| ObjectiveAtrial fibrillation (AF) is the most common sustained cardiac arrhythmia in the clinical practice and the exact pathophysiological mechanisms of AF are still unclear. This study used the spontaneous AF transgenic mice model, to explore the changes of the ion channel currents in the atrial myocytes. We aimed to elucidate the mechanisms associated with the atrial fibrillation in this model.MethodsThis study used spontaneous AF transgenic mice model as experiment object, and age matched wild-type C57BL/6 mice as control group. Echocardiographic images were obtained with a Visual Sonics Vevo 770 system, and Surface electrocardiogram (ECG) recording were using PCLAB UE Physiograph, with the limb leads were place subcutaneously in accordance to lead Ⅱ. Heart from mice was performed on an in vitro Langendorff perfusion system, and atrial myocytes were obtained by double enzymes digestion method. Whole-cell patch-clamp recording technique was used to record action potential (AP) and ion currents (transient outward potassium current,Ito, inward rectifier potassisum current,IK1 and funny current, If).ResultsEchocardiographic analysis revealed that structurally normal hearts in transgenic AF (abbreviated as TG) mice comparing with wild-type (abbreviated as WT) mice, and there were no significant differences in left ventricular dimensions and systolic function in TG mice compared with age-matched WT mice. In respect of electrocardiogram data, the resting heart rate (HR) between the TG was slower comparing to the WT mice (391±61 beats per min [bpm] vs.436±50 bpm,p=0.048). The QT interval was longer in TG mice (42.3±8.6 vs.34.2±8.6ms,p=0.02), but the corrected QT interval (34±6.8 vs.29.1±7.1ms, p=0.076) was not significantly difference among the TG and WT mice. The duration of the action potential (APD) among the WT and TG was not statistic difference (46.0±12 vs.59.1±14.3ms,p=0.082) and the resting membrane potential was not significantly different between the TG and WT mice (-65.3±4.2mV vs.-67.9±5.0mV, p=0.350). Moreover, no significant changes were detected in the APD20 (4.9±0.8 vs.4.8±0.5, p=0.837), APD50 (9.6±4.5 vs.7.3±4.6, p=0.370) and APD90 (25.6±8.2 vs.29.2±8.8, p=0.437) for atrial myocytes from TG mice compared to the WT littermates.As to the ion currents, the transient outward potassium current (Ito) was weaker in TG mice atrial myocytes, the density of Ito was significantly lower in the atrial myocytes of TG mice compared with WT mice from voltages -30 to +70mV (at+70mV:TG, 11.0±0.7pA/pF vs.12.2±0.6pA/pF, p=0.036), but the steady activation curves among them were not significant difference. Furthermore, there was no difference of the density of Inward rectifier potassium current (IK1) in atrial myocytes between WT and TG mice. Compared the density of funny current(If) in atrial myocytes from WT and TG mice, the density of If was significantly higher in the atrial myocytes of TG mice compared with WT mice from voltages of -70 to -170mV (at-170mV:TG,-39.6±4.6pA/pF; WT,-26.9±3.0pA/pF, p<0.001). A Boltzmann function fitted to the normalized conductances of atrial myocytes from TG mice yielded a voltage at half-maximal activation (V1/2) of-109.45±1.35 mV and a slope factor of 14.41±0.78 mV (n=15). The activation curve of atrial myocytes from WT mice showed a significant more negative V1/2 value (-128.20±1.65 mV) and a slope factor of 13.91±0.98 mV.ConclusionsEchocardiographic analysis revealed that structurally normal hearts in TG mice comparing with WT mice. Comparing to the WT mice, the resting heart rate was slower in TG mice and he QT interval was longer in TG mice, but the corrected QT interval was not significantly difference among the TG and WT mice. The action potential characteristic among the WT and TG mice in atrial myocytes was not statistic difference. The Ito was weaker in TG mice atrial myocytes compared to WT mice, but the steady activation curves among them were not significant difference. There was no difference of the density of IK1 in atrial myocytes between WT and TG mice. Most important, the density of If was significantly higher in the atrial myocytes of TG mice compared with WT mice from voltages of -70 to -170mV, as well as the If was easier to activation in TG mice myocytes.ObjectiveThe ion channel alteration were the important mechanisms underlying electrophysiology remodeling in atrial fibrillation (AF), the ion channel protein were important molecule substrate for AF initiating and maintenance. This study used the spontaneous AF transgenic mice model, to explore the changes of the ion channel protein in the atrial myocytes. We aimed to elucidate the molecular biological mechanisms associated with the atrial fibrillation in this model.MethodsThis study used spontaneous AF transgenic mice model as experiment object, and age matched wild-type C57BL/6 mice as control group. The quantitative RT-PCR analysis was performed in detecting mRNA level of HCN (HCN2 and HCN4), and Western blot analysis was used for detecting HCN (HCN2 and HCN4) and Kv4 (Kv4.2 and Kv4.3) protein.ResultsIn respect to the results of qRT-PCR quantification in the atrial tissue samples, a significant increase (p<0.01) in mRNA levels for HCN2 was observed in atrial tissue from TG mice (1.10±0.40, N=6) compared to control (0.36±0.17, N=6). Likewise, a significant increase (p<0.01) of HCN4 expression also found in TG atria