombinant human EPO pretreatment was found to protect myocardium against functional damage and electrophysiological disruption induced by acute DOX exposure in an isolated rat heart model (Ramond et al., 2008). However, the mechanism of EPO-mediated cardioprotection is still unclear. A growing number of studies suggest an intimate linkage between mitochondria and EPO’s cardioprotective action. EPO can modulate mitochondrial morphology to protect the heart from ischemia-reperfusion injury (Ong et al., 2015). EPO has also been found to mitigate hypoxic injury in cardiomyocytes by enhancing mitochondrial biogenesis (Qin et al., 2014). A proper function of mitochondrial biogenesis is critical for maintaining an optimal number of mitochondria and regulating mitochondrial homeostasis (Nisoli et al., 2007; Ren et al., 2010; Patten and Arany, 2012). Peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) has 6

emerged as a master regulator of mitochondrial biogenesis. In mammalian cells, PGC-1α regulates mitochondrial biogenesis by activating, among others, nuclear respiratory factor-1 (NRF-1) and mitochondrial transcription factor A (TFAM). TFAM in turn regulates the transcription of mitochondrial DNA-encoded genes involved in mitochondrial transcription and translation, mitochondrial DNA (mtDNA) repair, and maintenance of mitochondrial structural integrity and normal functions (Cantó and Auwerx, 2009). Silent mating type information regulation 2 homolog 1 (SIRT1), an NAD+-dependent protein deacetylase, can interact with PGC-1α physically and regulate PGC1α via deacetylation both in vitro and in vivo (Nemoto et al., 2005).Activation by reagents such as resveratrol or overexpression of SIRT1 has been shown to enhance cell resistance and survival from stress through activating PGC-1α (Lagouge et al., 2006; Planavila et al., 2011; Yuan et al., 2012). In recent studies where DOX administration induced mitochondrial biogenesis dysfunction in cardiomyocytes, a reduction of SIRT1 expression and its deacetylase activity were observed (Zhang et al., 2011; Ruan et al., 2015; Sin et al., 2015; Sin et al., 2016). Meanwhile, EPO has been reported to modulate SIRT1 expression, activity, and cellular trafficking to the nucleus (Jinling Hou et al., 2011). Recent studies demonstrated that EPO stimulated the SIRT1 pathway to promote mitochondrial function and protect against oxidative stress in white adipose tissue (Wang et al., 2013b). Taken together, we hypothesized that EPO protects cardiomyocytes from DOX-induced oxidative 7

stress and subsequent cytotoxicity through improving mitochondrial function and mitochondrial biogenesis in a SIRT1-dependent way.

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2. Material and Methods 2.1 Cell culture and drug treatments AC16 cells (ATCC, VA, USA), a human cardiomyocyte cell line, were maintained in Dulbecco’s Modified Eagle Medium supplemented with 10% fetal bovine serum (FBS), and 100 U/ml penicillin/streptomycin solutions. Cultures were incubated at 37 °C in a mixture of 5% CO2 and 95% atmospheric air, and the medium was replaced every other day. Protein expression of EPOR in AC16 cells was confirmed with immunoblot (Fig. S1A) Cells were pretreated with or without EPO (Sigma-Aldrich, USA) for 24 h, and then washed with culture media. Afterward, cells were treated with doxorubicin hydrochloride (Sigma-Aldrich) at various concentrations for 24 h. 2.2 Cell viability assay Cell viability was quantified by using a commercial Cell Counting Kit-8 (CCK8) (Dojindo Molecular Technologies, Japan) according to the manufacturer’s instruction. Briefly, immediately after drug treatments, CCK-8 solution was added to each well in a 96-well plate followed by 2-h incubation. The absorbance at 450 nm was determined using a microplate reader. Data were normalized to control cultures which were considered as 100% cell survival. 2.3 Analysis of mitochondrial membrane potential (ΔΨm) and mitochondrial superoxide

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ΔΨm was assessed by using a cationic dye JC-1 which exhibits potentialdependent accumulation in mitochondria. ΔΨm is indicated by a fluorescence emission shift from green (~525 nm) to red (~590 nm) and calculated as the ratio of red/green fluorescence intensities. After drug treatments, cells were incubated in dark at 37 °C with a JC-1 working solution at a final concentration of 2.5 μM for 30 min. Thereafter, cells were washed thrice with PBS and then visualized by SpectraMax M5 Microplate Reader (Molecular Devices, USA) to determine the fluorescence of green (Ex/Em=485/538 nm) and red (Ex/Em=544/590 nm). Data were normalized to cells in control group after background subtraction. Mitochondrial superoxide was measured with a mitochondrial superoxide indicator, MitoSOXTM Red (Molecular ProbesTM, Invitrogen Detection technologies), which selectively targets mitochondria and is oxidized by superoxide to emit red fluorescence. After drug treatments, cells were harvested and resuspended in a working solution containing 5 μM MitoSOX Red and incubated in dark for 10 min at 37 °C. Afterward, cells were centrifuged and rinsed thrice with PBS and then resuspended in PBS. The fluorescence of MitoSOXTM Red was analyzed by a FACSCalibur flow cytometer at Ex/Em=510/580 nm (Becton Dickinson, US). 2.4 Reverse transcription PCR and quantitative real-time PCR

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Total RNA was isolated from AC16 cells using TRIzol reagent (Life technologies). cDNA was synthesized from total RNA by reverse transcription using RevertAid first strand cDNA synthesis kit (Thermo scientific, US). The forward and reverse primers used were: human β-actin (5’CATGTGCAAGGCCGGCTTC-3’ and 5’-CTGGGTCATCTTCTCGCGGT-3’); human SIRT1 (5’-CAGATCCTCAAGCGATGTTT-3’ and 5’CTGTTCCAGCGTGTCTATGTT-3’). Taking cDNA as template, real-time PCR was performed with an IQ5 real-time PCR detection system (Bio-Rad, USA) using the SYBR Premix Ex Taqmix (TaKaRa, Japan). PCRs were conducted using the following conditions for 45 cycles after 2 min of pre-denaturation at 95°C: denaturation at 94°C for 30 s, annealing at 58°C for 30 s, and elongation at 72°C for 20 s. Changes in mRNA abundance were calculated using the ddCt method and β-actin was served as standard. 2.5 mtDNA copy number Total DNA was isolated from AC16 cells using TIANamp Genomic DNA Kit (TIANGEN, China) according to the manufacturer’s instruction. mtDNA copy number was measured by real-time PCR. The following primers for nuclear DNA (nDNA) were used: human GAPDH: forward primer 5’AAGGTGGAGGAGTGGGTGT-3’, reverse primer 5’TCAAGAAGGTGGTGAAGCAG-3’. The following primers for mtDNA were used: human ND1: forward primer 5’-GGAGTAATCCAGGTCGGT-3’, reverse primer 5’-TGGGTACAATGAGGAGTAGG-3’. To amplify mtDNA and nDNA 11

