Cell Biology International ISSN 1065-6995 doi: 10.1002/cbin.10205

RESEARCH ARTICLE

Erythropoietin enhances mitochondrial biogenesis in cardiomyocytes exposed to chronic hypoxia through Akt/eNOS signalling pathway Chuan Qin*, Shengkai Zhou, Yingbin Xiao and Lin Chen Department of Cardiovascular Surgery, Xinqiao Hospital, The Third Military Medical University, Chongqing, China

Abstract Adaptation of cardiomyocytes to chronic hypoxia in cyanotic patients remains unclear. Mitochondrial biogenesis is enhanced in myocardium from cyanotic patients, which is possibly an adaptive response. Erythropoietin (EPO) in blood and its receptor (EPOR) on cardiomyocytes are upregulated by chronic hypoxia, suggesting that EPO–EPOR interaction is increased, which is inferred to positively regulate mitochondrial biogenesis through protein kinase B (Akt)/endothelial nitric oxide synthase (eNOS) signalling pathway. H9c2 cardiomyocytes were exposed to hypoxia (1% O2) for 1 week and treated with different doses of recombinant human erythropoietin (rhEPO). Mitochondrial number, mitochondrial DNA (mtDNA) copy number and peroxisome proliferator activated receptor gamma coactivator alpha (PGC-1a) mRNA expression increased in a dosedependent manner induced by rhEPO. Akt and eNOS were significantly phosphorylated by rhEPO. Both blocking Akt with Wortmannin and silencing eNOS expression with shRNA plasmid decreased the mtDNA copy number and PGC-1a mRNA expression induced by rhEPO. Blocking Akt was associated with the decreased phosphorylation of Akt and eNOS. RNA interference led to a reduction in the total and phosphorylated proteins of eNOS. Thus EPO enhances mitochondrial biogenesis in cardiomyocytes exposed to chronic hypoxia, at least partly through Akt/eNOS signalling, which might be an adaptive mechanism of cardiomyocytes associated with the increased EPO–EPOR interaction in patients with cyanotic congenital heart disease (CCHD). Keywords: caridiomyocyte; chronic hypoxia; endothelial nitric oxide synthase; erythropoietin; protein kinase B

Introduction The incidence of congenital heart diseases (CHD) varies from about 4/1,000 to 50/1,000 live births (Hoffman and Kaplan, 2002), among which cyanotic congenital heart diseases (CCHD) are still a great threat to human health and lives. All patients with CCHD have to experience a common pathophysiological process, which is chronic hypoxia (Essop, 2007). The heart is very susceptible to acute hypoxia or ischaemia, because it is an organ with a high oxygen consumption. How the hearts of the patients with CCHD can adapt to chronic hypoxia is of much interest, and elucidating its underlying mechanism will be helpful in the treatment of CCHD. Energy metabolism is changed by hypoxia because oxygen is deficient. How myocardium can acquire sufficient energy to maintain its function during chronic hypoxia remains

unclear. Mitochondria are the key organelle for energy metabolism, which have been found to have increased respiratory function and ATP synthesis in animals exposed to chronic hypoxia (Essop, 2007). Mitochondrial biogenesis is enhanced in myocardium from patients with CCHD (Xiao et al., 2012), which might be an adaptive response to chronic hypoxia related to the changes of mitochondrial bioenergetic capacity, but the underlying regulatory mechanism remains unclear. Erythropoietin (EPO) is a haematopoietic cytokine that has direct non-haematopoietic effects on the myocardium (Fu and Arcasoy, 2007; Mao et al., 2008; Lombardero et al., 2011; Yamada et al., 2013). EPO concentration in blood and its receptor expression (EPOR) in myocardium increase significantly during chronic hypoxia (Stockmann and Fandrey, 2006; Hasnaoui-Saadani et al., 2013), suggesting that EPO–EPOR interaction is enhanced. However, its role in



Corresponding author: e-mail: [email protected] Chuan Qin and Shengkai Zhou contributed equally to this work.

