Corticosterone-stress Induces Myocardial Hypertrophy By Ubiquitination And Degradation Of Mfn2 On Mitochondria

Epidemiological studies found that adverse uterus environment in fetal development period can increase the risk of hypertension,type ? diabetes,cognitive disorder and myocardial hypertrophy in fetus.Pregnant women under prenatal stress release a large amount of glucocorticoids,which can be deliveried into the uterus through the placental barrier and affect the development of embryos.The previous studies of our group found that glucocorticoid can induce the ventricular hypertrophy of the chicken embryo.However,how the glucocorticoid causes myocardial hypertrophy still remains unclear.Therefore,in this study,we use H9C2 cell model to evaluate the effect of stress hormone corticosterone(CORT)on H9C2 myocardial cells,and further explore its underlying mechanism using chicken embryo model.Firstly,we used H9C2 cells to establish myocardial hypertrophy model by CORT treatment for 48 h at the dose of 200 ?M,and then verify the validity of the model by detecting the expression level of hypertrophy-related genes and observing cell morphology.Experiment results showed that CORT could increase the expression of hypertrophy-related genes ANP and BNP with remarkably enlarged cross-sectional area and growing sarcomere.Meanwhile,we found the short and broken mitochondria with disorganized reticular structure through mitored tracker tagging.And cells in CORT group showed darker mitochondrial matrix,more swelling crest structure and nebulous boundary and smaller size compared with cells in the control group under the transmission electron microscope.Furthermore,we detected the activity of mitochondrial enzyme complexes,ATP content and level of MDA,both of which are related to mitochondrial function.The results showed that CORT could inhibit activity of mitochondrial enzyme complex ?,? and ?,decrease ATP content and increase MDA level.All these results suggested that CORT may lead to myocardial hypertrophy in H9C2 by destroying mitochondria structure and function.Mfn2 is the key protein in the fusion of mitochondria,which regulates the dynamic balance of mitochondria.Our previous study found that Mfn2 is overexpressed in CORT induced myocardial hypertrophy in chick embryo model,which may correlates to its underlying mechanism.Here we first examined the expression of Mfn2 in H9C2 cells by western blot assay and found that the expression of Mfn2 was significantly higher than that of the normal group,and we found that Mfn2 protein on the mitochondria decreased while the endoplasmic Mfn2 protein increased,At the same time,we also use protein synthesis inhibitors CHX inhibiting protein synthesis and protein degradation pathway opening up,and found that CORT can accelerate the degradation rate of mitochondrial Mfn2 protein,so we speculated that Mfn2 on the mitochondria might have been degraded.The main pathways of protein degradation include ubiquitin-proteasome pathway and lysosomal autophagy pathway.We adopt degradation proteasome inhibitor MG132 and autophagy inhibitor 3-MA to pre-protect and found that autophagy inhibitors was unable to recover the decrease of Mfn2 induced by CORT,so we speculated that the mitochondria Mfn2 may be degraded by ubiquitin-proteasome pathway.By detecting the corresponding marker protein,we found the mitochondria Mfn2 ubiquitin level increases,while autophagy related protein expression showed no obvious change,these results suggested that Mfn2 degradation occurred in mitochondria,and the ubiquitin of Mfn2 did not occur on the endoplasmic reticulum.The activation of the ubiquitin-proteasome system requires E3 to identify the exposed Lys sites in the substrate molecule,which may cause a change in the ubiquitin level when the conformation of the substrate molecules changes.So we further speculated that there maybe some Mfn2 specific binding proteins in mitochondria rather than endoplasmic reticulum,leading to the difference of Mfn2 ubiquitin level.Through the NCBI protein database and the CELLO subcellular localization prediction system,we found that SLC25A38 protein(Appoptosin)could interact with Mfn2 and distributes almost on the mitochondria instead of the endoplasmic reticulum.We further found that CORT could upregulate the expression of appoptosin and increase the interaction between Mfn2 and appoptosin protein by Western blotting,CO-IP and Immunofluorescence confocal confocal localization assays.In the Appoptosin-knockout cells,after CORT treatment,the expression level of mitochondrial Mfn2 was higher,and mitochondrial Mfn2 ubiquitin level was much lower than that of non-knockout group,with the increasing of the activity of mitochondria complexes and the downregulation of hypertrophy-related genes,which suggested that CORT induced the ubiquitin degration of Mfn2 by AppoptosinTo sum up,CORT can induce cardiomyocyte hypertrophy with decreased mitochondrial complex activity and damaged mitochondrial structure and function.At the same time,CORT can induce the up-regulation of Appoptosin and promote the interaction between Mfn2 and Appoptosin,leading to mitochondrial Mfn2 ubiquitination.After silencing Appoptosin,it can inhibit CORT-induced Mfn2 reduction,recover mitochondrial damage and cardiac hypertrophy.These results suggested that CORT can induce mitochondrial structure and function damage by regulating appoptosin-mediated mitochondrial Mfn2,and eventually causes cardiac hypertrophy.