products, conditions were initial denaturation at 94 °C for 30 s; 45 cycles at 55 °C for 30 s each and final extension at 72 °C for 20 s. Relative amount of mtDNA and nDNA copy numbers was compared and normalized to control. 2.6 Western blot Cells were lysed with RIPA (Applygen, China) buffer containing protease and phosphatase inhibitors. Proteins were separated on 8% or 10% SDS-PAGE and then transferred to PVDF membranes. The membranes were blocked with 5% nonfat milk in TBS containing 0.05% Tween 20 (TBST) for 6 h, and incubated with different primary antibodies overnight at 4 °C. After washing thrice with TBST, the membranes were hybridized with goat anti-rabbit IgG H&L (HRP) or goat anti-mouse IgG H&L (HRP) (Abcam) for 1 h at room temperature. The blots were developed by using a Super Enhanced chemiluminescence detection kit (Applygen) and detected with Tanon Immunoassay System (Tanon, China). The relative signal intensity of bands was determined by Image J software and protein expression levels were standardized by normalizing to GAPDH. Antibodies against EPOR, β-actin, SIRT1 and NRF1 were purchased from Abcam; PGC-1α from Santa Cruz Biotechnology; acetylated-Lysine (Ac-Lys), citrate synthase (CS), VDAC and complex IV (cyclooxygenase, COX IV) from Cell Signaling; GAPDH, SOD2 and TFAM from Proteintech Group. 2.7 Nuclear extraction and SIRT1 activity assays 12

Nuclei were isolated by using a commercial kit (Applygen). Briefly, harvested cells were homogenized before cytosolic and nuclear extraction reagents were added to separate nuclei from cytoplasm. Nuclear extract was tested for protein concentration with BCA protein assay kit. SIRT1 activity of the isolated nuclei samples was measured using SIRT1 activity Assay Kit (Abcam) according to the manufacturer’s instruction. Extracted protein samples were mixed with reaction mixture of fluoro-substrate peptide, NAD and developer in duplicate in 96-well plate. Fluorescence intensity was read for 30 min at 2-min intervals using SpectraMax M5 Microplate Reader (Molecular Devices, USA) at Ex/Em=350/445 nm until the rate of reaction remained constant. 2.8 Immunoprecipitation Equal amounts of cell extracts were incubated with anti-PGC-1α antibodies or normal IgG (un-conjugated isotype control) at 4 °C overnight on a rotating incubator. Then the samples were added with 20 μl of resuspended volume of Protein A/G PLUS-Agarose and incubated at 4 °C for 2 h with rotation. Immunoprecipitates were collected by centrifugation and the pellets were washed 4 times before diluted with SDS-PAGE sample loading buffer. Samples were boiled before immunoblot assay was performed. 2.9 siRNA transfection Transfection of SIRT1 siRNA was performed using transfection reagents and medium according to the manufacturer’s instruction (Invitrogen). Cells were 13

transfected with 10 μM SIRT1 stealth RNAiTM siRNA duplex oligoribonucleotides or stealthTM RNAi negative control duplexes using LipofectamineTM RNAiMAX reagent (all from Invitrogen). Transfection efficiency was monitored by quantitative real-time PCR and western blotting. Transfected AC16 cells were then incubated for an additional 24 h for further experiments. 2.10 ATP measurements Cellular ATP content was determined by an ATP Colorimetric/Fluorometric Assay Kit (BioVision, USA) according to the supplier’s instruction. In brief, cells werelysed and pretreated using Deproteinizing Sample Preparation Kit (BioVision). 30 μl of supernatant and 20 μl ATP assay buffer were added to 96-well plate in duplicate, along with Reaction Mix of ATP assay buffer, ATP probe, ATP converter and developer. After incubation and protection from light at room temperature for 30 min, absorbance at 570 nm was measured in a micro-plate reader. Absorbance of no-ATP control was subtracted from each reading. ATP concentration was calculated by using an ATP standard curve which was run in parallel to the samples. The result of each sample was normalized by its protein content. 2.11 Statistical analysis All results are expressed as means ± SE. Comparison of groups was accomplished using one-way ANOVA followed by SNK test for evaluating 14

differences between groups of interest and the corresponding control. All statistical analyses were conducted using SPSS (SPSS Statistics 17.0, US). P < 0.05 was considered statistically significant.

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3. Results 3.1 EPO attenuated DOX-induced cytotoxicity and mitochondrial dysfunction in AC16 cells DOX-induced cytotoxicity was first evaluated with a cell viability assay as detailed in Methods. AC16 cells treated with DOX for 24 h showed a concentration-dependent decrease in viability (Fig. 1B). A slightly over 20% decrease in cell viability was observed at 125 nM DOX, which was the concentration we chose for testing the protective effects of EPO. A 24-h pretreatment with EPO at various concentrations (0.1, 0.2, 0.4, 0.8, and 1.6 ng/ml) showed protection against DOX-induced cytotoxicity, and the maximal benefit was attained when EPO was at 0.2 ~ 0.8 ng/ml (Fig. 1A). Cells treated with DOX at various concentrations with a pretreatment of 0.2 ng/ml of EPO showed significantly improved viability compared with those treated with DOX only (Fig. 1B). To evaluate the beneficial effects of EPO against DOX, mitochondrial biogenesis (Fig. 2A), mitochondrial membrane potential (Fig. 2B), and intramitochondrial ROS (Fig. 2C-2D) were assessed. We found that pretreatment of AC16 cells with EPO at 0.2 ng/ml alone resulted in a 20% increase in mtDNA copy number. Treatment with 125 nM DOX inhibited mtDNA content by about 40%. Pretreatment with EPO completely blocked the inhibitory effect of DOX, bringing mtDNA copy number back to the control level (Fig. 2A). 16

