Effects Of Purified Antibody Against α-1(138-177) Of Na+/Ca2+Exchanger On Cardiac Function In Isolated Rat Hearts And On Ionic Currents In Rat Ventricular Myocytes

ObjectiveTo investigate the effects of antibody against α-1(138-177) of Na+/Ca2+exchanger after Protein A Purification on cardiac function in isolated rat hearts, and to explore the effects of antigen affinity-purified antibody against α-1(138-177) of Na+/Ca2+exchanger on Na+/Ca2+exchange current (INa/Ca). Effects of the antibody on L-type calcium current (ICa-L), inwardly rectifier potassium current (IK1) and transient outward potassium current (Ito) were also investigated in rat ventricular myocytes.Methods1. Langendorff-perfused isolated hearts:Effects of the antibody against α-1(138-177) on cardiac function were observed in Langendorff-perfused isolated hearts.2. Antigen affinity purification:Antibody against α-1(138-177) of Na+/Ca2+exchanger was purified by antigen affinity purification, and ELIS A was used to determine antibody titer.3. Whole-cell patch clamp technique:Whole-cell patch clamp technique was used to investigate the effects of the affinity-purified antibody against α-1(138-177) of Na+/Ca2+exchanger on INa/Ca, ICa-L,Ito and IK1in rat ventricular myocytes.Results1. Effects of antibody against α-1(138-177) on cardiac function in isolated rat hearts:Compared with control group, the antibody at1-10nmoL/L increased left ventricular developed pressure (LVSP-LVDP) and the maximal rate of rise LVDP (+dP/dtmax) obviously(P<0.01)(Table1, Fig1.1,1.2,1.3,1.4), and the maximal rate of fall LVDP (-dP/dtmax) decreased in Langendorff-perfused isolated hearts (P<0.01)(Table1, Fig1.1,1.2,1.3,1.4). The above effects of α-1antibody (1nmoL/L) were further strengthened by application of KB-R7943(2μmoL/L). At the concentration of102-103nmoL/L of the antibody, however, values of LVSP-LVDP and±dP/dtmax all diminished (P<0.01)(Table1, Fig1.1,1.2,1.3,1.4).2. Titer and concentration of the antibody against α-1(138-177)Titer of the antibody against α-1(138-177) reached1:32000by SA-ELISA. Concentration of antibody against α-1(138-177) was0.5mg/ml after affinity purification.(Table2.1)3. Effects of the antibody against α-1(138-177) of Na-/Ca2-exchanger on INa/Ca, ICa-L, Ik1and Ito inrat ventricular myocytesThe results showed that at the concentration of0.1-102μg/mL, antibody against a-1(138-177decreased Na+/Ca2+exchange currents in rat ventricular myocytes concentration-dependently (Table2.2, Fig2.1). Investigations on their selectivity indicated that at the concentration of102μg/mL, the antibody also inhibited L-type calcium current (Table2.3, Fig2.2). Moreover, this antibody had no significant effects on Uk1and Ito in rat ventricular myocytes(Fig2.3,2.4).Conclusions1. The present data suggested that the antibody against a-1(138-177) of Na+/Ca2+exchanger promoted cardiac contraction via inhibiting Na+/Ca2+exchange activity specifically in Langendorff-perfused rat hearts at low concentration of1-10nmoL/L, whereas at high concentration of102-103nmoL/L, the antibody depressed both cardiac contraction and relaxation.2. Antibody against a-1(138-177) was successfully affinity-purified with a-1(138-177) peptide, providing the solid basis for further investigation on its function.3. At concentrations of0.1-102μg/mL, the antigen affinity-purified antibody against α-1(138-177of Na+/Ca2+exchanger inhibited INa/Ca.102μg/mL of this antibody also can decreased ICa-L. In the concentration range from0.1to102μg/mL, this antibody had no significant effects on IK1and Ito in rat ventricular myocytes.