Study On The Mechanism Of Antiarrhythmic Effects Of ShenSongYangXin

Study on the mechanism of antiarrhythmic effects of ShenSongYangXinBackground: Cardiac arrhythmia is a kind of common cardiovascular diseases in the clinical practice, which have great effects on life quality and safety of the patients. People have been continuously devoted to investigation and development of antiarrhythmic drugs in the recent decades. Along with the progress in science and technology, non-pharmacotherapy (such as defibrillation, pacing, radio frequency ablation, surgery and so on) plays more and more roles in clinical treatment, but pharmacotherapy is still the essential way in control cardiac tachy-arrhythmia.Cardiac arrhythmias result from abnormalities of impulse generation, conduction, or both. It is difficult, however, to illustrate the underlying mechanisms for all the clinical arrhythmias. At present, antiarrhythmic drugs have been classified as sodium channel blockers, [3-receptor blockers, action potential duration prolong agents (mainly potassium channel blockers) and calcium channel blockers. The abnormalities of expression and function of the membrane ion channels can impact on action potential duration, amplify dispersion of repolarization, induce triggered activity, and thus contribute to the development of cardiac arrhythmias. Although the classification of antiarrhythmic drugs are different, modulation the ion channels function is the basic mechanism. Besides the antiarrhythmic efforts, many antiarrhythmic agents have potential risk for proarrhythmia. The adverse side effects of drug may induce polymorphic ventricular tachycardia or torsade de pointes (TdP), which can increase mortality of the cardiovascular diseases. Since CAST and CASTâ…¡study published, it attached more attention to value the benefit risk ratio when antiarrhythmic drugs were applied. People begin to revalue the safety of antiarrhythmic drugs currently used in clinic and to develop new agents with fewer side effects.Compared to Western medicine, some antiarrhythmic Chinese herbs with many components can multi-target modulate the disorder of ion channel function. It might exert antiarrhythmic efforts with lower incidence of adverse effects. Screening antiarrhythmic Chinese herbs might lead to a new trend in pharmacotherapy.Shensong Yongxin (SSYX) is one of the compound recipe of Chinese materia medica including 12 ingredients such as Panax ginseng, dwarf lilyturf tuber, Nardostachys root, etc. Previous studies on animal model showed that SSYX significantly inhibited the arrhythmias induced by toxic chemical compounds or ischemia-reperfusion injury. Small random double-blind clinical trials also suggested that SSYX reduced the number of ventricular extra beats in patients with or without structure heart disease. However, the antiarrhythmic mechanisms of SSYX have not been studied.Objectives: To determine the effects of ShenSongYangXin(SSYX) on L-type calcium channels( I_Ca, L), sodium channels(I_Na), transient outward potassium current (I_to), delayed rectifier current (I_K), and inward rectifier potassium currents(I_K1) in isolated ventricular myocytes, and on hERG channel(I_Kr) in transfected HEK293 cells. To investigate the pharmacological mechanism of SSYX on the ion channels.Methods: Single ventricular myocytes of guinea pigs and rats were obtained by enzymatic dissociation method. Cultured HEK293 cells were transfected transiently with pcDNA3.1-hERG by a lipofectamine method and green fluorescent protein cDNA (pEGFP-C1) was co-transfected to serve as an indicator. 24～48h after transfection, electrical studis were applied in cells with green fluorescence. Whole cell patch-clamp technique was used to record ion channel currents. I_Ca,L, I_Na, I_K were studied in ventricular myocytes of guinea pigs; I_to,I_K1 were studied in ventricular myocytes of rats; and I_Kr were studied in transfected HEK293 cells.Results: The action potential and ion currents could be recorded in the isolated ventricular myocytes of guinea pigs and rats, which indicated the myocytes had satisfactory electrophysiological properties. Approximate 40～60 percent transfected HEK293 cells took green fluorescence, in which I_Kr could be recorded.1. The effect of SSYX on action potential. Before and after 0.5% drug application, the rest membrane potential of rat ventricular myocytes were -65.02±5.96mV and -62.67±7.59mV (n＝6,P＞0.05); the action potential amplitude were 101.31±8.78mV and 84.84±5.47mV(n=6, P＜0.05); 50% action potential duration(APD₅₀) were 23.92±5.50ms and 41.99±5.39ms(n=6, P＜0.05); 90% action potential duration(APD₉₀) were 46.97±7.89ms and 65.3±5.84ms(n=6, P＜0.05).2. The effect of S SYX on I_Ca,L. At the test potential of＋10 mV, S SYX inhibited L-type calcium current in a dose-dependent manner. At 0.25%, 0.5%, 1% of concentration, the peak I_Ca,L was reduced