Similar results were observed for mitochondrial membrane potential ΔΨm, which was suppressed by DOX by more than 40% and EPO normalized it nearly completely (Fig. 2B). Moreover, mitochondrial ROS increased by 2.5 fold in the DOX group, while EPO pretreatment significantly attenuated this increase by nearly 50% (Fig. 2C-2D). Our data indicated that EPO treatment markedly prevented DOX-induced mitochondrial energetic disruption, oxidative stress, and biogenesis stalling in cardiomyocytes, which have been considered as a cause of mitochondrial dysfunction in cardiac diseases (Goldenthal, 2016). 3.2 EPO rescued DOX-inhibited PGC-1α-mediated mitochondrial biogenesis pathway Since PGC-1α plays a cardinal role in governing the transcriptional control of mitochondrial biogenesis, we examined, using western blotting, the expression of proteins related to mitochondrial biogenesis and function, such as NRF1, TFAM, VDAC, CS, SOD2 and COXIV. NRF1 is a protein that homodimerizes and functions as a transcription factor to activate the expression of a number of genes involved in mitochondrial functions and biogenesis, including TFAM. TFAM is a key mitochondria-located transcription factor controlling mitochondrial DNA replication, transcription and repair. As shown in Fig. 3A and 3B, in keeping with the cytotoxicity observed with DOX at 125 nM, the same treatment resulted in a marked suppression of the protein expression of PGC-1α, NRF1 and TFAM in AC16 cells. A 24-h 17

pretreatment with EPO at 0.2 ng/ml (which alone increased the protein levels slightly) alleviated partially the suppression of protein expression. VDAC and COXIV, commonly used as mitochondrial loading controls, were measured to reveal mitochondrial content. CS is a rate-limiting enzyme in the first step of the citric acid cycle; SOD2 binds to the superoxide byproducts of the electron transfer chain and converts them to hydrogen peroxide and diatomic oxygen. The two proteins were measured to gauge mitochondrial metabolic function and antioxidative capacities respectively. As shown in Fig. 3C and 3D, similar suppression of these proteins induced by DOX and mitigation by EPO pretreatment were observed. 3.3 EPO mitigated DOX-induced suppression of SIRT1 and PGC-1α deacetylation It has been reported that SIRT1, as a deacetylase, was involved in mitochondrial biogenesis through its posttranslational regulation of PGC-1α protein (Nemoto et al., 2005). As noted in Fig. 4A, the deacetylase activity of SIRT1 was markedly reduced by 125 nM DOX, while pretreatment with EPO (0.2 ng/ml) brought the activity back to normal levels. The immunoblot of SIRT1 measuring its protein abundance showed a similar trend (Fig. 4B). Measuring PGC-1α acetylation level using IP pull-down assay and western blotting, we found that while DOX at 125 nM significantly decreased PGC-1α protein abundance (in accordance with the results in Fig. 3A and 3B), PGC-1α acetylation was substantially increased (Fig. 4C and 4D). Pretreatment with

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EPO, which preserved SIRT1 activity, was able to suppress the elevated acetylation of PGC-1α induced by DOX. 3.4 SIRT1 deficiency aggravated DOX-induced cytotoxicity and disruption of mitochondrial biogenesis and function To confirm whether the mitochondria-protective action of EPO against DOXinduced cardiotoxicity is SIRT1-dependent, SIRT1 was knocked down by transfecting AC16 cells with SIRT1-specific siRNA. Compared to nontransfected and non-specific siRNA control cells, SIRT1 mRNA and protein in knockdown cells were nearly wiped out for at least 72 h (Fig. S1B and S1C). Compared to control cells, cells with SITR1 knocked down were more vulnerable to DOX-induced cytotoxicity with the cell viability curve shifting to the left significantly (Fig. 5A). Similarly, the mitochondrial DNA copy number in SIRT1 deficient cells was also further suppressed compared to control cells under DOX treatment (Fig. 5B). These results indicated that SIRT1 plays an important role in supporting cell survival and mitochondrial biogenesis in the face of DOX. As further demonstrated in Fig. 6A-6B, SIRT1 knockdown cells exhibited generally higher mitochondrial ROS levels in all treatment groups compared with control cells of the same groups. With respect to ATP content and mitochondrial membrane potential, SIRT1 knockdown cells treated with DOX exhibited lower levels (Fig. 6C and 6D). We expected that SIRT1 knockdown would 19

eliminate the protective effects of EPO on the above cellular measures, including ROS, ATP and ΔΨm. However, we found that EPO could still restore the responses to some extent, suggesting that other mechanisms independent of SIRT1 may also be involved in EPO’s protective effects. 3.5 SIRT1 deficiency eliminated the protective effects of EPO on PGC1α-mediated mitochondrial transcriptional pathway To further dissect the role of SIRT1 in mediating the protective action of EPO, we examined the effects of SIRT1 deficiency on protein expression of PGC-1α and its downstream genes.We found that, while these proteins, including PGC-1α (Fig. 7A, 7C), NRF1 (Fig. 7A, 7D), TFAM (Fig. 7A, 7E) and VDAC (Fig. 7A, 7F), were repressed by DOX and rescued by EPO in control cells, they remained at low levels in SIRT1 knockdown cells regardless of EPO pretreatment. These data suggested that SIRT1 plays a key role in mediating the rescuing effects of EPO on PGC-1α-mediated mitochondrial transcriptional pathway in AC16 cells.