Cell Biol Int 38 (2014) 335–342 ß 2013 International Federation for Cell Biology

335

C. Qin et al.

Erythropoietin enhances mitochondrial biogenesis

the adaptation of cardiomyocytes to chronic hypoxia is unclear. Protein kinase B(Akt) is a very important part of EPO’s intracellular signalling network, which has been repeatedly shown to be the most important kinase mediating cardioprotection of EPO (Xu et al., 2009). Akt can activate endothelial nitric oxide synthase (eNOS) by phosphorylating its 1177 ser (Liang et al., 2009), which is the most important activation pathway of eNOS (Schleicher et al., 2009). eNOS is the key regulatory kinase of mitochondrial biogenesis by producing nitric oxide (NO), which switches on the classic pathway of mitochondrial biogenesis by activating peroxisome proliferation activated receptor gamma coactivator alpha (PGC-1a) (Nisoli et al., 2004). Therefore, we inferred that EPO participates in the regulation of mitochondrial biogenesis in cardiomyocytes exposed to chronic hypoxia through Akt/eNOS signalling pathway. We established a chronic hypoxia model of H9c2 cardiomyocytes, checked the effect of EPO on mitochondrial biogenesis represented with mitochondrial number, mitochondrial DNA (mtDNA) copy number and PGC-1a mRNA expression, and investigated the role of Akt/eNOS signalling pathway. Materials and methods

Chronic hypoxia model of H9c2 cells H9c2 cardiomyocytes (ATCC, USA) were cultured in the thermostatic incubator (378C) with 94% N2, 5% CO2 and 1% O2 for 1 week. The medium contained 10% fetal bovine serum, which was changed every 2 days. When recombinant human EPO (rhEPO) (Sansheng, China) was added to the medium at 0, 5, 10 or 20 U/mL, cardiomyocytes were exposed to chronic hypoxia.

Detection of mitochondria with green fluorescent probe After chronic hypoxia for 1 week, the slides with cells were washed with PBS buffer three times. MitoTracker (Invitrogen, USA) was diluted to 1 mmol/L with dimethl sulfoxide (DMSO), which was further diluted to 100 nmol/L with Dulbecco’s modified Eagle’s medium (DMEM). This prepared MitoTracker fluid was used to incubate with the slides with cells for 25 min at room temperature, which were observed by confocal laser scanning microscope. Mitochondrial number was expressed as the optical density of green fluorescence.

China) Beta-actin (b-actin) from nuclear genome was chosen as the reference gene. The primers for mitochondrial cytochrome-c and beta-actin were designed with Primer Premier 5.0 software as follows: for the former, 50 CCGGAGCAATCCAGGTCGGTT-30 and 50 -TGGTTGGGAGCACTTATGGTAAGGA-30 ; for the latter, 50 -TCCCGGCCCCTAGGGTGTAGA-30 and 50 -GCCGCACCGGCTCTCCAAAT-30 . Real-time PCR was performed with a SYBRgreen Premix EX Taq II (TaKaRa, China) using ABI PRISM 7900 Sequence Detection System (Applied Biosystems, USA). This PCR amplification was repeated at least 3 times for each DNA sample with 2 steps: 958C for 60 s and then 45 cycles of 958C for 15 s, 608C for 15 s and 728C for 45 s. A dissociation curve was generated at the end of the reaction to verify the specificity of the amplified products, including a 136 bp fragment of cytochrome-c and a 97 bp fragment of b-actin. The mtDNA copy number relative to nuclear DNA was calculated with the 2DDCT method (Trinei et al., 2006).

mRNA expression by quantitative real-time reverse transcriptase PCR PGC-1a mRNA expression was determined with quantitative real-time reverse transcriptase PCR. Briefly, total RNA was isolated from the treated cardiomyocytes by homogenisation using Trizol reagent (Invitrogen). First strand cDNA was generated with BioRT cDNA First Strand Synthesis Kit (Bioer, China). Primers were designed with Primer Premier 5.0 software as follows: 50 -CCAAATGACCCCAAGGGTTC30 and 50 -TATGAGGAGGAGTGGTGGGTG-30 for PGC-1a; 50 -CAGCAGCACTGTGACGA-30 and 50 -TGTGAGCAGGGAAGGGA-30 for ribosomal protein L10A (RPL10A) as an internal control. Real-time PCR amplification was performed in a 20 mL reaction mixture containing 1 mL cDNA sample, 10 mL SYBR Green Real-time PCR Master Mix (Applied Biosystems), and 0.25 mM primer using ABI PRISM 7900 Sequence Detection System (Applied Biosystems). This realtime PCR amplification was performed in triplicate including two steps: 958C for 60 s and then 45 cycles of 958C for 15 s, 608C for 15 s and 728C for 45 s. A dissociation curve was generated at the end of the reaction to identify the specificity of the amplified products, including a 109 bp fragment for PGC-1a and a 207 bp fragment for RPL10A. Relative quantity of PGC-1a mRNA expression was compared after nomalisation to the values of RPL10A. Fold change in mRNA expression was calculated using the 2DDCT method.

