The Mechanism Of PKA Signaling Pathway In Cardiac Hypertrophy Induced By Urotensin â…¡

Research background:With an aging population and the acceleration of urbanization process, the cardiovascular diseases(Cardiovascular diseases, CVD) prevalence and mortality is still in the rising stage. At present, the CVD deaths accounted for the total urban and rural residents the first cause of death, in the rural areas is 44.8%, the city was 41.9%,has been a serious threat to human health. CVD patients will increase by 2.13 million by the end of year of 2030. The death of CVD patients will increase 7.70 million. Therefore, CVD has gradually become a serious health problem toward all of us. It is really urgent for us to understand the pathogenesis of CVD and take measures to target corresponding pathways to prevent or cure it. Among all of complex etiologies, heart failure is usually the final outcome of many CVD patients. With the gradual research of this disease, more and more evidence has revealed that myocardial remodeling was the foundation of the occurrence of heart failure. The main causes of heart failure are malignant hypertrophy of myocardial cells, the extracellular matrix accumulation and fibrosis. Therefore it has drawn more and more attention for us to focus on the research of myocardial remodeling and the downstream mechanisms.Urotensin II(UII) is a somatostatin cyclic peptides which was first separated from the teleost spinal cord caudal neural secretion system. The human UII was also cloned in 1998.UII and its receptor are widely expressed in human tissues, especially in the cardiovascular system. At present, UII has been found to be the strongest vasoconstrictor. But the role of vasoconstrictor would be different in different parts of body in different species. Some studies have shown that the myocardial function can be adjusted by UII. In vitro experiment, UII showed a positive inotropic effect in human atrial and ventricular. UII showed a positive inotropic effect in a dose-dependent manner toward right atrial trabecular contraction. Douglas et al showed that compared with patients in early-stage of congestive heart failure, h UII and the m RNA of its receptor increased significantly in myocardial cells in end-stage heart failure patients. Lapp et al. found that h UII increased significantly in human plasma and positively related to the damage of heart function. Our previous study also found that UII exerts dose-dependent inhibition of cardiac function in rats. The understanding of mechanisms under the cardiac hypertrophy will provide new ideas for the prevention and the treatment of the disease.Another study suggests that a variety of signaling pathways are involved in the process of hypertrophy in myocardial cells, such as G protein, small G protein, mitogen activated protein kinase(MAPK), protein kinase C, TGF-β. But the potential mechanism of cardiac hypertrophy induced by UII has not been fully elucidated yet. Our laboratory preliminary experimental results showed that PKA signaling pathway played an important role in heart failure rats induced by UII. In vivo rat heart, specific blocker of PKA inhibitor KT-5720 could block the effect of heart failure induced by UII. UII may inhibit the cardiac function through PKA signaling pathway in rats. Chronic pressure overload is one of the important causes of cardiac hypertrophy or heart failure. It remains unclear that whether PKA signaling pathway is involved and plays a key role in the process of myocardial hypertrophy caused by UII in rats.The contraction and relaxation of myocardial cells are regulated by the concentration of Ca2+ which was regulated by sarcoplasmic reticulum. The balance of Ca2+ enables its concentration in a certain range and involves in multiple biological effects of heart. It needs to be investigated that whether UII and its receptor involved in the myocardial hypertrophy mediated by the Ca2+ in cytoplasm. Our laboratory has proved that UII can inhibit the heart function induced by chronic pressure overload, thus we speculated in this paper that PKA signaling pathway can be activated by UII and make effects on myocardial cells through the regulation of intracellular Ca2+ concentration.this experiment is planed to divide into three parts. First, we need to observe the heart function and myocardial cell morphology, detect the expression of UII and other related proteins caused by chronic pressure overload model in rats. Next, neonatal rat myocardial cells were cultured in different concentrations of UII and in different treatment time. The expression changes of intracellular UII, Ca2+ and related proteins is planned to be detected.Thirdly, the specific inhibitor of PKA, KT-5720, was used to observe whether blocks the UII induced myocardial cell hypertrophy in vitro cultured neonatal rat myocardial cells.Part 1 The expression changes of UII in cardiac tissues of chronic pressure overload ratsObjective: To observe the heart function of chronic pressure overload in rats, and the expression changes of collagen fibers, UII, PKA and related protein.Methods: The chronic pressure overload models in rats were established. 15 rats were randomly selected for normal control group, another 15 rats as sham operated group, the remaining 45 rats as experiment model group. After modeling, all observation index will be detected at the time of 2w, 4w, 6w, 8w.(1) Evaluating heart function by echocardiography.