Effects Of Prostaglandin E₁ Pre-treament On Potassium Channels In Ischaemia/reperfusion Cardiac Myocyte

Target thrombolytic therapy,percutaneous transluminal angioplasty,coronary artery bypass surgery and cardiac surgery extracorporeal circulation are widely used in clinical practices for treating cardioischemia diseases.Evidences from animal studies and clinical practices,however,with the relief of local ischemia situation, further damage to myocardial cells can be often observed from some patients and animals,which is called ischemia/reperfusion injury,involving in myocardial functional,metabolic and ultrastructural changes.One manifestation of myocardial dysfunction changes is reperfusion arrhythmia,especially ventricular arrhythmia which has been considered the main cause of sudden death after relief of coronary ischemia.Molecular basis of cadiocyte bioelectricity action is membrane flow of ions exterior and interior through definite ion channels.It has been well known that K⁺ channels are ubiquitous membrane proteins.They play critical roles in a wide variety of biological processes,from electrophysiological actions to metabolic actions,such as regulating heart rate,muscle contraction,neurotransmitter release,neuronal excitability,insulin secretion,epithelial electrolyte transport,cell volume regulation and cell proliferation.Therefore,K⁺ channels have been recognized as potential therapeutic target in many fields.Previous studies have been shown that pre-treating ischemia/reperfusion cardiac myocytes with Prostaglandin E1(PGE1) have potential cardioprotective effects,such as increasing remarkably antioxygen cardiac myocytes, inhibiting cardiac myocyte apoptosis,diminishing infarction areas,promoting vascular proliferation in ischemia areas,improving hemodynamics and function of ischaemia/reperfusion myocardium,reducing reperfusion arrhythmia frequencies and duration.However,the mechanism of PGE1's cardioprotective effects is still unknown. And there were seldom reports about whether pretreatment with PGE1 have effects on subtype potassium channels such as ATP-sensitive potassium,transient outward potassium channel,inward rectifying potassium channel and delayed rectifying potassium channel.So,using patch-clamp technique,we investigated whether PGE1 exert its cardioprotective effects through regulating the activity of K⁺ channels in vitro ischemia/reperfusion myocardium cells.Objective:To study the effects of PGE₁ pre-treament on ATP-sensitive potassium current(I_K(ATP)),transient outward potassium current(I_to),delayed rectifying potassium current(I_k),inward rectifying potassium current(I_K1)on ischaemia/ reperfusion ventricular myocytes of rat or guinea pig.Methods:Isolated heart perfusion according to langendorff to built ischaemia/reperfusion model.Single ventricular myocyte was obtained from rat or guinea pig using enzymatic dissociation techniuqes.The whole-cell Patch clamp method was used to record the I_K(ATP),I_to,I_K1 and I_K at room temperature 18～28℃.Experiments were divided into five groups:normal control group;ischaemia/reperfusion group; PGE₁ low-dose pre-treament group(14μg/L);PGE₁ middle-dose pre-treament group(42μg/L);PGE₁ high-dose pre-treament group(126μg/L);Data were analyzed by spss 10.0 software and demonstrated with mean±sd,Mean value were compared by one-factor analysis of variance,P＜0.05 was considered statistically significant level. Practical cells of currents obtained come from different animals in each group were taken as statistical sample capacity and represented with the letter of "n".Results:1.Effects of PGE₁ pre-treament on I_K(ATP) in guinea pig ischaemia/reperfusion ventricular myocyte.K_ATP channel was closed in normal physiological condition and I_K(ATP) was not nearly observed.At 0mV of ramp pulse protocol,I_K(ATP) of ischaemia/reperfusion group was 1.31±0.11pA/pF(n=9);compared with ischaemia/reperfusion