Erythropoietin Attenuates Neonatal Rat Cardiomyocyte Hypertrophy In Vitro And The Association With TGF β1-TAK1-p38MAPK Pathway

Background and aims:Hypertrophy is a principal response of the heart to overload from any causes,including hypertension,myocardial infarction,valvar heart disease,and dilated cardiomyopathy. At the cellular level,cardiac hypertrophy is characterized by an increase in cell size and protein synthesis and the reactivation of a set of fetal cardiac genes which are normally expressed in the heart only before birth. The moleculear mechanisms that regulate cardiac hypertrophy remain poorly understood. This experiment was to observe the effects of erythropoietin (EPO) on angiotensin Ⅱ (Ang Ⅱ) induced neonatal rat cardiomyocyte hypertrophy and the association with TGF β1-TAKl-p38MAPK signaling pathway.METHODS:1、Ventricular myocardial tissues were isolated from new-born Sprague-Dawley rats and dissociated with0.1%trypsin and0.5g/L collagenase type Ⅱ. Cardiomyocyte were further purified by differential adhesion and with Bromodeoxyuridine. To observe the morphological characteristics of the cultured cardiomyocyte and identify cardiomyocyte.2、The cells were divided into5groups:Control、EPO、Ang Ⅱ、 AngⅡ+EPO、Ang Ⅱ+EPO+SB203580. cardiomyocytes of the groups were exposed to with Angll (1×10-6mol/L)、EPO(20U/mL) or/and SB203580(15umol/L).3、The cell surface area,[3H]-leucine incorporation,mRNA expression of atrial natriuretic factor(ANF) and β-myosin heavy chain(B-MHC) of cardiomyocytes were employed to detect cardiomyocyte hypertrophy.4、The mRNA levels of TGF-B1and p38MAPK was analyzed by quantitative real-time PCR.5、The protein expression of TGF-β1、TAK1、p38MAPK and the phosphorylation of TAK1and p38MAPK were analyzed by Western blot. RESULTS:1.. The cultured myocardiocytes began to adhere and grow after4-6h,they showed spherical,fusiform and polygon shape in the visual field of inverted microscope.24h after inoculating most cardiac myocytes had attached and partly of them began to beat sponta-neously,and they got together and beat synchronously in clusters72h after inoculating. Immunocytochemistry staining analysis showed that myocardiocytes expressed a-actin.2、EPO attenuated hypertrophy of cardiomyocytes induced by AngⅡ as shown by decreased cell surface area, protein synthesis and the expression of ANF and β-MHC. These effects could be partially reinforced by cotreatment with SB203580(P<0.05).3、Expression of TGF-β1and p38MAPK mRNA was up-regulated induced by AngⅡ by RT-Q-PCR, and this effect could be significantly decreased by EPO or/and cotreatment with SB203580(all P<0.05).4^The protein expression of TGF-β1、TAK1、p38MAPK and the phosphorylation of TAK1and p38MAPK was up-regulated induced by AngⅡ by Western blot., and this effect could be significantly attenuated by EPO or/and cotreatment with SB203580(all P<0.05).CONCLUSION:EPO attenuates Ang Ⅱ induced cardiomyocytes hypertrophy,and it might be associated with TGF-β1-TAKl-p38MAPK signaling pathway.