The Molecular Mechanisms Of CaMKⅡ/PLN/SERCA2a Signaling Pathway In Cardiomyocyte Hypertrophy Induced By Urotensin Ⅱ

Cardiac hypertrophy is a series of adaptive changes by acting to meet the long-term pressure overload of heart, which is one of the most common clinical complications of cardiovascular disease. Although compensatory hypertrophy can maintain or increase cardiac output in the short term, the long-term cardiac hypertrophy can cause heart dilation, leading to clinical heart failure and sudden cardiac death. Today, there are some side effects in the clinical treatment of cardiac hypertrophy and heart failure program.Therefore, it’s of great significance in medical value to actively explore mechanisms of the disease that provides new target for the development of ideal drugs to treat the disease.As a common disease, cardiac hypertrophy can be induced by many factors such as urotensin Ⅱ(UII). UII is a new kind of polypeptide, which was isolated from the urophysis of teleost fish originally. In 1998, this UII was cloning out for the first time in human. UII has very extensive biological effects, such as regulating cardiovascular function, central nervous system, endocrine system organizations and movements. So far,UII is considered as one of the strongest vasoconstrictor substance in vivo. Studies have shown that UII has some main biological effects. It plays important role in mediating vasoconstriction, inducing myocardial ischemia and cardiomyocyte hypertrophy,contributing to proliferation of myocardial fibroblast and vascular smooth muscle cells.As a result it can lead to hemodynamic changes, prompt the dysfunction of heart and myocardial remodeling, eventually leading to heart failure. Currently, UII has been proved to closely relate to heart failure in many fields, such as cell experiments, animalVII disease models and clinical research. One of the basic performance of myocardial remodeling is myocardial hypertrophy, which is an important leading factor of heart failure. Therefore, this study was to further consider the mechanism of myocardial hypertrophy induced by UII and UII‘s effect in the process of heart failure. Thus, this study results would provide a new foothold for the treatment of heart failure.The increasing concentration of intracellular free calcium is one of the main reasons for cardiac cell contraction. It is found that a variety of cardiac biological effects processed by UII were mediated by Ca2+. It is still unclear that how UII mediates the Ca2+signal transduction in cardiomyocytes. Thus, further research is needed. As intracellular second messengers, Ca2+ signal transduction plays an important role in intracellular signal transduction. Calcium signaling system is a ubiquitous signal transduction mechanism in cells. Cardiac hypertrophy is often occurred with abnormal intracellular Ca2+ concentration, thereby causing a series of physiological and pathological reactions.Cell contraction, rhythm, growth and apoptosis are directly affected by the spatial distribution, concentration and phase change of myocardial intracellular Ca2+. Some studies have pointed that the cell life processes such as growth, metabolism, secretion and apoptosis are affected by various downstream signaling pathways which were mediated by intracellular cytoplasmic Ca2+ concentration. One of the basic conditions of myocardial contractility is calcium reserving in the sarcoplasmic reticulum( SR).Therefore, the changing contents of calcium in the SR is accompanied by the development of heart failure, ischemia and other diseases. Ca2+ and calmodulin-dependent protein kinase Ⅱ(Ca MKII) involved in the process of cardiac diseases, so that Ca MKII signal transduction pathway may play a key role in the development of cardiac hypertrophy and left ventricular hypertrophy in ventricular arrhythmias. So, we hypothesize that the UII induces or promotes myocardial hypertrophy, Ca MKII may be a key regulatory target.In summary, this study uses a rat model of chronic pressure overload in order to observe cardiac hypertrophy effects induced by UII and possible involved targets. Itexplores the molecules mechanism of UII in promoting cardiomyocytes hypertrophy and its relationship with Ca2+-Ca MKII signal transduction pathways in rat neonatal cardiomyocytes cell culture techniques. The research provides an important basic intervention for the development of cardiac hypertrophy caused by UII and explores a new mechanism of myocardial fibrosis disease.Part 1: the expression changes of UII and calmodulin in chronic pressure overload in ratObjective:To observe the expression changes of UII, p-Ca MKII, Ca MKII, ANP, p-PLN(Thr17),PLN, SERCA2 a and Ry R2 in myocardial tissue from the chronic pressure overload rats’ heart.Methods:Adult male Wistar rats were randomly divided into sham group(control group) and abdominal aortic coarctation model group(model group). Then the indexes were measured in 2w, 4w, 8w, 12 w after modeling.