Stachydrine Hydrochloride Regulating β1-AR Desensitization And Internalization To Improve Isoproterenol-induced Cardiac Insufficiency

Objective:To investigate the mechanism of stachydrine hydrochloride(Sta)inhibiting Isoproterenol(ISO)-induced cardiac insufficiency by regulating the desensitization and internalization of β1-adrenergic receptor(β1-AR).Methods: In vitro,the Langendoff constant flow perfusion system is used to isolate single adult mouse cardiomyocytes;The ion imaging system detects cardiomyocyte calcium transients;The sarcomere length measurement system detects the diastolic and contractile functions of single cardiomyocytes;Immunity Fluorescence detection of protein distribution in cardiomyocytes.In vivo,ISO micro-osmotic pump implantation is used to prepare a mouse model of cardiac insufficiency;Vevo2100 small animal echocardiography detects cardiac function and structural parameters;Pressure-volume loops conductance technology detects hemodynamic parameters;HE and WGA staining to observe the heart and the morphological changes of myocardial cells;Western blotting and Co-IP were used to detect the expression levels and connection of protein related to desensitization and internalization of β-adrenergic receptor in adult mice.Results: In vitro,ISO could over activate sympathetic nervous system,promote myocardial over contraction and increase calcium transport(p<0.05);After continuous activation of β1-AR by ISO,the distribution of β1-AR shifted from cell membrane to Ttube region and cytoplasm.In vivo,in the heart tissue of mice in the ISO group,the expression of GRK2,GRK3,β-arrestin1,AP2β,Rab5 and Rab7 was significantly increased(p<0.05),while the expression level of β1-AR,p-β-arrestin1 and Rab11 a was markedly reduced(p<0.05),besides,the expression levels of β2-AR,GRK5,GRK6,β-arrestin2,AP2α and Rab4 A did not change significantly(p>0.05),and the binding of β1-AR to GRK3 was significantly increased,while the binding of β1-AR to other proteins was not significantly changed.Sta can significantly inhibit the expression of GRK2,GRK3,β-arrestin1,AP2β,RAB5 and RAB7 in the heart of Iso-induced cardiac insufficiency mice(p<0.05),significantly increase the expression of β1-AR,p-β-arrestin1 and Rab11A(p<0.05);Compared with Sham group,the heart shape of ISO group was significantly enlarged,HW/BW and HW/TL significantly increased(p<0.05),while Sta could significantly inhibit the cardiac morphology and cardiomyocyte enlargement of ISO group,HW/BW and HW/TL significantly decreased(p<0.05);IVSS,LVID,LVIDS and LVPWS of ISO group were significantly higher than those of Sham group(p<0.05),while Sta could inhibit the increase of LVID(p=0.552),and significantly decreased IVSS,LVPWS and LVIDS(p<0.05);ISO can increase the PV-loop area and make it shift to the right,while Sta can inhibit the PV-loop area increase and shift to the right in ISO-induced cardiac dysfunction mice;ISO significantly increased cardiac hemodynamic indexes ESP,ESV,PVA,PE and Tau Mirsky(p<0.05),significantly decreased ESPVR(p<0.05),while Sta could significantly inhibit the increase of ESP,ESV,PVA,PE and Tau Mirsky(p<0.05),significantly increase ESPVR(p<0.05).Conclusions: Sta improved the cardiac structure and function of ISO-induced cardiac insufficiency model mice,mainly by down-regulating Rab7 and up-regulating Rab11,promoting the recirculation of β1-AR and maintaining the quantity of β1-AR.Sta can also inhibit the binding of GRK3 and β-arrestin1 to β1-AR,inhibit the expression of AP2β,inhibit the endogenization of β1-AR,and make it distributed on the membrane,and maintain the physiological function.