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4. Discussion DOX is an antitumor anthracycline antibiotic, which presents a dosedependent, cumulative cardiotoxicity as a fatal side effect. Though the mechanisms of DOX-induced cardiotoxicity remain to be elucidated, multifactorial and complex causes have been implicated. Among them, ROSelicited oxidative stress has been proposed as a critical mechanism (Segredo et al., 2013). DOX has a strong affinity for cardiolipin, a lipid located in the inner membrane of mitochondria, which leads to the formation of a drug-lipid complex that can impede normal oxidative phosphorylation in cardiomyocytes (Goormaghtigh et al., 1990; Parker et al., 2001). DOX can undergo a oneelectron reduction that is catalyzed by NAD(P)H reductases in mitochondria to form semiquinone, which initiates a redox cycle and releases ROS as a byproduct (Doroshow, 1983). ROS can directly attack the components of mitochondria such as lipid, protein, and mtDNA. As a result, the opening of mitochondrial membrane permeability transition pore occurs, leading to organelle swelling and dropping of the mitochondrial membrane potential; moreover, oxidative phosphorylation may also be disrupted, leading to mitochondrial depolarization, structural fragmentation and cytoskeleton disorganization (Sardao et al., 2009). mtDNA damages including rearrangements, deletions, and decreased copy numbers, have been reported to occur specifically in cardiac tissues (Lebrecht et al., 2003; Lebrecht et al., 2005). Our present study confirmed many of these toxicities in AC16 cells, 21

where treatment with DOX resulted in higher mitochondrial ROS, lower MMP, ATP content, mtDNA copy number, and cell viability. It has been proved that EPO can protect against DOX cardiotoxicity both in vitro and in vivo in animal models and humans (Hamed et al., 2006; Li et al., 2006; Longhu Li et al., 2006; Kim et al., 2007). A growing number of studies have reported that mitochondria are critically involved in the cardioprotection furnished by EPO (Carraway et al., 2010; Qin et al., 2014; Ong et al., 2015). Attenuation of oxidative stress and improved myocardial angiogenesis are believed to be behind its beneficial effects (Maiese et al., 2008; Ammar et al., 2011). Here we demonstrated that EPO pretreatment can improve the cardiomyocytes viability (Fig. 1), decrease ROS production and relieve the cells from MMP loss (Fig. 2), confirming a mitochondrial protection effect of EPO against DOX treatment of AC16 cells. Remarkably, DOX-induced decrease of mtDNA copy number was greatly attenuated by EPO (Fig. 2), prompting us to further explore the relationship between EPO protection and mitochondrial biogenesis. Mitochondrial biogenesis is the growth and division of pre-existing mitochondria which require numerous processes, involving the expression of nuclear-encoded key proteins for the replication of mtDNA and maintenance of mitochondrial mass (Wenz, 2013). Previous studies have shown that in acute or subchronic models of DOX administration, mtDNA oxidative damage resulted in impairment of mitochondrial biogenesis (Marechal et al., 2011; 22

Guo et al., 2015). Through activating endothelial NO synthase (eNOS), EPO can support the pro-survival program of mitochondrial biogenesis in the heart and thus significantly enhance mitochondrial biogenesis in myocardium (Carraway et al., 2010; Martinez-Bello et al., 2012). PGC-1α is one of the main regulators of mitochondrial biogenesis and oxidative metabolism. PGC-1α-mediated transcriptional network containing, among others, PGC-1α, NRF1, TFAM, CS and SOD2 underpins mitochondrial energetic function and biogenesis. Our previous studies have shown that PGC-1α and its transcriptional network are critically involved in protecting against DOX-induced cardiotoxicity (Guo et al., 2014; Guo et al., 2015; Yuan et al., 2016). The present study showed that DOX-induced suppression of PGC-1α-mediated transcriptional induction of mitochondrial function-related genes, including NRF1, TFAM, SOD2, CS, VDAC, and COXIV, can be largely reversed by pretreatment of AC16 cells with EPO (Fig. 3). As a transcriptional coactivator, the molecular basis of PGC-1α activation has been extensively studied and its activity can be regulated at both transcriptional and posttranslational levels (Nemoto et al., 2005). Besides being phosphorylated by kinases such as AMPK (AMP-activated protein kinase), an important posttranslational regulation of PGC-1α is acetylation, in which SIRT1 is a key NAD+-dependent deacetylase (Gerhart-Hines et al., 2007; Rodgers et al., 2008). Similar to AMPK which is activated when the mitochondrial energetics is compromised and ATP/ADP ratio decreases, 23

SITR1 activity is increased also when cells are in a low-energy state where NAD+ as a co-substrate accumulates to a high level. Deacetylation of PGC-1α by SIRT1 has been proposed as an important regulatory mechanism for mitochondrial function as well as other cellular processes including oxidative stress response and apoptosis (Olmos et al., 2013; Huang et al., 2014). Recent studies demonstrated that SIRT1 plays a protective role against DOXinduced mitochondrial biogenesis dysfunction and cardiomyocytes toxicity (Zhang et al., 2011; Ruan et al., 2015; Sin et al., 2015; Sin et al., 2016). There were studies suggesting that the cardioprotective action of EPO is associated with regulation of SIRT1 (Jinling Hou et al., 2011). Our results showed that EPO pretreatment increased both the activity and protein expression of SIRT1 and ameliorated DOX-induced inhibition and the PGC-1α protein level changed in correlation with SIRT1. What’s more, by immunoprecipitation and western blotting of PGC-1α we found that the acetylation level of PGC-1α was significantly increased by DOX and eliminated by EPO. These data suggested that in the course of EPO protection against DOX, PGC-1α was not only posttranslational modified by SIRT1 activation, but also was transcriptionally regulateded (Fig. 4). To further investigate the potential role of SIRT1 in mediating the protective action of EPO, we silenced SIRT1 with specific siRNA. Silencing of SIRT1 sensitized AC16 cells to DOX-induced cell death, decrease in mtDNA copy number, ROS accumulation, ATP depletion, and mitochondrial membrane 24

potential loss (Figs. 5&6). We found the protective effects of EPO in ROS, ATP and ΔΨm were not totally eliminated by knocking down SIRT1 (Fig. 6), suggesting there may be other mechanisms involved in EPO’s protection against mitochondrial dysfunction. Growing studies have linked AMPK/SIRT1/PGC-1α in a cross-talk network regulating cellular metabolism and mediating the beneficial effects of dietary supplements (Cantó and Auwerx, 2009; Price et al., 2012). Studies have shown that EPO may activate AMPK to activate PGC-1α to regulate metabolism and specification of skeletal muscles (Wang et al., 2013a). Further studies are needed to investigate whether AMPK is also involved in mediating EPO protection against DOXinduced mitochondrial dysfunction. While EPO could still reverse the alterations of mitochondrial functions to some extent, it failed to rescue the suppressed induction of PGC-1α and its downstream transcriptional network underpinning mitochondrial function and biogenesis (Fig. 7). These results suggest SIRT1 may play a critical role in mediating the cardioprotective action of EPO against DOX-induced mitochondrial distress. This is in keeping with early studies showing that EPO protected cerebral microvascular endothelial cells from oxidative damage through regulating SIRT1 (Jinling Hou et al., 2011). In summary, our data provided further evidence supporting the beneficial effects of EPO in the cardiovascular system, and implicated SIRT1 as a promising target for prevention/treatment of DOX-induced cardiovascular 25

injury. To our knowledge, this is the first study reporting critical involvement of SIRT1-mediated deacetylation of PGC-1α in EPO protection against DOXinduced mitochondrial dysfunction and impairment of biogenesis.