tissure (1.48±0.63, N=6) compared to WT atrial tissue (0.52±0.11, N=6). The relative HCN2 protein expression was not significantly different among the TG (0.034±0.010) and WT (0.035±0.017).As for HCN4, protein changes paralleled those observed for mRNA, with HCN4 being significantly higher in TG atrial tissue (0.09±0.05) than in control samples (0.01±0.009) (p<0.05).The relative Kv4.2 protein expression was lower in TG mice atrium compared to WT mice (0.27±0.22 vs.0.68±0.26, both N=6,p=0.015), likewise, a significant decrease of Kv4.3 protein expression also found in TG atrium (0.23±0.08 vs.1.04±0.13, N=6, p=0.002).ConclusionsThe mRNA levels for HCN2 and HCN4 were significant increase in atrial tissue from TG mice compared to WT mice, and HCN4 protein was significantly higher in TG atrial tissue than in control samples. The relative Kv4.2 and Kv4.3 protein expression was lower in TG mice atrium compared to WT mice.ObjectiveCurrent studies show that ivabradine, a specific heart rate lowering drug, it selective inhibitor of funny (If), performing anti-arrhythmic effect of some disease condition, such as heart failure, myocardial ischemia, and so on. But little is known regarding the long-term ivabradine treatment on If current and prevention of atrial fibrillation (AF).MethodsBeginning at 6 months of age, both transgenic (TG) mice and wild-type (WT) C57BL/6J mice were randomly assigned to ivabradine group or control group, the ivabradine group mice were treated with ivabradine (7 mg/kg per day orally) given in drinking water ad libitum for 4 month, surface electrocardiogram recording were conducted every 2 weeks to monitor the change of heart rate and incidence of AF.Echocardiographic images were obtained with a Visual Sonics Vevo 770 system, and Surface electrocardiogram (ECG) recording were using PCLAB UE Physiograph, with the limb leads were place subcutaneously in accordance to lead II. Heart from mice was performed on an in vitro Langendorff perfusion system, and atrial myocytes were obtained by double enzymes digestion method. Whole-cell patch-clamp recording technique was used to record action potential (AP) and ion currents. The quantitative RT-PCR analysis was performed in detecting mRNA level of HCN, and Western blot analysis was used for detecting HCN protein.ResultsAfter treatment with ivabradine for 4 months (7mg/kg per day orally), we found that ivabradine dramatically reduced the number of episodes of spontaneous AF in TG+IVA group mice comparing to Control TG mice (41.2% vs.16.7%,p<0.01). In respect of electrocardiogram data, we found ivabradine reduced resting heart rate by 50±9 bpm (-13%,p<0.05) in TG+IVA group mice compared to Control TG mice (N=15 for TG, and 12 for TG+IVA). And also reduced by 49±11 bpm (-11%, p<0.05) in WT+IVA group mice compared to Control WT mice (N=13 for WT, and 6 for WT+IVA), and the QT interval and QTc were not significant difference among the two group. After treatment, the echocardiographic data among the Control WT and WT+IVA group were not significantly difference. But the LVDs (2.57±0.22 vs.3.28±0.21), FS%(39.9±3.07 vs. 28.9±5.36), and EF%(70.7±3.67 vs.55.2±8.25) of TG+IVA group were significant difference from the Control TG groups.Long-term treatment with ivabradine on the electrophysiological properties of myocytes isolated from TG+IVA atrial, and we found ivabradine did not significantly affect the resting membrane potential in TG mice atrial myocytes, and there were no significant differences in APD, APD20 and APD90 in TG+IVA mice compared to TG and WT compared to WT+IVA mice. After 4 months treatment, it partially reverses the electrophysiological remodeling occurring in TG+IVA group mice, and If density was significantly reduced in the atrial myocytes (at -170mV:TG+IVA,-17.7±3.0pA/pF; TG,-39.6±4.6pA/pF, p<0.001), and also had a faster activation kinetics (half activation voltage:TG+IVA,-124.4±3.25mV, TG,-109.45±0.72mV,p<0.001; slope factor k: TG+IVA,22.58±2.75mV vs.14.40±0.78mV,p<0.001).In respect of molecule experiment, treatment with ivabradine downregulated HCN isoforms expression in TG+IVA mice, by which HCN2 was decreased by 50% in the atrium (TG+IVA 0.55±0.34 vs. TG 1.10±0.40, p=0.029), HCN4 expression was decreased by 63.5% in the atrium (TG+IVA 0.54±0.41 vs. TG 1.48±0.63,p=0.012). In WT+IVA mice, no decrease in HCN2 or HCN4 expression was observed compared with WT mice. The effect of 4 months ivabradine treatment on the HCN protein expression, the HCN2 protein was nearly absent in atrial tissue in both conditions, the relative HCN2 protein expression among both group was not significantly different (WT+IVA, 0.0097±0.0062; TG+IVA,0.02±0.0078, both N=4). No difference of the relative HCN4 protein expression between WT+IVA (0.22±0.15, N=4) and TG+IVA (0.18±0.10, N=4) group.ConclusionsLong-time ivabradine treatment partially reverses electrophysiological cardiac remodeling in TG mice by reducing If gain-of-function; ivabradine treatment can significantly reduce the incidence of AF in TG mice; the anti-arrhythmic of ivabradine was beyond the hate rate reduction, may partially due to counteract the HCN overexpression in TG mice. Moreover, long-term treatment with ivabradine can improve the cardiac function of the mice, and it did not infect the QTc interval, suggest the safety of ivabradine were favorable. |
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