| Objective:1. To prepare monoclonal antibodies against α-2(840-877) of Na+-Ca2+ exchanger, and to explore effects of the acquired monoclonal antibodies on rat myocardial Na+-Ca2+exchange and other ionic channel activities.2. To investigate the effects of monoclonal antibodies against α-2(840-877) of Na+-Ca2+ exchanger on ischemia and reperfusion induced cardiac arrhythmia using ischemia and reperfusion induced rat arrhythmic model, and to analyze the antiarrhythmic mechanism.Methods:1. Preparation of monoclonal antibodies against α-2(840-877) of Na+-Ca2+ exchanger.BALB/c mice were immunized by NCX α-2(840-877) coupled peptide, spleen cells with SP2/0 bone marrow tumor cell fusion and, by positive clones were selected from the strains and three times after cloning can be obtained stably secreting monoclonal antibody hybridoma cell, expanded in culture, the animals and induce method to prepare ascites and by protein A affinity column on the income of ascites were purified and monoclonal antibody. Using indirect ELISA method for the detection of monoclonal antibody titer purified. SDS-PAGE of purified monoclonal antibody purity.2. Preparation of rat single ventricular myocytes(enzymatic dissociation procedure modified).Rat single left ventricular myocytes were isolated from normal rats. SD rats were anesthetized with pentobarbital sodium(40 mg·kg-1, i.p.), the heart was rapidly excised,and mounted via the aorta on a Langendorff retrograde perfusion apparatus. Firstly, the heart was perfused with 100% O2 saturated Ca2+-free Tyrode’s solution(at 37°C, 75 cm H2O) for approximately 8 minutes to wash out the blood. Secondly, the perfusate was switched to enzyme solution(contained with Collagenase P 0.07~0.1 g·L-1) for 15~20minutes until the heart were well digested. The left ventricle was then separated and minced in the KB solution. The dispersed cells were filtrated in KB solution at room temperature(25 °C) at least 3 hours before use.3. Whole-cell patch clamp recording.Whole-cell patch clamp recording technique was used to investigate INa/Ca, ICa-L, IK1,INa, Ito.4. Establishment of rat arrhythmic models by ischemia and reperfusion(I/R).(1) Arrhythmia induced by ischemia-reperfusion in Langendorff-perfused rat heart.SD rats were anesthetized with pentobarbital sodium(40 mg·kg-1, i.p.), the heart was rapidly excised, and mounted via the aorta on a Langendorff retrograde perfusion apparatus,record ECG, ligation of left anterior descending coronary artery after the heart systolic stability, caused local ischemia for 30 min, loosen the wire reperfusion for 30 min. The incidence of ventricular tachycardia, duration, incidence of ventricular fibrillation, and duration of ischemia for 30 min and reperfusion for 30 min were accumulated,respectively.(2) Arrhythmia induced by ischemia-reperfusion in rat heart in vivo.SD rats were anesthetized with pentobarbital sodium(40 mg·kg-1, i.p.), open the chest,BL-420 F biological function experiment system was used to record the electrocardiogram,Ligation of the left anterior descending branch of the coronary artery, caused local ischemia for 15 min, loosen the wire reperfusion for 15 min. Antibody 2D2, 3F10(50μg·kg-1), Lidocaine(7.5 mg·kg-1), tail vein injection before 3 min of ischemia or reperfusion. The rate of premature contraction, the incidence rate of ventricular tachycardia,the duration of ventricular fibrillation, the incidence of ventricular fibrillation, the duration,and the changes of ST segment were observed.5. Calcium transient detection.The isolated adult rat ventricular myocytes were isolated by enzymatic hydrolysis,add 5 μmol·L-1 Fura2-AM, avoid light, Tyrode’s solution perfusion. Ion imaging analysis system exhaust air in the device, preheating equipment, turn the cells was loaded with fluorescent dye into cell suspension, adherent in the groove for 10 min, with Tyrode’s solution continuous perfusion flow, velocity about 1~2 ml min-1, a 10 V voltage with a 0.5Hz frequency were given field stimulation triggers cell shrinkage, in high magnification to selected the ventricular muscle cells which were texture clear, with stimulation contraction to stabilize as the observation object. Application of ion imaging analysis system records calcium transients.Results:1. Screening and setting up of monoclonal hybrid cell lines.ELISA was used to detect the antibody in culture supernatant and then positive clones were selected. Following positive clone screening and continuous subcloning for three times, two strains of hybridoma cell lines, which stably secreted monoclonal antibodies against NCX α-2(840-877) were set up, and were named as #2D2, #3F10.2. Detection of the titer and purity of the acquired NCX monoclonal antibodies.