Stress Hormone, Corticosterone, Caused Cardiac Dysplasia In Chicken Embryo Through Inhibiting Mitofusin 2-mediated Mitochondrial Function

Epidemiological studies demonstrated that an adverse fetal environment is closely linked to increased risk of hypertension, cardiac hypertrophy, insulin resistance, type 2 diabetes and mental psychological disorders and so forth. In prenatal maternal stress, fetus exposed to plenty of maternal corticosteriodes and increased vasoconstriction, which in turn would decrease blood flow to the fetus via uterine arteries and caused malnutrition. Increasing evidences from animal experiments and clinical studies have shown that fetal overexposure to glucocorticoids in pregnancy is associated with growth retardation and development of cardiovascular and endocrine metabolic disease in adult. There is short-term and long term side effect in heart and thus become one of the focuses in the present studies. Given that corticosteriodes have various of biological functions, the mechanism of corticosteriodes induced cardiac defects is still unknow. Therefore, in the present study, we will establish related pathological models in vivo and in vitro, aiming to evaluate heart morphological characteristic and studying the underlying mechanism.Firstly, we chose chicken embryos to establish in vivo model with corticosterone(CORT) load. In order to mimic a foetal overexposure to corticosteriods environment, a series concentration of avian stress hormone–corticosterone(CORT) were administrated via air sac of EDD3 chicken embryo on every other day, until EDD14. Recording body weight(BW) and heart weight(HW) and counting death rate and the HW: BW ratio. The heart external characteristics were observed and photographed via stereomicroscope. The internal characteristics were observed via paraffin embedding and H&E staining. Results showed that though CORT load caused increased mortality, growth retardation, a marked increase in HW: BW ratio and in a dose-dependent manner. The CORT concentration at 2.5 nmol and 5 nmol were optimized for the following experiments eventually. The embryonic plasma CORT levels were detected by HPLC-UV and the results showed that CORT levels was increased significantly. Furthermore, morphological results showed that there is an increased heart size with ventricular wall thinning, reduced cardiomyocyte density and increased spacing among cells following CORT overexposure. Otherwise, the CORT hearts also exhibited increased cell area with Wheat germ agglutinin(WGA) staining. The Echocardiography analysis demonstrated that CORT load caused embryos cardiac dysfunction, such as lower ejection fraction and fraction shorting capacity, less Posterior wall thickness in diastole and systole. In addition, we also found that CORT load could inhibit cell proliferation, while cardiac structural gene and protein expression levels are significantly up-regulated the methods of RT-PCR or immunofluorescence techniques. It concluded that CORT could inhibit myocyte hyperplasia while promote apoptosis, and stimulate the hypertrophic myocardial growth pattern, which caused cardiac dysplasia and dysfunction.Concerning that the characteristic of metabolic actively, highly energy demands and riched mitochondrias in cardiac tissue, the occurring of various cardiac disease is related with mitochondrial dysfunction. Related studies have demonstrated that mitochondria dysfunction has been regarded as one of the main pathogenesis of cardiac hypertrophy and shift into heart failure as follows. Hence, we collected cardiac tissue of EDD14 old chicken embryos and assayed the mitochondrial functions via separating myocardial mitochondria with tissue mitochondria isolated kit, including mitochondrial membrane potential evaluation by flow cytometry, real-time monitored mitochondrial ROS generation rate via a fluorescence microplate reader and HPLC-UV methods for determination of hypertrophic myocardial ATP content. Moreover, we determined oxidative and anti-oxidative index and mitochondrial enzyme activity by using commercial assay kit. Our results showed that CORT load could significantly reduce mitochondrial functions with reduction of mitochondrial membrane potential, increment of ROS generation rate and energy product ATP content decreased. In addition, increased MDA levels and decreased anti-oxidative index Mn-SOD activity and GSH/GSSG relative content are also occurring in CORT cardiac tissue. Besides that, the activity of mitochondrial respiratory complexs were significantly inhibited, include complex III, IV, V. It was suggested that CORT overexposure could induced cardiac mitochondrial dysfunction in chicken embryos.Mitofusin2(Mfn2) is one of mitochondrial fusion protein family which mediated mitochondrial fusion and played an important role for keeping mitochondrial dynamic balance and structural homeostasis. In depression models, GC was found to influence mitochondrial dynamics and caused mitochondrial network damage and dysfunction, in which Mfn2 regulation was involved. Therefore, we further speculated that CORT caused embryonic heart developmental disorder and mitochondrial dysfunction was involved with Mfn2 expression anomalies. Our Western Blotting results shown that CORT heart Mfn2 protein levels were significantly up-regulated, compared with vehicle heart. It also found that CORT load facilitate cell apoptosis and increase apoptotic protein expression, while lower proliferative protein levels. Meanwhile, it appears clustered and small round mitochondrias following CORT exposure by electron microscopy.Moreover, aimed to elucidate the influence of CORT on cardiomyocyte and associated underlying mechanism, we established a glucocorticoid overexposure in vitro model through CORT(200 μM, 48 h) load to H9C2 cell line. The experiment was divided into three groups, Control, CORT and CORT+Ru486 respectively. First of all, the influence of CORT on H9C2 cell morphology, Mfn2 protein levels and mitochondrial transmembrane potential were determined. Additionally, we evaluated mitochondrial apoptotic pathway protein expressions by western blotting. Finally, it was aimed to further verify that Mfn2 protein was involved in CORT induced cardiac hypertrophy. We determined the effects of Mfn2 overexpression on caridomyocyte structure and apoptosis, by transfecting Mfn2 plasmid. The results were consistent with chicken embryo experiment, CORT load could lead to H9C2 cell volume enlargement, up-regulate Mfn2 expression in cell level and reduced cell mitochondrial transmembrane potential. Under CORT conditions, it increased apoptotic pathway protein Bax: Bcl-2 ratio and promoted cell apoptosis. However, those alterations could be ameliorated with GR antagonist Ru486 treatment in advance. It suggested that CORT might be mediated cardiomyocyte structural and apoptotic alterations through GR signalings. Simultaneously, it happening that Bcl-2 protein family Bax: Bcl-2 ratio and active caspase3 protein were markedly increased after Mfn2 overexpression, besides the significant increment of hypertrophic marker SKA mRNA. It implied that Mfn2 may be play an important role for mediating CORT caused mitochondrial damage, promote cell apoptosis and ultimately lead to cardiac dysplasia and hypertrophy.To sum up, CORT mediated mitochondrial dysfunction via regulating Mfn2, caused myocardial energy metabolic abnormality, ROS overproduction and cardiomyocyte apoptosis, lead to cardiac dysplasia and hypertrophy eventually. Then provide new targets and theoretical basis for preventing and controling of such heart disease.