Studies Of Electrophysiology On Rat Cardiocytes And Coronary Artery Smooth Muscle Cells By Docosahexaenoic Acid

Objective Recently,more attentions have been paid on the beneficial effects of docosahexaenoic acid(DHA) on prevention and reduction of cardiovascular diseases, especially in anti-arrhythmia,vasodilatation,lowering blood pressure,improvement of coronary artery blood flow,prevention of sudden death,and so on.However,the molecular mechanisms underlying thatω-3 PUFAs exert their cardioprotective effects are not fully understood.The study was to investigate DHA effects on resting potentials(RP),action potentials (AP),ionic currents of rat ventricular myocytes and effects on ionic currents of rat in rat coronary artery smooth muscle cells(CASMCs) by patch clamp technique.Investigating DHA mechanisms from the level of cell,molecule,and ion can provide theoretical evidences for applying rationally in clinical practice.Methods(1) RP,AP,sodium current(I_Na),L-type calcium current(I_Ca-L),transient outward potassium current(I_to),delayed rectification potassium current(I_k),and inwardly rectified potassium current(I_kl) of normal rat ventricular myocytes were respectively recorded by patch clamp after "four-step" enzyme digestion method,i.e.,perfusion with Tyrode's solution without Ca²⁺,perfusion with 50μmol/L Ca²⁺,perfusion with 200μmol/L Ca²⁺,incubation with KB solution in room temperature.Effects on RP,AP,I_Na,I_Ca-L,I_to,I_k and I_kl were observed by addition of 20,40,60,80,100 and 120μmol/L DHA respectively.(2) BK_Ca and K_V currents in individual CASMC were recorded by patch-clamp technique after CASMCs were isolated by "three-step" enzyme digestion,i.e.,incubated in buffer soluition containing 0.125%BSA for about 10min at room temperature,digested in enzymeâ… for about 20min and then for about 15 min in enzymeâ…¡.Effects on BK_Ca and K_V currents were studied by addition of 10,20,40,60 and 80μmol/L DHA respectively.Results(1) 70～90%calcium-tolerant ventricular myocytes from rat were obtained by "four-step" enzyme digestion method.(2) RP,AP and ionic currents of normal rat ventricular myocytes:a.RP of ventricular myocytes was -75.96±4.52mV(n=100).b.AP maximal velocity(Vmax),AP amplitude (APA) and AP overshoot(OS) were 226.37±15.36V/s,116.59±11.63mV,31.79±6.35mV (n=50) respectively,c.25%,50%and 90%of action potential durations(APD₂₅,APD₅₀, and APD₉₀) of ventricular myocytes were 4.85±1.36ms,11.76±2.23ms and 52.81±5.33ms(n=50),respectively,d.I_Na:I_Na currents at -30mV were-1183.71±315.62pA, and corresponding current densities were -7.86±2.11pA/pF(n=50).50%stably activated potential was -44.12±2.68mV;50%stably inactivated potential was -81.52±2.07mV;the fast and slow recovered time from inactivation wasÏ„₁=0.66±0.32ms,Ï„₂=10.56±2.51ms.e. I_Ca-L:I_Ca-L currents at 0mV were-949.52±213.51pA,and corresponding current densities were -6.30±1.42 pA/pF(n=60).50%stably activated potential was-16.67±2.46mV;50% stably inactivated potential was -34.03±0.57mV;recovered time from inactivation was 91.41±10.26 ms.f.I_to:I_to currents at +70mV were 4936.52±653.61pA,and corresponding current densities were 32.91±4.36 pA/pF(n=50).50%stably activated potential was36.39±4.17 mV;50%stably inactivated potential was -25.68±1.01 mV;recovered time from inactivation was 83.07±10.25ms.g.I_k:I_k currents at +60mV were 1530.15±512.2pA, and corresponding current densities were10.20±3.41pA/pF(n=30).50%stably activated potential was35.71±3.81mV;50%stably inactivated potential was -33.36±1.07mV; recovered time from inactivation was 168.18±16.67ms.h.Peak currents and current densities of I_kl:I_kl currents at -120mV were -4735.5±721.6pA,and corresponding current densities were -31.57±4.81pA/pF(n=28).