| Long QT syndrome (LQTS) is a genetic heart disorder characterized by an abnormal heart rhythm (long QT interval) where the heart takes time longer than the normal. The main reason of the arrhythmia is of delayed repolarization of the heart. And patients are apt to display torsades de pointes (TDP), ventricular fibrillation, even fainting and sudden death. Moreover, young people with familial genetic genes often appear cardiogenic shock, and the mortality rate is higher.Studies have shown that LQTS can be divided into14species which are LQT1-12and JLN1-2according to the etiology and pathogenesis. The pathogenesis of LQT4relates to the ankyrin-B gene mutation on chromosome4q25-27which leds to encoding ankyrin-B protein dysfunction. Ankyrin-B is required to the integrity of the plasma membranes and to anchor specific ion channels, ion exchangers and ion transporters in the plasma membrane, and the structure of which can be divided into the membrane-binding domain, spectrin-binding domain, the carboxy terminal domain and "death" domain. The evidences indicate that the main reason of abnormal ECG disorder phenotype, such as ventricular tachycardia and sick sinus syndrome are ankyrin-B dysfunction due to mutation. Recently, a large number of animal model studies also found that ankyrin-B protein play an extremely important role in the maintenance of normal heart function. So far, it has been found that the majority of the ankyrin-B gene nucleotide point mutation occurs in the carboxy terminal domain and the spectrin-binding domain, but the "death" domain point mutation is rarely reported. Therefore, in this study, we investigated the molecular mechanism of the mutation in "death" domain of ankyrin-B gene leading to LQT4.In the first part, we determined the method of the adenovirus transfection in myocardial tissue.28rats were randomly divided into4groups:The control group (group Ⅰ), myocardium injecting group (group Ⅱ), coronary artery infusion group (group Ⅲ) and coronary artery post-asystole infusion group (group IV). The transfection efficiency was measured by detection of expression of EGFP in myocardium under fluorescence microscope and by western blotting. Inflammation was observed by HE stain of paraffin sections of the myocardial tissue. The expression of EGFP of extracardiac tissues (liver, kidney and lung) was detected by western blot, we also observed the relationship between expression of EGFP and transfection time in group IV.In the second part, we explored the impact of ANKB W1535R in the rat ECG.24rats were randomly divided into3groups:the control group (control), transfected with Ad-EGFP group (EGFP group) and transfected with Ad-EGFP-ANKB W1535R (ANKB W1535R group). We made the model of rat LQT4with the method of coronary artery post-asystole infusion, and observed the impact of ANKB W1535R in the rat ECG. The expression of ANKB、molecular、NCX1、Ip3R、EGFP of acardiac tissues were detected by western blot.In the third part, adult rat cardiomyocytes were isolated and cultured in M199medium. The rat cardiomyocytes were transfected with EGFP or Ad-EGFP-ANKB W1535R. Then we observed the changes of the [Ca2+]i transient under these conditions, and The expression and location of ANKB, Na+/K+ATP α1/α2, NCX1and IP3R of cardiomyocytes were detected by immunofluorescence.The results were as follows:In the first part:The availability of adenoviruses in vivo transfection in rat heart 1. The comparison of EGFP fluorescence intensity and transfection efficiency in myocardial tissue of rats each groupMyocardial tissue of group I displayed weak background fluorescence under a fluorescence microscope. Myocardial tissue4days after transfection were displayed significantly aneven distributed green fluorescent, in group Ⅱ showed uneven, and the fluorescence in group Ⅳ are even distributed, but in group Ⅲ displayed only a little flaky fluorescent. The lowest level of transfection efficiency was in group Ⅲ (5.29±4.9%), and it reduced significantly compared to group Ⅱ (61.9±9.7%) and group IV(52.9±7.4%)(P<0.01), but no difference was found between group Ⅱ and group IV (P>0.05)2. The comparison of the expression of EGFP in the left ventricleThe expression of EGFP the left ventricle in group Ⅱ and group IV were significantly stronger than group Ⅲ (P<0.01), while it showed no significant difference between group Ⅱ and group Ⅳ (P>0.05)3. The expression of EGFP in extracardiac tissue (kidney, lung and liver)The EGFP expression of heart in group Ⅱ and group Ⅳ was significantly higher than that of the kidney, lung and liver(P<0.01); and the EGFP expression of liver in group Ⅲ was significantly higher than that of heart, kidney and lung (P<0.05), but no significant differences of EGFP expression was found among the heart, kidney and lung (P>0.05)5. The comparison of EGFP in the arrest coronary perfusion group (Ⅳ) of different time pointsCompared with the control group, the EGFP expression was significantly increased (2.484±0.315, P<0.01) in4d after transfection. But in group Ⅳ, the EGFP expression after transfection in7d (0.234±0.040),10d (0.132±0.038) and14d(0.045±0.008)decreased significantly