Prenatal Testosterone Exposure Induces Cardiac Hypertrophy In Adult Female Rats And Its Mechanisms

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Establishment of Rat Model of Maternal testosterone exposure and effects of treatment with Maternal propionate testosterone on body weight and heart structure and function of female adult offspringObjctive:To study the effects of Maternal testosterone propionate(Maternal-TP)treatmemt on cardiac structure and function in offspring female rats.Material and methods:All pregnant rats were divided into two groups:animals in the Maternal-TP group were injected with 0.5 mg/kg/day TP dissolved in corn oil,and those of the control group were injected with an equal amount of corn oil.The daily injections were done on gestational days 15 to 19.Each rat heart was collected and weighed at age of 8W,16W and 24W.The width of the left ventricle wall and of the intraventricular septum was measured in mid-cardiac sections.Cardiomyocyte crosssectional area and the extent of cardiac fibrosis were measured by staining with hematoxylin and eosin(HE),FITC-conjugated wheat germ agglutinin(WGA),and picrosirius red(PSR)respectively.Cell size and fibrotic area were analyzed using digital image.At the age 8W,16W and 24W,cardiac function of the female offspring from both groups was measured via an echocardiographic imaging system under continuous anesthesia with 2.5%isoflurane.Left ventricular ejection fraction,fractional shortening,left ventricular posterior wall thickness at end-diastole,interventricular septal wall thickness at end-diastole and left ventricular end-diastolic diameter were measured and calculated.ELISA kits were used to measure the concentrations of serum and cardiac tissue T,DHT and endogenous estradiol of female offspring at 6 months of age.Results:The pregnant rats in Maternal-TP group showed a 2.5-fold increase in circulating T level and a 2.0-fold increase in circulating DHT level on gestation day 21.5 compared with control rats.However,in adult female offspring,there were no significant differences in circulating T,DHT and E2 levels between Maternal-TP and control groups at age of 8W,16W and 24W.From birth to 8 weeks old,the mean body weight of female offspring in Maternal-TP group was significantly less than that in control group.However,after 8 weeks,there was no significant difference in the mean body weight between two groups.The measurements of heart weight and the ratio of heart weight to body weight showed that the 16w and 24w old female offspring in Maternal-TP group had a significant increase in heart weight,compared with control offspring.Staining with HE revealed that both left ventricle wall and interventricular septum wall of the 16w and 24w old female offspring in Maternal-TP group were significantly thicker in the Maternal-TP group than those of the control group.Both HE and wheat germ agglutinin staining showed that the cross-sectional area of cardiomycytes of the 16w and 24w old female offspring in Maternal-TP group was significantly larger than that in the control group.Furthermore,collagen accumulation was significantly greater in the 16w and 24w old female offspring of Maternal-TP group than controls.Left ventricular mass/body weight,left ventricular posterior wall thickness at end-diastole,interventricular septal wall thickness at end-diastole and left ventricular end-diastolic diameter of the 16w and 24w old female offspring in MaternalTP group were significantly greater than those of the control group.Left ventricular ejection fraction and fractional shortening of the 16w and 24w old female offspring in Maternal-TP group were significantly lower than those of the control group.Heart rate was similar between two groups.Blood pressure was significantly higher in the Maternal-TP group at 24w old.Conclusion:Maternal-TP adult female offspring presented cardiac hypertrophy,interstitial fibrosis,and reduced cardiac function,which is associated with increased local DHT level in cardiac tissue.Part ? The expression of genes related to cardiac hypertrophy in ventricular from female adult offspring of Maternal-TP treatmentObjctive:To explore the effects of Maternal-TP treatment on the expression profile of genes related to cardiac hypertrophy in rat cardiac and to investigate the mechanism underlying the cardiac hypertrophy induced by prenaltal androgen exposure.Material and methods:Cardiac tissue samples were obtained from the hearts of two female offspring rats at 16w and 24w old.Total RNA was extracted by Trizol method and reverse transcribed using reverse transcription kit.SYGR-Green real-time quantitative PCR was used to detect the cardiac