Effects Of Antibodies Targeting Extracellular Peptides Of Human K_Ach Channel On I_KAch And Action Potential In Human Atrial Myocytes

Part Ⅰ Preparation and verification of antibodies targeting extracellular peptides of human KAch channel in human atrial myocytesObjective:To prepare antibodies targeting extracellular peptides of human KAch channel (hKAch) by immunizing rabbits as a novel and potent IkAch blocker and verify whether the antibodies could specifically recoganize hKAch in human atrial myocytes.Methods:Two extracellular peptides of human KAch channel were chosen as antigenic determinants according to its constituent amino acid antigenic index calculated by the software DNAStar and the corresponding amino acid alignments were synthesized by PSSM8Peptide Synthesizer. Adult male New Zealand white rabbits were fortnightly immunized by subcutaneous injection of a mixture of the synthetic peptides conjugated with bovine serum albumin (BSA). Serum were collected before each immunization and the rabbits were sacrificed on63d of the experiment. Harvested serum were screened by enzymelinked immunosorbent assay (ELISA) and purified by affinity chromatography. The specific binding between antibodies and KAch channel in human atrial myocytes were detected by immunostaining and Western blotting technique.Results:By immunizing rabbits with the hapten, we. generated the polyclonal antibodies against the hKAch Kir3.1-E112and Kir3.4-E113peptides with a high titre. After three immunizations, the antibody titre in serum was markedly boosted and reached a high and stable level at the termination of the immunization. By immunostaining and Western blotting, we observed that antibodies could specifically recoganized hKAch protein expressed in human atrial myocytes, whereas they were not able to recognize Navl.5%Cavl.2、HERG、 hKCNQ1and Kv1.5proteins stably expressed in human atrial myocytes and HEK293cells. Specific recognition of hKAch protein could be completely blocked by preincubated antibodise with an excess of corresponding peptides.Conclusion:we sucesesfully generated antibodies targeting extracellular peptides of human KAch channel which could specifically recoganized hKAch protein expressed in human atrial myocytes. Part Ⅱ Effects of antibodies targeting extracellular peptides of human KAch channel on IKAch and action potential in human atrial myocytesObjective:To observe effects of antibodies targeting extracellular peptides of human KAch channel on IkAch and action potential duration (APD) in human atrial myocytes, evaluate whether the antibodies could as a novel and potent IKAch blocker for atrial fibrillation treatment.Methods:Atrial myocytes were isolated from specimens of human right atrial appendage obtained from patients receiving cardiac surgery with sinus rhythm and atrial fibrillation. The effects of antibodies targeting extracellular peptides of human KAch channel on IKAch in human atrial myocytes with sinus rhythm were recorded by using whole-cell patch clamp techniques. The antibody potently inhibiting IKAch was selected as a IkAch blocker and further investigated its effects on IKAch in human atrial myocytes with atrial fibrillation. The effects of this antibody on action potential of human atrial myocytes with sinus rhythm and atrial fibrillation were recorded by current-clamp mode with perforated patch clamp technique.Results:The Kir3.1-E112antibody had no inhibiting effect on IKAch in human atrial myocytes with sinus rhythm; The Kir3.4-E113antibody with a concentration of120,60,30nM significantly decreased IKAch in human atrial myocytes with sinus rhythm and the inhibition showed a concentration-dependence; The120nM Kir3.4-E113antibody significantly decreased IKAch in human atrial myocytes with atria fibrillation; The120nM Kir3.4-E113antibody significantly prolonged APD50and APD90in human atrial myocytes both with sinus rhythm and atria fibrillation. The inhibited effect of Kir3.4-E113antibody on IKAch and action potential could be completely blocked by preincubated the antibody with an excess of Kir3.4-E113peptide.Conclusion:The Kir3.4-E113antibody significantly inhibited IKAch and prolonged action potential in human atrial myocytes both with sinus rhythm and atria fibrillation which could be used as a potent IKAch blocker for atrial fibrillation treatment. Part Ⅲ Verification of the specificity of Kir3.4-E113antibodyObjective:To evaluate specific effects of the Kir3.4-E113antibody on IKAch, investigate the effects of this antibody on other human functional cardiac ion channelsMethods:Atrial myocytes were isolated from specimens of human right atrial appendage obtained from patients receiving cardiac surgery with sinus rhythm and atrial fibrillation. HEK293cell lines stably expressing Ikr and Iks channels were established sucesesfully. The effects of the Kir3.4-E113antibody on INa、ICa-L、IKr、Iks、Itol and IKur stably expressing in human atrial myocytes and HEK293cells were recorded by using whole-cell patch clamp and perforated patch clamp techniques. Results:Compared with control group, the Kir3.4-E113antibody had no inhibiting effect on INa、Ica-L、Ikr、 Iks、Itol and Ikur stably expressed in human atrial myocytes and HEK293cells.Conclusion:The Kir3.4-E113antibody significantly inhibited IKAch, prolonged action potential in human atrial myocytes both with sinus rhythm and atria fibrillation and had no inhibiting effect on other human functional cardiac ion channels which could be used as a novel, potent and specific IKAch blocker for atrial fibrillation treatment.