光遗传技术终止大鼠心房颤动的在体研究

The termination of atrial fibrillation by optogenetics in rats in vivo

目的 验证光遗传技术能否实现大鼠在体心房颤动(简称房颤)终止。方法 选取12只SD大鼠经右颈静脉系统性注射50μl重组腺相关病毒[rAA V9-CAG-hChR2 (XXM)-eYFP]。7周后,大鼠随机分为房颤组和对照组,每组6只。房颤组大鼠经尾静脉注射Ach-CaCl_(2)混合物(Ach 66 ug/kg, CaCl_(2)10 mg/kg),连续给药7天。对照组使用等量PBS液代替Ach-CaCl;混合溶液。病毒注射8周后,大鼠经麻醉后开胸充分暴露右心房,使用S_(1)S_(1)刺激心房并记录单相动作电位(MAP)和体表心电图(ECG),分析并统计RR间期、P波幅度、心率及APD_(90)。然后用频率为8 Hz的473 nm蓝光脉冲照射右房,并同步记录光照输出信号和体表心电图,寻找能完全夺获心房节律的最小光功率密度。大鼠通过Burst刺激(6 V,波宽20 ms,持续2~4 s)诱发房颤后,对心房施加4个间隔1 s的光脉冲,观察光照能否有效终止房颤。实验结束后对心房组织进行HE染色、Masson三色染色和免疫荧光染色观察。结果 与对照组比较,房颤组RR间期延长,P波幅度、心率和APD;均减少(P均<0.05).完全夺获心率的最小光照功率密度为(0.057±0.016) MW/mm^(2)。光照后房颤终止率为(80.7%±5.8%)远高于未光照的(30%±8.6%);光照后房颤的持续时间为(10.9±1.1)s远低于未光照的(41.6±10.4)s。免疫荧光染色显示转染目标光敏感蛋白ChR2成功。结论 光遗传技术可以有效地终止房颤并减少持续时间。

Objective To verify whether cardiac optogenetics can terminate atrial fibrillation(AF) in vivo in SD rat. Methods Twelve SD rats were injected of 50 μl of recombinant adeno-associated virus [rAAV9-CAG-hChR2(XXM)-eYFP] via right jugular vein systemically. After seven weeks, AF group was injected Ach-CaCl_(2)mixture(Ach 66 ug/kg, CaCl_(2)10 mg/kg) via tail vein for 7 d, control group was injected an equivalent volume of PBS within the identical performance. Eight weeks post to the AAV injection, the right atria of anesthetized rats, after open-chest, were performed S_(1)S_(1)stimulation(at 150 ms-baseline), and monophasic action potential(MAP) and body surface electrocardiogram(ECG) were recorded. Then the right atria were irradiated with 473 nm blue light at 8 Hz frequency, the output signal of light and surface electrocardiogram(ECG) were recorded simultaneously to find the minimum light power density that could completely capture the atrial rhythm. Four-light pulses were applied with a delay of 1 second to AF group, after AF was induced by burst stimulation(6 V, Duration 20 ms, lasting 2-4 s). HE staining, Masson′s trichrome staining and immunostaining observation were performed at the end of all experiments. Results The minimum illumination intensity that could completely capture heart rate was(0.057±0.016) MW/mm^(2).The termination rates with or without illumination were(80.7%±5.8%)and(30%±8.6%), respectively, the recovery times with or without illumination were(10.9±1.1)s and(41.6±10.4)s. Immunostaining showed the successful transfection of ChR2. Conclusion Cardiac optogenetics can terminate AF in ChR2-expressing rats effectively.