Erythropoietin Attenuates Hypertrophy Of Neonatal Rat Cardiac Myocytes Induced By Angiotensin-Ⅱ In Vitro

Background and ObjectivesCardiac hypertrophy is the heart's compensatory response to a variety of extrinsicand intrinsic stimuli including pressure or volume overload,mutations of sarcomericproteins,or loss of contractile masses from previous myocardial infarction.Sustainedcardiac hypertrophy can ultimately become deleterious and result in cardiomyopathy,heart failure,and sudden death.There is increasing evidence showing that preventionof cardiac hypertrophy may represent a new therapeutic target to improve patientsurvival.Erythropoietin (EPO) is a hypoxia-induced hormone that is essential for normalerythropoiesis and has been broadly used in patients with anemia caused by variouskinds of etiologies.As the erythropoietin receptor (EPOR) has been detected in thecardiovascular system,there was evidence confirming that EPO was a novelcardio-protective agent in addition to its hematopoietic effects.Preclinical studieshave established EPO to be a pleiotropic cytokine with anti-apoptotic activities andtissue-protective actions in the cardiovascular system.A remarkable fact is that bothclinical trials and animal experiments have shown us the phenomenon that EPO caninhibit ventricle hypertrophy and attenuate cardiac remodeling besides its otherbenefits,such as enhancement of exercise performance and increase of ejectionfraction.Although this amelioration could be partly attributed to the hemoglobinelevation and thus the increased oxygen-binding capacity of blood,the non-hematopoietic effects of EPO on cardiomyocytes should also be considered.AsEPORs have been found in the cardiovascular system widely,there is a possibilitythat EPO can inhibit cardiac hypertrophy directly.However,very few studies weredesigned to explore the relationship between EPO and cardiomyocyte hypertrophy exvivo and the cellular mechanism underlying the potential effect was also notunderstood.Extension of these mechanistic insights into the role of EPO in cardiacremodeling would further the development of novel therapeutic strategies related tocardiac failure.Nitric oxide (NO) is an important and potential regulator of cardiac remodeling.Low levels and transient release of NO by endothelial nitric oxide synthase (eNOS)exert beneficial effects on the remodeling process by reducing cardiomyocytehypertrophy,cavity dilation,and mortality.Several recent studies have demonstratedthat EPO increased eNOS expression,caused eNOS phosphorylation and resulted inNO release through the phosphatidylinositol 3'-kinase (PI3K)-Akt (PKB)-dependentpathway.However,to date,the potential connection between the anti-hypertrophiceffects and PI3K-Akt-eNO-NO stimulatory effects of EPO has not been identified.Furthermore,it is noteworthy that Li Y,et al recently showed that EPO treatmentreduced inflammation and cytokine production in the failing heart,such astransforming growth factor-beta1 (TGF-beta1),interleukin (IL)-1beta,IL-6 and tumornecrosis factor-alpha.Importantly,TGF-beta1,which can activate its downstreamsignal,such as Smad2 and Smad3,plays a central role in cardiac hypertrophy.Thus itis reasonable to postulate that EPO may act in anti-hypertrophy throughdown-regulating TGF-betal expression in cardiomyocytes.In this regard,we hypothesized that EPO may inhibit cardiac myocyte hypertrophyvia the PI3K-Akt-eNOS-NO signal pathway and the TGF-betal-Smad2 signalpathway.To test this idea,the classical pharmacological inducer for cardiachypertrophy angiotensin-Ⅱ(Ang-Ⅱ) was used in this study;we evaluated the effectsof the pretreatment with EPO.We examined NO levels,myocardial eNOS protein levels,Akt levels,and its phosphorylation.To further elucidate the role of Akt andeNOS,we also tried to confirm whether the effects of EPO on cardiomyocytes wereblunted by LY294002 or NG-nitro-L-arginine methyl ester (L-NAME).Moreover,todelineate the role of TGF-betal and Smad2 in EPO-modulated cardiac hypertrophy,we measured these protein alternations and analyzed the relationship among them.Part one:Isolation and identification