| Objective In order to offer a new idea and experimental basis for the treatment and prevention of myocardial hypertrophy, we observed effects of 17p-estradiol on phenylephrine-induced hypertrophy in cultured neonatal rat cardiomyocytes and explored its mechanisms at cellular and molecular levels.Methods Cardiomyocytes were isolated with trypsin and collagenase digestion and purified by differential attachment technique and mitomydn C, cardiomyocytes were identified by immunofluorescence staining with cardiac specific antibodes against myosin heavy chain α/β (MHCα/β) and troponin I (Tn I), the model for cardiomyocytes was established; After cultured with a serum-free maintenance media for 24h, cardiomyocytes were treated with different agents: control group(no agent), PE group (phenylephrine 10-5mol/L), E group(17β-estradiol 10-8mol/L), PE+E group(17β-estradiol 10-8mol/L and phenylephrine 10-5mol/L); We measured following indexes at different hours according to experimental need: ①surface area of cardiomyocytes were measured by computer photograph analysis software, ②the protein synthesis of cardiomyocytes was assayed with [3H]leucine intake method, ③ the proto-oncogene c-fos protein expression was assessed with immunocytochemistry method, ④ the markers of cardiomyocyte hypertrophy, embryonic genes such as p-myosin heavy chain(β-MHC), α-skeletal-actin(a-skA) and atrial natriuretic factor(ANP) mRNA expressions were assessed with semiquantitative reverse transcription-PCR.Results 1. Immunocytochemistry with cardiac specific antibodes against MHCα/β and Tn I showed the purify of cardiomyocytes was above 95% and cross striation could be observed clearly. 2. After stimulated with PE for 48h, cardiomyocytes surface area increased by 2.6-fold compared with control group(P<0.05). 17β-estradiol inhibited obviously phenylephrine-induced increase of cell surface area(P<0.05). 3. After stimulated with PE for 24h, the protein synthesis rate of cardiomyocytes increased by3.1-fold compared with control group(P<0.05). 17p-estradiol inhibited obviously phenylephrine-induced increase of the protein synthesis rate(P<0.05). 4. After stimulated with PE for 2h, the expression level of proto-oncogene c-fos protein was markedly enhanced, the positive yields of immunoreactivity were annularly located in the peripheral area of nucleus of cardiomyocytes. 17P-estradiol reduced the c-fos protein expression of hypertrophic cardiomyocytes. 5. After stimulated with PE for 48h, the expression levels of P-MHC\ a-skA and ANP mRNA increased significantly compared with control group(P<0.05). 17|3-estradiol down-regulated |3-MHC> a-skA mRNA expressions and up-regulated ANP mRNA expression(P<0.05).Conclusions 17P-estradiol inhibits hypertrophy of cardiomyocytes induced by phenylephrine and its mechanisms might be associated with the suppression of c-fos protein expression, the reversion of the embryonic switching of contractile protein genes (P-MHC and a-skA), the increase of ANP mRNA expression.
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