(3) Effects on basic electrophysiology of rat ventricular myocytes by DHA:RP,Vmax, APA and OS were not significant(P＞0.05,n=10),but APD changed after application 20, 40,60,80,100 and 120μmol/L DHA.APD₂₅ were 4.85±1.36ms,5.76±1.92ms, 7.28±2.21ms,10.97±3.25ms,13.26±4.19ms,14.35±4.96ms and 15.83±5.26ms respectively (P＜0.05,n=10).APD₅₀ were 11.76±2.23ms,13.21±3.02 ms,14.59±3.37ms,18.92±4.16ms, 20.53±4.77ms,22.19±5.34ms and 23.87±5.66ms respectively(P＜0.05,n=12).APD₉₀ were 52.81±5.33ms,56.64±5.73ms,59.16±6.11ms,88.45±8.92ms,103.37±10.25ms, 121.57±11.81ms and 133.65±12.34ms respectively(P＜0.05,n=10).(4) Effects on ionic currents of ventricular myocytes by DHA:when 20,40,60,80,100 and 120μmol/L DHA were gradually applicated:a.I_Na were gradually blocked,â… -â…¤curves were upward,stably inactivated curves were shifted to the left,and recovered time from inactivation was prolonged(P＜0.05,n=10),and stably activated curves were no remarkable significance(P＞0.05,n=10).I_Na was blocked to 1.51±1.32%,21.13±4.62%, 51.61±5.73%,67.62±6.52%,73.49±7.59%and 79.95±7.62%under manding potential equal to -30mV(P＜0.05,n=10),and 50%inhibition concentration(IC₅₀) of DHA was 47.91±1.57μmol/L.c.I_Ca-L were gradually blocked,â… -â…¤curves were upward,stably inactivated curves were shifted to the left,and recovered time from inactivation was prolonged with augmentation of DHA(P＜0.05,n=10),and stably activated curves were no remarkable significance(P＞0.05,n=10).I_Ca-L was blocked to 2.72±1.63%,21.97±3.35%, 44.16±4.59%,67.89±4.87%,70.51±5.39%and 72.32±5.57%under manding potential equal to 0mV(P＜0.05,n=10),and IC₅₀ of DHA was 52.01±3.24μmol/L.d.I_to were gradually blocked,â… -â…¤curves were downward,stably inactivated curves were shifted to the left,and recovered time from inactivation was prolonged with augmentation of DHA (P＜0.05,n=10),and stably activated curves were no remarkable significance(P＞0.05, n=10).I_to was blocked to 2.61±0.26%,21.79±4.85%,63.11±6.57%,75.52±7.26%, 81.82±7.63%and 84.33±8.25%under manding potential equal to +70mV(P＜0.05,n=10), and IC₅₀ of DHA was49.11±2.68μmol/L,e.I_K were gradually blocked,â… -â…¤curves were downward,stably inactivated curves were shifted to the left,and recovered time from inactivation was prolonged with augmentation of DHA(P＜0.05,n=10),and stably activated curves were no remarkable significance(P＞0.05,n=10).I_to was blocked to 2.78±0.26%,27.23±3.97%,64.18±6.73%,77.59±7.36%,83.26±8.31%and 87.93±9.35% under manding potential equal to +60mV(P＜0.05,n=10),and IC₅₀ of DHA was47.52±2.32μmol/L,f.DHA at different concentrations did not have any effect on I_Kl (P＞0.05,n=10).(5) BK_Ca and K_V currents of normal rat CASMCs:a.Open probability(Po) of BK_Ca channel in individual CASMCs were observed using a patch-clamp technique in an inside-out configuration.Under manding potential equal to +20,+40,+60,+80 and +100mV,Po of BK_Ca channel were 0.009±0.001,0.017±0.002,0.072±0.003,0.362±0.043 and 0.637±0.071 respectively(P＜0.05,n=10),and half activated voltage and slope rate of BK_Ca channel was 79.47±4.68mV and 8.53±0.14.Conductance of BK_Ca channel was 230.42±20.13pS(n=10).b.Peak currents and current densities of IBK_Ca under whole-cell configuration:BK_Ca currents at +150mV were 784.81±261.63pA,and corresponding current