Western blots Detection of mtDNA copy number by quantitative realtime polymerase chain reaction (PCR) Total genomic DNA was isolated from the treated cardiomyocytes with the TIANamp Genomic DNA kit (Tiangen, 336

Akt, phosphorylated Akt, eNOS and phosphorylated eNOS were detected by Western blot. Briefly, the cardiomyocytes were collected and treated with the mixture of 1 mL sodium dodecyl sulphate (SDS) lysate (Biyuntian, China), 10 mL Cell Biol Int 38 (2014) 335–342 ß 2013 International Federation for Cell Biology

C. Qin et al.

phenylmethylsulfonyl fluoride (PMSF) and 1 mL inhibitor of degradation of phosphorylated proteins for 30 min at 08C, followed by the use of ultrasonic cell disruptor to separate the total protein. After centrifugation at 12,000 rpm for 5 min, the total protein was isolated, which was subjected to SDS– PAGE in 8% polyacrylamide gels for 2 h at voltage of 100 V. After electrophoresis, proteins were electrotransferred from gels to polyvinylidene fluoride (PVDF) membrane (Biyuntian) at 250 mA constant current (45 min for Akt and phosphorylated Akt and 100 min for eNOS and phosphorylated eNOS), which was blocked for 2 h at room temperature. The primary rabbit anti-rat antibodies (1:1,000, Cell Signalling Technology, USA) were used to conjugate with the target proteins overnight at 48C, followed by the use of the secondary peroxidase-conjugated antibody (1:5,000, Biyuntian). b-actin was used as the internal control. Immunocomplexes were detected by chemiluminescent reaction (ECL kit, Biyuntian), followed by densitometric analysis with Quantity One imaging system version 4.62 (Bio-Rad, USA).

Interference on the Akt/eNOS signalling pathway To inhibit the activation of Akt, 100 nmol/mL Wortmannin (Cell Signalling Technology) was added to the medium along with 20 U/mL rhEPO. To silence the expression of eNOS, the shRNA plasmid(Nos3-RNAi(2984-1)) with the sequence was used: 50 -GCGTGGAGTGTTTGGACAAGTCC-30 (Jikai, China). This plasmid contained anti-neomycin and green fluoresence protein (GFP) genes for the further screening. Lipofectin transfection was performed. Briefly, when the cardiomyocytes in a 6-well plate became 90% confluent, they were washed three times with PBS buffer. The preparation of the transfection reagent was as follows: mixing 12 mg shRNA plasmid or emtpy plasmid (without the shRNA sequence) with 12 mL Lipofectamine 2000 (Invitrogen) in 288 mL serum-free medium. This transfection reagent was added to every well of the plate combined with 700 mL serum-free medium. After incubation of 5 h at 378C, the transfection reagent was replaced with serum medium. The transfection efficiency after 24 h was only 30–40%. The cells were collected and further screened for those GFP-positive cells by flow cytometry. The transfection efficiency after 24 h culture increased to 70%, maintained with the medium containing neomycin (Figure 6A). The transfected cells were treated with 20 U/mL rhEPO to generate the study group. Statistical analysis The results are expressed as means  SD. For the results of PCR and Western blots, one-way ANOVA or LSD were used to compare between the groups. For the results of mitochondrial fluorescent probe assay, because the heteroCell Biol Int 38 (2014) 335–342 ß 2013 International Federation for Cell Biology

Erythropoietin enhances mitochondrial biogenesis

geneity of variance existed, Kruskal–Wallis test was used. P < 0.05 was considered significant. All the statistical analyses were performed with IBM SPSS Statistics 20 software. Results

Mitochondria observed by scanning electron microscope After hypoxia, regional accumulation of mitochondria with inequality of size and shape and intramitochondrial deposits were detected in both the control cells without rhEPO treatment (Figure 1A) and the cells treated with 20 U/mL rhEPO (Figure 1B). Visually, the cells treated with rhEPO had more and larger mitochondria compared to the control.

