| Erythropoietin(Epo),a hypoxia-induced,lineage-specific hormone,is essential for normal erythropoiesis.Recombinant human Erythropoietin(rh-Epo) is widely used in the treatment of various kinds of anemia,such as end-stage renal disease,Human Immunodeficiency Virus infection,and in patients with non-myeloid malignancies on chemotherapy.Recently it is found out that Epo receptor is widely existed in cardiovascular system.Animal studies demonstrated that Epo can potentially protect myocardium against ischemia-reperfusion-induced injury in and restrict infarcted size. However,little is known about the role of Epo in the development of cardiac hypertrophy.In this study,we examined the in vitro effects of Epo on hypertrophic responses of cultured cardiomyocytes of neonatal rats.PART ONE:Epo-induced morphological changes in cultured cardiomyocytes of neonatal ratsObjective:To observe the morphological changes of cultured cardiomyocytes after addition of Epo for various times.Methods:Primary cardiac myocytes were isolated from neonatal Sprague-Dawley rats and cultured in DMEM culture medium containing 10%fetal bovine serum(FBS) for the first 24 hours,and then in 2%FBS-containing medium for synchronization in the next 24 hours.Three groups were included in these experiments:control group;AngⅡtreated group and Epo treated group.The final concentration of AngⅡwas 1×10-6M; and the final concentration of Epo was 10U/ml.Culture cells were fixed at 24h,48h and 72h,respectively,and immunohistochemical stained with cTnI.The sizes of the cardiomyocytes were measured with light microscopy.Low serum culture medium containing 0.1%FBS was used for myocardium ultrastructure study with transmission electron microscope.Three groups were included as describe before. Cells were fixed at 24h,48h and 72h,and myocardium ultrastructure was observed by a transmission electron microscope.Results:1) After 24 hours exposure to different stimulating factors,the mean surface area of myocardial cells was 2135.6±524.8μm2 for control group,3202.3±661.2μm2 for AngⅡtreated group,and 2586.2±165.3μm2 for Epo group.Comparing to the control group and the Epo treated group,the mean size of the ceils was significantly increased for AngⅡtreated group(P＜0.05).After 48 hours exposure to different stimulating factors,the mean surface area of myocardial cells was 1844.5±323.3μm2 for control group,3321.0±428.8μm2 for AngⅡtreated group,and 3318.9±321.3μm2 for Epo treated group.Comparing to the control group,the mean surface area of the cells is significantly increased in AngⅡtreated group and in Epo treated group(P＜0.05).The difference between AngⅡtreated group and Epo treated group was not significant.After 72 hours exposure to different stimulating factors,the mean surface area of myocardial cells was 2213.90±149.5μm2 for control group,3222.20 ±225.3μm2 for AngⅡtreated group,and 3317.20±196.1μm2 for Epo treated group.Compared to control group,the mean surface area of the cells was significantly increased in AngⅡtreated group and in Epo treated group(P＜0.05);however,no significant difference was seen between AngⅡtreated group and Epo treated group. Epo intervention for 48h and 72h significantly increased the mean surface area of the cells comparing to the 24h treated group(P＜0.05).2) Transmission electron microscopy observation showed that in the control group myofilament dissolved gradually,and most of organelle was damaged when exposure to 72hs low-FBS culture medium.Cardiac myocytes in AngⅡtreated group and the Epo treated group had irregular nuclei,distinct nucleolus,faint chromatin,recessed nuclear membrane,lots of swollen mitochondria and prospersous secretion and synthesis related organelles such as endoplasmic reticulum and Golgi body,showed vacuolization occurred within sub-cellular structures when exposed to AngⅡand Epo for 72h.Conclusion:Addition of Epo to culture medium can induce enlargement of cardiomyocytes and ultrastructural changes in cardiomyocytes.PART TWO:Epo-induced upregulation of ERK activation and hypertrophy-related gene expression in cultured cardiomyocytesObjective:To investigate whether Epo upregulates ERK activation and some specific gene expression,such as ANP,BNP and c-fos,in cultured cardiomyocytes.Methods:Primary cardiac myocytes were isolated from natal Sprague-Dawley rats and cultured in DMEM culture medium containing 10%fetal bovine serum(FBS) for the