(1) The titer of purified 2D2 antibody was 1 : 81920, with the affinity constant of9.33E+08. SDS-PAGE analysis showed that there are only two bands of 25 KD and 50 KD in electrophoresis lane of 2D2 antibody, which were consistent with the standard Ig G.After purification, the concentration of 2D2 antibody was 3.21 mg·ml-1.(2) The titer of purified 3F10 antibody was 1 : 40960, with the affinity constant of3.8E+09. SDS-PAGE analysis showed two clear bands with the same molecular weight of standard Ig G in electrophoresis lane of 3F10 antibody. The concentration of 3F10 antibody was 1.62 mg·ml-1.3. Effect of NCX α-2(840-877)-3F10 monoclonal antibody on INa/Ca in isolated rat ventricular myocytes.INa/Ca. was measured in voltage-clamp mode. The results showed that from 5 to 40μg·ml-1 antibody 3F10 dose-dependently inhibited INa/Ca(P < 0.05). At holding potential of+50 and-100 m V, IC50 for outward and inward currents were 11.15 and 11.69 μg·ml-1,respectively.4. Effect of NCX α-2(840-877)-3F10 monoclonal antibody on ICa-L in isolated rat ventricular myocytes.The results showed that in the range of 5~40 μg·ml-1, 3F10 antibody also had an inhibitory effect on L-type calcium channel current(P < 0.05).5. Effect of NCX α-2(840-877)-3F10 monoclonal antibody on IK1,Ito and INa in isolated rat ventricular myocytes.In the range of 5~40μg·ml-1, 3F10 antibody showed no significant effect on the inward rectifier potassium current(IK1), transient outward potassium current(Ito) and voltage-gated sodium current(INa) in adult rat single ventricular myocytes.6. Inhibitory effects of NCX α-2(840-877)-2D2and-3F10 monoclonal antibodies on ischemia reperfusion(I/R) induced cardiac arrhythmia.(1) 10 μg·ml-1 antibody 2D2 or 3F10 showed inhibitory effects on ischemic-induced cardiac arrhythmia in Langendorff-perfused rat heart. In ischemic rat group, 100% rats showed ventricular tachycardia, and 77.78% rats had ventricular fibrillation. After administration of antibody 2D2 or 3F10 5 min before ischemia, rats ventricular tachycardia rate significantly dropped to 22.2% and 25%, respectively. The total duration of ventricular tachycardia also shorten remarkably after two antibodies administration(P < 0.05).(2) Antibody 2D2 or 3F10 at 10 μg·ml-1 suppressed reperfusion-induced arrhythmia in Langendorff-perfused rat heart. During reperfusion in I/R group, 100% rats showed ventricular tachycardia, and 88.89% rats had ventricular fibrillation. After administration of antibody 2D2 or 3F10 5 min before reperfusion, rat ventricular tachycardia occurrence rate reduced to 20.09% and 44.43%, respectively, and total duration of ventricular tachycardia also decreased significantly(P < 0.05).(3) After using 2D2 or 3F10 antibody at 50 μg·kg-1 3 min before ischemia, the two antibodies both showed significantly antiarrhythmic effects in ischemic heart inanesthetized rat in vivo. Compared to ischemia model group, incidence and total duration of ventricular tachycardia, ventricular fibrillation all decreased significantly, and total number of ventricular premature beats also reduced(P<0.05). Moreover, the antiarrhythmic potential of antibody 2D2 is more comparable to lidocaine than 3F10 antibody.(4) After administrating 2D2 or 3F10 antibody at 50 μg·kg-1 3 min before reperfusion,the number of ventricular premature beats, incidence and total duration of ventricular tachycardia and ventricular fibrillation all decreased significantly during reperfusion,compared with those in I/R rat model in vivo(P<0.05). Compared with 3F10 antibody,2D2 antibody have similar antiarrhythmic effect on reperfusion-induced arrhythmia as lidocaine.7. Effect of the two monoclonal antibodies against α-2(840-877) of Na+-Ca2+ exchanger on calcium transient in isolated rat ventricular myocytes.At concentration of 5~40 μg·ml-1, antibody 2D2 and 3F10 significantly reduced amplitude of calcium transient in rat single ventricular myocytes dose dependently(P<0.05). Additionally, 2D2 antibody showed a more effective antiarrhythmic action than3F10 antibody.Conclusions:1. Using chemically synthesized NCX α-2(840-877) peptide as antigen for active immunization, two strains of anti-NCX monoclonal antibodies of 2D2 and 3F10 with high titration and binding affinity were acquired and purified for further functional investigation.2. In the range of 5~40 μg·ml-1, 2D2 and 3F10 NCX antibodies both showed dose-dependent inhibitory effect on NCX current, and the latter antibody also has a inhibitory effect on L-type calcium current. Both of the two antibodies has no obvious effects on inward rectifier potassium current, sodium current and transient outward potassium.3. The two NCX antibodies of 2D2 and 3F10 showed significant antiarrhythmiceffects on ischemia-reperfusion induced arrhythmia in rats both in vitro and in vivo, the underlying mechanism of which is mainly attributed to NCX inhibition and calcium overload reduction by the NCX antibody. |