(1) Observe the spirit, eating, drinking,activity, etc of all experimental rats;(2) Monitor ventricular mass index and hemodynamic in all groups;(3) Detect expression the myocardial pathological changes by HE staining;(4)Detect the expression changes of UII, p-Ca MKII, Ca MKII, p-PLN(Thr17), PLN, SERCA2 a and Ry R2 in myocardial tissue by immunofluorescence staining and DAB staining;(5)Detect the expression changes of UII, p-Ca MKII, Ca MKII,ANP, p-PLN(Thr17), PLN, SERCA2 a and Ry R2 in myocardial tissue expression by Western blot assay.Results:Compared with the control group,(1) left ventricular end-diastolic pressure(LVEDP)increased with time increasing. It showed more than 15 mm Hg after 8w in the model group;(2) the echocardiography showed rat ventricular start gradually thickening after modeling for 4w. Interventricular septum diastolic thickness(IVSTd), left ventricular posterior wall diastolic thickness(LVPWTd) and left ventricular and diastolic diameter(LVDd) increased significantly and the ventricular chamber enlarged after modeling for4 w. The results were more significant in 8w and 12 w after modeling;(3) the results of HE staining showed that rat cardiomyocytes diameter in the model group gradually increased in a time-dependent manner, especially in 8w and 12 w after surgery;(4) the results of immunofluorescence staining and DAB staining showed the expression of UII,p-Ca MKII, p-PLN(Thr17), SERCA2 a and Ry R2 in myocardial tissue of the model group were significantly increased in a time-dependent manner, while PLN’ and Ca MKII’s expression did not change significantly.(5) Western Blot results also showed that the expression of UII, p-Ca MKII, p-PLN(Thr17), SERCA2 a, ANP and Ry R2 in the myocardial tissue of the model group increased with time prolonged, while PLN’ and Ca MKII’ expression did not change significantly.Conclusion:(1) there is a obvious effect of cardiac hypertrophy in chronic pressure overload rat established by abdominal aortic coarctation heart failure model, and the degree of myocardial hypertrophy increased gradually with time prolonged;(2) UII may play an important role in myocardial hypertrophy of the model group;(3) Ca2+-Ca MKII signal transduction pathway may be involved in the development of pressure overload myocardial hypertrophy.Part 2:the expression of primary cardiomyocytes and calmodulin affected by Urotensin ⅡObjective:to observe the changes of different concentrations and different time of UII treatment in primary myocardial cells, and further discuss the role of Ca2+-Ca MKII signal transduction pathway in this process.Methods:Primary cardiomyocytes of neonatal rat were randomly divided into control and model groups, the model groups treated with different concentrations of UII(10-10 mol / L;10-9mol / L; 10-8mol / L; 10-7mol / L and 10-6mol / L)(1)after treating with different concentrations of UII, the changes of myocardial cell viability was detected by MTT assay in different time treatment;(2) the OD value of DNA and total protein in myocardial cell treated with different concentrations of UII was analyzed in different time.At the same time changes in cell area was measured;(3) monitor changes of Ca2+concentration in cardiomyocytes treated by UII with Fura-3 AM fluorescent probe;(4)Western blot assay was used to detect the expression of p-Ca MKII, Ca MKII, ANP,p-PLN(Thr17), PLN, SERCA2 a and Ry R2 in myocardial cells treated by UII.Results:Compared with the control group,(1) with UII concentration increasing and treatment time extension, primary myocardial viability and the cell area gradually increased;(2)OD value shows that, total protein of primary cardiomyocytes and the ratio of protein/DNA significantly increased with the UII concentration increasing and time extension, but the total DNA content did not change significantly;(3) with the time of UII treatment prolonged, the concentration of Ca2+ in myocardial cells significantly increased;(4)the results of Western Blot also showed the protein expression of p-Ca MKII, p-PLN(Thr17),SERCA2 a, ANP and Ry R2 in primary cardiomyocytes gradually increasing with the time of UII treatment, while PLN and Ca MKII expression did not change significantly.Conclusion:(1) UII may cause the hypertrophy of primary cardiac myocyte, and the degree of cardiomyocyte hypertrophy gradually increased with the increasing concentration of UII and extension of time;(2)Ca2+-Ca MKII signal transduction pathway may be involved in the process of UII induced cardiomyocyte hypertrophy in vitro.Part 3:Research on the signal transduction pathway of cardiomyocyte hypertrophy induced by urotensin IIObjective:To further explore the function of Ca2+-Ca MKII signal transduction pathway in the process of UII-induced myocardial hypertrophy and illuminate the molecular mechanisms of UII-induced cardiac hypertrophy.Methods:Primary neonatal rat cardiomyocytes were randomly divided into control group, UII group(10-8mol / L), UII + KN-93 group and KN-93 group.(1) OD value of cardiomyocytes DNA and total expression of protein were analyzed. What’s more, the changes in cell area were measured too;(2)Fura-3 AM fluorescent probe was used to monitor changes of myocardial intracellular Ca2+ concentrations after the treatment with UII;(3)Western Blot assay was used to detect the changes of p-Ca MKII, Ca MKII, p-PLN(Thr17), PLN and SERCA2 a in myocardial cells after UII treatment.Results:Compared with the control group,(1) UII significantly induced cardiomyocyte hypertrophy, and KN-93 can significantly reduce the myocardial hypertrophy caused by UII treatment;(2)OD value showed that KN-93 can significantly reduce the increasing of total protein and the ratio of protein/DNA in primary cardiomyocytes caused by UII, and total cellular DNA content is not affected;(3)Fura-3 AM fluorescent probe test showed KN-93 can significantly reduce the increasing of Ca2+ concentration in primary cardiomyocytes caused by UII;(4)The results of Western Blot also showed that KN-93 treatment can significantly reduce the increasing protein expression of p-Ca MKII, p-PLN(Thr17) and SERCA2 a in primary cardiomyocytes, and the expression of Ca MKII and PLN were not affected by the treatment of KN-93.Conclusion:Ca MKII-PLN-SERCA2 a signaling pathway may play an important role in the process of UII-induced cardiomyocyte hypertrophy.