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Acknowledgments Funding: This work was supported by National Natural Science Foundation of China [grant numbers 81470167, 81430090]; Beijing Nova Program [grant number Z171100001117103]; AMMS Innovative Foundation [grant number 2017CXJJ13]; and Unilever International Collaborative Project [grant number MA-2015-00410].

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References Ammar, H.I., Saba, S., Ammar, R.I., Elsayed, L.A., Ghaly, W.B., Dhingra, S., 2011. Erythropoietin protects against doxorubicin-induced heart failure. Am J Physiol Heart Circ Physiol 301, H2413-2421. Arola, O.J., Saraste, A., Pulkki, K., Kallajoki, M., Parvinen, M., Voipio-Pulkki, L.M., 2000. Acute doxorubicin cardiotoxicity involves cardiomyocyte apoptosis. Cancer Res 60, 1789-1792. Ashley, N., Poulton, J., 2009. Mitochondrial DNA is a direct target of anti-cancer anthracycline drugs. Biochem Biophys Res Commun 378, 450-455. Bai, P., Mabley, J.G., Liaudet, L., Virag, L., Szabo, C., Pacher, P., 2004. Matrix metalloproteinase activation is an early event in doxorubicin-induced cardiotoxicity. Oncol Rep 11, 505-508. Berthiaume, J.M., Wallace, K.B., 2007. Adriamycin-induced oxidative mitochondrial cardiotoxicity. Cell Biol Toxicol 23, 15-25. Cai, Z., Manalo, D.J., Wei, G., Rodriguez, E.R., Fox-Talbot, K., Lu, H., Zweier, J.L., Semenza, G.L., 2003. Hearts from rodents exposed to intermittent hypoxia or erythropoietin are protected against ischemia-reperfusion injury. Circulation 108, 79-85. Cantó, C., Auwerx, J., 2009. PGC-1α, SIRT1 and AMPK, an energy sensing network that controls energy expenditure. Current Opinion in Lipidology 20, 98-105. Carraway, M.S., Suliman, H.B., Jones, W.S., Chen, C.W., Babiker, A., Piantadosi, C.A., 2010. Erythropoietin Activates Mitochondrial Biogenesis and Couples Red Cell Mass to Mitochondrial Mass in the Heart. Circulation Research 106, 1722-1730. Carvalho, F.S., Burgeiro, A., Garcia, R., Moreno, A.J., Carvalho, R.A., Oliveira, P.J., 2014. Doxorubicin-Induced Cardiotoxicity: From Bioenergetic Failure and Cell Death to Cardiomyopathy. Medicinal Research Reviews 34, 106-135. Doroshow, J.H., 1983. Anthracycline antibiotic-stimulated superoxide, hydrogen peroxide, and hydroxyl radical production by NADH dehydrogenase. Cancer Res 43, 4543-4551. Fu, P., Arcasoy, M.O., 2007. Erythropoietin protects cardiac myocytes against anthracycline-induced apoptosis. Biochemical and Biophysical Research Communications 354, 372-378. Gerhart-Hines, Z., Rodgers, J.T., Bare, O., Lerin, C., Kim, S.H., Mostoslavsky, R., Alt, F.W., Wu, Z., Puigserver, P., 2007. Metabolic control of muscle mitochondrial

28

function and fatty acid oxidation through SIRT1/PGC-1αlpha. EMBO J 26, 19131923. Gobe, G.C., Morais, C., Vesey, D.A., Johnson, D.W., 2013. Use of high-dose erythropoietin for repair after injury: A comparison of outcomes in heart and kidney. J Nephropathol 2, 154-165. Goldenthal, M.J., 2016. Mitochondrial involvement in myocyte death and heart failure. Heart Fail Rev 21, 137-155. Goormaghtigh, E., Huart, P., Praet, M., Brasseur, R., Ruysschaert, J.M., 1990. Structure of the adriamycin-cardiolipin complex. Role in mitochondrial toxicity. Biophys Chem 35, 247-257. Guo, J., Guo, Q., Fang, H., Lei, L., Zhang, T., Zhao, J., Peng, S., 2014. Cardioprotection against doxorubicin by metallothionein Is associated with preservation of mitochondrial biogenesis involving PGC-1αlpha pathway. Eur J Pharmacol 737, 117-124. Guo, Q., Guo, J., Yang, R., Peng, H., Zhao, J., Li, L., Peng, S., 2015. Cyclovirobuxine D Attenuates Doxorubicin-Induced Cardiomyopathy by Suppression of Oxidative Damage and Mitochondrial Biogenesis Impairment. Oxid Med Cell Longev 2015, 151972. Hamed, S., Barshack, I., Luboshits, G., Wexler, D., Deutsch, V., Keren, G., George, J., 2006. Erythropoietin improves myocardial performance in doxorubicin-induced cardiomyopathy. Eur Heart J 27, 1876-1883. Hanlon, P.R., Fu, P., Wright, G.L., Steenbergen, C., Arcasoy, M.O., Murphy, E., 2005. Mechanisms of erythropoietin-mediated cardioprotection during ischemiareperfusion injury: role of protein kinase C and phosphatidylinositol 3-kinase signaling. FASEB J 19, 1323-1325. Hixon, S.C., Ellis, C.N., Daugherty, J.P., 1981. Heart mitochondrial DNA synthesis: preferential inhibition by adriamycin. J Mol Cell Cardiol 13, 855-860. Huang, B., Cheng, X., Wang, D., Peng, M., Xue, Z., Da, Y., Zhang, N., Yao, Z., Li, M., Xu, A., Zhang, R., 2014. Adiponectin promotes pancreatic cancer progression by inhibiting apoptosis via the activation of AMPK/Sirt1/PGC-1αlpha signaling. Oncotarget 5, 4732-4745. Jinling Hou, Shaohui Wang, Yan Chen Shang, Zhao Zhong Chong, Maiese, K., 2011. Erythropoietin Employs Cell Longevity Pathways of SIRT1 to Foster Endothelial Vascular Integrity During Oxidant Stress. Curr Neurovasc Res 8, 220–235.

