| Backgrounds and Objectives:Cytokine VEGF(165)is a potential target for the treatment of ischemic heart disease and heart failure.Studies have confirmed that VEGF165 not only promotes the formation of ischemic regional neovascularization,but also plays a role in inhibiting myocardial cell apoptosis,reducing fibrous tissue formation and improving the success rate of stem cell transplantation.These results suggest that VEGF165 can affect the activity of cardiomyocytes themselves as well.When using different concentrations of VEGF165 protein,the mechanical activity and electrical activity of cardiomyocytes may change.L-type calcium channel is an important voltage-dependent calcium channel on the cardiomyocyte membrane,and calcium ions that enter through the L-type calcium channel are of great importance to cardiac excitation-contraction coupling(EC coupling)by initiating calcium-induced calcium release(CICR)and contributes to action potential plateau.The effect of VEGF165 on the electrical activity of cardiomyocytes was investigated by studying the changes of L-type calcium channel currents and action potentials in cardiomyocytes under the intervention of different concentrations of VEGF165 protein.Methods:The guinea pig ventricular myocytes were isolated.The current clamp and the voltage clamp were performed by patch clamp techniques.1.Voltage clamp testsFirstly,nifedipine was used to examine the voltage clamp set to get isolated ventricular myocyte membrane L-type calcium current(whole-cell).The isolated ventricular myocytes were divided into 10,40,100 and 300 ng/ml VEGF165 intervention groups.The current-voltage curve,the maximum current value,the L-type calcium channel steady-state activation curve and inactivation curve were recorded at different concentrations of VEGF165.At the levels of 100 ng/ml and 300 ng/ml VEGF165,the current-voltage curve and the normalized current were detected and compared before and after using SU5416,a VEGF type-2 receptor inhibitor.2.Current clamp testsUnder the intervention of 300 ng/ml VEGF165,the resting membrane potential,the action potential duration 50,the action potential duration 90,the action potential amplitude and the maximum voltage change rate were observed and compared with the control.Results:1.The voltage clamp protocol for ICa,L was confirmed successfully.The normalized current was 0.04 ± 0.02 in nifedipine group(P < 0.05 versus control group).2.VEGF165 increased L-type calcium current by a dose-dependent manner in guinea pig ventricular myocytes.Compared with the control group without VEGF165,the standardized L-type calcium current was significantly increased in 40(-1.070 ± 0.049),100(-1.170 ± 0.042)and 300 ng/m L(-1.220 ± 0.060)VEGF165 intervention groups(P < 0.05).Compared with 40 ng/m L group,the standardized L-type calcium current was significantly increased in 100 and 300 ng/m L VEGF165 intervention groups(P < 0.05).3.The steady-state activation and steady-state inactivation curves of L-type calcium channels in ventricular myocytes were compared between 100 ng/m L VEGF165 intervention and control groups.The results showed no significant difference between the half maximally activated potential(V1/2)and the slope factor(k).4.VEGFR2 inhibitor significantly eliminated 100 ng/m L(-1.170 ± 0.019 vs-0.923 ± 0.033,P < 0.05)and 300 ng/m L(-1.220 ± 0.025 vs-0.911 ± 0.030,P < 0.05)VEGF165-mediated L-type calcium current increase.5.Before and after high concentrations of VEGF165 intervention,APD90 and other parameters of AP did not change significantly in isolated ventricular myocytes.Conclusions:In the range of 10-100 ng/m L,VEGF165 can enhance the L-type calcium current in ventricular myocytes by binding to VEGFR2 on the cell membrane in a dose-dependent manner.VEGF165-mediated calcium current enhancement has nothing to do with the voltage-gated characteristics of L-type calcium channels in ventricular myocytes,and does not affect the action potential of cardiomyocytes,which contributes to the stability of cardiac electrical activity. |
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