Monoclonal Antibody NCX-2D2 Inhibited Isoproterenol-induced Arrhythmias In Adult Rats And Investigation On Its Mechanism

Objective:1.To establish an isoproterenol（ISO）-induced adult rat model of arrhythmia in vitro and in vivo,and to observe the effect of sodium-calcium exchanger NCX-2D2 antibody on arrhythmia in model rats.2.The whole-cell patch clamp technique was used to investigate effects of the NCX-2D2 antibody on Na⁺/Ca²⁺exchange current(I_Na/Ca),L-type calcium current(I_Ca-L)in voltage-clamp mode and DADs in current-clamp mode in single rat ventricular myocyte to analyze its possible electrophysiological mechanism.Methods:1.Establishing isoproterenol-induced rat model of arrhythmia in vitro and in vivo.（1）Establishment of isoproterenol-induced rat cardiac arrhythmia model in vitro.The healthy SD rats were randomly selected and anesthetized by intraperitoneal injection of 1%pentobarbital sodium(40 mg·kg^-1).The heart was removed quickly after opening the chest.The heart was excised and suspended on a Langendorff perfusion device.The aorta retrogradely was perfused by pure oxygen-saturated thermostatic（37°C）with high calcium Tyrode’s solution through.The flow rate was controlled by a peristaltic pump at a rate of 8 ml·min^-1.After ECG waveform was recorded 30 minutes to make the heart constraction stable,starting to use drug pump administration.The electrocardiogram recorded the number of premature ventricular contractions,the incidence and duration of ventricular tachycardia,and the incidence and duration of ventricular fibrillation within 30 minutes after drug intervention.Grouped as follows:1)Control group:administrating CaCl₂(2.5 mmol·L^-1);2)2D2 group:administrating NCX-2D2 antibody(10μg·ml^-1)and CaCl2(2.5mmol·L^-1);3)ISO group:administrating ISO(1μmol·L^-1)and CaCl₂(2.5 mmol·L^-1);4)2D2+ISO group:administrating NCX-2D2 antibody(10μg·ml^-1),ISO(1μmol·L^-1)and CaCl₂(2.5 mmol·L^-1).（2）Establishment of isoproterenol-induced rat cardiac arrhythmia model in vivo.The healthy SD rats were randomly selected and anesthetized by intraperitoneal injection of 1%pentobarbital sodium(40 mg·kg^-1),the rats was fixed on a rat board in a supine position.The BL-420F bio-functional experimental system was used to record the electrocardiogram of rat standard limb lead.The electrocardiogram was continuously observed for more than 30 minutes and then administered drug through the tail vein.The body surface electrocardiogram was observed in each group.The number of ventricular premature beats,the incidence and duration of ventricular tachycardia,the incidence and duration of ventricular fibrillation within 1 hour after drug intervention were recorded.Grouped as follows:1)Control group:normal saline（NS 1 ml）was injected into the tail vein;2)2D2 group:NCX-2D2 antibody(80μg·kg^-1)was dissolved in 1 ml NS and was administered to tail vein;3)ISO group:ISO(1.28 mg·kg^-1)was dissolved in 1 ml NS and was administered to tail vein;4)2D2 antibody+ISO group:NCX-2D2 antibody(80μg·kg^-1)was dissolved in 0.5ml NS and was administered to tail vein,5 minutes later,ISO(1.28 mg·kg^-1)was dissolved in 0.5 ml NS and was administered to tail vein;5)Amiodarone+ISO group:Amiodarone(5 mg·kg^-1)was dissolved in 0.5 ml NS and was administered to the tail vein of and 5 minutes later,ISO(1.28 mg·kg^-1)was dissolved in 0.5 ml NS and injected into the tail vein.2.Isolation of single rat ventricular myocytes.Healthy SD rats were randomly selected and anesthetized by intraperitoneal injection of 1%pentobarbital sodium(40 mg·kg^-1).The carotid artery bled and the heart was removed immediately after thoracotomy and placed on pure oxygen saturated calcium-free Tyrode’s solution in 4°C.Extra tissue was trimmed and suspended in the Langendorff perfusion device through the retrograde aorta.After perfusion of the heart with Ca²⁺-free Tyrode’s solution for 8 to 10 minutes,perfusion with an enzyme solution was performed for 15 to 20 minutes.After the cardiac enzyme was digested,the left ventricular muscle tissue was cut and put it into the KB solution and squirted it with a straw for 3 to 5 minutes.Filters（100 mesh pore size）were used to obtain single ventricular myocyte,which were placed in KB solution and kept stable.3.The whole-cell patch clamp was used to record ion channels.Pipette the cell suspension 1² drops in the cell of the inverted microscope（about 1ml）,adhere to the wall for 3⁵ min,and refill with calcium containing 1.8 mmol·L^-1CaCl₂ solution(flow rate 2 ml·min^-1).The glass electrode was drawn and filled with an electrode drawing device to record the current in the electrode.The electrode resistance is controlled at 3⁵ MΩ.Under