compared with4d (P<0.01), but there were no significant difference of the EGFP expression of10d,14d after transfection compared with the control group (P>0.05).4. The comparison of morphology in myocardial tissue each groupThe myocardial tissue in group I showed:regular myocardial fibers, normal morphology, integrity structure and clear transverse striation. There were a large number of inflammatory cell infiltration, and some necrosis in myocardial tissue of group Ⅱ; But myocardial tissue of group Ⅲ and group Ⅳ showed morphological and structural integrity, and no inflammatory cells infiltration were found.In the second part:The effects of ANKB-W1535R on ECG and the expression of interrelated proteins in vivo1. The effects of ANKB-W1535R on ECGs of rats(1) The influence of ANKB-W1535R in ECGs morphology of ratsECGs morphology of rats in three groups (control group, EGFP group and ANKB-W1535R group) did not change significantly at4d after transfection.1rat’s ECG of ANKB W1535R group exhibits the occasional heart leakage stroke after exercise.3rats’heart rate increased and4rats’ ECGs exhibited a high variability of heart-rate in ANKB-W1535R group after exercise. We mimic emotional stress by injection with epinephrine, no rat of three groups were died. ANKB W1535R rats (4out of7) displayed polymorphic ventricular arrhythmia at10-15min after exercise combined with epinephrine infection, but no one was died after exercise combined with epinephrine infection, while no arrhythmic episodes but only increasing heart rate in ECGs of control or Ad-EGFP rats after exercise or exercise plus epinephrine. There were significantly differences in QTc between the different treatment groups after exercise plus epinephrine (F=7.769, P=0.004); The QTc was longest (206.99±32.70ms) in ANKB W1535R group, From LSD pairwise comparisons, compared with the control group (155.17±13.96ms) and EGFP group (168.83±21.91ms), the QTc in ANKB-W1535R group was increased significantly (P values were0.002and0.014), And it didn’t change in the control group compared with EGFP group (P=0.353). PR, QRS interval and P-wave duration in ANKB group were increased compared with the control group and EGFP group, but there were no significant difference between the different treatment groups (F values were0.674,2.031,0.257; the P values0.524,0.164,0.777)(2) The influence of ANKB-W1535R on HR (heart rate) of ratsThere were no significantly differences in heart rate between the different treatment groups after exercise or exercise plus epinephrine (F=0.619, P=0.551); the rats exhibit a high degree of heart-rate variability at25min after exercise plus epinephrine, the heart rate peaked at586BPM and the lowest only at249BPM. The rats’HR of control group and EGFP group didn’t exhibit a high degree of heart-rate variability, the heart rate change from394BPM to430BPM in control group and from381BPM to478BPM in EGFP group.2. The Expression of ANKB, Na+/K+ATP α1/α2, NCX1and IP3R proteins detected by western blot(1) The ANKB expression of myocardial tissue was significantly different among the three groups (F=6.382, P=0.033). ANKB expression in the myocardial tissue of ANKB-W1535R rats(0.489±0.091)was significantly lower compared with that in control group(0.793±0.139)and EGFP group(0.764±0.110)(P values were0.018,0.026). However, there was no significantly difference between control group and EGFP group (P=0.766)(2) The Na+/K+ATPase α1expression of myocardial tissue was significantly different among the three groups (F=13.875, P=0.006). Na+/K+ATPase α1 expression in the myocardial tissue of ANKB-W1535R rats (0.620±0.119) was significantly lower compared with that in control group (1.291±0.193) and EGFP group (1.031±0.151)(P values were0.002,0.019). However, there was no significantly difference between control group and EGFP group (P=0.090).(3) The Na+/K+ATPase α2expression of myocardial tissue was significantly different among the3groups(F=566.5, P=0.042). Na+/K+ATPase α2expression in the myocardial tissue of ANKB-W1535R rats(0.101±0.021)was significantly lower compared with that in control group (0.196±0.040) and EGFP group (0.201±0.055)(P values were0.030and0.024). However, there was no significantly different between control group and EGFP group (P=0.883)(4) The NCX1expression of myocardial tissue was significantly different among the3groups (F=7.033, P=0.027). NCX1expression in the myocardial tissue of ANKB-W1535R rats(0.370±0.151)was significantly lower compared with that in control group (0.741±0.141) and EGFP group (0.694±0.098)(P values were0.014and0.024). However, there was no significantly difference between control group and EGFP group (P=0.682)(5) The IP3R expression of myocardial tissue was significantly different among the3groups (F=7.033, P=0.027). IP3R expression in the myocardial tissue of ANKB-W1535R rats (0.370±0.151) was significantly lower compared with that in control group (0.741±0.141) and EGFP group (0.694±0.098)(P values were0.014and0.024). However, there was no significantly difference between control group and EGFP group (P=0.682)In the third part:The effects of ANKB-W1535R on Ca2+transient and the