Chiang David Y, Verkerk Arie O, Victorio Rachelle, Shneyer Boris I, van der Vaart Babet, Jouni Mariam, Narendran Nakul, Kc Ashmita, Sampognaro James R, Vetrano-Olsen Franki, Oh John S, Buys Eva, de Jonge Berend, Shah Disheet A, Kiviniemi Tuomas, Burridge Paul W, Bezzina Connie R, Akhmanova Anna, MacRae Calum A
Cardiovascular Medicine, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, MA (D.Y.C., R.V., N.N., A.K., J.R.S., F.V.-O., J.S.O., E.B., C.A.M.).
Department of Experimental Cardiology, Heart Center (A.O.V., C.R.B.), Academic Medical Center, Amsterdam UMC, the Netherlands.
Circ Res. 2024 Jan 5;134(1):46-59. doi: 10.1161/CIRCRESAHA.123.323231. Epub 2023 Dec 14.
Brugada syndrome is associated with loss-of-function variants, yet these account for only ≈20% of cases. A recent genome-wide association study identified a novel locus within , which encodes EB2 (microtubule end-binding protein 2), implicating microtubule involvement in Brugada syndrome.
A knockout zebrafish model was generated using CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeat-associated protein 9) and validated by Western blot. Larval hearts at 5 days post-fertilization were isolated for voltage mapping and immunocytochemistry. Adult fish hearts were used for ECG, patch clamping, and immunocytochemistry. Morpholinos were injected into embryos at 1-cell stage for knockdown experiments. A transgenic zebrafish line with tandem fluorescent timer was used to study adherens junctions. Microtubule plus-end tracking and patch clamping were performed in human induced pluripotent stem cell derived cardiomyocytes (iPSC-CMs) with knockdown and knockout, respectively.
Voltage mapping of knockout hearts showed a decrease in ventricular maximum upstroke velocity of the action potential and conduction velocity, suggesting loss of cardiac voltage-gated sodium channel function. ECG showed QRS prolongation in adult knockout fish, and patch clamping showed decreased sodium current density in knockout ventricular myocytes and arrhythmias in knockout iPSC-CMs. Confocal imaging showed disorganized adherens junctions and mislocalization of mature Ncad (N-cadherin) with loss of function, associated with a decrease of detyrosinated tubulin. knockdown in iPSC-CMs led to an increase in microtubule growth velocity and distance, indicating changes in microtubule dynamics. Finally, knockdown of encoding tubulin tyrosine ligase in knockout larvae rescued tubulin detyrosination and ventricular maximum upstroke velocity of the action potential.
Genetic ablation of led to a decrease in voltage-gated sodium channel function, a hallmark of Brugada syndrome, associated with disruption of adherens junctions, decrease of detyrosinated tubulin as a marker of microtubule stability, and changes in microtubule dynamics. Restoration of the detyrosinated tubulin fraction with knockdown led to rescue of voltage-gated sodium channel-related functional parameters in knockout hearts. Taken together, our study implicates microtubule dynamics in the modulation of ventricular conduction.
Brugada综合征与功能丧失性变异相关，但这些变异仅占病例的约20%。最近一项全基因组关联研究在[具体区域]内鉴定出一个新位点，该位点编码EB2（微管末端结合蛋白2），提示微管参与Brugada综合征。
使用CRISPR/Cas9（成簇规律间隔短回文重复序列/成簇规律间隔短回文重复序列相关蛋白9）构建了[基因名称]敲除斑马鱼模型，并通过蛋白质免疫印迹法进行验证。在受精后5天分离幼虫心脏用于电压标测和免疫细胞化学分析。成年鱼心脏用于心电图、膜片钳和免疫细胞化学分析。在1细胞期将吗啉代寡核苷酸注射到胚胎中进行敲低实验。使用带有串联荧光定时器的转基因斑马鱼品系研究黏附连接。分别在[基因名称]敲低和敲除的人诱导多能干细胞衍生心肌细胞（iPSC-CMs）中进行微管正端追踪和膜片钳实验。
[基因名称]敲除心脏的电压标测显示动作电位的心室最大上升速度和传导速度降低，提示心脏电压门控钠通道功能丧失。心电图显示成年敲除鱼的QRS波增宽，膜片钳显示敲除心室肌细胞的钠电流密度降低，敲除iPSC-CMs出现心律失常。共聚焦成像显示黏附连接紊乱，成熟的Ncad（N-钙黏蛋白）定位错误，[基因名称]功能丧失，同时去酪氨酸化微管蛋白减少。iPSC-CMs中[基因名称]敲低导致微管生长速度和距离增加，表明微管动力学发生变化。最后，在[基因名称]敲除幼虫中敲低编码微管蛋白酪氨酸连接酶的基因可挽救微管蛋白去酪氨酸化和动作电位的心室最大上升速度。
[基因名称]的基因敲除导致电压门控钠通道功能降低，这是Brugada综合征的一个标志，同时伴有黏附连接破坏、作为微管稳定性标志物的去酪氨酸化微管蛋白减少以及微管动力学变化。通过敲低[相关基因]恢复去酪氨酸化微管蛋白部分可挽救[基因名称]敲除心脏中与电压门控钠通道相关的功能参数。综上所述，我们的研究表明微管动力学参与心室传导的调节。