the microscope,the long rod-shaped cells with clear texture without self-contraction were selected as the experimental subjects.After the negative pressure seal was given with the glass electrode,the negative pressure was applied to break the membrane,and after membrane capacitance compensation and series resistance compensation were performed,whole cells were harvested.In the voltage clamp mode,Na⁺/Ca²⁺exchange currents(I_Na/Ca)and L-type calcium currents(I_Ca-L)were recorded in the voltage-clamp mode of single rat ventricular myocyte,and the action potential（AP）of single rat ventricular myocyte were recorded in the current clamp mode.Conditioned stimuli（15 ms 3 Hz）were used to induce delayed afterdepolarization（DADs）.Clampex10.3 software was used for data acquisition and analysis.Results:1.Monoclonal antibody NCX-2D2 can significantly inhibit isoproterenol-induced arrhythmia in vitro rats.The NCX-2D2 antibody(10μg·ml^-1)significantly inhibited isoproterenol-induced isolated heart arrhythmias in rats.In isolated rat heart isoproterenol group,the incidence of ventricular tachycardia was 100%,and the incidence of ventricular fibrillation was57.14%;after intervention with NCX-2D2(10μg·ml^-1)antibody,during the observation period（30 minutes）,ventricular fibrillation completely disappeared;the incidence of ventricular tachycardia decreased to 42.8%,and the duration was significantly shortened from（187.81±23.14）s to（11.29±15.84）s（P<0.01）;the number of premature beats decreased from 328±22 to 104±9（P<0.01）.2.Monoclonal antibody NCX-2D2 can significantly inhibit the occurrence of arrhythmias induced by isoproterenol in anesthetized rats.NCX-2D2 antibody(80μg·kg^-1)significantly inhibited the occurrence of arrhythmias in isoprenaline-induced anesthetized rats.In the isoproterenol group,ventricular tachycardia occurred in 100%of the rats and ventricular fibrillation occurred in 57.14%of the rats.After intervention with 80μg·kg^-1 of NCX-2D2 antibody 5 minutes earlier,during the observation period（1 hour）ventricular fibrillation completely disappeared in rats;the incidence of ventricular tachycardia decreased to 42.8%,and duration decreased significantly from（40.73±3.06）min to（0.90±1.13）min（P<0.01）;the number of ventricular premature beats decreased from 774±31 to 168±33（P<0.01）.After comparison,it was found that 80μg·kg^-1 of NCX-2D2 antibody inhibits isoproterenol-induced ventricular arrhythmias in anesthetized rats similar to amiodarone,and is even slightly better than amiodarone in inhibiting ventricular premature beats.3.Monoclonal antibody NCX-2D2 can partially inhibit the increase of sodium-calcium exchange current(I_Na/Ca)induced by isoproterenol in isolated rat ventricular myocyte.The I_Na/Ca of isoproterenol group was 8.71±0.12 and-7.59±0.11（pA/pF）at+50mV and-100mV respectively,which was significantly increased（P<0.01）than that of control group（3.95±0.16 and-3.85±0.12 pA/pF）.The outward and inward currents of I_Na/Ca at+50 mV and-100 mV were reduced to 6.31±0.12 and-6.11±0.10（respectively）,after 5μg·ml^-1 NCX-2D2 antibody was administered with isoproterenol elution（pA/pF）,which was significantly lower than the isoproterenol group（P<0.01）,but did not return to normal control group level.4.Monoclonal antibody NCX-2D2 can partially inhibit the increase of L-type calcium current(I_Ca-L)induced by isoproterenol in a single rat ventricular myocyte.The results showed that the I_Ca-L of isoproterenol group was-6.75±0.22（pA/pF）,which was significantly higher than that of the control group-4.33±0.20（pA/pF）（P<0.01）.I_Ca-L decreased to-5.50±0.20（pA/pF）with 5μg·ml^-11 NCX-2D2 antibody and significantly decreased compared with isoproterenol group,but did not return to normal control group level（P<0.01）.5.Monoclonal antibody NCX-2D2 can effectively inhibit isoproterenol-induced delayed afterdepolarization in single rat ventricular myocyte.The results showed that the incidence of DADs in single rat ventricular myocyte was 85.71%in isoproterenol group,and the incidence of DADs in single rat ventricular myocyte was 14.29%after intervention with 5μg·ml^-1 NCX-2D2 antibody.Compared with isoproterenol group,the incidence of DADs in single rat ventricular myocyte was significantly decreased（P<0.05）in antibody intervention group（isoproterenol and 2D2antibody group）.Conclusion:The sodium-calcium exchanger monoclonal antibody NCX-2D2 significantly inhibits isoproterenol-induced ventricular arrhythmias in rats.The mechanism against ventricular arrhythmias is mainly due to its inhibition of cardiomyocyte sodium-calcium exchanger and L-type calcium channel and marked suppression of DADs in rat ventricular myocytes.