Mitochondrial number by fluorescent probe assay After hypoxia for 7 days, cells treated with 5, 10 or 20 U/mL rhEPO presented stronger green fluorescence as compared to the controlled cells not receiving rhEPO treatment (P < 0.05), suggesting that rhEPO increased the mitochondrial number in cardiomyocytes exposed to chronic hypoxia. More mitochondria were found in the cells treated with 10 or 20 U/mL rhEPO than in those treated with 5 U/mL rhEPO (P < 0.05; Figure 2).

MtDNA copy number relative to nuclear DNA by real-time PCR Seven days after hypoxia, MtDNA copy number of the cells treated with different doses of rhEPO was higher than in the control cells without rhEPO treatment (P < 0.05). Cells treated with 10 or 20 U/mL rhEPO had the higher mtDNA copy number compared to those treated with 5 U/mL rhEPO (P < 0.05; Figure 3A).

PGC-1a mRNA expression by real-time reverse transcriptase PCR As the key regulatory transcriptional factor of mitochondrial biogenesis, PGC-1a mRNA level reflects the level of mitochondrial biogenesis. We found that after hypoxia for 7 days, PGC-1a mRNA level was elevated by rhEPO treatment (P < 0.05). Doses of 10 or 20 U/mL rhEPO induced higher expression of PGC-1a mRNA than 5 U/mL rhEPO (P < 0.05; Figure 3B).

Changes of Akt/eNOS signalling detected by Western blot Both Akt and eNOS are activated by phosphorylation on their particular amino acid residues. Akt and eNOS were both significantly phosphorylated by treatment of rhEPO after 337

C. Qin et al.

Erythropoietin enhances mitochondrial biogenesis

Figure 1 Mitochondria in H9c2 cardiomyocytes observed by transmission electron microscope after hypoxia for 1 week (8,900, bar 1 mm). (A) Control cells not receiving recombinant human erythropoietin (rhEPO). (B) Cells treated with 20 U/mL rhEPO.

hypoxia for 7 days (P < 0.05). The cells treated with 10 or 20 U/mL rhEPO had increased phosphorylated Akt and eNOS compared to those treated with 5 U/mL rhEPO (P < 0.05; Figure 4). The total proteins of Akt and eNOS did not alter across all the groups.

Effects of the inhibition of Akt Blocking Akt by Wortmannin reduced mitochondrial biogenesis in the cells treated with 20 U/mL rhEPO, represented by decreased mtDNA copy number and reduced PGC-1a mRNA expression (P < 0.05; Figures 5A and 5B). Phosphorylation of Akt and eNOS was significantly inhibited by Wortmannin (P < 0.05); however, the total

Figure 2 Changes of mitochondrial number in H9c2 cardiomyocytes treated with different doses of recombinant human erythropoietin (rhEPO) (0, 5, 10 or 20 U/mL) and exposed to hypoxia for 1 week, detected by mitochondrial fluorescent probe and calculated based on the mean optical density (MOD) of green fluorescence (1,000, confocal laser scanning microscope). # P < 0.05 versus control  versus cells treated with 5 U/mL rhEPO.

338

protein content of both molecules was not altered (Figures 5C and 5D).

Effects of silencing eNOS expression by shRNA plasmid Cells treated with shRNA plasmid and 20 U/mL rhEPO showed reduced mtDNA copy number and PGC-1a mRNA expression compared to those treated with only 20 U/mL rhEPO (P < 0.05; Figures 6B and 6C). Western blot indicated that both the total and the phosphorylated proteins of eNOS were significantly decreased by shRNA plasmid (P < 0.05; Figure 6D). Discussion How the hearts of CCHD patients can adapt to the condition of chronic hypoxia remains unclear, although this is very important in relation to the improvement of the treatment of CCHD. Cardiomyocytes are highly dependent on the sufficient oxygen supply, which provides the necessary energy for their survival and function by oxidative phosphorylation in mitochondria. Under hypoxia, energy metabolism of cardiomyocytes is greatly deficient, with oxidative phosphorylation being weakened and anaerobic glycolysis taking the predominant position to provide relatively less ATP, and increasing metabolic acidosis by producing lactate. If this status persists, cardiomyocytes become irreversibly injured. Therefore, during chronic hypoxia, cardiomyocytes have to make adaptive changes to survive. It has been reported that mitochondrial biogenesis is enhanced in myocardia from patients with CCHD, accompanied by an increased volume and number of mitochondria (Xiao et al., 2012). Enhanced mitochondrial biogenesis has the increased mitochondrial respiratory efficiency and production of ATP (Funk et al., 2010), which can also induce the expression of antioxidant proteins, such as uncoupling proteins (UCPS), superoxide dismutase 2 (SOD2) and glutathione peroxidase-1 (St-Pierre et al., 2003). These biological effects are beneficial to the Cell Biol Int 38 (2014) 335–342 ß 2013 International Federation for Cell Biology

