Effects And Mechanisms Of The Hypothalamic Pituitary Gonadal Axis On Gender Related Cardiovascular Phenotypes With Genetically Modified Mice

Background and the purpose: Gender-related cardiovascular diseases have been reported by a large number of clinical observations and genetically modified animal models.In general,estrogen plays as a protective role in heart,whereas androgen tends to aggravate gate cardiac diseases.However,the cardioprotective effect of estrogen in estrogen replacement therapy（ERT）and androgen deprive therapy has been challenged by WHI and MWS trials that found null to even harmful effects on the risk of cardiovascular events because of imbalance of sex hormone.This illuminated that ER and AR didn’t be fully responsible for the gender differences in cardiac pathology,and the therapy shoudn’t fully depend on ER and AR signaling and the downstream signaling.We observed that there were gender-related cardiac hypertrophy or cardiac functions in two gene-targeted mouse models FKBP12.6 and CD38 null mice.Interestingly,we also detected that serum 17β-estradiol（E2）,testosterone（T）and LH were increased in CD38 null mice,indicating that the hypothalamic-pituitary-gonadal axis（HPGs）was up-regulated in the knock-out mice.Based upon above,we hyposised that HPGs was the most important regulator in gender related cardiovascular phenotypes from various reasons.In the present study,we aimed to open up a new possibility that sex dimorphism cardiovascular diseases relied on the disorder of regulation in HPGs signaling which led to hormonal imbalance based upon studies of tet-on inducible hypothalamic specific Gn RH1 transgenic mice and deeply studies of FKBP12.6-/-and CD38^-/-mice.Methods:1.In order to confirm the role of FKBP12.6 deficiency in heart,the expressions of calcium signaling related protein such as calcineurin（Ca N）,MCIP1,MCIP1.4,Ca MKⅡ,and hormone receptor were analyzed by western blot.2.In order to confirm the role of sex hormone in FKBP12.6-/-mice,we compare of heart weight/body weight ratio（HW/BW）in WT male mice and age matched FKBP12.6-/-male mice with their testicles surgically removed or not.The concentrations of serum testosterone and 17β-estradiol in FKBP12.6-/-were measured by radioimmunoassay and LH and FSH secretion were detected by ELISA.3.In order to find out which part was involved in regulation of HPGs by FKBP12.6,FKBP12.6 expressions in LβT2 and GT1-7 cells were detected by western blot and the expression of pro-Gnrh1 and m RNA of Gn RH1 were detected by western blot and RT-QPCR with a si RNA-mediated FKBP12.6 transfected GT1-7 cells.4.Ca²⁺ concentration([Ca²⁺]i)was determined by measuring the fluorescence of Fluo-3 in FKBP12.6 knockdown LβT2 cells and the m RNA expressions of LHβ,Nur77,SF-1 were analyzed by RT-QPCR.The protein expressions of the pERK1/2,MCIP1.4,Ca N,FKBP12.6,NFAT3,Nur77,pin1,p-pin1 were detected by western blot in the cytosol and nuclues of FKBP12.6 knockdown LβT2 cell lines.5.In order to figure out the role of Sirt1 in the hypothalamus of CD38^-/-mice,the expressions of CD38,pro-Gn RH1,Otx2,Ac-p53 and Sirt1 were detected by western blot after treated with or without EX527 in CD38 knockdown GT1-7 cell lines.6.In order to further confirm the role of HPGs in gender-related cardiovascular diseases,the tet-on inducible hypothalamic specific Gn RH1 transgenic mice were generated by microinjection in which the Gn RH1 was specifically expressed in the hypothalamus.The expressions of Gn RH1 were measured by immunofluorescence and western blot and the concentrations of serum Gn RH1 and FSH secretion were detected by ELISA,and the left ventricular wall thickness and cardiac function were measured by echocardiogram.Results:1.There were no significant changes in phosphorylation of Ca MKⅡ and MCIP1,an inhibitor of Ca N,were up-regulated in FKBP12.6 KO female mice.However,the expression of MCIP1.4 which is sensitively related to the activity of Ca N was upregulated in male mice,indicating that the activity of Ca N was increased in male mice and was inhibited in female mice.2.The castrated adult male null mice would compeletely rescue the phenotype of cardiac hypertrophy,indicating that androgen may play a role in the cardiac hypertrophy of FKP12.6-/-male mice.We also observed that the serum E2,T,LH and FSH were increased in FKBP12.6-/-mice compared with WT mice,indicating that hypothalamic-pituitary-gonadal axis（HPGs）was up-regulated in the knockout mice.3.We found that the expressions of FKBP12.6 were much higher in mouse pituitary gonadotroph LβT2 cells than in the GT1-7 cell lines which produced the Gn RH.In addition,although FKBP12.6 was also expressed in GT1-7 it didn’t affect the expression of Gn RH1,indicating that the fluctuation of sex hormone in FKBP12.6-/-mice is greatly on count of pituitary.4.We found that there were the elevations of [Ca²⁺]i and the m RNA expressions of hormone-related genes（LHβ,SF-1,Nur77）in FKBP12.6 knockdown LβT2 cell lines（F12.6 SI2）campared with negative control LβT2 cell lines（NC）.Moreover,nucleus translocation of NFAT3,Nur77 and Pin1 were significantly increased in F12.6 SI2 campared with NC.5.Protein expressions of pro-Gn RH1 and Otx2 were increased in CD38 knockdown GT1-7 cell lines which could be inhibit by EX527,indicating that CD38 regulates the expressions of Gn RH1 through promoting the activity of Sirt1.6.We successfully induced the over-expression of Gn RH1 in Gn RH1 TG mice by DOX feeding and we also observed that the concentrations of serum Gn RH1 and FSH and the phosphorylation of m TOR were elevated only in the male mice.The Echo results showed that the thickness of IVS and LV mass were increased only in Gn RH1 TG male mice on the days of 30 and 60 after feeding with DOX,indicated that gender-related cardiac hypertrophy was only induced in male Gn RH1 transgenic mice.Conclusion Disruption of FKBP12.6 or CD38 gene could produce cardiovascular phenotypes by activating the hypothalamic-pituitary-gonadal axis（HPGs）through regulating the Ca N/NFAT3 signaling pathway in hypothalamus or Sirt1 signaling pathway in pituitary in FKBP12.6 and CD38 knockout mice,respectively.More importantly,the overexpression of hypothalamic specific Gn RH1 resulted in the gender-related cardiac hypertrophy in the hypothalamus-specific Gn RH transgenic mice.Therefore,based our findings,we concluded that the functional disorder of the hypothalamic-pituitarygonadal axis（HPGs）is a major reason for the production of the gender-related cardiovascular phenotypes.