Changes Of Kv4.2 And KChIP2 Proteins In Ventricular Myocardium During The Development Of Cardiac Hypertrophy And Failure

Cardiac hypertrophy and failure is the common complication for such diseases as hypertension, ischemic heart disease, and heart valve disease, etc. Cardiac hypertrophy and failure often accompany with arrhythmias. Heart failure in particular is associated with a significant increase in the risk of sudden cardiac death (SCD). Despite considerable progresses have been made in the treatment of heart failure in recent years, the mortality of patients with heart failure remains high. Therefore, it is of significance to elucidate the molecular mechanism underlying the arrhythmias companying cardiac hypertrophy and failure and seek for the effective medication.Up to now, the reduction of transient outward potassium current (Ito) density has been observed in many experimental animal models with cardiac hypertrophy and failure. Ito plays an important role in cardiac early repolarization. The Ito channel is composed ofαsubunits (Kv4.2, Kv4.3) andβsubunit (KChIP2). The reduction of Ito results from the changes of the kinetics properties and/or the molecular expression of the channel and contributes to the prolongation of action potential duration (APD), which is believed to predispose the heart to afterdepolarization and reentrant arrhythmias.In our previous study we found a progressing prolongation of APD in mouse papillary muscle as the development of cardiac hypertrophy and the failure in a mouse cardiac pressure overload model. The electrical remodeling in this model is a dynamic process and perhaps depends on the degree and the state of ventricular functional compensation. It is known that Ito is main component for the repolarization in mouse myocardium. However, little is known about the changes ofαandβsubunits of the ion channel and underlying mechanism during the pathological process of cardiac hypertrophy and failure.In this study, we quantified the titation of Kv4.2 and KChIP2 proteins in subepicardial and subendocardial ventricular myocardium either in hypertrophied or failing hearts in the mouse cardiac pressure over-loaded model. By treating animals with calcineurin inhibitor cyclosporin A(CsA), We evaluated the possible role of calcineurin pathway in the regulation of Kv4.2 and KChIP2 protein levels in heart failure.Part 1. Changes of Kv4.2 and KChIP2 protein level in subepicardial and subendocardial ventricular myocardium during the development of pressure over-loaded mouse cardiac hypertrophy and failureObjective: To understand the molecular mechanism of ion channel remodeling in the pathological condition by determining Kv4.2 and KChIP2 protein level in subepicardial and subendocardial ventricular myocardium in hypertrophied or failing hearts.Methods: (1) Mouse model of cardiac pressure overload: Transverse aorta was banded by using microsurgical techniques to create cardiac pressure overload, as described by others. Briefly, Kunming male mice, 4-5 weeks old (provided by Experimental Animal Center of Hebei Province), were anesthetized with ketamine (25mg/kg, intraperitoneal injection). The mice were orally intubated and ventilated (Harvard Apparatus). The chest cavity was opened in the second intercostal space and transverse aortic banding was performed by tying a nylon suture against a 27-gauge needle to produce an aortic narrowing 0.4 mm in diameter when the needle was removed. The procedure resulted in a reproducible transverse aortic banding of 65-70%. Age-matched mice were subjected to a sham operation in which the aortic arch was visualized but not banded. Mice were then maintained for 13 weeks after operation. Hemodynamic variables in some TAB or sham-operated mice were measured by placing a catheter in the isolated right carotid artery, which was advanced as far as the aorta and left ventricle. Cardiac mass index was also assessed and compared to the sham-operated animals at the different time after operation. (2) Immunofluorescence staining: Immunofluorescence labeling was performed in isolated subepicardial and subendocardial ventricular myocytes. The cells were treated with anti-Kv4.2 or KChIP2 antibody. Immunofluorescence labeling for confocal microscopy were done by treatment with fluorescein isothiocyanate-conjugated goat anti-rabbit antibody. Immunofluorescence-labeled samples were examined using a confocal laser scanning microscopy. Identical settings were used for all the specimens. (3) Western blot analysis: Immunoblots were performed using membrane fraction from mouse subepicardial and subendocardial ventricular myocardium at week 2, 5, 13 after operation. Anti-Kv4.2 and anti-KChIP2 were used as primary antibodies. Anti-GAPDH antibody was used as an internal loading control. Quantification of the signals was performed by densitometry. The protein bands were normalized to the GAPDH band in each sample. The value was then averaged from all the different sets of experiments.Results: (1) A mouse pressure over-loaded cardiac hypertrophy and failure model was established by aorta banding. The results showed that first 7 week hearts in banded mice were manifested the progressive increase in heart mass index and contractile function, which we defined the stage of compensatory hypertrophy. The following was the stage of heart failure, characterized by declining contraction. (2) Immunofluorescence labeling revealed that Kv4.2 and KChIP2 were both intensely observed on cell membrane