first 24 hours,then in 0.1%FBS-containing medium for synchronization for the next 24 hours, before Epo intervention.1)Epo treated cardiomyocyte with final concentration of Epo 10U/ml,Epo intervention was ended at 0min(control group),10min,15min,30min, 60min and 120min respectively.Total cellular protein was extracted and Western blot detection was used to quantify the myocardial p-ERK1/2;2) Epo treated cardiomyocyte for 30min,with the final concentration 0(control group),5 U /ml,10 U/ml and 100 U/ml respectively.Total cellular protein was extracted and western blot detection was used to quantify the myocardial p-ERK1/2 with different Epo concentration treatment;3) Cardiomyocytes were divided into two groups,control group and the Epo treated group(Epo10U/ml treated for 30min).The total RNA of the cardiomyocytes were extracted and RT-PCR was introduced to semi-quantify c-fos mRNA expression.Similarly,ANP and BNP mRNA was extracted from the cardiomyocytes with Epo treatment or blank control for 2hs and gene expression level was measured by RT-PCR.Results:1) p-ERK2 expression in cardiac myocytes first increased and then decline in 120min after Epo 10U/ml intervention,and the expression level was at peak when harvested from Epo 10U/ml treatment for 30min,comparing to the control group and the 120min-treated group(P＜0.05).The similar changes of p-ERK1 expression were also seen but the differences were not significant.2) Increasing concentration of Epo intervention resulted in increased p-ERK2 expression levels in cardiac myocytes, p-ERK2 expression was significantly higher in the group of Epo 10 U/ml and 100 U/ ml than that of the control group(P＜0.05).The p-ERK1 expression was also increased with the increasd Epo concentration,yet the differences were not significant. 3) With Epo10U/ml intervention in cardiac myocytes,c-fos mRNA expression was not increased significantly,yet ANP and BNP mRNA expression were significantly higher than that of control group(P＜0.05).Conclusion:1) Epo induces ERK1/2 activation in a time- and concentration-dependent manner;2) Treatment with Epo at 10U/ml for 30min can significantly increase ERK1/2 phosphorylation;3) Epo can stimulate the expression of ANP and BNP genes,and prone to increase c-fos gene expression.PART THREE:Involvement of angiotensinⅡin Epo-induced hypertrophic responses of cardiomyocytesObjective:1) To examine the impact of Epo on angiotensinogen(AGT) mRNA expression in cardiomyocytes;2) To examine whether RAS is involved in Epo-induced ERK activation in cardiomyocytes.Methods:Primary cardiac myocytes were isolated from natal Sprague-Dawley rats and cultured in DMEM culture medium containing 10%FBS for the first 24h,and then in 0.1% FBS-containing medium for synchronization for the next 24h.1) Epo treatment with final concentration of 10U/ml was terminated at 0min(control group),1h,2h,6h,12h,24h respectively.Total cellular RNA were extracted and RT-PCR was introduced to quantify the AGT mRNA expression.2) 6 groups were included in this study:a) Control group;b) AngⅡgroup:AngⅡ1×10-6M for 8min;c) ARB+AngⅡgroup:Telmisartan 1×10-6M blockage for 30min then AngⅡ1×10-6M for 8min;d) Epo Group:Epo 10U/ml for 30min;e) ACEI+Epo group:Enalapril 1×10-6M blockage for 2h then Epo 10U/ml for 30min;f) ARB+Epo groups:Telmisartan 1×10-6M blockage for 30min then Epo 10U/ml for 30min.Total cellular protein was extracted and western blot detection was used to quantify the myocardial p-ERK1/2 from the groups described above.3) The cardiomyocytes were divided into 3 groups:a) Control group; b) Epo Group:Epo 10U/ml for 6h;c) PD98059+Epo group:PD98059 50×10-6M blockage for 30min then Epo 10U/ml for 6h.Total cellular RNA was extracted and RT-PCR was introduced to quantify the AGT mRNA expression.Results:1) Cardiomyocytes AGT mRNA expression increased after 2 hours(2h,6h,12h,24h Vs control,P＜0.05) treatment of Epo 10U/ml with the highest expression level at 6hrs treatment.After that,its expression was inclined to decrease.2) The increase of p-ERK1/2 caused by Epo treatment could be reversed by Enalapril or Telmisartan treatment.3) p-ERK1/2 pathway blockage with PD98059 could not stop the increase of AGT mRNA expression on the treatment of Epo.Conclusion:1) Epo upregulates AGT gene expression in cultured cardiomyocytes.2) RAS may be involved in Epo-induced ERK activation. |
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