C. Qin et al.

Erythropoietin enhances mitochondrial biogenesis

Figure 3 Effects of erythropoietin (EPO) on mitochondrial biogenesis in H9c2 cardiomyocytes exposed to hypoxia for 1 week. (A) Changes of mtDNA relative copy number in cardiomyocytes treated with different doses of recombinant human EPO (rhEPO) (0, 5, 10 or 20 U/mL), calculated on the basis of the cytochrome c/b-actin ratio. (B) Changes of peroxisome proliferator activated receptor gamma coactivator alpha (PGC-1a) mRNA expression, normalized by ribosomal protein L10A (RPL10A) mRNA. #P < 0.05 versus control  versus cells treated with 5 U/mL rhEPO.

adaptation of cardiomyocytes to chronic hypoxia. However, the underlying mechanism regulating mitochondrial biogenesis in cardiomyocytes in chronic hypoxia has to be understood, and eNOS is thought to be involved (Nisoli et al., 2003). EPO may have been seen as a haematopoietic cytokine for a long time (Gregory and Eaves, 1978). However, the nonhaematopoietic effects of EPO have been identified more recently, including a direct cardioprotective effect mediated by EPO–EPOR interaction on cardiomyocytes. EPO concentration in blood and EPOR expression in the myocardium

significantly increase under chronic hypoxia, suggesting that EPO–EPOR interaction on cardiomyocytes is enhanced, but the related biological effects need to be identified. Exogenous rhEPO can significantly enhance the mitochondrial biogenesis of the myocardium in the condition of normoxia (Carraway et al., 2010), providing the evidence of the regulatory effect of EPO on the mitochondrial biogenesis. From our results, rhEPO seems to enhance mitochondrial biogenesis and increase the number of mitochondria in cardiomyocytes exposed to chronic hypoxia in a dosedependent way, suggesting that EPO is an important factor

Figure 4 Changes of the expression and phosphorylation of protein kinase B (Akt) and endothelial nitric oxide synthase (eNOS) induced by different doses of recombinant human EPO (rhEPO) (0, 5, 10 or 20 U/mL) in H9c2 cardiomyocytes exposed to hypoxia for 1 week, which were detected by Western blots and normalised by b-actin protein p-Akt: phosphorylated Akt; p-eNOS: phosphorylated eNOS. #P < 0.05 versus control with 0 U/mL rhEPO  versus cells treated with 5 U/mL rhEPO.

Cell Biol Int 38 (2014) 335–342 ß 2013 International Federation for Cell Biology

339

Erythropoietin enhances mitochondrial biogenesis

C. Qin et al.

Figure 5 Influence from inhibiting protein kinase B (Akt) with Wortmannin in H9c2 cardiomyocytes exposed to hypoxia for 1 week. (A) Comparison of mtDNA copy number between the cells treated only with 20 U/mL recombinant human erythropoietin (rhEPO) (EPO control)and those treated with 20 U/mL rhEPO and 100 nmol/mL Wortmannin (EPO þ wort), based on the cytochrome c/b-actin ratio. (B) Comparison of peroxisome profilerator activated receptor gamma coactivator alpha (PGC-1a) mRNA expression, normalised by ribosomal protein L10A (RPL10A) mRNA. (C) Comparison of the expression and phosphorylation of Akt. (D) Comparison of the expression and phosphorylation of endothelial nitric oxide synthase (eNOS). p-Akt: phosphorylated Akt; p-eNOS: phosphorylated eNOS. #P < 0.05 versus cells treated only with 20 U/mL rhEPO.

