Impact of microtubule detyrosination on subcellular distribution of Nav1.5 in mdx cardiomyocytes

Abstract

Abstract Background Subcellular trafficking and distribution of the cardiac sodium channel Nav1.5 in cardiomyocytes is dependent on the microtubule network as well as Nav1.5 interacting proteins. Duchenne muscular dystrophy (DMD) is associated with loss of dystrophin, a Nav1.5-interacting protein localized at the lateral membrane of cardiomyocytes, leading to sodium current (INa) reduction in this microdomain. Cardiomyocytes of DMD (mdx) mice also display increased microtubule detyrosination, but its impact on INa and Nav1.5 distribution is unclear. Purpose To investigate the effect of reducing microtubule detyrosination by the compound parthenolide (PTL) on INa and subcellular Nav1.5 distribution in mdx cardiomyocytes (CMs). Methods and Results Isolated ventricular CMs from wild type (WT) and mdx (DMD) mice were incubated with either 10 µM PTL or DMSO for 3-5 hours. INa properties were assessed using the patch-clamp technique, confocal microscopy was used to investigate microtubule detyrosination, and stochastic optical reconstruction microscopy (STORM) was employed to assess Nav1.5 cluster density and size, as well as Nav1.5 cluster distribution (crest vs groove) at the cardiomyocyte lateral membrane. Compared to WT. mdx CMs displayed increased levels of detyrosinated tubulin and decreased whole-cell INa. Incubation with PTL decreased the fraction of detyrosinated tubulin and significantly increased whole-cell INa magnitude in mdx CMs, but had no effect in WT CMs. No effect of PTL on INa gating properties were observed, indicating that it increased INa by enhancing Nav1.5 membrane trafficking. Indeed, STORM analysis showed that PTL increased Nav1.5 cluster density at both the lateral membrane and intercalated disc region in mdx CMs while it had no effect on WT CMs. Furthermore, Nav1.5-α-actinin cluster distance analysis showed that PTL increased Nav1.5 clusters specifically at the crest of the lateral membrane in mdx CMs. Conclusions In mdx CMs, reduction of MT detyrosination rescues INa density and increases Nav1.5 cluster density both at the intercalated disc and lateral membrane. Hence, in addition to loss of the Nav1.5-interacting protein dystrophin, MT remodelling contributes to INa reduction in DMD. The PTL-induced increase in Nav1.5 at the crest of the lateral membrane of mdx CMs is likely due to a dystrophin-independent mechanism since dystrophin is normally absent from this microdomain. Overall, our findings identify MT detyrosination as a potential therapeutic target for modulating INa and subcellular Nav1.5 distribution during pathophysiological conditions.