| Background and Objective: Malignant arrhythmia is one of the major causes of sudden cardiac death(SCD). The core mechanism of malignant arrhythmia is still largely unknown. Current clinical prevention and treatment are not effective. The human ether-a-go-go-related gene(h ERG) is the major molecular component of the rapidly activating delayed rectifier K+ current(Ikr), which is a crucial determinant of cardiac repolarization. Impairment of h ERG function is commonly believed to be a mechanism causing long QT syndromes(LQTS), a lethal ventricular tachyarrhythmia. Growing evidence have shown that micro RNAs(mi RNAs) are involved in functional modulation of the h ERG pathway. The purpose of this study was to validate mi R-103a-1that regulate the h ERG pathway. The mi R-103a-1 identified in this study will provide new tools to assess the mechanism of LQTS. The mi R-103a-1 could facilitate the development of new drugs for patients with arrhythmic.Methods:Electrophysiological recordings were performed in HEK293 T cells, a cell line that is amenable to patch clamp analysis. HEK293 T cells with stable expression of h ERG were further transfected with cel-mi R-67, mi R-103a-1 or mi R-103a-1 plus AMO-103a-1. The p RK5-GFP was used to monitor transfection efficacy. Cel-mi R-67 was taken as random negative controls. To assess the effect of mi R-103a-1 on the elctrophysiological characteristics of Ikr of the h ERG protein channel by the whole-cell patch-clamp technique, HEK293 T cells were harvested at 24 hours(h ERG plasmid only), 48 hours(h ERG followed by mi R-103a-1 or cel-mi R-67) or 72 hours(h ERG plasmid followed by mi R-103a-1 and corresponding AMO-103a-1) after transfection.Results:1. Effect of mi R-103a-1 on h ERG Current Amplitudes.Before and after cells co-expressing h ERG with mi R-103a-1, the maximal current amplitudes were 660.67 ± 159.55 p A and 253.72 ± 47.35 p A,respectively. This corresponds to a statistically significant decreased by 61.60%( n=6, P<0.05). Similarly, the peak Itail amplitudes were 981.28 ± 5.94 p A and 376.23± 2.84 p A This also corresponds to a statistically significant decreased by 61.66%(n=6,P<0.05). In contrast, the negative control, cel-mi R-67, failed to affect Ikr current amplitude. The addition of cel-mi R-67 had minimal effect on the maximal current amplitudes(691.30 ± 143.57 p A with cel-mi R-67 vs 660.67 ± 159.55 p A without cel-mi R-67; 4.6% [n = 6]) and tail currents(985.79 ± 3.96 p A with cel-mi R-67 981.28 ± 5.94 p A without cel-mi R-67; 0.45% [n=6]). As expected, co-transfection of AMO-103a-1 nearly abolished the effects of mi R-103a-1. Cells transfected with mi R-103a-1 and AMO-103a-1 restored the maximal current amplitude to 595.75 ± 99.08 p A(n=6)(p < 0.05), from 253.72 ± 47.35 p A(n = 6) in cells treated with mi R-103a-1 alone. Similarly, the tail current amplitude was also restored from 376.23± 2.84 p A in cells treated with mi R-103a-1 alone, to 984.58 ±6.49 p A(n = 6)(p < 0.05) in cells transfected with mi R-103a-1 and AMO-103a-1. Compared with h ERG expressing cells, the cells co-transfected with mi R-103a-1 and AMO-103a-1 showed minimal differences in maximal current amplitudes.