group, pre-treament with different dose of PGE1 all significantly increased I_K(ATP),and I_K(ATP) density respectively were 2.10±0.14pA/pF(n=11,P＜0.01),2.81±0.29 pA/pF(n=10, P＜0.01) and 4.06±0.29pA/pF(n=12,P＜0.01),additionally,there were significant differences between PGE₁ pre-treatment groups.At 0mV of voltage- clampe rectangular pulse protocol.I_K(ATP) of ischaemia/ reperfusion group was 0.24±0.34pA/pF(n=11),compared with ischaemia/reperfusion group,pre-treament with different doses of PGE₁ all remarkably increased I_K(ATP) current,I_K(ATP) respectively were 0.57±0.14pA/pF(n=13,P＜0.01),0.82±0.20pA/pF(n=11,P＜0.01),1.12±0.22 pA/pF(n=13,P＜0.01).In addition there were differences between groups of PGE₁ pre-treament(P＜0.05).Although assay methods of I_K(ATP) were different,conclusions were consistent with each other.2.Effects of PGE₁ pre-treament on I_to in rat ischaemia/reperfusion ventricular myocytes.At +60mV of voltage-clamp rectangular pulse protocol.I_to decreased from 15.54±2.24pA/pF(n=16) of normal control group to 9.99±2.03pA/pF of ishaemia/ reperfusion group(n=16,P＜0.01).Compared with ishaemia/reperfusion group, pretreatment with different doses of PGE1 all significantly increased I_to,and all of which were 14.24±1.97pA/pF(n=17,P＜0.01),18.41±1.39 pA/pF(n=13,P＜0.01), 21.63±3.20pA/pF(n=12,P＜0.01) respectively;additionally,significant difference were found between groups of PGE₁ pre-treament(P＜0.05).The steady-state inactivation curve of ishaemia/reperfusion was shifted to the depolarizing direction and the half-inactivation voltage(V_1/2) of Ito changed from -25.50±6.68mV of normal control group(n=16) to -18.61±7.81mV(n=10,P＜0.05),compared with ischemia/ reperfusion group,different doses of PGE1 pre-treament all shifted steady-state inactivation curve of Ito to left and V_1/2 were -27.95±8.00mV(n=11,P＜0.05),-31.34±7.59mV(n=16,P＜0.05) and -39.50±7.38mV(n=13,P＜0.05) respectively.3.Effects of PGE₁ pre-treament on I_k1 in guinea pig ischaemia/reperfusion ventricular myocytes.At -120mV of voltage-clamp rectangular pulse protocol.I_k1 of normal group ventricular myocytes was -33.93±9.71 pA/pF(n=24),however,it markedly decreased to -11.68±3.82pA/pF in ischaemia/reperfusion group(n=6,P＜0.01).Compared with ischaemia/reperfusion group,pre-treament with different doses of PGE₁ all significantly increased components of I_k1,values of which were -31.89±8.83 pA/pF (n=7,P＜0.01),-34.71±8.99pA/pF(n=13,P＜0.01),-32.36±9.13 pA/pF(n=11,P＜0.01) respectively.But there were no differences among PGE₁ pre-treament groups.4.Effects of PGE₁ pre-treament on I_K in guinea pig ischemia/reperfusion ventricular myocytes.At +60mV of depolarization and -30mV repolarization of Voltage-clamp rectangular pulse protocol.I_K-peak and I_K-tail were reduced from 6.01±0.76pA/pF and 2.47±0.13pA/pF of normal group(n=12) to 2.82±0.25pA/pF and 1.7±0.11pA/pF of ischaemia/reperfusion group(n=9,P＜0.05) respectively.I_K-peak and I_K-tail of PGE₁ pretreament groups were 3.30±0.18pA/pF and 1.81±0.07 pA/pF(n=14),2.98±0.13 pA/pF and 1.81±0.15pA/pF(n=13),3.10±0.26pA/pF and 1.82±0.07pA/pF(n=11). However,there was no distinction compared with ischemia/reperfusion group.Conclusions:1.In myocardial cells,I_to,I_K1 and I_K are inhibited after ischemia/reperfusion compared with normal physiological state.2.Treating myocardial cells with PGE₁ before ischemia/reperfusion lead to increased I_K(ATP) and Ito in a dose dependent manner,suggesting PGE₁ potentiating the opening of K_ATP and I_to channels after ischemia/reperfusion.3.PGE₁ pre-treatment also increased components of I_K1 after ischemia/reperfusion, Though it don't change the inward rectification property of I_K1 even at high dose.4.PGE₁ pre-treatment don't show a significant impact on I_K after ischemia/ reperfusion.5.PGE₁ pre-treatment may exert its cardioprotective effects through regulating the activities of distinct potassium channels.