29

Jun, J.H., Jun, N.H., Shim, J.K., Shin, E.J., Kwak, Y.L., 2014. Erythropoietin protects myocardium against ischemia-reperfusion injury under moderate hyperglycemia. Eur J Pharmacol 745, 1-9. Kim, K.H., Oudit, G.Y., Backx, P.H., 2007. Erythropoietin Protects against DoxorubicinInduced Cardiomyopathy via a Phosphatidylinositol 3-Kinase-Dependent Pathway. Journal of Pharmacology and Experimental Therapeutics 324, 160169. Lagouge, M., Argmann, C., Gerhart-Hines, Z., Meziane, H., Lerin, C., Daussin, F., Messadeq, N., Milne, J., Lambert, P., Elliott, P., Geny, B., Laakso, M., Puigserver, P., Auwerx, J., 2006. Resveratrol Improves Mitochondrial Function and Protects against Metabolic Disease by Activating SIRT1 and PGC-1α. Cell 127, 1109-1122. Lebrecht, D., Kokkori, A., Ketelsen, U.P., Setzer, B., Walker, U.A., 2005. Tissue-specific mtDNA lesions and radical-associated mitochondrial dysfunction in human hearts exposed to doxorubicin. J Pathol 207, 436-444. Lebrecht, D., Setzer, B., Ketelsen, U.P., Haberstroh, J., Walker, U.A., 2003. Timedependent and tissue-specific accumulation of mtDNA and respiratory chain defects in chronic doxorubicin cardiomyopathy. Circulation 108, 2423-2429. Li, L., Takemura, G., Li, Y., Miyata, S., Esaki, M., Okada, H., Kanamori, H., Khai, N.C., Maruyama, R., Ogino, A., Minatoguchi, S., Fujiwara, T., Fujiwara, H., 2006. Preventive effect of erythropoietin on cardiac dysfunction in doxorubicininduced cardiomyopathy. Circulation 113, 535-543. Longhu Li, Genzou Takemura, Yiwen Li, Shusaku Miyata, Masayasu Esaki, Hideshi Okada, Hiromitsu Kanamori, Ngin Cin Khai, Rumi Maruyama, Atsushi Ogino, Fujiwara, S.M., Fujiwara, a.H., 2006. Preventive Effect of Erythropoietin on Cardiac Dysfunction in Doxorubicin-Induced Cardiomyopathy. Circulation 113, 535-543. Maiese, K., Chong, Z.Z., Hou, J., Shang, Y.C., 2008. Erythropoietin and oxidative stress. Curr Neurovasc Res 5, 125-142. Marechal, X., Montaigne, D., Marciniak, C., Marchetti, P., Hassoun, Sidi M., Beauvillain, Jean C., Lancel, S., Neviere, R., 2011. Doxorubicin-induced cardiac dysfunction is attenuated by ciclosporin treatment in mice through improvements in mitochondrial bioenergetics. Clinical Science 121, 405-413. Martinez-Bello, V.E., Sanchis-Gomar, F., Romagnoli, M., Derbre, F., Gomez-Cabrera, M.C., Vina, J., 2012. Three weeks of erythropoietin treatment hampers skeletal muscle mitochondrial biogenesis in rats. J Physiol Biochem 68, 593-601. 30

Moon, C., Krawczyk, M., Ahn, D., Ahmet, I., Paik, D., Lakatta, E.G., Talan, M.I., 2003. Erythropoietin reduces myocardial infarction and left ventricular functional decline after coronary artery ligation in rats. Proc Natl Acad Sci U S A 100, 11612-11617. Mukhopadhyay, P., Rajesh, M., Batkai, S., Kashiwaya, Y., Hasko, G., Liaudet, L., Szabo, C., Pacher, P., 2009. Role of superoxide, nitric oxide, and peroxynitrite in doxorubicin-induced cell death in vivo and in vitro. Am J Physiol Heart Circ Physiol 296, H1466-1483. Nemoto, S., Fergusson, M.M., Finkel, T., 2005. SIRT1 Functionally Interacts with the Metabolic Regulator and Transcriptional Coactivator PGC-1 Journal of Biological Chemistry 280, 16456-16460. Nisoli, E., Clementi, E., Carruba, M.O., Moncada, S., 2007. Defective mitochondrial biogenesis: a hallmark of the high cardiovascular risk in the metabolic syndrome? Circ Res 100, 795-806. Olmos, Y., Sanchez-Gomez, F.J., Wild, B., Garcia-Quintans, N., Cabezudo, S., Lamas, S., Monsalve, M., 2013. SirT1 regulation of antioxidant genes is dependent on the formation of a FoxO3a/PGC-1αlpha complex. Antioxid Redox Signal 19, 1507-1521. Ong, S.B., Hall, A.R., Dongworth, R.K., Kalkhoran, S., Pyakurel, A., Scorrano, L., Hausenloy, D.J., 2015. Akt protects the heart against ischaemia-reperfusion injury by modulating mitochondrial morphology. Thromb Haemost 113, 513521. Pacher, P., Liaudet, L., Bai, P., Mabley, J.G., Kaminski, P.M., Virag, L., Deb, A., Szabo, E., Ungvari, Z., Wolin, M.S., Groves, J.T., Szabo, C., 2003. Potent metalloporphyrin peroxynitrite decomposition catalyst protects against the development of doxorubicin-induced cardiac dysfunction. Circulation 107, 896904. Parker, M.A., King, V., Howard, K.P., 2001. Nuclear magnetic resonance study of doxorubicin binding to cardiolipin containing magnetically oriented phospholipid bilayers. Biochim Biophys Acta 1514, 206-216. Parsa, C.J., Matsumoto, A., Kim, J., Riel, R.U., Pascal, L.S., Walton, G.B., Thompson, R.B., Petrofski, J.A., Annex, B.H., Stamler, J.S., Koch, W.J., 2003. A novel protective effect of erythropoietin in the infarcted heart. J Clin Invest 112, 9991007. Patten, I.S., Arany, Z., 2012. PGC-1 coactivators in the cardiovascular system. Trends Endocrinol Metab 23, 90-97. 31