in subepicardial and subendocardial ventricular myocytes. Kv4.2 protein appeared more strongly expressed in subepicardial myocytes than in subendocardial myocytes, while expression of KChIP2 protein was similar in subepicardial and subendocardial myocytes. (3) Quantification of channel proteins showed that there was a transmural gradient of Kv4.2 protein level in sham operated mice, i.e. higher amount of protein in subepicardium than in subendocardium with a ratio 1.82. The quantity of Kv4.2 protein was significantly decreased both in endocardium and epicardium in banded mice ( P<0.05 ) . Compared to sham-operated animals, amount of the protein in aorta-banded mice was decreased by 25.37%,41.33%,62.67% (P < 0.05) in subepicardium at week 2, 5, 13 after operation respectively, and in endocardial myocytes was respectively decreased 26.32%,41.46%,66.67% (P < 0.05). There were no significant differences in Kv4.2 protein level either in subendocardium or subepicardium between band 2w and band 5w groups. However, Kv4.2 protein level in 13w banded mice was less both in subendocardium and subepicardium than it in band 2w and band 5w ( P<0.05 ) mice. (4) Amount of KChIP2 protein in subepicardium is similar to that in subendocardium in sham-operated mice at week 2, 5, 13 after sham operation. Compared to sham-operated mice, no significant changes of KChIP2 level were observed in band 2w and 5w mice. However, KChIP2 protein was down-regulated at week 13 after operation(P<0.05), and the degree of reduction in subendocardium and subepicardium was 61.11% and 48.08%.Conclusions: (1) It was in the early phase that amount of Kv4.2 protein started to be decreased both in subepicardium and subendocardium in hypertrophied hearts, but KChIP2 expression was not changed in compensated hypertrophy stage. (2) The expression of Kv4.2 protein was kept reduced in heart failure phase. KChIP2 protein level was decreased in failing hearts, and the reduction degree was higher in subendocardium than in subepicardium. The results suggest that the changes of protein expression level are the molecular basis contributing to abnormal repolarization of cardiomyocyte in the pathological condition. Both Kv4.2 and KChIP2 proteins were down-regulated in heart failure, showing the possible reason for the propensity for arrhythmias in this stage.Part 2. The possible role of calcineurin pathway in down- regulation of Kv4.2, KChIP2 protein expression in failing heartsObjective: To evaluate the role of calcineurin signaling pathway in down-regulation of Kv4.2, KChIP2 protein expression in failing heartsMethods: Sham operated (Sham) or aorta banded (Band) mice were randomized to receive CsA (25mg/kg, injected subcutaneously twice daily, North China Pharmaceutical Group Corporation) or vehicle (Veh) for 2 weeks. Treatments were started from 9 weeks after operation, which represented heart failure phases. At the end of treatment, i.e. 11 weeks after operation, the amount of Kv4.2 and KChIP2 proteins from subepicardium and subendocardium were determined by using Western blot technique in CsA or Veh treatment mice, which including following groups: Band 11w + CsA, Band 11w + Veh, Sham 11w + CsA and Sham 11w + Veh. Meantime, calcineurin activity in subepicardium and subendocardium, cardiac mass index and cardiac histopathology were assessed.Results: (1) There were no significant differences in appearances between the Band 11w + CsA group and Band 11w + Veh group, also in Sham 11w + CsA and Sham 11w + Veh groups. The mortality was 8% in Band 11w + CsA group, which was similar to that in Band 11w + Veh mice. There was no mortality in sham-operated mice. The cardiac mass index of Band 11w + CsA mice was significantly decreased compared to Band 11w + Veh group (P<0.01),but not down to the level in Sham 11w + Veh group. (2) Cardiac histopathology: cardiac myocytes from Sham 11w + CsA, Band 11w + CsA and Sham 11w + CsA had well-arranged appearance, with similar sizes and clear transverse lines. There was little collagen between the cells. However, cells from Band 11w + Veh were lightly dyed and had blurry-transverse lines, with bigger size and disorganized appearance. There were a lot of denatured cells with dark-dyed and blurry nucleus. (3) Calcineurin activity was significant higher in subendocardium than in subepicardium (P<0.01) in Sham mice. Compared to Sham 11w + Veh group, calcineurin activity in Band 11w + Veh mice was greatly increased both in subepicardium and subendocardium (P<0.01), and subendocardium with higher activity than subepicardium. CsA could completely revised the increase of calcineurin activity since there was no difference in calcineurin activity between Band 11w + CsA and Sham 11w + Veh. (4) CsA treatment partially up-regulated Kv4.2 protein level both in subepicardium and subendocardium in banded mice. The expression of KChIP2 was no sigificiant differences in epicardium or endocardium between Band 11w + CsA and Band 11w + Veh groups. (5) In sham-operated mice, there was no effect of CsA on general conditions, cardic mass index, calcineurin activity and the expressions of Kv4.2, KChIP2 proteins.Conclusions: Inhibition of calcineurin signaling pathway attenuated the changes of cardiac histopathology and partially reversed the reduction of Kv4.2 protein level in heart failure, but it didn't affect the expression of KChIP2 proteins. The result suggests that calcineurin pathway may down regulate Ito by inhibiting the expression of Kv4.2 protein.