| Objection To establish rats model of hypertensive heart disease induced chronic heart failure(HHD-induced CHF), carried out in accordance with the group cardiac myocytes’ s separate quickly and observation calcium release events-calcium spark and calcium signal under confocal microscope, at the same time, through the changes of the excitation contraction coupling related proteins Junctophilin- 2(JP- 2) and the calcium taken back related proteins SERCA2 a to research the correlation with calcium sparks. Explore the evolution process of hypertensive heart disease induced chronic heart failure and calcium signal changes and the related mechanism of calcium cycle changes in the pathological state.Methods1. The experimental rats were divided into two groups, HF model group(n = 40) and sham operation group(n = 26).2. The model group rats by transverse abdominal aortic constriction(TAC) induced hypertension to establish pressure overload model of HF. The sham operation group only open threading, without ligation, the other by the same method.3. Take blood pressure monitoring of rats tail vein on a regular basis and enzyme-linked immune ELISA method to detect markers BNP expression quantity on postoperative survival of rats,used to judge the determine whether high blood pressure caused by heart failure model is successful.4. Two groups of rats in 4 weeks, 6 weeks, 8 weeks, 10 weeks, 12 weeks after the operation are acute digestived the heart and obtain single ventricular muscle cells, and used for the subsequent testing.5. The laser confocal microscope and patch clamp together, observe and record unit release calcium activity and the occurrence of calcium sparks in ventricular myocytes.6.Used Western Blotting to detect myocardial cell JP2 excitation-contraction coupling structure of protein and the calcium taken back related proteins SERCA2 a expression with the development of heart failure, assessment of structural changes in the process of developing high blood pressure induced heart failure.Results1. The postoperative symptoms and success rate of high blood pressure building in rats.Successful operation of rats to restore clear within 3 ~ 6 h, 12 h can eat, and good mental state.Heart failure model group survival rate of 70.0%, high blood pressure in the model rats of survival replication success rate is 89.2%;Control rat survival rate was 96.1%.2. The rat tail arterial pressure monitoring results. Normal rats blood pressure at about 13 ~15 kpa,。Two groups of rats blood pressure changes In the initial measurement, no difference between two groups(P > 0.05);7 days, 14 days after the surgery, blood pressure and heart failure model group control is not obvious difference(P > 0.05), after 21 days, blood pressure heart failure model group was obviously higher than that of control group(P > 0.05);After 28 days, the heart failure model group blood pressure rising blood pressure(P < 0.05), and higher than the control group(P < 0.05);To 49 days postoperatively, model group blood pressure to maintain a certain pressure value(P > 0.05), significantly higher than the control group(P <0.05);56 days, 63 days after model group decreased(P < 0.05), blood pressure and blood pressure value is higher than the control group(P < 0.05);After 84 days, the model group rats blood pressure continues to decrease(P < 0.05), blood pressure and higher than the control group(P < 0.05).Contrast between the way is no difference(P > 0.05).3. Heart failure markers b-type brain natriuretic peptide(BNP) test results in heart failure rat model induced by group BNP levels increased significantly in 4 weeks, hf plasma BNP levels of model group was obviously higher than that of control group(P < 0.05), BNP levels as a group of heart failure process of heart failure increased(P < 0.05).4. The rat acute myocardial cell separation results are in good shape individual cardiomyocytes rods, cell survival rate at about 80%.Under a microscope, more than 60% of cells show obvious long rod, activity is good.5. Myocardial cell calcium sparks morphology observation results compared with control group, the difference of calcium sparks was observed in the first two groups is not obvious (P > 0.05);Along with the development of heart failure, heart failure model group of calcium sparks of myocardial cell observed have obvious differences in time and space form, the margin of calcium sparks heart failure model group decrease(P < 0.05), prolonged(P < 0.05),peak duration(P < 0.05).Along with the development of heart failure, the cellular structure change, heart muscle cells for potential gradually lose their reaction, structure decision phenomenon, change of calcium sparks has its structure change.6.Western blot test results. Western blot test results show that compared with the control group, model group of hf in 4 weeks JP2 protein expression level in rats the same(P > 0.05),and 8 weeks model group of protein expression protein expression is lower than the control group(P < 0.05);With the process development of heart failure, heart failure model group rats amount of protein expression were decreased(P < 0.05), to 10 weeks, 12 weeks, the protein expression has no difference(P > 0.05);Stable expression between the control group, no difference(P > 0.05).SERCA2 a protein imprinting, according to the results of heart failure in rats of model group at 4 weeks SERCA2 a protein expression levels(P > 0.05), 8 weeks model group of protein expression protein expression is lower than the control group(P < 0.05);With the process development of heart failure, heart failure model group rats amount of protein expression were decreased(P < 0.05), to 10 weeks, 12 weeks, the protein expression has no difference(P > 0.05);Stable expression between the control group is no difference(P > 0.05).Conclusion:1.TAC method is a good way to duplicate rat hypertension, which is caused by heart failure.In this study, comparing with previous reports, the rat survival rate after operation and the am ount model is high.2. The blood pressure of rat heart failure model is a rule change, and it keeps a high value in a certain period of time within the heart failure occurs. Heart failure rats BNP expression level increased with the severity of heart failure gradually.3. In the process of Heart failure, the injury of myocardial cell decreases the number of rod-sh aped cells.4. Comparing calcium sparks in the myocardial cell between HF group and normal group, the amplitude of calcium sparks decreases, also the time to peak and FDHM prolonged obviously.These proposed that myocardial cells in the whole calcium cycle is frustration, including calcium inspire and taken back of calcium.5. Calcium cycle is the basis of contraction diastole function in myocardial cell. In the process of heart failure, the calcium release parts related to excitation contraction coupling-JP- 2protein and calcium taken back protein in the sarcoplasmic reticulum-SERCA2 a protein were gradually decreased, and protein changes over time is correspond with calcium sparks. The two changes, which have some relationship, indicate that a variety of mechanisms can cause heart failure. |
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