| Apelin, the endogenous ligand for the APJ receptor, was firstly extracted from bovine stomachs by Tatemoto in 1998 using the reverse pharmcological method. The gene encoding APJ is located on band q12 of chromosome 11, and codes for a protein of 377 amino acids and 50 to 60 kDa, depending on its glycosylation state.The apelin gene, located on band q25 to 26.1 of the X chromosome, encodes an initially translated 77-amino acid pre-proprotein, which is cleaved by proteases to shorter forms, such as apelin-36,apelin-31,apelin-17,apelin-13 and so on.Messenger RNA (mRNA) of apelin and APJ is expressed extensively in body tissues. There are particularly high concentrations in the cerebellum, vascular endothelium, heart, lung and kidney, playing important physiological roles, for instant regulating homostasis of cardiovascular system, keeping equilibrium on water and salt intake, regulating immune reactions.It is currently thought that the apelin–APJ pathway is capable of juxtacrine,paracrine and autocrine signalling.The APJ receptors are aboundantly distributed in heart and they are expressed on a number of cell types including endothelium, smooth muscle and the myocyte. APJ receptors are expressed in heart at a similar density to AT1 receptors and using radioligand-binding assays human heart tissue demonstrated a high binding affinity for apelin. In cardiovascular system, apelin is responsible for functions such as enhancing the myocardium contraction, relaxing blood vessels, and lowering blood pressure, suggesting that apelin may be an important factor in the regulation of vascular tone and cardiovascular function. In the current study, isolated rat ventricular myocytes and whole cell patch clamp were used, and the effects of apelin on INa/Ca, ICa,L and IKATP were observed in isolated rat ventricular myocytes,in order to furtherly identify the ion channel mechanism of positive inotropic effect of apelin on myocardium.Objective The current study is aimed to study the effect of apelin-13 on ion channels current such as INa/Ca, ICa,L and IKATP, in order that the ion channel mechanism of inotropic effect of apelin-13 on myocardium could be furtherly identified. Methods Male SD rat(250-300g) were scarified by decapitation after anesthesiaed to isolate the hearts. The aorta of the isolated heart was connected to a Langendorff apparatus and perfused with collagen solution. Membrane currents of the isolated rat ventricular myocytes were recorded by the whole cell patch clamp technique. INa/Ca, ICa,L and IKATP of the myocytes perfused with or without apelin-13 were recorded and analyzed.Results1.Effects of apelin-13 on INaCa in isolated rat ventricular myocytes. Apelin-13 increased both inward and outward INaCa in isolated rat ventricular myocytes. A 5±4% decrease inward INaCa and a 2±7% decrease outward INaCa were observed in the control group without apelin-13 treatment, while a 20±4% (P <0.05, n=8), 33±7% (P <0.01. n=8) and 54±10% (P <0.01, n=8) increase inward INaCa at 0.01 nmol/L, 0.1 nmol/L and 1 nmol/L, and a 1±6% (P >0.05, n=8), 7±2% (P <0.01, n=8) and 30±7% (P <0.01, n=8) increase outward INaCa were observed respectively in the group perfused with apelin-13.2.Effects of apelin-13 on ICa,L in isolated rat ventricular myocytes. Apelin-13 increased ICa, L in rat ventricular myocytes in a dose-dependent manner. After perfusion with apelin-13 at the concentration of 0.01 nmol/L, 0.1 nmol/L and 1nmol/L for 5 min, ICa, L increased by 45%±9%, 20±6% and 3±3% respectively. Apelin-13 at 0.1 nmol/L and 1 nmol/L in the perfusion group was significantly higher than that in the control group, where apelin-13 decreased by 4%±2% because of rundown (P <0.01, n=8), while there was no significant difference in apelin-13 in the 0.01 nmol/L group vs the control group. Treatment with 1nmol/L apelin-13 for 5 min shifted I-V curve downward, but it did not alter the I-V relationship and reversal potential of ICa,L. The value at V1/2 of the normalized activation conductance curve was -19.43±2.76 mV with a slope factor (k) of 4.76±0.33 mV for the control group, while it changed to -18.93±3.05 mV with a k value of 5.21±0.38 mV at V1/2 after perfusion with 1 nmol/L apelin-13. In the group without apelin-13 treatment, V1/2 of the steady-state inactivation was -23.62±1.79 mV with a k value of 5.35±0.21 mV. In the presence of apelin-13, V1/2 of the steady-state inactivation changed to -24.6±2.14 mV with a k value of 5.47±0.23. These results suggest that apelin-13 did not alter the activation and inactivation gating property of the cardiac L type calcium channel.3. Effects of apelin-13 on IKATP in isolated rat ventricular myocytesThe membrane potential was clamped -40 mV, and a ramp pulse protocol of 125 ms form 50 mV to -100 mV was used to record the quasi-steady-state current (IKATP). Pinacidil brought about a manifest increase to the current, which was completely inhibited by glibenclamide, indicating that the outward current recorded was IKATP. No change to IKATP was observed after perfusion with 1nmol/L and 10 nmol/L apelin for 5 min, indicating that apelin-13 could not open the KATP channel of cardiomyocytes in the resting condition.Conclusions Using isolated rat ventricular myocytes and the whole cell patch calmp technique, we found that apelin-13 could enhance INa/Ca and ICa,L in isolated rat ventricular myocytes, but it had no effect on IKATP, suggesting that the positive inotropic effect of apelin-13 on the myocardium may be partly attributed to its enhancing effect on INa/Ca and ICa,L in cardiomyocytes, thus directly increasing calcium influx. |