hypertrophy in the two groups Gene expression.RIPA protein lysis solution was used to extract the total protein,Western blot was used to detect the expression of cardiac tissue-related protein in both groups.Results:There were no significant differences in ANP and BNP(molecular markers of cardiac hypertrophy),Col1a1 and Col3a1(markers of cardiac interstitial fibrosis),Esrl and Esr2 expression between two groups.The Maternal-TP group female offspring at 16W,AR,PKC? and Myh7(cardiac hypertrophy molecular marker)gene expression was significantly higher than the control group.The BNP,Myh7,collal,AR and PKC?gene levels in cardiac tissue of Maternal-TP female rats at 24w were significantly higher than that of the control group.At 24w,the AR,PKC? and Myh7 protein expression in cardiac tissue of offspring of Maternal-TP group were significantly higher than that of control group.Conclusion:Increased AR and PKC? expression in myocardial tissue may play an important role in cardiac hypertrophy in adult female offspring induced by maternal androgen treatment.Part ? The effects and mechanisms of high androgen exposure on primary neonatal rat ventricular cardiomyocytes sizeObjctive:To investigate the effects and mechanisms of AR and PKC? on cardiomyocyte size by in vitro hyperandrogenic cell exposure model.Material and methods:We isolated primary neonatal rat ventricular cardiomyocytes(NRCMs)from heart samples of 1-to 3-day-old Sprague-Dawley rats.NRCMs were plated on gelatin-coated 6-well plates at a density of?1×106 cells/well and then cultured for 24 h.On the day after isolation,cells were transferred to maintenance serum-free medium and were incubated for 3 days with different concentrations of DHT(1 nM/L,5 nM/L,10 nM/L,50 nM/L and 100 nM/L),after which cells were lysed for preparation of RNA or protein samples.Some of the NRCMs treated with 1?M/L DHT were treated in concert with flutamide(10?M/L)for 24 h,and those cells were then prepared for immunofluorescence staining.In addition,primary NRCMs were treated with PMA(0.2 ?M),rottlerin(0.1 ?M),DHT(1 ?M)or vehicle with or without PMA(0.2 ?M)and/or rottlerin(0.1 ?M)for 24 h.Cells were kept in serum-free medium for 4 hours before administration.Cell extracts were used to determine Myh7 protein levels by western blotting after stimulation PMA(0.2 ?M)for 1 h,rottlerin(0.1 ?M)for 30 min,DHT(50 nM)for 48 h or vehicle with or without the addition of PMA(0.2 ?M)and/or rottlerin(0.1 ?M)as indicated.,which was normalized to Gapdh.Western blot was used to detect the protein expression of AR,PKC? and Myh7 in cardiomyocytes after different drug treatment.The mRNA expression of AR,PKC? and Myh7 in cardiomyocytes were detected by RT-PCR.Immunofluorescence Immunofluorescence was used to observe the size of NRCMs.Chromatin immunoprecipitation was used to detect the binding of AR and PKC?promoter regions.Results:Treatment of primary NRCMs with DHT at the concentrations of 1,5,10,50 and 100 nM for 3 days significantly upregulated the levels of AR,PKC?,and Myh7 in cells in a dose-dependent manner.We also found that DHT at 50 nM not only increased the mRNA and protein levels of intracellular PKC? and Myh7,but also significantly increased cardiomyocyte size.Pretreatment of primary NRCMs with flutamide,an inhibitor of AR significantly blocked the effects of DHT on PKC?,and Myh7 expression and cell structure.Bioinformatics analysis revealed 6 putative AR binding sites located at the promoter of PKC?.We used ChIP to confirm the interaction between AR and these sites in the PKC? promoter in vitro.We found that ARE1,ARE2,ARE5,and ARE6,but not ARE3 and ARE4,interacted with AR in a DHT-dependent manner.Treatment with DHT,PMA and DHT with PMA induced significant cardiomyocyte hypertrophy with increased cell size compared to the control(1.6-fold;1.8-fold;1.6-fold),and these increasements were significantly attenuated by rottlerin(0.4-fold;0.3-fold;0.5-fold;).Moreover,the intracellular level of Myh7 protein was significantly increased by treatment with DHT(1.4-fold),PMA(1.9-fold),DHT plus PMA(1.7-fold)compared with vehicle,and there were approximate 29.2%,53.1%,49.1%reduction in DHT-upregulated,PMA-upregulated,DHT with PMA-upregulated Myh7 level in NRCMs pretreated with rottlerin.Conclusion:DHT stimulation of primary NRCMs can cause increased expression of cardiac hypertrophy-related genes.Flutamide,an AR inhibitor,alleviated DHTinduced cardiomyocyte hypertrophy through down-regulating the expression of PKC?.PKC? may be a downstream target of AR.DHT may positively regulate the transcription of PKC? by AR binding to the promoter region upstream of the PKC?gene.PKC? can regulate cardiomyocyte growth.