Treatment Of Angiotensin Ii-induced Hypertensive Cardiac Hypertrophy By Inhibition Of The Ubiquitin-activating Enzyme

Background and Objective:Cardiac hypertrophy mainly occurs in hypertensive cardiomyopathy, pulmonary hypertension, and chronic congestive heart failure. It increase the rate of arrhythmia, heart failure, and sudden death.Meanwhile,ubiquitin proteasome system(UPS) are highly involved in regulation of cardiac hypertrophy. Approximately 80%-90% of proteins were degraded through UPS. Inhibitors of the proteasome block cardiac hypertrophy.Ubiquitin-activating enzyme1(UBA1,E1), as the start site of the UPS pathway, initiates the degradation of proteins.Therefore,we hypothesized that E1 inhibition might possess the same or even stronger anti-hypertrophy ability. To test our hypothesis, we analyzed the effects of E1 inhibitor PYR-41 in the angiotensinII-induced cardiac hypertrophy in vivo and in vitro experiments.E1 inhibitor may provide a profound understanding and a new treatment strategy for cardiac hypertrophy and remodeling.Methods:We dyed clinical biopsies by immunohistochemical staining to test the expression of E1 in human heart tissue. In vivo experiments, we subjected age 6-8 weeks C57Bl/6 male mice to subcutaneous implantation of micro pump with angiotensinII infusion(1000ng/kg per minute) for 3-14 days to induce cardiac hypertrophy. Animals were treated with E1 inhibitorPYR-41 at 5mg/kg and 10 mg/kg in saline by intraperitoneal injection or saline control alone.In vitro experiments, neonatal rat cardiomyocytes(NRCs) were set serum-free for 24 hours and cultivated in DMEM medium in the presence of angiotensionII or phenylephrine for 24 hours, either with solvent control or with E1 inhibitor.The expression changes of E1 and one hypertrophy related signaling pathway were test by western blot analysis. Cardiac structures and functions were evaluated by M-mode echocardiogram. Cardiomyocyte size were calculated after wheat germ agglutinin(WGA)staining or immunofluorescent staining.Results data are presented as mean±SEM. Statistical differences between groups were calculated significance by 1-way ANOVA or Kruskal Wallis where appropriate. Results p<0.05 was regarded as significant.Results:1.The expression of ubiquitin-activating enzyme 1(E1)were widely found in both human and animal heart tissue or cardiomyocytes.2. Compared with control group, E1 were upregulated in the hypertensive cardiac hypertrophy after 3,7and 14 days angiotensinII infusion pump implantation(p<0.05).In vitro experiments,E1 expression in different concentration of angiotensinII groups(50nM and100nM) increased significantly compared with solvent control(p<0.05).3.After two weeks of angiotensinII infusion,The thickness of the heart wall and cross-section area of cardiomyocytes markedly increased and the hert functions were jeopardized(p<0.05 vs control). The cardiac structures and functions of E1 inhibitor-treated groups did not deteriorate of which group high blood pressure established(p>0.05 vs control).In vitro studies,The nearly 2-fold angiotensinII-induced or phenylephrine-induced increase in cardiomyocyte size was markedly suppressed by treatment with 5uM and 10 uM dose of E1 inhitor.4. After one week of angiotensinII infusion, the systolic blood pressure of mice increased significantly in the presence or absence of E1 inhibitor treatment(p<0.05 vs control). The saline-only group and inhibitor-only group blood pressure did not differ(p>0.05).5.Western blot of extracellular signal-regulated kinase(ERK1/2) was analysed, After 2weeks of angiotensinII infusion, ERK1/2 was activated notably(increase in phosphorylated/total ERK)(p<0.05 vs control).This increase of phosphorylated ERK1/2(p-ERK1/2)was suppressed by E1 inhibitor treatment.Conclusions:1.AngiotensinII-induced cardiac hypertrophy significantly elevated the expression of E1 in the hear.There is an obvious positively related concentration-response relationship between AngiotensinII and E1.2.Inhibited E1 by specific inhibitor PYR-41 suppressed chamber and myocyte hypertrophy. It also improved heart functions in mice exposed to blood pressure overload induced by angiotensinII.3.Blood pressure elevation could not be suppressed by intraperitoneal injection of E1 inhibitor. The mechanism of anti-cardiac hypertrophy by E1 inhibitor was not related to hypotensive effect.4.E1 inhibition blocked the process of neonatal rat cardiomyocytes hypertrophy in the presence of angiotensionII or phenylephrine in vitro.5.E1 inhibition deactivates a hypertrophy signaling pathway triggered by pressure load(ERK signaling pathway).