regulating the mitochondrial biogenesis in cardiomyocytes exposed to chronic hypoxia. The Akt/eNOS signalling pathway is also significantly phosphorylated and activated by rhEPO with a dosedependent mode. Akt is an important intracellular signalling molecule closely related to the direct cardioprotection of EPO, mainly by inhibiting apoptosis (Kim et al., 2008). This molecule is a serine/threonine protein kinase that is phosphorylated and activated by phosphatidylinositol 3kinase (PI3K). The phosphorylated Akt can activate eNOS by phosphorylating its 1177 ser and the latter is the key kinase regulating mitochondrial biogenesis. Blocking Akt with Wortmannin reduced mitochondrial biogenesis enhanced by rhEPO and the lower phosphorylation of both Akt and eNOS, demonstrating that Akt is the upstream regulatory kinase of eNOS and participates in the regulation of EPO on mitochondrial biogenesis. Silencing the expression of eNOS gene with shRNA plasmid also 340

negatively regulated the mitochondrial biogenesis and reduced the expression and phosphorylation of eNOS protein, suggesting that eNOS plays a key role in regulating EPO in mitochondrial biogenesis in cardiomyocytes exposed to chronic hypoxia. It can be concluded that EPO enhances mitochondrial biogenesis in cardiomyocytes exposed to chronic hypoxia in a dose-dependent way. The Akt/eNOS signalling pathway at least partly mediates this regulatory effect by sequential activation. These findings provided some valuable information about the role of the enhanced EPO–EPOR interaction in the adaptation of cardiomyocytes to chronic hypoxia. Mitochondria from the myocardium of CCHD patients had a higher efficiency of ATP synthesis compared to those from patients with acyanotic congenital heart disease (Eells et al., 2000), which might be one of the biological effects produced by enhanced mitochondrial biogenesis. This can also lead to the increase of mitochondrial number and Cell Biol Int 38 (2014) 335–342 ß 2013 International Federation for Cell Biology

C. Qin et al.

Erythropoietin enhances mitochondrial biogenesis

Figure 6 Effects of silencing endothelial nitric oxide synthase (eNOS) gene with shRNA plasmid (A) H9c2 cardiomyocytes were successfully transfected with shRNA plasmid of eNOS, which presented with green fluorescence (200, fluorescence microscope). (B) Comparison of mtDNA copy number between the cells treated only with 20 U/mL recombinant human erythropoietin (rhEPO) (EPO control) and those treated with 20 U/ mL rhEPO and shRNA plamid (EPO þ shRNA), based on the cytochrome c/b-actin ratio. (C) Comparison of peroxisome profilerator activated receptor gamma coactivator alpha (PGC-1a) mRNA expression, normalized by ribosomal protein L10A (RPL10A) mRNA. (D) Comparison of the expression and phosphorylation of endothelial nitric oxide synthase (eNOS). p-eNOS: phosphorylated eNOS. #P < 0.05 versus cells treated only with 20 U/mL rhEPO.

volume, which may provide more surface area for oxygen diffusion, increasing the efficiency of oxygen utilization under hypoxia. However, how enhanced mitochondrial biogenesis by EPO helps cardiomyocytes adapt to the chronic hypoxia needs further investigation. Acknowledgments and funding This study was supported by Chinese National Natural Science Foundation, with the approval number of 81100120. References Carraway MS, Suliman HB, Jones WS, Chen CW, Babiker A, Piantadosi CA (2010) Erythropoietin activates mitochondrial biogenesis and couples red cell mass to mitochondrial mass in the heart. Circ Res 106: 1722–30.

Cell Biol Int 38 (2014) 335–342 ß 2013 International Federation for Cell Biology

Eells JT, Henry MM, Gross GJ, Baker JE (2000) Increased mitochondrial K(ATP)channel activity during chronic myocardial hypoxia: is cardioprotection mediated by improved bioenergetics? Circ Res 87: 915–21. Essop MF (2007) Cardiac metabolic adaptations in response to chronic hypoxia. J Physiol 584: 715–26. Fu P, Arcasoy MO (2007) Erythropoietin protects cardiac myocytes against anthracycline-induced apoptosis. Biochem Biophys Res Commun 354: 372–8. Funk JA, Odejinmi S, Schnellmann RG (2010) SRT1720 induces mitochondrial biogenesis and rescues mitochondrial function after oxidant injury in renal proximal tubule cells. J Pharmacol Exp Ther 333: 593–601. Gregory CJ, Eaves AC (1978) Three stages of erythropoietic progenitor cell differentiation distinguished by a number of physical and biologic properties. Blood 51: 527–37. Hasnaoui-Saadani RE, Marchant D, Pichon A, Escoubet B, Pezet M, Hilfiker-Kleiner D, Hoch M, Pham I, Quidu P, Voituron N,