densities were 68.24±22.75pA/pF(n=20).BK_Ca tail currents at +90mV were 1046.73±113.67pA,and corresponding current densities were 91.02±13.52pA/pF(n=20).d. Peak currents and current densities of IK_V under whole-cell configuration:K_V currents at +50mV were 553.85±106.77pA,and corresponding current densities were 48.16±9.28pA/pF(n=20).K_V tail currents at +40mV were 388.24±81.26pA,and corresponding current densities were 33.76±7.68pA/pF(n=20)(6) Effects on BK_Ca and K_V currents of normal rat CASMCs by DHA:a.Po of BK_Ca channel at +60mV were 0.072±0.003,0.077±0.004,0.136±0.070,0.436±0.083, 0.592±0.109 and 0.676±0.115(P＜0.05,n=10) by addition of 0,10,20,40,60 and 80μmol/L DHA respectively,and IC₅₀ of DHA on BK_Ca channel was 36.30±2.15μmol/L. Curves of relative Po and manding potential relation were shifted to the left under manding potential equal to +20,+40,+60,+80 and +100mV respectively,and half activated voltage and slope rate of BK_Ca channel was 69.16±3.57mV and 10.16±0.20 after addition of 35μmol/LDHA,b.IBK_Ca were gradually increased,â… -â…¤curves were upward,and BK_Ca tail currents were gradually increased,BK_Ca tail currentsâ… -â…¤curves were upward with augmentation of DHA(P＜0.05,n=10),and stably activated curves were no remarkable significance(P＞0.05,n=10).IBK_Ca were increased to 6.1±0.3%,76.5±3.8%,248.0±12.3%, 374.2±18.7%and 443.1±22.1%(P＜0.05,n=10) with 20,40,60,80 and 100μmol/L of DHA under manding potential equal to +150mV,and IC₅₀ of DHA was 36.22±2.17μmol/L. BK_Ca tail currents were increased to 10.12±0.33%,146.12±4.97%,305.62±9.89%, 461.85±14.92%and 669.22±20.37%with augmentation of DHA under manding potential equal to +90mV(P＜0.05,n=10),c.IK_V and K_V tail currents were gradually blocked,â… -â…¤curves were downward,stably activated curves were shift to the right,and stably inactivated curves were shifted to the left.IK_V were decreased to 4.57±0.23%, 10.77±0.54%,35.32±1.76%,70.25±3.51%and 78.51±3.93%(P＜0.05,n=10) with 20,40, 60,80 and 100μmol/L of DHA under manding potential equal to +50mV,and IC₅₀ of DHA was 42.19±1.59μmol/L.K_V tail currents were decreased to 6.65±0.27%,15.22±2.76%, 43.59±7.82%,73.80±11.79%and 83.02±13.65%with augmentation of DHA under lnanding potential equal to +40mV(P＜0.05,n=10).Conclusion(1) Effects on Electrophysiology of rat ventricular myocytes:a.APDs are gradually prolonged with augmentation of DHA,but DHA has no effects on Vmax,APA and OS.b.DHA have blocked effects on I_Na,I_Ca-L,I_to and I_K,stably inactivated curves of these currents were shifted to the left,and recovered time from inactivation were prolonged with augmentation of DHA,and stably activated curves were no remarkable significance,c. DHA with different concentrations has no effects on I_Kl and its channel dynamics,d.The blocked effects of DHA on I_Na,I_Ca-L,I_to and I_K,are beneficial to prevent arrhythmia and sudden death.(2) Effects on Electrophysiology of rat CASMCs:a.DHA actived BK_Ca channel in an inside-out configuration.Curves of relative Po and manding potential relation were shifted to the left.b.DHA actived BK_Ca channel in an whole-cell configuration,and stably activated curves were no remarkable significance,c.DHA blocked K_V channel,tably activated curves were shift to the right,and stably inactivated curves were shifted to the left.d.The activation effects on BK_Ca channel by DHA are larger than its inhibitation effects on K_V channel.The complex action on BK_Ca channel and K_V channel induced by DHA result in vasodilatation.