by 19.22%±1.10 %, 44.82%±6.50 % and 50.69%±5.64%, respectively (n=5, all P＜0.05). In the presence of SSYX 0.5%, the current density-voltage curve was moved up and activation potential, the potential of peak current, and the shape of the I-V curve did not change. The avtivation time constant(Ï„) was measure as the monoexponential fit to the curret curve induced at＋10 mV, andÏ„increased from 1.04±0.17ms to 1.52±0.19ms(n=6, P＜0.05) after 0.5% drug application. The steady-state inactivation curve was fitted by a Boltzmann function. It was moved to more negative potential, the half inactivation potential (V_1/2) was -15.38±2.4 mV and -20.44.24±5.01 mV(n=6, P＜0.05) in control and SSYX 0.5% respectively. Monoexponential function was best fit the curve of recovery time from inactivation. After 0.5% drug use, the channel recovery time constant increased, from 85.24±20.18ms to 158.42±38.23 ms(n＝5, P＜0.05).3. The effect of SSYX on I_Na. At the test potential of -20 mV, SSYX 0.5% decreased peak I_Na by 44.84%±7.65 % from 27.21±5.35 to 14.88±2.75 pA/pF (n=5, P＜0.05). SSYX up shifted the I-V curve of I_Na without changing the threshold, peak and reverse potentials. Vt -20mV, before and after SSYX 0.5% application, the time constant of activation were 0.444±0.15 ms and 0.364±0.11 ms (n=5, P＞0.05); the time constant of inactivation were 1.84±0.34ms and 2.054±0.51 ms (n=5, P＞0.05). Before and after SSYX 0.5% use, steady-state activation V_1/2 were -34.27±2.38mV and -31.40±1.63mV (n=5, P＜0.05); steady-state inactivation V_1/2 were -73.68±4.80mV and -75.15±4.50mV(n=5, P＞0.05); channel recovery time constant were 19.03±1.13 ms and 21.754±1.48 ms(n=5, P＞0.05).4. The effect of SSYX on I_to. At 0.5% of concentration, the drug blocked the transient component of I_to by 50.60 % at membrane voltage of 60mV, from -19.82±7.10(pA/ pF) to -10.02±3.93 ( pA/ pF). SSYX 0.5% could accelerate the inactivation of I_to, the time constant were 29.06±4.66ms and 21.4 4±3.12ms (n=6, P＜0. 05). SSYX 0.5% negatively shifted the inactivation curve, the inactivation V_1/2 were -15.67±2.52 mV and -26.45±3.88 mV (n=7, P＜0.05) respectively in control and in drug group. SSYX 0.5% delayed the channel recovery from inactivation. Before and after drug use, the recovery time constant were 12.86±0.31ms and 18.52±3.76ms (n=5, P＜0.05).5. The effect of SSYX on I_K1. SSYX 0.5% inhibited the I_K1 from -10.78±1.80 (pA/pF) to -7.18±2.05 (pA/pF)by 33.10 %±16.85 % (n＝11, P＜0.05) at the test potential of-100mV with little effect on reversal potential and the rectification property.6. The effect of SSYX on/Ks. Holding potential at -40mV and test potential at +50mV last 5s could elicit time dependent increased I_Ks in isolated guinea pig ventricular cells. SSYX 0.5% inhibited I_Ks from 4.02±0.27(pA/pF) tol.39±0.30(pA/pF), by 65.21%±8.5%(n=5, P＜0.05), and inhibited I_Ks,tail by 30.77%±1.11%(n=5, P＜0.05).7. The effect of SSYX on I_Kr. I_Kr was recorded in hERG-transfected HEK293 cells. During the depolarizing steps, the outward current was activated at voltages positive to -20mV, and the current amplitude was increased to reach a maximum at +20inV. With further depolarization, the current amplitude decreased progressively, because of the inward rectification. Test potential at +20mV, after 0.5% SSYX use the I_Kr amplitude changed from 18.56±2.47 pA/pF to16.57±3.57 pA/pF (n＝5, P＞0.05). Activation curve mearured with I_Kr,tail and fitted to a Boltzmann relationship. Before 0.5%SSYX use, the V_1/2 and K were -3.78±5.30 mV and 8.14±1.26, and after drug use the V_1/2 and K were -5.65±5.29 mV and 8.43±1.79 (n=5,P＞0.05). Test potential at -20mV, before and after 0.5% SSYX use, the I_Kr,tail inactivation time constant were 16.85±3.78 and 14.76±1.34 (n=5, P＞0.05); the recovery time constant from inactivation were 6.29±0.84 and 5.94±0.50 (n=5, P＞0.05).Conclusions: It reveals that SSYX could block multiple ion channels includeI_Ca,L I_Na, I_to, I_K1 and I_Ks. SSYX could inhibit I_Ca,L peak current, slow down time dependent activation and inactivation, and promote channel voltage dependent inactivation. SSYX could decrease I_Na peak current, and right shift the steady-state activation curve. SSYX could depress I_to peak current, promote channel time dependent and voltage dependent inactivation. SSYX could inhibit the inward current component of I_K1. SSYX could inhibit the slowly-activating component of I_K on guinea pig ventricular myocytes. SSYX had few effects on I_Kr current amplitude, time dependent inactivation and recovery from inactivation. The integrated effects of SSYX may change the action potential duration and contribute to some of its antiarrhythmic effects. Moreover, the block effect on multiple ion channels would be beneficial to reduce proarrhythmic side effects. SSYX may be expected to have a wild spectrum of antiarrhythmic effect with less proarrhythmic potential.