341

C. Qin et al.

Erythropoietin enhances mitochondrial biogenesis

Journe C, Richalet JP, Favret F (2013) EPO deficiency alters cardiac adaptation to chronic hypoxia. Respir Physiol Neurobiol 186: 146–54. Hoffman JI, Kaplan S (2002) The incidence of congenital heart disease. J Am Coll Cardiol 39: 1890–900. Kim KH, Oudit GY, Backx PH (2008) Erythropoietin protects against doxorubicin-induced cardiomyopathy via a phosphatidylinositol 3-kinase-dependent pathway. J Pharmacol Exp Ther 324: 160–9. Liang C, Ren H, He Z, Jiang Q, Wu J, Zhen Y, Fan M, Wu Z (2009) Rosiglitazone via upregulation of Akt/eNOS pathways attenuates dysfunction of endothelial progenitor cells, induced by advanced glycation end products. Br J Pharmacol 158: 1865–73. Lombardero M, Kovacs K, Scheithauer BW (2011) Erythropoietin: a hormone with multiple functions. Pathobiology 78: 41–53. Mao W, Iwai C, Liu J, Sheu SS, Fu M, Liang CS (2008) Darbepoetinalfa exerts a cardioprotective effect in autoimmune cardiomyopathy via reduction of ER stress and activation of PI3K/Akt and STAT3 pathways. J Mol Cell Cardiol 45: 250–60. Nisoli E, Clementi E, Moncada S, Carruba MO (2004) Mitochondrial biogenesis as a cellular signaling framework. Biochem Pharmacol 67: 803. Nisoli E, Clementi E, Paolucci C, Cozzi V, Tonello C, Sciorati Bracale R, Valerio A, Francolini M, Moncada S, Carruba MO (2003) Mitochondrial biogenesis in mammals: the role of endogenous nitric oxide. Science 299: 896–9. Schleicher M, Yu J, Murata T, Derakhshan B, Atochin D, Qian L, Kashiwagi DI, Lorenzo A, Harrison KD, Huang PL, Sessa WC

342

(2009) The Akt1-eNOS axis illustrates the specificity of kinasesubstrate relationships in vivo. Sci Signal 2: ra41. Stockmann C, Fandrey J (2006) Hypoxia-induced erythropoietin production: a paradigm for oxygen-regulated gene expression. Clin Exp Pharmacol Physiol 33: 968–79. St-Pierre J, Lin J, Krauss S, Tarr PT, Yang R, Newgard CB, Spiegelman BM (2003) Bioenergetic analysis of peroxisome proliferator-activated receptor gamma coactivatiors 1alpha and 1beta in muscle cells. J Biol Chem 278: 26597–603. Trinei M, Berniakovich I, Ppelicci PC, Giorgio M (2006) Mitochondrial DNA copy number is regulated by cellular proliferation: a role for Ras and p66(Shc). Biochim Biophys Acta 1757: 624–30. Xiao J, Chen L, Wang X, Liu M, Xiao Y (2012) eNOS correlates with mitochondrial biogenesis in hearts of congenital heart disease with cyanosis. Arq Bras Cardiol 99: 780–8. Xu X, Cao Z, Cao B, Li J, Guo L, Que L, Ha T, Chen Q, Li C, Li Y (2009) Carbamylated erythropoietin protects the myocardium from acute ischemia/reperfusion injury through PI3K/Aktdependent mechanism. Surgery 146: 506–14. 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 sialyl Lewis X repairs infarcted myocardium in rabbits. Am J Physiol Heart Circ Physiol 204: H1124–33. Received 21 August 2013; accepted 27 October 2013. Final version published online 16 January 2014.

Cell Biol Int 38 (2014) 335–342 ß 2013 International Federation for Cell Biology

eNOS signalling pathway.

Adaptation of cardiomyocytes to chronic hypoxia in cyanotic patients remains unclear. Mitochondrial biogenesis is enhanced in myocardium from cyanotic...
2MB Sizes 0 Downloads 0 Views