Gene transfer of connexin43 mutants attenuates coupling in cardiomyocytes -: Novel basis for modulation of cardiac conduction by gene therapy
Gene transfer of connexin43 mutants attenuates coupling in cardiomyocytes -: Novel basis for modulation of cardiac conduction by gene therapy
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DOI：
10.1161/circresaha.106.144956
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发表时间：
2007-06-08
影响因子：
20.1
通讯作者：
Marban, Eduardo
中科院分区：
文献类型：
--
作者：
Kizana, Eddy;Chang, Connie Y.;Marban, Eduardo
电传导的改变将是预防或治疗某些心律失常，特别是室性心动过速（VT）的有用原理。在这里，我们追求一种新的基因转移方法，通过减少间隙连接细胞间通讯（GJIC）来调节电传导，从而潜在地改变心律失常的底物。最终目标是发展一种非破坏性的方法，通过局灶基因转移来解耦慢传导区。制备了编码连接蛋白43 （Cx43）内环突变体的慢病毒载体并进行了体外研究。新生大鼠心室肌细胞（nrvm）的转导揭示了突变基因产物的亚细胞定位。荧光染料转移研究显示转基因nrvm中GJIC显著降低。此外，相邻突变基因修饰的nrvm显示延迟钙瞬态，表明电解偶联。在基因修饰的NRVM单层中，动作电位（AP）传播的多位点光学图谱显示，相对于未转导的NRVM，传导速度（CV）减慢了3倍。综上所述，Cx43突变体的慢病毒载体介导的基因转移降低了nrvm中的GJIC。相邻NRVM的钙瞬态延迟和NRVM单层的CV降低也证明了电荷转移减少。这些数据验证了一种分子工具，为基因转移靶向间隙连接作为调节心脏传导的方法开辟了前景。
Modification of electrical conduction would be a useful principle to recruit in preventing or treating certain arrhythmias, notably ventricular tachycardia (VT). Here we pursue a novel gene transfer approach to modulate electrical conduction by reducing gap junctional intercellular communication (GJIC) and hence potentially modify the arrhythmia substrate. The ultimate goal is to develop a nondestructive approach to uncouple zones of slow conduction by focal gene transfer. Lentiviral vectors encoding connexin43 (Cx43) internal loop mutants were produced and studied in vitro. Transduction of neonatal rat ventricular myocytes (NRVMs) revealed the expected subcellular localization of the mutant gene product. Fluorescent dye transfer studies showed a significant reduction of GJIC in NRVMs that had been genetically modified. Additionally, adjacent mutant gene-modified NRVMs displayed delayed calcium transients, indicative of electrical uncoupling. Multi-site optical mapping of action potential (AP) propagation in gene-modified NRVM monolayers revealed a 3-fold slowing of conduction velocity (CV) relative to nontransduced NRVMs. In conclusion, lentiviral vector-mediated gene transfer of Cx43 mutants reduced GJIC in NRVMs. Electrical charge transfer was also reduced as evidenced by delayed calcium transients in adjacent NRVMs and reduced CV in NRVM monolayers. These data validate a molecular tool that opens the prospect for gene transfer targeting gap junctions as an approach to modulate cardiac conduction.