调控calpain-ankyrin-G-Nav1.5通路防治心房颤动心房电重构的研究

Atrial ?brillation (AF) by itself induces electrical remodeling favoring its recurrence and persistence. However, the precise mechanism of electrical remodeling in AF is unknown. Previous studies have shown that AF is accompanied by intracellular calcium overload, which causes activation of the calcium-dependent neutral proteases-calpain. Cytoskeletal protein ankyrin-G can be degraded by calpain. Ankyrin-G is required critically for voltage-gated sodium channels (Nav) targeting to cardiac emembrane domains. Ankyrin-G dysfunction has been linked with abnormal ion channel membrane organization. Nav1.5 is the primary voltage-gated sodium channel in heart and generates the rapid upstroke of the cardiomyocyte action potential. Human Nav1.5 gene (SCN5A) loss-of-function mutations may lead to many cardiac phenotypes, including Brugada syndrome, sick sinus syndrome and atrial ?brillation. The overall hypothesis is that calpain-ankyrin-G-Nav1.5 pathway is involved in electrical remodeling in atrial ?brillation: calpain can degrade ankyrin-G and disturb localization and expression of Nav1.5, resulting in inbalance of ions and atrial ?brillation. The objective of this proposal is to elucidate the role of and mechanisms underlying calpain-ankyrin-G-Nav1.5 pathway in electrical remodeling during atrial ?brillation. We will use a canine model of atrial ?brillation produced by prolonged atrial pacing and rapidly stimulated HL-1 atrial myocytes to observe the possibility of preventing electrical remodeling by regulating calpain-ankyrin-G-Nav1.5 pathway. This is a highly innovative proposal that focuses on a novel concept. The outcomes will enhance the understanding of the pivotal mechanisms of electrical remodeling in atrial ?brillation and lead to new therapeutic targets for this important problem.

心房电重构在房颤促进房颤发生与持续过程中发挥重要作用。本课题组前期研究发现房颤时钙依赖的中性蛋白酶calpain激活，可降解锚蛋白ankyrin-G。最近文献报道ankyrin-G可与心肌细胞膜钠离子通道Nav1.5相结合而调节钠内流；Nav1.5基因突变可引起心房颤动等遗传性心律失常。故提出假设：房颤心房电重构存在正反馈环路- - 房颤时calpain激活并降解ankyrin-G，影响ankyrin-G与Nav1.5结合，Nav1.5表达减少和运输障碍，跨膜钠电流失衡，导致心房电重构，使房颤持续。但ankyrin-G-Nav1.5通路在心房电重构中的作用与机制尚不明确。本课题拟构建心房快速起搏犬和HL-1心房细胞系模型，应用calpain抑制剂和转染calpastatin进行干预，来验证上述假设。本研究将为心房颤动心房电重构的药理学干预提供新的靶点与方向。

心房电重构在房颤促进房颤发生与持续过程中发挥重要作用。本课题组前期研究发现房颤时钙依赖的中性蛋白酶calpain激活，可降解锚蛋白ankyrin-G。最近文献报道ankyrin-G可与心肌细胞膜钠离子通道Nav1.5相结合而调节钠内流；Nav1.5基因突变可引起心房颤动等遗传性心律失常。故提出假设：房颤心房电重构存在正反馈环路——房颤时calpain激活并降解ankyrin-G，影响ankyrin-G与Nav1.5结合，Nav1.5表达减少和运输障碍，跨膜钠电流失衡，导致心房电重构，使房颤持续。但ankyrin-G—Nav1.5通路在心房电重构中的作用与机制尚不明确。30只健康兔分为3组，A组（对照组）、B组（起搏器组）、C组（起搏器+Caipain抑制剂组）。C组及B组给予心房起搏8周，C组腹腔内注射Calpain抑制剂（1mg/kg/d）2周,B组给予腹腔内注射MDSO溶剂（1mg/kg/d）2周。随后A、B、C组分别进行电生理检测，经电镜观察心房超微结构的变化，经免疫组化及western-blot检测心房calpain及ankyrin-G蛋白的变化。电生理检测：起搏后，B组兔均能诱发出房颤，房颤诱发率和AF平均持续时间较A组兔显著增加。calpain抑制剂组兔起搏后也都能诱发出AF，但与B组兔相比，房颤诱发率明显降低，房颤平均持续时间显著缩短。各组兔Western及免疫组化结果：房颤组较对照组Calpain蛋白含量明显增加，calpain抑制剂可抑制calpain的上调；房颤组较对照组ankyrin-G、Nav1.5、spectrin蛋白含量明显下降，calpain抑制剂可抑制ankyrin-G、Nav1.5、spectrin的下调；房颤组较对照组calpastatin蛋白含量明显下降，calpain抑制剂可抑制calpastatin的下调。本研究证实房颤心房电重构存在正反馈环路—房颤时心房肌细胞内钙浓度增加，激活calpain并降解锚蛋白ankyrin-G，干扰ankyrin-G与Nav1.5相互作用，使Nav1.5表达、定位、功能异常，钠离子跨膜电流失衡，易于形成折返引起心房电重构，形成正反馈，促进房颤发作并持续。为心房颤动心房电重构的药理学干预提供新的靶点与方向。