Background The microtubule (MT) network plays a major role in the transport of the cardiac sodium channel Nav1.5 to the membrane, where the latter associates with interacting proteins such as dystrophin. Alterations in MT dynamics are known to impact on ion channel trafficking. Duchenne muscular dystrophy (DMD), caused by dystrophin deficiency, is associated with an increase in MT detyrosination, decreased sodium current (INa), and arrhythmias. Parthenolide (PTL), a compound that decreases MT detyrosination, has shown beneficial effects on cardiac function in DMD, but its impact on INa has not been investigated. Methods and Results Ventricular cardiomyocytes (CMs) from wild-type (WT) and mdx (DMD) mice were incubated with either 10 µM PTL, 20 µM EpoY or DMSO for 3-5 hours, followed by patch-clamp analysis to assess INa and action potential (AP) characteristics in addition to immunofluorescence and stochastic optical reconstruction microscopy (STORM) to investigate MT detyrosination and Nav1.5 cluster size and density, respectively. In accordance with previous studies, we observed increased MT detyrosination, decreased INa and reduced AP upstroke velocity (Vmax) in mdx CMs compared to WT. PTL decreased MT detyrosination and significantly increased INa magnitude (without affecting INa gating properties) and AP Vmax in mdx CMs, but had no effect in WT CMs. Moreover, STORM analysis showed that in mdx CMs, Nav1.5 clusters were decreased not only in the grooves of the lateral membrane (LM; where dystrophin is localized), but also at the LM crests. PTL restored Nav1.5 clusters at the LM crests (but not the grooves), indicating a dystrophin-independent trafficking route to this subcellular domain. Interestingly, Nav1.5 cluster density was also reduced at the intercalated disc (ID) region of mdx CMs, which was restored to WT levels by PTL. Treatment of mdx CMs with EpoY, a specific MT detyrosination inhibitor, also increased INa density, while decreasing the amount of detyrosinated MTs, confirming a direct mechanistic link. Conclusions Attenuating MT detyrosination in mdx CMs restored INa and enhanced Nav1.5 localization at the LM crest and ID. Hence, the reduced whole-cell INa density characteristic of mdx CMs is not only the consequence of the lack of dystrophin within the LM grooves, but is also due to reduced Nav1.5 at the LM crest and ID secondary to increased baseline MT detyrosination. Overall, our findings identify MT detyrosination as a potential therapeutic target for modulating INa and subcellular Nav1.5 distribution in pathophysiological conditions.

中文翻译：

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减少微管去酪氨酸化调节 Nav1.5 亚细胞分布并恢复 mdx 心肌细胞中的钠电流

背景 微管 (MT) 网络在心脏钠通道 Nav1.5 向细胞膜的转运中发挥着重要作用，后者与肌营养不良蛋白等相互作用的蛋白质结合。已知 MT 动力学的改变会影响离子通道的运输。由肌营养不良蛋白缺乏引起的杜氏肌营养不良症 (DMD) 与 MT 去酪氨酸增加、钠电流 (INa) 减少和心律失常有关。小白菊内酯 (PTL) 是一种减少 MT 去酪氨酸化的化合物，已显示出对 DMD 患者心脏功能有益的作用，但其对 INa 的影响尚未得到研究。方法和结果 将野生型 (WT) 和 mdx (DMD) 小鼠的心室心肌细胞 (CM) 与 10 µM PTL、20 µM EpoY 或 DMSO 一起孵育 3-5 小时，然后进行膜片钳分析以评估 INa 和除了免疫荧光和随机光学重建显微镜 (STORM) 之外，还使用动作电位 (AP) 特征分别研究 MT 去酪氨酸和 Nav1.5 簇大小和密度。根据之前的研究，我们观察到与 WT 相比，mdx CM 中 MT 去酪氨酸增加，INa 减少，AP 上冲速度 (Vmax) 降低。PTL 降低了 mdx CM 中的 MT 去酪氨酸作用并显着增加了 INa 强度（不影响 INa 门控特性）和 AP Vmax，但对 WT CM 没有影响。此外，STORM 分析表明，在 mdx CM 中，Nav1.5 簇不仅在侧膜（LM；肌营养不良蛋白位于此处）的凹槽中减少，而且在 LM 嵴处也减少。PTL 恢复了 LM 波峰（但不是凹槽）的 Nav1.5 簇，表明通往该亚细胞结构域的不依赖肌营养不良蛋白的运输路线。有趣的是，mdx CM 的闰盘 (ID) 区域的 Nav1.5 簇密度也有所降低，通过 PTL 恢复到 WT 水平。用 EpoY（一种特定的 MT 去酪氨酸抑制剂）治疗 mdx CM 也增加了 INa 密度，同时减少了去酪氨酸 MT 的量，证实了直接的机制联系。结论 减弱 mdx CM 中的 MT 去酪氨酸作用可恢复 INa 并增强 Nav1.5 在 LM 嵴和 ID 处的定位。因此，mdx CM 的全细胞 INa 密度特征降低不仅是 LM 沟内缺乏抗肌营养不良蛋白的结果，而且还归因于 LM 波峰和 ID 处的 Nav1.5 降低，继发于基线 MT 脱酪氨酸增加。总的来说，我们的研究结果确定 MT 去酪氨酸化是调节病理生理条件下 INa 和亚细胞 Nav1.5 分布的潜在治疗靶点。