of the culturedneonatal cardiac myocytesObjective To introduce a steady and reliable method of cardiomyocytes culture ofneonatal rats.Methods Ventricular myocardial tissues from wild-type rats born within 24 to 72 hwere minced in a Ca2+- and Mg2+-free Hanks' balanced solution.Cardiomyocyteswere digested with 0.1% collagenaseⅠ.Purification of cardiomyocytes was done bydifferential adhesion and the supplement of 5-bromodeoxyuridine,andcardiomyocytes were identified by immunohistochemistry staining ofα-actin.Results①Just half an hour later of plantation,partly cardiomyocytes wereattached to the bottom of the dishes.After 24 hours,some single myocytes began tobeat spontaneously.After 48 hours,cardiomyocytes got together in clusters and beatsynchronously.②All cells used in experiments were confirmed as cardiomyocyte byimmunohistochemistry staining ofα-actin.Conclusion The method as an ideal one for cardiomyocytes culture from ourlaboratory has been proved steady and reliable.Part two:Erythropoietin inhibits the hypertrophy of cardiacmyocytes in vitro via the activation of PI3K/Akt-eNOSpathwayObjective To test the hypothesis that erythropoietin (EPO) inhibits angiotensinⅡ (AngⅡ)-induced hypertrophy in cultured neonatal rat cardiomyocytes and to explorethe roles of PI3K/Akt-eNOS signaling pathway on the related effects.Methods Cardiomyocytes were isolated from new-born Sprague-Dawley rats andwere used to establish the model of hypertrophy by AngⅡin vitro.The cell surfacearea and mRNA expression of atrial natriuretic factor (ANF) of myocytes wereemployed to detect cardiac hypertrophy.Different concentrations of EPO were used toinvestigate its impact on hypertrophic cardiomyocytes.A phosphatidylinositol3'-kinase (PI3K) inhibitor LY294002 and a nitric oxide synthase (NOS) inhibitorL-NAME were also employed to detect the underlying mechanism of EPO.Intracellular signal molecules,such as Akt,phosphorylated Akt,eNOS andphosphorylated eNOS protein expression were determined by Western blot.Nitricoxide (NO) levels in the supernatant of cultured cardiomyocytes were assayed byusing an NO assay kit.Results 20U/ml of EPO significantly inhibited cardiac myocytes hypertrohyinduced by AngⅡ,which can be confirmed by the decreases of cell surface area(3221.68±434.28 vs.2375.13±369.50μm~2,P＜0.05) and ANF mRNA expression (P＜0.05);however,low concentrations of EPO have no effects of attenuation.EPO canalso activated Akt and enhanced the expression of eNOS and its phosphorylation (P＜0.05).Therefore,compared with the control group,the NO production of co-treatmentwith EPO group was also significantly increased (P＜0.01).Adversely,LY294002 orL-NAME partially abolished the antihypertrophic effect of EPO,accompanied by adecrease of Akt activation,eNOS protein expression and/or a reduction of eNOSactivation (P＜0.05).LY294002 and L-NAME also suppressed NO expression (P＜0.05).Conclusion EPO attenuates cardiac hypertrophy induced by AngⅡvia theactivation of PI3K-Akt -eNOS pathway and the increase of NO production. Part three:The TGF-beta1-Smad2 signal pathway mediatesthe anti-hypertrphic effect of erythropoietinObjective To explore the effect of TGF-betal-Smad2 signal pathway inerythropoietin (EPO)-mediated cardiac hypertrophy attenuation.Methods Cardiomyocytes were isolated from new-born Sprague-Dawley rats andwere used to establish the model of hypertrophy by AngⅡin vitro.The cell surfacearea and mRNA expression of atrial natriuretic factor (ANF) of myocytes wereemployed to detect cardiac hypertrophy.Different concentrations of EPO were used toinvestigate its impact on hypertrophic cardiomyocytes.Intracellular signal molecules,such as transforming growth factor-beta1 (TGF-beta1),Smad2 and phospho-Smad2(P-Smad2) protein expression were determined by Western blot.Results EPO attenuated hypertrophy of cardiomyocytes,which accompanied by thephenomenon that EPO could reversed AngⅡ-induced pro-hypertrophic signalmolecules TGF-betal,Smad2 and P-Smad2 protein expression.Conclusion EPO attenuates cardiac hypertrophy induced by AngⅡand this effectsis associated with TGF-beta1- Smad2 pathway.