| Research background:Urotensin ??U??is a potent vasoactive “somatostatin-like” cyclic peptide which originated from the spinal cord of the Goby fish,and has recently been discovered in Mammals.U? and its specific receptor which is a independent G-protein coupled receptor 14?GPR14,UT?express in many organs,and mainly present in heart and arterial vessels,which suggests that U? participates in the process of regulating the homeostasis of cardiovascular system.The finding that the combination of U? and UT resulted in many biology effects has been showed in many studies and indicates that U? may involve in development and progression of multiple cardiovascular diseases.In previous studies,serum U? levels increased in heart failure patients,higher expression of U? presented in myocardial tissue of end-stage heart failure patients,especially.The increasing expression of U? and its receptor were time-dependent in myocardial infarction rat model induced by coronary artery ligation,which suggested that U? might play a crucial role in ventricular remodeling and then resulting in heart failure.Some studies showed that U? participated in ventricular remodeling,and the mechanism might involve PKC,MAPK and Ca N pathway but have not been completely clarified.c AMP-PKA pathway is one of the primary signal transduction pathway,which involve in cardiac hypertrophy.In previous study,we found that the effect of U? on inhibiting rat heart function was time-dependent and the effect was greater in the isolated Langendorff-perfused rat heart model.KT5720,a specific inhibitor of PKA,could prevent the effect.We conclude that c AMP-PKA signaling pathway may be involved in the inhibitory effect of U? on cardiac function.Chronic pressure overload isone of important reasons inducing heart failure.Whether U? participates in the emergence and development of myocardial fibrosis or not in myocardial tissues of chronic pressure overload rat and underlying mechanism remain unclear.Therefore,we deduced that c AMP-PKA pathway might participate in the development and progression of myocardial fibrosis during the heart failure induced by chronic pressure overload.The contractibility of myocardial cell is mainly attribute to increasing intracellular free calcium,and Ca2+ mediate many effects on heart which were generated by U? binding with it receptor.However,the signal transduction mechanism of myocardial cell Ca2+ influx need to be unraveled further.Our previous findings indicated that U ? could inhibit the heart function of abdominal aortic constriction heart failure in rats,so we conjectured that inhibitory effect of U? on left ventricle in rat was related with Ca2+.In conclusion,this study is aimed to detect the effect of U?/UT system on promoting myocardial fibrosis and underlying target in chronic pressure overload rat model,and investigate the relationship between the molecular mechanism and c AMP-PKA signal transduction pathway by incubating cardiac fibroblasts of newborn rat and whole-cell patch clamp technique,which can provide important basis for exploring new drug to interfere the effects of U? on promoting myocardial fibrosis and new mechanism about emergence of myocardial fibrosis.Part 1 The expression changes of U?/UT system and protein kinase A in cardiac tissues of chronic pressure overload ratsObjective: To observe the expression changes of U?/UT system,type ?,? Collagen and protein kinase A.Methods:The pressure overload animal model was established in rats by abdominal aortacoarctation,15 rats were randomly selected as sham-operated and 30 rats were made to be abdominal aorta coarctation model.Dynamic detection of various functions was performed after modeling for 4,8,12 weeks:1)Evaluating heart function by echocardiography.2)Measuring left ventricular pressure by right carotid artery intubation;3)Observing the pathologic changes of cardiac tissues by HE and Masson stain;4)Detecting the expression changesof U?,GPR14,col-? and col-?in cardiac tissues by immunohistochemistry;5)Measuring the level of c AMP by ELISA;6)Observing the expression changes of U?,GPR14,col-?,col-?,and PKA by Western blots.Results: Left ventricular end-systolic pressure?LVSP?presented obvious upward trend with the passage of modeling time.After modeling for 12 weeks,LVSP was higher than 180 mm Hg.Echocardiography showed that ventricular wall of model rat was raised,and interventricular septal thickness at end diastole?IVSTd?,left ventricular posterior wall thickness at end diastole?LVPWTd?and left ventricular end diastolic diameter?LVDd?were markedly increasing,and ventricular chamber was enlarged with the passage of modeling time,which was obvious after modeling for 12 weeks.HE and Masson stain displayed that the expression of collagen fiber in cardiac tissues of model rat gradually increased with the passage of modeling time,which was prominent after modeling for 8 and 12 weeks.The result of immunohistochemistry showed that the expression of U?,col-?,col-? increased markedly in myocardial interstitium of model rat,and the expression of GPR14 was also raised,which were time-dependent.In model rat plasma,the concentration of c AMP increased time dependently?P<0.01?.Compared with the control,the expression of U?,GPR14,col-?,col-?,and PKA increased significantly?P<0.01?in a time-dependent manner.Conclusion:There was prominent myocardial fibrosis in chronic pressure overload rat modelconstructed by abdominal aorta coarctation.With the passage of heart failure time,the degree of myocardial fibrosis gradually enhanced.U?/UT system may played an important role in the process of myocardial fibrosis in pressure overload rat,in which may involve c AMP-PKA pathway.Part 2 Study on signal transduction pathway of Urotensin ? improving collagen synthesis in cardiac fibroblastsObjective: To explore the effect of c AMP-PKA signal pathway on Urotensin ? improving collagen synthesis in cardiac fibroblasts.Methods: After subcultured 3-5 generations,cardiac fibroblasts of neonatal rat were randomly divided four groups as follows: In control group cells were without any stimulation;?n U? group cells were stimulated with U?(10-8mol/L);In U? and KT-5720 group cells were treated with U?(10-8mol/L)and KT-5720?1?mol/L?;In U? and SB-611812 group cells were treated with U?(10-8mol/L)and SB-611812?1?mol/L?.The amount and expression of col-?and col-? in cell supernatant were detected by ELISA and western blots,respectively.Results:In comparison to control group,the amount and expression of col-? and col-? prominently increased?P<0.01?after stimulating with U?(10-8mol/L).However,the amount and expression of col-? and col-? marked decreased?P<0.01?.When cells were treatedwith U? receptor antagonist?SB-611812?and specific blocking agent of PKA?KT-5720?,comparingwith U? group.Conclusion: c AMP-PKA signal pathway played an important role in the process of Urotensin ? inducing collagen synthesis in cardiac fibroblasts.Part 3 The electrophysiology mechanism about the effect of Urotensin ? on rat heartObjective: To explore the effect of c AMP-PKA signal pathway on Urotensin ? improving collagen synthesis in cardiac fibroblasts.Methods: Rat ventricular myocytes were acutely isolated by collagenase method.After treated with different dosages of U? and KT5720,the change of L-type calcium current density in rat ventricular myocytes were detected by whole cell patch clamp technique.Results:When the single myocardial cell was perfused with different concentrations of U?(10-910-5mol/L),the peak value of Ica-Ldependent decreased as 6.70±1.78,5.93±2.02,5.40±2.15,4.54±2.00,3.80±1.82,which has statistical difference?P<0.05?in comparison to control group;2)After perfused with U?(EC50=10-8mol/L),cells were then treated with KT5720,the changes of the peak value of Ica-L were reversed by KT5720?P>0.05?;3)Give U??EC50?on the basis of perfusion KT5720,the peak value of Ica-L were inhibited by KT5720 based on 10-8mol/L U?.U? did not further inhibit Ica-L?P>0.05?,suggesting that KT5720 could block the inhibitory effect of U? on Ica-L by inhibiting PKA signal pathway.Conclusion: U? attenuated time dependently L-type calcium current density in ventricular myocytes,but KT5720 could block the inhibition,which indicated that U? might inhibit L-type calcium current density in ventricular myocytes via PKA pathway and then perform the inhibition of cardiac function. |
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