Calcium-dependent Nedd4-2 upregulation mediates degradation of the cardiac sodium channel Nav1.5: implications for heart failure
Calcium-dependent Nedd4-2 upregulation mediates degradation of the cardiac sodium channel Nav1.5: implications for heart failure
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DOI：
10.1111/apha.12872
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发表时间：
2017-09-01
影响因子：
6.3
通讯作者：
Wang, T.
中科院分区：
文献类型：
--
作者：
Luo, L.;Ning, F.;Wang, T.
目的电压门控钠通道 (Nav1.5) 功能/表达的减少为心律失常提供了减慢传导的底物。 Nedd4-2 被钙激活，在翻译后调节 Nav1.5。我们的目的是研究细胞内钙 ([Ca2+](i)) 升高是否通过 Nedd4-2 降低 Nav1.5 及其在心力衰竭 (HF) 中的作用。方法采用生化、电生理、细胞和体内方法相结合，我们测试了钙对 Nedd4-2 进而 Nav1.5 的影响和机制。结果 [Ca2+](i) 增加，如下 在稳定表达 Nav1.5 的新生大鼠心肌细胞 (NRCM) 和 HEK 293 细胞中，24 小时离子霉素处理可降低钠电流 (I-Na) 密度和 Nav1.5 蛋白，但不改变其 mRNA。钙螯合剂 BAPTA-AM 恢复了经离子霉素预处理的 NRCM 中降低的 Nav1.5 和 I-Na。 Nav1.5 因 Nedd4-2 转染而降低，并因 6 小时离子霉素处理而进一步降低。在用催化失活突变体 Nedd4-2 C801S 或用含有受损 Nedd4-2 结合基序的 Y1977A-Nav1.5 突变体转染的细胞中没有观察到这些效应。此外，[Ca2+](i) 升高会增加 Nedd4-2、Nedd4-2 和 Nav1.5 之间的相互作用以及 Nav1.5 泛素化。在容量超负荷的心衰大鼠心脏中，Nav1.5 蛋白减少，而 Nedd4-2 增加，免疫荧光染色表明 Nav1.5 与泛素或 Nedd4-2 的共定位增加。 BAPTA-AM 可以挽救由异丙肾上腺素或血管紧张素 II 诱导的肥大 NRCM 中减少的 Nav1.5 蛋白 I-Na 和增加的 Nedd4-2。结论钙介导的 Nedd4-2 增加通过泛素化下调 Nav1.5。在衰竭大鼠心脏中，Nav1.5 下调并与 Nedd4-2 和泛素共定位。这些数据表明 Nedd4-2 在 HF 中 Nav1.5 下调中发挥作用。
AimReductions in voltage-gated sodium channel (Nav1.5) function/expression provide a slowed-conduction substrate for cardiac arrhythmias. Nedd4-2, which is activated by calcium, post-translationally modulates Nav1.5. We aim to investigate whether elevated intracellular calcium ([Ca2+](i)) reduces Nav1.5 through Nedd4-2 and its role in heart failure (HF).MethodsUsing a combination of biochemical, electrophysiological, cellular and invivo methods, we tested the effect and mechanism of calcium on Nedd4-2 and in turn Nav1.5.ResultsIncreased [Ca2+](i), following 24-h ionomycin treatment, decreased sodium current (I-Na) density and Nav1.5 protein without altering its mRNA in both neonatal rat cardiomyocytes (NRCMs) and HEK 293 cells stably expressing Nav1.5. The calcium chelator BAPTA-AM restored the reduced Nav1.5 and I-Na in NRCMs pre-treated by ionomycin. Nav1.5 was decreased by Nedd4-2 transfection and further decreased by 6-h ionomycin treatment. These effects were not observed in cells transfected with the catalytically inactive mutant, Nedd4-2 C801S, or with Y1977A-Nav1.5 mutant containing the impaired Nedd4-2 binding motif. Furthermore, elevated [Ca2+](i) increased Nedd4-2, the interaction between Nedd4-2 and Nav1.5, and Nav1.5 ubiquitination. Nav1.5 protein is decreased, whereas Nedd4-2 is increased in volume-overload HF rat hearts, with increased co-localization of Nav1.5 with ubiquitin or Nedd4-2 as indicated by immunofluorescence staining. BAPTA-AM rescued the reduced Nav1.5 protein, I-Na and increased Nedd4-2 in hypertrophied NRCMs induced by isoproterenol or angiotensin II.ConclusionCalcium-mediated increases in Nedd4-2 downregulate Nav1.5 by ubiquitination. Nav1.5 is downregulated and co-localizes with Nedd4-2 and ubiquitin in failing rat heart. These data suggest a role of Nedd4-2 in Nav1.5 downregulation in HF.