(2)Measuring left ventricular pressure by right carotid artery intubation.(3)Observe the pathologic changes of cardiac tissue by HE stain.(4)Observe changes in the expression of UII, Ry R2, PKA, p-PKA, PLN,ser16p-PLN, SERCA2 a in myocardial tissue by immunohistochemical method.Results :(1)Echocardiography plots showed that IVST(interventricular septal thickness),LVPWTd(left ventricular posterior wall thickness), LVDD(left ventricular diastolic diameter) were significantly thicker after operation, especially in the time of 12 w. The ventricular cavity expands largely.(2) Hemodynamic results show that left ventricular diastolic end diastolic pressure(LVEDP) increased, left ventricular systolic pressure(LVSP), left ventricular pressure rise / decrease the maximum rate(+dp/dtmax,-dp/dtmax)decreased gradually, which was obvious after modeling for 12 weeks.(3) HE stain displayed that the expression of collagen fiber in cardiac tissue of model rat gradually increased, the myocardial cell becomes hypertrophic, cells arrange disorder with the passage of modeling time, which was prominent after modeling for 8 and 12 weeks.(4) The results of immunohistochemistry showed that the expression of UII, Ry R2, p-PKA,PKA,p-PLN, PLN and SERCA2 a protein increased significantly and showed time dependent.Conclusion:(1)The establish of chronic pressure overload rat model constructed by abdominal aorta coarctation caused the increasing ventricular cavity, myocardium hypertrophy, myocardial fibrosis in a time-dependent manner.(2) the expression of UII,Ry R2, p-PKA,PKA, p-PLN, PLN and SERCA2 a protein increased significantly and showed time dependen.(3) Ca2+ regulatory proteins in downstream of PKA signaling pathway may play an important role in myocardial hypertrophy caused by UII in rats.Part 2 The effect in the myocardial hypertrophy induced by UIIObjective: To further explore the effect and mechanism of different concentrations and reaction time of UII in vitro cultured myocardial cells of neonatal rat.Methods: Isolate and culture the neonatal rat myocardial cells. The primary myocardial cells were randomly divided into two groups:(1) the normal control group;(2) UII group(the concentration of UII are 10-10、10-9、10-8、10-7、10-6mol/L), each concentration of UII was treated in 12 h, 24 h and 48 h. Myocardial cell proliferation was detected by MTT method. Cell planimetric area was measured by analyzing the image which obtained from inverted phase contrast microscope. BCA method was used to detect total intracellular protein in myocardial cells and Hoechst 33258 staining to detect total intracellular DNA.Then the protein/DNA ratio was calculated.Fura-3AM fluorescence staining was used to detect myocardial cell cytoplasm Ca2+ concentration. Intracellular ANP, p-PKA, PKA,Ry R2, p-PLN, PLN, SERCA2 a protein content was measured by Western blot assay.Results:(1) the MTT assay results showed that under the same treatment time, UII concentrations from 0 to 10-6 mol/L, live number of cardiac cells were in a significantly increased trend. With the increasing of treatment time, the number of live cells increased significantly in time-dependent manner.(2) The planimetric area and total amount of protein increased significantly in the vitro culture of neonatal rat myocardial cells(p<0.05).At the same time the quantity of total DNA did not change. So it is obvious that the ratio of protein/DNA showed a increased manner(p<0.05).(3)with the increase of concentration of UII(from 0 to 10-6 mol/L),myocardial cell intracellular Ca2+ concentration was significantly rising trend.(4)The expression of ANP、p-PKA、Ry R2、p-PLN、SERCA2a all showed in a increased and UII dose-dependent manner. The amount of proteins all exhibited significant difference in the treatment with concentration in 10-9mol/L(p<0.05).Conclusion:(1) UII treatment can lead to increased activity and induce the hypertrophy of myocardial cells in a dose-dependent manner.(2)UII may be related to PKA signaling pathway on mycardial hypertrophy.Part 3 The effect of PKA signaling pathway in processing UII induced myocardial hypertrophyObjective: To further explore the role of PKA signaling pathway in UII induced cardiomyocyte hypertrophy.Methods: Isolate and culture the neonatal rat myocardial cells. The primary myocardial cells were randomly divided into 4 groups:(1) the normal control group;(2) the UII10-8mol/L group;(3) KT-5720+ UII 10-8mol/L group;(4) KT-5720 group. Cell planimetric area was viewed using an inverted fluorescent microscope. BCA method was used to detect total intracellular protein in myocardial cells and Hoechst 33258 staining to detect total intracellular DNA. Then the protein/DNA ratio was calculated. Fura-3AM fluorescence staining was used to detect myocardial cell cytoplasm Ca2+ concentration. Intracellular p-PKA, PKA, p-PLN, PLN, SERCA2 a protein content was measured by Western blot assay.Results:(1) UII induced myocardial hypertrophy in the concentration of 10-8mol/L.Pretreatment with KT-5720 could inhibit the hypertrophy of large extent.(2) UII could lead to sharp increase in intracellular Ca2+ concentration; KT-5720 pretreatment also attenuated this increase degree.(3) KT-5720 pretreatment significantly increased the expression of p-PKA, p-PLN and SERCA2 a protein which were induced decreasing by UII.Conclusion: PKA signaling pathway plays an important role in UII induced hypertrophy ofmyocardial cells.