Planavila, A., Iglesias, R., Giralt, M., Villarroya, F., 2011. Sirt1 acts in association with PPARalpha to protect the heart from hypertrophy, metabolic dysregulation, and inflammation. Cardiovasc Res 90, 276-284. Price, Nathan L., Gomes, Ana P., Ling, Alvin J.Y., Duarte, Filipe V., Martin-Montalvo, A., North, Brian J., Agarwal, B., Ye, L., Ramadori, G., Teodoro, Joao S., Hubbard, Basil P., Varela, Ana T., Davis, James G., Varamini, B., Hafner, A., Moaddel, R., Rolo, Anabela P., Coppari, R., Palmeira, Carlos M., de Cabo, R., Baur, Joseph A., Sinclair, David A., 2012. SIRT1 Is Required for AMPK Activation and the Beneficial Effects of Resveratrol on Mitochondrial Function. Cell Metabolism 15, 675-690. Qin, C., Zhou, S., Xiao, Y., Chen, L., 2014. Erythropoietin enhances mitochondrial biogenesis in cardiomyocytes exposed to chronic hypoxia through Akt/eNOS signalling pathway. Cell Biol Int 38, 335-342. Ramond, A., Sartorius, E., Mousseau, M., Ribuot, C., Joyeux-Faure, M., 2008. Erythropoietin pretreatment protects against acute chemotherapy toxicity in isolated rat hearts. Exp Biol Med (Maywood) 233, 76-83. Ren, J., Pulakat, L., Whaley-Connell, A., Sowers, J.R., 2010. Mitochondrial biogenesis in the metabolic syndrome and cardiovascular disease. J Mol Med (Berl) 88, 9931001. Rodgers, J.T., Lerin, C., Gerhart-Hines, Z., Puigserver, P., 2008. Metabolic adaptations through the PGC-1α and SIRT1 pathways. FEBS Letters 582, 46-53. Ruan, Y., Dong, C., Patel, J., Duan, C., Wang, X., Wu, X., Cao, Y., Pu, L., Lu, D., Shen, T., Li, J., 2015. SIRT1 suppresses doxorubicin-induced cardiotoxicity by regulating the oxidative stress and p38MAPK pathways. Cell Physiol Biochem 35, 11161124. Saher Hamed, Iris Barshack, Galia Luboshits, Dov Wexler, Varda Deutsch, Gad Keren, George, J., 2006. Erythropoietin improves myocardial performance in doxorubicin-induced cardiomyopathy. European Heart Journal 27, 1876–1883. Sardao, V.A., Oliveira, P.J., Holy, J., Oliveira, C.R., Wallace, K.B., 2009. Morphological alterations induced by doxorubicin on H9c2 myoblasts: nuclear, mitochondrial, and cytoskeletal targets. Cell Biol Toxicol 25, 227-243. Segredo, M.P.d.F., Salvadori, D.F., Rocha, N., Moretto, F.F., Correa, C., Camargo, E., Almeida, D.d., Reis, R.S., Freire, C.M., Braz, M., Tang, G., Matsubara, L., Matsubara, B., Yeum, K.J., Ferreira, A., 2013. Oxidative stress on cardiotoxicity after treatment with single and multiple doses of doxorubicin. Human & Experimental Toxicology 33, 748-760. 32

Sin, T.K., Tam, B.T., Yu, A.P., Yip, S.P., Yung, B.Y., Chan, L.W., Wong, C.S., Rudd, J.A., Siu, P.M., 2016. Acute Treatment of Resveratrol Alleviates Doxorubicin-Induced Myotoxicity in Aged Skeletal Muscle Through SIRT1-Dependent Mechanisms. J Gerontol A Biol Sci Med Sci 71, 730-739. Sin, T.K., Tam, B.T., Yung, B.Y., Yip, S.P., Chan, L.W., Wong, C.S., Ying, M., Rudd, J.A., Siu, P.M., 2015. Resveratrol protects against doxorubicin-induced cardiotoxicity in aged hearts through the SIRT1-USP7 axis. J Physiol. Tokarska-Schlattner, M., Zaugg, M., Zuppinger, C., Wallimann, T., Schlattner, U., 2006. New insights into doxorubicin-induced cardiotoxicity: the critical role of cellular energetics. J Mol Cell Cardiol 41, 389-405. Tramontano, A.F., Muniyappa, R., Black, A.D., Blendea, M.C., Cohen, I., Deng, L., Sowers, J.R., Cutaia, M.V., El-Sherif, N., 2003. Erythropoietin protects cardiac myocytes from hypoxia-induced apoptosis through an Akt-dependent pathway. Biochem Biophys Res Commun 308, 990-994. Von Hoff, D.D., Rozencweig, M., Layard, M., Slavik, M., Muggia, F.M., 1977. Daunomycin-induced cardiotoxicity in children and adults. A review of 110 cases. Am J Med 62, 200-208. Wallace, K.B., 2003. Doxorubicin-induced cardiac mitochondrionopathy. Pharmacol Toxicol 93, 105-115. Wang, L., Jia, Y., Rogers, H., Suzuki, N., Gassmann, M., Wang, Q., McPherron, A.C., Kopp, J.B., Yamamoto, M., Noguchi, C.T., 2013a. Erythropoietin contributes to slow oxidative muscle fiber specification via PGC-1α and AMPK activation. The International Journal of Biochemistry & Cell Biology 45, 1155-1164. Wang, L., Teng, R., Di, L., Rogers, H., Wu, H., Kopp, J.B., Noguchi, C.T., 2013b. PPARalpha and Sirt1 mediate erythropoietin action in increasing metabolic activity and browning of white adipocytes to protect against obesity and metabolic disorders. Diabetes 62, 4122-4131. Watson, A.J., Gao, L., Sun, L., Tsun, J., Jabbour, A., Ru Qiu, M., Jansz, P.C., Hicks, M., Macdonald, P.S., 2013. Enhanced preservation of the rat heart after prolonged hypothermic ischemia with erythropoietin-supplemented Celsior solution. J Heart Lung Transplant 32, 633-640. Wenz, T., 2013. Regulation of mitochondrial biogenesis and PGC-1αlpha under cellular stress. Mitochondrion 13, 134-142. Yamada, Y., Kobayashi, H., Iwasa, M., Sumi, S., Ushikoshi, H., Aoyama, T., Nishigaki, K., Takemura, G., Fujiwara, T., Fujiwara, H., Kiso, M., Minatoguchi, S., 2013. Postinfarct active cardiac-targeted delivery of erythropoietin by liposomes with 33

