The Role Of Immunoproteasome β5i Subunit In The Regulation Of Cardiac Remodeling And The Underlying Mechanism In Deoxycorticosterone Acetate(DOCA)-Salt Mice

BackgroundCardiac remodeling is characterized by a increase of cardiomyocyte volume and collagen deposition after various pathological stimuli such as growth factors and mechanical stretch.Deoxycorticosterone acetate(DOCA)is a synthetic mineralocorticoid derivative that has a potent mineralocorticoid effect.Administration of DOCA and high-sodium induces an angiotensin-independent hypertension and cardiac remodeling with low-renin level in different animal models.The ubiquitinproteasome system(UPS)is the main pathway for promoting intracellular proteins for degradation in eukaryotic cells.The 26 S proteasome contains three standard catalytic subunits,including β1,β2,and β5,which are responsible for caspase-like,trypsin-like and chymotrypsin-like activities,respectively.After inflammatory stimuli such as IFN-γ,three of the standard catalytic subunits are replaced with three inducible subunits,including β1i,β2i,and β5i to form the immunoproteasome.Increasing evidence has demonstrated that the UPS plays critial roles in cardiac hypertrophy,apoptosis and sarcomere protein quality control.Recent studies indicate that immunoproteasome β2i subunit has been implicated in regulating hypertension and cardiac remodeling in DOCA-salt-treated mice.However,the role of another immunosubunit β5i in the development of DOCA-salt-induced cardiac remodeling in mice remains unclear.Aims To explore the functional role of immunosubunit β5i in DOCA-salt-induced cardiac remodeling in mice and the underlying molecular mechanism.To examine whether β5i may represnt a potential therapeutic target for the treatment of cardiac remodeling.Methods: 1.Animal model Male C57BL/6J wild-type(WT,8-10 weeks of age)mice,β5i knockout(β5i KO)mice,WT mice were treated with a β5i-specific inhibitor(PR-957)were subjected to uninephrectomy and DOCA-salt treatment for three weeks.2.Blood pressure and echocardiography The systolic blood pressure of all mice was detected by the tail-cuff system.Echocardiography was performed with a high-resolution micro imaging system(Vevo1100;VisualSonics Inc.)to evaluate cardiac structure and function.3.Examination of immune cells and tissue pathology The inflammatory cells in the heart were analyzed by Hematoxylin and eosin(H&E)staining.Masson’s trichrome staining was performed to analyze tissue fibrosis.Immunohistochemistry was performed to analyze macrophage recruitment and myofibroblast differentiation.4.Analysis of gene and protein expression: The expression levels of genes and proteins associated with hypertrophy,fibrosis and inflammation were examined by quantitative real-time PCR or Western blot analysis,respectively.5.Statistical analysis All data are expressed as mean ± SEM.If data were normally distributed,the student t test was used to determine the significant difference between two groups.If the data were not normally distributed,the Mann-Whitney test was used.More than three groups of data were analyzed by ANOVA.Results: 1.DOCA-salt treatment for 3 weeks significantly upregulated β5i expression and the chymotrypsin-like activity in the heart compared with sham control.2.Compared with sham control mice,DOCA-salt treatment markedly induced elevation of systolic blood pressure,cardiac contractile dysfunction(increased EF% and FS%),cardiac hypertrophy [increased heart size,left ventricular wall thickness,heart weight/body weight(HW/BW)and heart weight to tibial length(HW/TL)ratios,cross-sectional area of myocytes and expression hypertrophic markers(ANF,BNP,and β-MHC)],fibrosis(enhanced collagen deposition area and expression of collagen I/III),inflammation [increased infiltration of inflammatory cells including Mac-2+ macrophages and expression of proinflammatory cytokines(IL-1β,IL-6,TNF-α and MCP-1)],and oxidative stress [increased DHE densitity and expression of NADPH oxidases(NOX1 and NOX2)].Conversely,these effects were remarkably reduced in β5i-KO mice.3.The benefical effect of β5i-KO on DOCA-salt-induced hypertension,cardiac dysfucntion,hypertrophy,fibrosis and inflammation were also confirmed in β5i specific inhibitor(PR-957)-treated mice compared with vehicle-treated controls.4.DOCA-salt treatment induced downregulation of PTEN and activation of the downstream targets(AKT/mTOR、TGF-β1/Smad2/3 and NF-κB-p65).Conversely,these effects were markedly reversed in β5i-KO mice.5.β5i KO-mediated beneficial effects on cardiac dysfunction,hypertrophy,fibrosis,inflammation and oxidative stress were also attenuated in PTEN inhibitor(VO-OHpic)treated β5-KO mice.Accordingly,β5i KO-mediated inhibition of AKT/mTOR、TGF-β1/Smad2/3 and NF-κB-p65 was also reversed in VO-OHpic-treated β5-KO mice.These results suggest that PTEN is involved in the protective effect of β5iKO on cardiac remodeling.Conclusions: The present study indicates an important role of β5i in DOCA-salt-induced cardiac remodeling.Mechanistically,DOCA-salt tratment induces upregulation of β5i,which promotes PTEN degradation that results in activation of the downstream mediators(AKT/mTOR,TGF-β1/Smad2/3 and NF-kB)thereby leading to cardiac remodeling.Thus,inhibition of β5i may be a promising therapeutic target for treating hypertensive cardiac remodeling.