The Acute And Chronic Effects Of Amiodarone On Currents In Isolated Ventricular Myocytes Of Hypertrophied Rats

Objective Amiodarone is the most promising drug in the treatment of life-threatening supraventricular and ventricular tachyarrhythmias. The pharmacological actions of this compound are very complex and the profile of amiodarone is different between its acute and chronic effects. In the past we always used normal myocytes as research objects, but didn't know if there were differences between normal and pathosis myocytes. So in our research we compared the acute and chronic effects of amiodarone on I_Na, I_Ca-L, I_k, I_Ks, I_Kl, I_to between normal and hypertrophied myocytes, and hoped to demonstrate electrophysiologic changes in cardiac hypertrophy and its clinical meanings .Methods The pressure overload hypertrophy models of rats were established by partial ligation of ascending aorta for 4 weeks. To observe the short-term effects of amiodarone , cells were exposed acutely to 0.01, 0.1, 1,10, 50μmol/L amiodarone. To observe the long-term effects of amiodarone, the hypertrophied and normal rats were administered oral amiodarone (100mg/kg/d) for 4 weeks before cell isolation. Then whole cell patch-clamp techniques were used to study the acute and chronic effects of amiodarone on the transmembrane ionic currents in rat single ventricular myocyes isolated from normal ,pressure overload rats and the rats treated with amiodarone for 4 weeks .Results (1) acute research:1. L-type Calcium current(Ica-L): ①Amiodarone decreased I_Ca-L in a concentration-dependent manner on normal myocytes, and its IC50 was 10.38umol/L. In cells exposed to acute amiodarone (1 , 10μmol/L) superfusion, the acute and chronic deactivation time constant didn't change, but activation time constant prolonged significantly. ?While for hypertrophied myocytes, activation voltage, peak voltage, summit current density were same as control, and the activation and acute deactivation time constants had also no significant differences. But the chronic deactivation time constant prolnged significantly (P<0.01) . (3) Amiodarne was insensitive to Ica-L of hypertrophied cells andthe current density was only reduced by 50umol/L amiodarone by 16.73±8.03%. But amiodarone superfusion can shorten chronic deactivation time constant.2. Sodium current(INa) : ?Amiodarone decreased INa in a concentration-dependent manner in normal myocytes, and its IC50 was 9.19 umol/L . In cells exposed to acute amiodarone (1, lOumol/L) superfusion, the deactivation time constant didn't change, but activation time constant prolonged significantly. ?While for hypertrophied myocytes, activation voltage> peak voltage, summit current density were same as control, and the activation and deactivation time constants had also no significant differences. (3) Amiodarne was more sensitive to INa on hypertrophied cells and its IC50 was 5.88 umol/L . Amiodarone superfusion can shorten activation time constant and had no effects on deactivation time constant.3. Delayed rectifier outward K+ current(Iic) and slowly activating delayed rectifier outward K+ current^): ?Amiodarone decreased Ik in a concentration-dependent manner in normal myocytes, 1 > 10 and 50 umol/L amiodarone decreased current density of Ik by 20.98±3.97°/ck 31.18 + 5.1% and 61.52+5.97%. (2) The peak current density of IKon hypertrophied myocytes (5.08±0.35 pA/pF) was lower than control (7.54±1.61 pA/pF) . (3)The sensibility of amidarone on hypertrophied cells was moderate lower than control, and K 10 and 50 umol/L amiodarone decreased 15.51 ±4.03%. 24.64±6.82% and 62.28 + 5.48% current density of Ik on hypertrophied myocytes. So we can see the inhibition percentages of 1 and 10 umol/L amiodarone on hypertrophied myocytes were lower than control. ?In normal myocytes, Iks occupied 78% the component of Ik. K 10 and 50 Umol/L amiodarone decreased current density of Iks by 13.24 ± 4.19% > 21.56 ± 5.62% and 48.25 ±5.15% on control myocytes. ?The current density of Iks on hypertrophied myocytes (3±0.82pA/pF) was lower than control (5.68+0.56pA/pF) significantly. Iks occupied 70% the component of Ik. ?The inhibition potent of amiodarone on hypertrophied cells was higher than control, and 1 > 10 and 50 umol/L amiodarone decreased 22.33 ± 3.77%> 42.7 ±3.9% and 65.14 ±4.74% current density of IKs on hypertrophied myocytes. Concentration-effective curve forecasted that concentration corresponding to a 50% current inhibition (IC50) of IKs was 10.2umol/L.4. Inward rectifier K+ current (Iki): ?High concentration amiodarone candecreased Iki on normal myocytes, 10u.mol/L amiodarone began to reduce inward current density of IKi by 13.43%, while 50u.mol/L amiodarone decreased about 24.01% .Samely 10, 50u.mol/L amiodarone decreased outward current density of IKi by 8.65%n 24.25% .(2) The current density of IKi on hypertrophied myocytes was lower than control, and the inward and outward current density of Iki on hypertrophied and normal cells were -22.82 ±5.34 pA/pF and -15.75 ± 1.19pA/pF (P=0.021), 10.16 ±0.2 pA/pF and 7.83 ± 1.35pA/pF (P=0.014) respectivelyo ?The sensibility of amidarone on hypertrophied cells was lower than control, and 0.0 K 0. K K 10u.mol/L amiodarone didn't change Iki , while 50u.mol/L amiodarone can only decrease 16.73% inward current density of Iki , and had no effects on outward current.5. Transient outward K+ current (It0): ? Hypo-concentration amiodarone had no effects on Ito on normal myocytes. Higher concentration amiodarone had inhibition action. 