The Molecular Mechanism Of β-arrestin2in Ventricular Remodeling Mediated By Pressure Overload-induced AT1Receptor Activation

Renin-angiotensin(RAS) system is the important signal regulator for in vivo pressure and volume overload, Angiotensin Ⅱ typel receptor (AT1-R) as one of the terminal baroreceptors in RAS system has become the research hotspot in recent years, especially for the critical role of AT1-R in cardiac hypertrophy and heart failure induced by compensatory ventricular remodeling. The clinical studies showed that plenty of angiotensin receptor antagonists (ARBs) have superior effects than angiotensin-converting enzyme inhibitors (ACEIs) in controlling the blood pressure. Our previous study also showed AT1-R could be activated without the involvement of Angll. For instance, mechanical stretch induced hypertrophic response in in-vitro cultured cardiomyocytes through Angll-independent manner. Transverse aortic constriction (TAC)—one of the pressure overload models, envoked ventricular remodeling and heart failure in ATG-/-mice. The potential mechanism for AngⅡ independent AT1-R activation and intercellular signaling pathway remained unknown. However, growing evidences indicated mechanical stress trigged GPCRs through a G protein independent signal transduction. As one of G protein-coupled receptor superfamily members, we hypothesized that AT1-R might has sensitive domains for mechanical stress, and they could be activated during stretching, which resulted in conformational switch of AT1-R and being activated. These activated domains might finally change the biological characters of cardiomyocytes through G protein independent pathway. Because, we have found one kind of scaffold proteins β-arrestin2, it was recruited by cell membrane during stretching. β-arrestins dependent signaling cascade was also confirmed to play important role in GPCRs activation, and possibly involved in the process of cardiac remodeling. Therefore, we investigated the mechanism of mechanical stress induced AT1-R activation from the beginning with research in screening the sensitive domains in AT1-R stimulated by mechanical stretch, and how they finally changed the biological traits of cardiomyocytes. Thus, we could elucidate the pathogenic mechanism of pressure overload induced ventricular remodeling and heart failure, provide basic theories for clinical prevention and treatment of pressure overload-induced cardiovascular diseases and shed light on the screening of potential drug targets.PartⅠMechanical stress induced conformational change of AT1-R and the molecular mechanism for receptor activationObjective:To investigate sensitive domains for mechanical stress-induced activation of the AT1-R and downstream signaling pathways.Methods:ATl-R transmembrane (TM) domain point mutant plasmids was co-transfected with exogenous FLAG-ERK2in COS7cells, the phosphorylation level of ERK2was examined in anti-FLAG immunoprecipitates after mechanical stretch for10minutes. Meanwhile, AT1-R mediated intracellular signaling pathways were determined in cells stimulated by Angll or mechanical stretch. The effect of mechanical stretch(10%) induced ERK1/2activation was compared with that induced by AngII(10-7mol/L) after blocking the critical effector molecules for G protein-coupling:(1) Inhibition of Gaq by GTP antagonist G0635or negative regulator RGS4;(2) blockade of PKC by GF109203X.Results:Both Angll and mechanical stretch could activate ERKl/2in wild-type AT1-R transfected COS7cells; AT1mutants located close to TM1-3region (C76A, C121A) had no response to AngⅡ stimulation; ERK1/2activation was not affected by mutation at TM4-5region (C149A, K199Q), but attenuated in K199Q transfected cells by Angll; AT1mutants located close to TM6-7region (H256A, C289A) had no response to mechanical stretch; mutation at the C terminus of AT1-R (G303R) abolished both stimulus induced ERK1/2activation. In addition, mechanical stretch stimulated time-dependent PKC expression and IPx release were significantly attenuated when compared with Angll stimulation. Furthermore, Angll mediated ATl-R signaling was inhibited after blocking G protein-coupled pathway (Gaq or PKC), but ERK1/2phosphorylation was not affected in mechanical stretch model.Conclusion:The sensitive domain to mechanical loading stimulus was located at TM7region close to C terminus of AT1-R, and regulated mechanical-biochemical signal transition via C terminal residues; mechanical stress-induced AT1-R and its effector molecule ERK1/2activation was not dependent on G protein-coupling, but through G protein independent pathway in cardiomyocytes.Part ⅡMechanical stretch induced AT1-R activation through G protein independent signaling pathwayObjective:To elucidate the intracellular signaling proteins responsible for mechanical stretch induced AT1-R and its effector molecule ERK1/2activation.Methods:Immunofluorescence(IF) and immunoprecipitation(IP) analyses detected the expression and location of scaffold protein after mechanical stretch and its relationship between AT1-R in cardiomyocytes or HEK-293-AT1cells. Echocardiography and hemodynamic analyses determined the cardiac morphology and function in ATG knockout mice (not express the angiotensinogen) underwent TAC-induced pressure overload, and examined the involved role of scaffold protein.Results:In vitro experiments showed that both mRNA and protein levels of scaffold protein—β-arrestin1/2were not affected by mechanical stretch for10minutes, but stretch induced β-arrestin2membrane recruitment. In exogenous β-arrestinl and2respectively, transfected into β-arrestins knock-down293-AT1cells, mechanical stretch envoked enhanced ERK1/2phosphorylation