expression of interrelated proteins in vitro1. The transfection efficiency of myocytes after transfection by recombinant adenoviral vectorThe survival rate of freshly isolated myocardial cell reached80%, and about70%of the myocardial cells were survived48h after transfection by recombinant adenovirus vector. The transfection efficiency was approximately100%.2. The fluorescence intensity of ANKB, Na+/K+ATP α1/α2, NCX1and IP3R(1) The result of the fluorescence intensity of ANKB:ANKB fluorescence localized along the T-tubule, distributed and stained regularly in the adult cardiomyocytes in control group and EGFP group. In ANKB W1535R group, ANKB fluorescence distributed foggy. The fluorescence intensity of ANKB was significantly different among the three groups CF=11.318, P=0.009). It was lowest in ANKB W1535R group (29.441±4.474), the difference was significantly compared with that in control group (52.425±7.619) and EGFP group (48.762±6.573)(P values were0.004and0.01). However, there was no significantly difference between control group and EGFP group (P=0.507)(2) The result of the fluorescence intensity of Na+/K+ATP on:Na+/K+ATP α1expression in the adult cardiomyocytes in control group and EGFP group is localized along the T-tubule, distributed and stained regularly. In ANKB-W1535R group, ANKB expression distributed foggy. The fluorescence intensity of ANKB was significantly different among the3groups (F=10.942, P=0.01). It was lowest in ANKB W1535R group (16.110±3.640), the difference was significantly compared with that in control group (42.083±11.637) and EGFP group (44.363±7.345)(P values were0.008and0.006). However, there was no significantly different between control group and EGFP group (P=0.746)(3) The result of the fluorescence intensity of Na+/K+ATP<α2:Na+/K+ATP α2expression in the adult cardiomyocytes in control group and EGFP group is locolized along the T-tubule, distributed and stained regularly. In ANKB-W1535R group, ANKB expression distributed foggy. The fluorescence intensity of ANKB was significantly different among the3groups (F=9.355, P=0.014). it was lowest in ANKB W1535R group (23.191±2.997), the difference was significantly compared with that in control group (42.029±7.672) and EGFP group (45.202±8.269)(P values were0.007and0.014). However, there was no significantly different between control group and EGFP group (P=0.585)(4) The result of the fluorescence intensity of NCX1:NCX1expression in the adult cardiomyocytes in control group and EGFP group is localized along the T-tubule, distributed and stained regularly. In ANKB W1535R group, ANKB expression distributed foggy. The fluorescence intensity of ANKB was significantly different among the3groups (F=31.281, P=0.000). It was lowest in ANKB W1535R group (10.219±3.330), the difference was significantly compared with that in control group (42.582±7.197) and EGFP group (39.483±5.372)(P values were0.000and0.001). However, there was no significantly different between control group and EGFP group (P=0.518)(5) The result of the fluorescence intensity of IP3R:IP3R expression in the adult cardiomyocytes in control group and EGFP group is localized along the T-tubule, distributed and stained regularly. In ANKB-W1535R group, ANKB expression distributed foggy. The fluorescence intensity of ANKB was significantly different among the3groups (F=17.037, P=0.003). it was lowest in ANKB W1535R group (12.386±3.862), the difference was significantly compared with that in control group(40.514±5.659)and EGFP group(40.509±9.610)(both of the P values were0.002). However, there was no significantly different between control group and EGFP group (P=0.999) 3. Intracellular Ca2+transientsThe results of contraction amplitude in cardiomyocytes were as follows: Control group was (9.21±1.23)%, EGFP group was (9.20±1.50)%, ANKB W1535R group was (8.97±0.99)%, there were no significantly differences between the3groups(F=1.348, P=0.261). The Ca2+transients amplitude(△F/F0)of ANKB W1535R group was highest(1.43±0.14), compared with control group(1.03±0.19) and EGFP group (0.98±0.21), the difference was significantly (P=0.036,0.023). The difference of the rise time of Ca2+transients in different treatment groups was not significantly (F=1.384, P=0.252); The half full duration of half maximum of Ca2+transients (FDHM) was significantly different among the3groups (F=79.19P=0.000), compared with control group (129.87±11.91ms) and EGFP group (126.59±13.77ms), it increased significantly in ANKB W1535R group (153.64±23.40ms)(both of P values were0.000)Conclusions1. This study suggest that coronary adenovirus infusion after asystole seem to be a better method of in vivo myocardial gene transfer for its good effect of transfection and little negative effects on myocardial tissue2. ANKB-W1535R transfection can lead to LQT4in rat in vivo. ANKB-W1535R cause ANKB, Na+/K+ATP α1/α2, NCX1and IP3R protein expression levels dropped and positioning disorder may be one of the reasons of LQT4rat arrhythmia.3. At the cellular level, the increased intracellular calcium transients may be one of the reasons for the arrhythmia in LQT4rats. |
PDF Full Text Request