10 and 50u.mol/L amiodarone decreased 37.17±6.42% and 55.88±5.5% summit current density of Ito. (D The peak current density on hypertrophied cells (27.71 + 1.93 pA/pF ) was lower than control (32.89 ±12.45 pA/pF). But activation voltage> activation time constant > acute and chronic deactivation time constants had no changes between them.(3) The inhibition action of amidarone on hypertrophied cells was lower than control, and 10, 50u.mol/Lamiodarone decreased (9.45±2.14)% and (23.03±2.83)% summit current density of Ito. Its IC50 must be higher than 50u.mol/L. Amiodarone superfusion(K 10u.mol/L) had no effects on chronic deactivation time constant and 10u.mol/L amiodarone can prolong acute deactivation time constant moderately.(2) chronic research: l.The chronic amiodarone treatment had no effects on current characteristic, peakcurrent density and channel kinetic constant of Inb^ Ica-L > Iki-2. Delayed rectifier outward K+ current and slowly activating delayed rectifier outward K+current: (l)The chronic amiodarone treatment (lOOmg ? kg'1 -d"1, 4w) didn't alter the channel characteristic , and after repolarization from -40 to 50mV began to active a series of outward currents in concentration and voltage dependent manner. But chronic amiodarone treatment decreased the current density of Ik from 7.27 ±0.95 pA/pF on normal myocytes to 5.45 ±0.77 pA/pF on amiodarone long-term treatment myocytes,and the loss was nearly 25.03%. (2)The chronic amiodarone treatment also didn't change the channel characteristic of hypertrophied myocytes. But long-term administration of amiodarone can decrease the current density of hypertrophied myocytes from 4.28±0.23pA/pF to 3.06±0.45pA/pF, and the loss was nearly 28.5%.? Compared with control, the chronic amiodarone treatment decreased Iks by 40.49%, and Iks occupied 62.02% the component of Ik. ? the chronic amiodarone treatment decreased Iks on hypertrophied myocytes by 43%, and Iks occupied 55.88% the component of Ik. So long-term administration of amiodarone can inhibite the outward current density of IKand Iks on normal and hypertrophied myocytes .3. Transient outward K+ current: ? The chronic amiodarone treatment had no effects on activation voltage^ peak voltage and reversal potential. But it can decreased the peak current density of It0 from 34.71 ±2.83 pA/pF on normal myocytes to 23.52 + 6.76 pA/pF on amiodarone long-term treatment myocytes> and the loss was nearly 32.24%. (2) The chronic amiodarone treatment also had no effects on current characteristics of hypertrophied myocytes. But the peak current density decreased from 23.35 +1.18pA/pF to 19.02±2.65pA/pF, and reduced by 18.54%.? The chronic amiodarone treatment didn't altered the kinetics constants of It0 on normal and hypertrophied myocytes.Conclusion(1) The currents amplitude of inward currents (In3 and Ica-0 on hypertrophied myocytes were higher than control, but the current densities were similar . It suggested that the augmentation of current amplitude were resulted by the swelling volume of hypertrophied myocytes. The currents density of outward current (Ik^ 1^ W Iki) were all lower than control. So electroreconstruction of hypertrophied myocytes mainly displayed the abnormality of repolarization and the lengthening of action potential.(2) Acute application of amiodarone mainly inhibited In8 and Ica-L- The inhibition on outward currents needed higher AM concentration. So AM intravenous injection didn't prolong APD primitively.(3) The acute effects of AM on hypertrophied myocytes were different from normal.? iNa on hypertrophied myocytes was more sensitive to amiodarone's inhibition than control.? Ica-L wasn't more sensitive to amiodarone's inhibition than control. (3)While for hypertrophied myocytes, the sensitivity decreased on Ik somewhat and increased on Iks- ? Acute amiodarone application nearly had no effects on Itoand Iki. Its showed that the acute effects of amiodarone were mainly due to the inhibition of Ins and Iks? and it had few effects on QT interval . It demonstrated the use-dependent and good therapeutic action of amiodarone on hypertrophied myocytes.(4 ) The chronic amiodarone treatment on hypertrophied myocytes was not same as control. The chronic amiodarone treatment had little effects on inward currents. For Ik and Iks , the inhibition on hypertrophied myocytes was higher than control,but for It0 the sensitivity was lower than control. So the predominant appearance of chronic amiodarone treatment was the prolongation of QT interval, which was more fit for antiarrythmias therapy.In conclusion, the reason for the differences between the acute and chronic actions of amiodarone wasn't clear. Maybe the acute effects were due to direct channel inhibition of amiodarone, while the effects by chronic amiodarone might reflect direct channel inhibition of the parent drug and /or its active metabolite (DEA) retained at the sites of action which disturbed the metabolization of thyroxin and the modulation of potassium channel gene expression.