level only in β-arrestin2transfected cells. Accompanied by the recruitment of β-arrestin2, Src kinase level was significantly increased. IP analysis showed that stretching promoted the binding of Src with β-arrestin2, simultaneously IF analysis revealed that stretch-induced membrane recruiting of β-arrestin2/Src was abrogated after mutation at the catalytic domain (V54D) of β-arrestin2. The similar effect was observed in stretched cells pre-treating with SU6656(Src kinase inhibitor). In vivo, mechanical stress (TAC) significantly enhanced the expression of cardiac AT1-R and β-arrestin2in tissue membrane proteins. Echocardiography and hemodynamic analyses showed that Left ventricular anterior wall at end-diastolic (LVAWd) and posterior wall (LVPWd) were markedly thickened; accompanied by increases of left ventricular ejection fraction (LVEF), aortic blood pressure (ABP), left ventricular end-systolic pressure (LVESP) and dp/dtmax. All these indexes were reversed by pretreatment with SU6656. In addition, the cardiac mRNA levels of ANP, BNP and SAA were significantly downregulated, but only SERCA2a upregulated after inhibition of Src kinase with SU6656, indicating a critical role of β-arrestin2/Src signal complex in regulating pressure overload-induced myocardial hypertrophy.Conclusion:Membrane recruitment of β-arrestin2was essential for Src kinase activation, and promoted ERK1/2phosphorylation, which might be the key intercellular mechanism for mechanical stretch induced AT1-R activation. Meanwhile, β-arrestin2mediated G protein independent signaling pathway also induced ventricular remodeling, which was improved by inhibiting β-arrestin2/Src signal complex.Part ⅢThe binding sites of AT1-R with β-arrestin2induced by mechanical stretchObjective:To verify the importance of TM7domain of AT1-R for β-arrestin2membrane recruitment and signal transduction.Methods:FLAG-β-arrestin2was co-transfected with AT1mutants (Cys289Ala or Gly303Arg) into COS7cells, and then the binding effect of β-arrestin2with AT1was measured by IP analysis. Second, the effect of stretch sensitive ARB (Candesartan) on inhibition of ATl-R mediated ERK1/2signaling was compared with the effect by AngII sensitive ARB (Telmisartan). Third, to explore the survival signal exerted by the regions close to TM7-C terminal of ATl-R, we examined the apoptosis indexes: Caspase3activity and Bcl-2/Bax ratio induced by mechanical stretch in Cys289Ala or Gly303Arg mutants transfected COS7cells was by Westernblot analyses.Results:The membrane recruitment of β-arrestin2was significantly reduced in collected membrane proteins using anti-AT-1R antibody in COS7cells transfected with Cys289Ala or Gly303Arg mutants. The inhibitory effect of Candesartan on conformational change of TM7helix led to blocking mechanical stretch-induced ERK1/2activation. Moreover, cell apoptosis was significantly increased in COS7cells transfected with Cys289Ala or Gly303Arg mutants.Conclusion:Conformational change of TM7helix is critically involved in mechanical-biochemical signal transition, which is important for recruiting of β-arrestin2and downstream ERK1/2activation. Mutation or inhibition of the switch of TM7helix caused activation of apoptotic signal.Part IVThe role of AT1-R-mediated β-arrestin2signal in myocardial fibrosisObjective:To investigate the effect of mechanical stress activated β-arrestin2signaling pathway on pressure overload-induced cardiac fibrosis.Methods:First, Westernblot analysis of TGF-β1signal associated proteins Cofilin and LIMK expressions in FLAG-β-arrestin2transfected HEK-293-AD cells and HEK-293-AT1cells underwent mechanical stretch. Second, determining the effect of Src on β-arrestin2mediated regulation of Cofilin/LIMK activity after transfecting domaint-negative β-arrestin2-V54D (mutation at catalytic domain) or pre-treating with SU6656. Third, we examined the possible role of Cofilin activated by β-arrestin2in regulating TGF-β1signaling molecules (TGF-βRI, ROCK and Smad3) after knocking-down Cofilin with siRNA. In vivo,β-arrestin2shRNA was transfected into ATG-/-mice using lentiviral plasmids. Then, myocardial fibrosis underwent pressure overload was measured by MassonⅢ staining; the mRNA levels of type Ⅰ and type Ⅲ collagens were detected by RT-PCR; ROCK and Cofilin expressions were examined by Westernblot.Results:β-arrestin2regulated the Cofilin/LIMK activity through AT1-R activation dependent manner, but Src activation independent; Cofilin inhibition resulted in blocking the regulation on TGF-β1signaling by β-arrestin2; TGF-β1signaling was significantly attenuated in β-arrestin2knock-down ATG-/-mice underwent pressure overload; simutanously reduced the collagen expression and prevented cardiac fibrosis. Conclusion:Mechanical stress mediated AT1-R/β-arrestin2signaling pathway plays a critical role in cardiac hypertrophy, also in myocardial fibrosis by regulating Cofilin/LIMK activity. Inhibition of β-arrestin2signaling might protect heart against pressure overload-induced ventricular remodeling.Conclusions:1. Mechanical stretch induced AT1-R activation and downstream ERK1/2phosphorylation through G protein independent signaling pathway, which is different from Angll induced AT1-R activation by G protein-coupled signaling mechanism.2. TM7helix of AT1-R is the stretch sensitive location, and conformational switch of TM7contributes to mechanical-biochemical signal transition.3. AT1-R mediated β-arrestin2/Src signaling cascade promotes pressure overload-induced cardiac hypertrophy.4. β-arrestin2regulates the Cofilin/LIMK activity through AT1-R activation dependent manner, and promotes the process of pressure overload-induced myocardial fibrosis.