(595.75 ± 99.08 p A vs 660.67 ± 159.55 p A, p > 0.05) and tail currents(984.58 ± 6.49 p A vs 981.28 ± 5.94 p A, p > 0.05).2.Effect of mi R-103a-1 on Gating Properties of h ERG Protein Channel(1) In h ERG-expressing cells, mi R-103a-1 treatment resulted in a significant shift of the activation curve in a negative direction. The half-maximal activation voltage(V1/2) for h ERGexpressing cells(V1/2 =-8.66 ± 0.38 m V; n=6) was statistically different from the corresponding value in cells treated with mi R-103a-1(V1/2 =-25.31 ± 0.52 m V; n = 6; p < 0.05). Similarly, statistically significant difference was seen in the slope factor k values of cells expressing h ERG(6.88 ± 0.44 m V with mi R-103a-1 vs 9.99 ± 0.34 m V without mi R-103a-1; n=6; p < 0.05). Interestingly, cel-mi R-67 treatment resulted in a significant change in activation voltage in h ERG expressing cells(V1/2 =-20.07 ± 3.30 m V vs-8.66 ± 0.38 m V control; n=6, p <0.05) but no change in slope factor(k = 9.49 ±0.27 m V vs 9.99 ± 0.34 m V control; n=6). Between cells treated only with mi R-103a-1 and cells treated with mi R-103a-1 plus AMO-103a-1, there was no significant change in either the activation voltage(V1/2 =-25.31 ± 0.52 m V with mi R-103a-1 vs-18.64 ± 0.45 m V with mi R-103a-1 puls AMO-103a-1; n=6, p > 0.05;) or the slope factor(k = 6.88 ± 0.44 m V with mi R-103a-1 vs 8.29 ± 0.39 m V with mi R-103a-1 puls AMO-103a-1; n=6, p > 0.05;). The half-maximal activation voltage was-8.66 ±0.38 m V for cells expressing h ERG, compared with-18.64 ± 0.45 m V for cells treated with mi R-103a-1 plus AMO-103a-1(n = 6, p < 0.05). On the other hand, the slope factor was 9.99 ± 0.34 m V for h ERG expressing cells and 8.29 ± 0.39 m V for cells treated with mi R-103a-1 plus AMO-103a-1(n = 6, p > 0.05).(2) Steady-state inactivation curve was mearured with Ikr and fitted with Boltzmann function.The half-maximal inactivation voltage(V1/2) for h ERG protein channel treated with mi R-103a-1 is-39.07 ±3.13 m V compared with V1/2 of-46.88 ± 4.35 m V for h ERG protein channel without mi R-103a-1 treatment(n=6,P>0.05). The slope factors for cells expressing h ERG and cells treated with mi R-103a-1 were 14.96 ± 2.88 m V and 11.30 ± 3.90 m V, respectively(n= 6, p > 0.05). Compared to untreated comtrol(V1/2 =-39.07 ±3.13 m V, k = 14.96 ± 2.88 m V, n=6, p > 0.05), the h ERG protein channels were largely unaffected by treatment with either cel-mi R-67(V1/2 =-40.07 ± 2.03 m V, k = 17.32 ± 1.94 m V) or mi R-103a-1 plus AMO-103a-1(V1/2 =-31.44 ± 1.75 m V, k = 19.00 ± 1.54 m V). Lastly, there was also no difference in the k or V1/2 value among cells expressing h ERG, cells treated with mi R-103a-1, and cells treated with mi R-103a-1 plus AMO-103a-1.(3) The inactivation of h ERG channels treated with mi R-103a-1 was faster than those without treatment(n = 6, p < 0.05). The addition of AMO-103a-1 nearly abolished the effect of mi R-103a-1 such that the time constants of inactivation of h ERG protein channel do not have significant deceleration at test potentials compared with those with mi R-103a-1 plus AMO-103a-1 treatment(n = 6, p > 0.05). Moreover, transfection of cel-mi R-67 failed to affect the time constants of inactivation of h ERG protein channel.(4) The recovery from inactivation properties and the traces of fast and slow time constants of deactivation of cells expressing h ERG were not affected by mi R-103a-1 or cel-mi R-67, and there was no statistically significant difference between h ERG with mi R-103a-1 treatment and h ERG with mi R-103a-1 plus AMO-103a-1 treatment.Conclusion:Our findings indicate that h ERG is one of the targets of mi R-103a-1. AMO-103a-1 may silence the effects of the mi R-103a-1. Our results provided new insights to better understand the functional mechanism of h ERG protein channel and further built the foundation for gene therapy as alternative treatment for LQTS. However, these results need to be further validated in animal models, preclinical studies, and clinical research. |
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