sialyl Lewis X repairs infarcted myocardium in rabbits. Am J Physiol Heart Circ Physiol 304, H1124-1133. Yuan, H., Zhang, Q., Guo, J., Zhang, T., Zhao, J., Li, J., White, A., Carmichael, P.L., Westmoreland, C., Peng, S., 2016. A PGC-1α-Mediated Transcriptional Network Maintains Mitochondrial Redox and Bioenergetic Homeostasis against Doxorubicin-Induced Toxicity in Human Cardiomyocytes: Implementation of TT21C. Toxicological Sciences 150, 400-417. Yuan, Y., Huang, S., Wang, W., Wang, Y., Zhang, P., Zhu, C., Ding, G., Liu, B., Yang, T., Zhang, A., 2012. Activation of peroxisome proliferator-activated receptorgamma coactivator 1alpha ameliorates mitochondrial dysfunction and protects podocytes from aldosterone-induced injury. Kidney Int 82, 771-789. Zhang, C., Feng, Y., Qu, S., Wei, X., Zhu, H., Luo, Q., Liu, M., Chen, G., Xiao, X., 2011. Resveratrol attenuates doxorubicin-induced cardiomyocyte apoptosis in mice through SIRT1-mediated deacetylation of p53. Cardiovasc Res 90, 538-545.





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Fig.1 EPO protected against DOX-induced cytotoxicity in AC16 cells. (A) Determination of protective EPO concentrations against DOX. Cells were pretreated with EPO at various concentrations for 24 h followed by another 24-h treatment with or without 125 nM DOX. (B) Effect of EPO on DOX-induced cytotoxicity. Cells were pretreated with EPO (0.2 ng/ml) for 24 h followed by another 24-h treatment with or without DOX at various concentrations. Data are presented as means ± SE. n=3. *P ＜0.05, compared with untreated control group; #P＜0.05, compared with DOX-only group with the same concentration.



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Fig.2 EPO protected against DOX-induced decrease of mtDNA copy number (A), mitochondrial membrane potential loss (B), and mitochondrial superoxide accumulation (C, representative flow cytometry histograms; D, quantitative analysis of C). Cells were pretreated with EPO (0.2 ng/ml) for 24 h followed by another 24-h treatment with or without 125 nM DOX. Data are presented as means ± SE from 3 independent experiments. *P＜0.05, compared with untreated control group; #P＜ 0.05, compared with DOX-only group.



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Fig.3 EPO mitigated DOX-induced suppression of mitochondrion-related proteins expression (PGC-1α, NRF1, TFAM, VDAC, SOD2, and COXIV). EPO pretreatment and subsequent DOX treatment of AC16 cells are the same as described in Fig. 2 legend. Proteins measured are related to mitochondrial biogenesis regulation (A, B) and function (C, D). A, C are representative blot images; B, D are quantitative analysis. Data are presented as means ± SE from 3 independent experiments. *P＜ 0.05, compared with untreated control group; #P＜0.05, compared with DOX-only group.

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Fig.4 EPO activated SIRT1 and PGC-1α in protection against DOX. EPO pretreatment and subsequent DOX treatment of AC16 cells are the same as described in Fig. 2 legend. SIRT1 activity (A) and expression (B), acetylation level of PGC-1α (C, representative image of immunoprecipitation blots; D, quantitative analysis of blots). Data are presented as means ± SE from 3 independent experiments. *P＜0.05, compared with untreated control group; #P＜0.05, compared with DOX-only group.



Fig.5 SIRT1 deficiency aggravated DOX-induced cytotoxicity and decrease of mtDNA copy number. Cells were transfected with SIRT1-specific siRNA (SIRT

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siRNA) or non-specific control siRNA (CON siRNA) and then treated for 24 h with DOX at various concentrations (A) or 125 nM (B). Non-transfected cells were subjected to the same drug treatments as control. Data are presented as means ± SE from 3 independent experiments. *P＜0.05, compared with untreated control group (CON); $P＜0.05, compared with control siRNA group (CON siRNA) with the same drug treatment.

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Fig.6 SIRT1 deficiency diminished EPO-mediated protection against DOX-induced mitochondrial dysfunction. A, Representative FCM histograms of and quantitative results of the fluorescence intensity of mito-ROS indicator MitoSOXTM Red in untransfected, control siRNA- and SIRT1 siRNA-transfected cells in the absence of any drug treatment. B, Representative FCM histograms of and quantification of the fluorescence intensity of MitoSOXTM Red in control siRNA- and SIRT1 siRNA40

transfected cells treated with EPO and/or DOX. C, ATP content and D, mitochondrial membrane potential level. In Fig. B, C and D, transfected cells (SIRT siRNA, Control siRNA) as well as control cells (CON) underwent EPO pretreatment and subsequent DOX treatment as described in Fig. 2 legend. Data are presented as means ± SE from 3 independent experiments. *P＜0.05, compared with untreated control group (CON); #P＜0.05, compared with DOX-treated group; $P＜0.05, compared with control siRNA group (CON siRNA) with the same drug treatments.

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Fig.7 EPO failed to reverse DOX-induced suppression of protein expression in SIRT1-deficient AC16 cells. Transfected cells (SIRT siRNA and Control siRNA) as well as control cells (CON) underwent EPO pretreatment and subsequent DOX treatment as described in Fig. 2 legend. Representative blot images of protein 42

expression (A) and quantitative analysis (B, SIRT1; C, PGC-1α; D, NRF1; E, TFAM; F, VDAC) are shown. Data are presented as means ± SE from 3 independent experiments. *P＜0.05, compared with untreated control group (CON); #P＜0.05, compared with DOX-treated group; $P＜0.05, compared with control siRNA group (CON siRNA) with the same drug treatments.

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