| Objective: Rabbits model of heart failure is established by injection ISO in their ear vein. To investigate the changes of INaL and the effect of KN-93 on INaL in cardiomyocytes in rabbits model of heart failure; at the same time, we detect the expression of CaMKII?, phosphorylated CaMKII?, Nav1.5, AnkyrinG in myocardial tissue and study the location of CaMKII? and AnkyrinG. In order to clarify CaMKII? may regulate late sodium channel by AnkyrinG and induced arrhythmias.Methods: The Japanese white rabbits, male or female, are randomly divided into HF group and control group. HF group rabbits are injected ISO(300?g ? kg-1 ? d-1) via ear vein for 15 days; control group rabbits are injected an equal volume of 0.9% saline(1ml ? kg-1 ? d-1) via ear vein for 15 days; after the completion of the injection, all rabbits have free access to food and water.(1) One months later, all rabbits are detected by echocardiography, ELISA is used to detect the content of BNP in rabbit plasma and HE staining to observe the pathological changes of myocardium to evaluate whether the HF model is established successfully.(2) The ventricular myocytes are isolated from the control group and the HF group rabbits, using whole cell patch clamp technique to record INaL.(3) We use Western Blotting to detect the protein expression of CaMKII, phosphorylated CaMKII?, Nav1.5, AnkyrinG in each group.(4) The q RT-PCR method is used to detect the expression of CaMKII, Nav1.5, AnkyrinGmRNA.(4) We use double immunofluorescence labeling of CaMKII and AnkyrinG to observe their location.Results: 1) General modeling results: In the process of modeling, HF group rabbits have 3 death, control group rabbits have no death, the success rate of model was 75.0%; and the heart rate of HF group is increasing. 2) Echocardiographic results show: compared to control group, HF group ventricular cavity enlargement and cardiac function decrease; HF group LVEF(46.52±7.18%) compare with the control group(65.52±4.09%) decreases significantly and less than 50%(P<0.01); HF group LVFS(21.14±3.70%) compares with the control group(32.58±2.84%) decreases significantly(P<0.01). 3) Rabbit plasma BNP content results: plasma BNP content of HF group(391.40±38.59pg/ml) than in the control group(234.49±31.68pg/ml) increases significantly(P<0.01). 4)HE staining is observed under the microscope, it is found that control group cardiomyocytes are long spindle shaped and arranged in neat rows, boundaries between cells are clear, myocardial stripes can be seen; HF group cardiomyocytes arrange in disorder, have no rules, can be seen vacuolar degeneration in the cardiomyocytes and myocardial interstitial edema, fibrous tissue is increasing. 5) Whole cell patch clamp results show: HF group INaL(-1.64±0.30pA/pF) compare with control group(-0.70±0.17pA/pF) have significantly increased(P<0.01); after adding ATXII(30nM), ATXII group vs blank control group INa L has increased significantly in the HF group, and the control group INaL also has increased significantly; however, the HF group increases obviously than in the control group(P<0.01); after ATXII induced current, we add KN-93(1?M) into control group and HF group, and we find that the ATXII increased-INaL decreasing from(-1.39±0.29pA/pF) to(-0.99±0.28pA/pF) in the control group(P<0.05) and also find the ATXII increased-INaL in the HF group decreasing from(-2.27±0.22pA/pF) to(-1.87±0.25pA/pF), P<0.01. 6) Blotting Western results are shown: the expression of CaMKII?, phosphorylated CaMKII?, Nav1.5 and AnkyrinG in HF group are significantly increased compared to control group(P<0.01). qRT-PCR results: the expression of CaMKII?, Nav1.5and AnkyrinG mRNA in HF group are significantly higher than the control group(P<0.01). Double immunofluorescence results showed: Control group and HF group have green fluorescence and red fluorescence, the green and red fluorescence of HF group are stronger fluorescence than control group; in each group, the green and red fluorescence are distributed in the cell, and the expression of both in the cell membrane is more obvious, which shows that CaMKII? and AnkyrinG are co expressed co-expressed on the cell membrane.Conclusions: 1. When Isoproterenol- induced heart failure in rabbits, not only the expression of Nav1.5 will enhance and the INaL will increase. 2. When HF occurs, the expression and activity of CaMKII? will increase, and we use KN-93 to inhibit CaMKII? can reduce INaL, indicating that CaMKII? can regulate INa L. 3. When HF occurs, the expression of AnkyrinG will increase; AnkyrinG and CaMKII? overlap in the same part of cardiomyocytes membrane; so, AnkyrinG may play a role during CaMKII regulates INaL, which may be one of mechanism when CaMKII? trigger arrhythmias. |
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