Decreasing microtubule detyrosination modulates Na_v1.5 subcellular distribution and restores sodium current in mdx cardiomyocytes

Aims 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. We here investigated its impact on I(Na )and Na(v)1.5 subcellular distribution. Methods and results Ventricular cardiomyocytes (CMs) from wild-type (WT) and mdx (DMD) mice were incubated with either 10 mu M PTL, 20 mu M EpoY, or dimethylsulfoxide (DMSO) for 3-5 h, followed by patch-clamp analysis to assess I(Na )and action potential (AP) characteristics in addition to immunofluorescence and stochastic optical reconstruction microscopy (STORM) to investigate MT detyrosination and Na(v)1.5 cluster size and density, respectively. In accordance with previous studies, we observed increased MT detyrosination, decreased I(Na )and reduced AP upstroke velocity (V-max) in mdx CMs compared to WT. PTL decreased MT detyrosination and significantly increased I-Na magnitude (without affecting I(Na )gating properties) and AP V-max 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 Na(v)1.5 clusters at the LM crests (but not at the grooves), indicating a dystrophin-independent trafficking route to this subcellular domain. Interestingly, Na(v)1.5 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. Conclusion Attenuating MT detyrosination in mdx CMs restored I-Na and enhanced Na(v)1.5 localization at the LM crest and ID. Hence, the reduced whole-cell I-Na 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 Na(v)1.5 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 I(Na )and subcellular Na(v)1.5 distribution in pathophysiological conditions. [GRAPHICS] . Graphical Abstract