Decreasing microtubule detyrosination modulates Nav1.5 subcellular distribution and restores sodium current in mdx cardiomyocytes

Abstract

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 INa and Nav1.5 subcellular distribution.

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 dimethylsulfoxide (DMSO) for 3-5 h, 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 at 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.

Conclusion: 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.

Keywords: SCN5A; Channel trafficking; Duchenne muscular dystrophy; Ion channels; Microtubule detyrosination.

Graphical Abstract

Figure 1

Effect of parthenolide (PTL) on MT detyrosination in WT and mdx cardiomyocytes. (A) Typical examples of confocal images showing immunolabelled total α-tubulin (red), detyrosinated α-tubulin (green), and overlay (yellow) in WT (left panel) and mdx (right panel) cardiomyocytes (CMs) after incubation with either dimethylsulfoxide (DMSO) or PTL (scale bar, 20 μm). (B) Quantification of total α-tubulin, detyrosinated α-tubulin, and ratio of detyrosinated α-tubulin on total α-tubulin in WT and mdx CMs after 3–5 h incubation with either DMSO or PTL (10 µM). n represents the number of cells measured. WT: N = 3 mice; mdx: N = 3 mice. *P < 0.05, ****P < 0.0001, one-way ANOVA followed by Holm–Sidak test for pairwise multiple comparison or Kruskal–Wallis followed by Dunn’s test.

Figure 2

PTL restores I_Na density in mdx cardiomyocytes. (A, B) Representative I_Na traces measured in WT (A) and mdx (B) cardiomyocytes (CMs) after incubation with either DMSO or PTL. (C, D) Average current–voltage (IV) relationships in WT (C) and mdx (D) CMs after 3–5 h incubation with either DMSO or PTL (10 µM). Insets: voltage-clamp protocols. n represents the number of cells measured. WT: N = 5 mice; mdx: N = 7 mice. *P < 0.05, two-way ANOVA repeated measures followed by Holm–Sidak test for post hoc analysis.

Figure 3

PTL does not affect I_Na gating properties in cardiomyocytes isolated from WT and mdx mice. (A–D) Average voltage dependence of (in)activation (A, B) and time course of recovery from inactivation (C, D) in WT and mdx cardiomyocytes after 3–5 h incubation with either DMSO or PTL (10 µM). Insets: voltage-clamp protocols. n represents the number of cells measured. WT: N = 3–5 mice; mdx: N = 5–7 mice.

Figure 4

Effect of PTL on action potential properties in WT and mdx cardiomyocytes. (A, C) Typical examples of action potentials (APs) recorded at the stimulation frequency of 2 Hz in WT (A) and mdx (C) cardiomyocytes (CMs). Insets: first derivatives (dV/dt) of the AP upstrokes. (B, D) Average data at 2 Hz for maximal upstroke velocity (V_max), AP amplitude (APA), resting membrane potential (RMP), AP duration at 20, 50, and 90% repolarization (APD₂₀, APD₅₀, and APD₉₀) in WT (B) and mdx (D) CMs after 3–5 h incubation with either DMSO or PTL (10 µM). n represents the number of cells measured. WT: N = 8 mice; mdx: N = 7 mice. **P < 0.01, *P < 0.05, unpaired Student’s t-test or Mann–Whitney test.

Figure 5

Effect of PTL on Na_v1.5 cluster density and cluster size at the lateral membrane and intercalated disc in WT and mdx cardiomyocytes. (A, B) Representative STORM images showing immunolabeled Na_v1.5 (green) and α-actinin (red) after 3–5 h incubation with either DMSO or PTL in WT (A) and mdx (B) cardiomyocytes (CMs) (scale bars: 10 µm). (C–F) Quantification of Na_v1.5 cluster density and cluster size at the lateral membrane (C, D) and intercalated disc (E, F) in WT and mdx CMs after 3–5 h incubation with either DMSO or PTL (10 µM). n represents the number of cells measured. WT: N = 4 mice, mdx: N = 3 mice. *P < 0.05, **P < 0.01, ***P < 0.001, **** < 0.0001, one-way ANOVA followed by Holm–Sidak test for pairwise multiple comparison.

Figure 6

PTL increases Na_v1.5 clusters at the crest of the lateral membrane in mdx cardiomyocytes. (A, B) Magnified sections of STORM images of the lateral membrane (LM) showing immunolabelled Na_v1.5 (green) and α-actinin (red) after 3–5 h incubation with either DMSO or PTL (10 µM) in WT (A) and mdx (B) cardiomyocytes (Scale bar 2 µm). (C) Frequency distribution plot of edge distances from any Na_v1.5 cluster to the closest α-actinin cluster. Na_v1.5 clusters within 50 nm from α-actinin are considered localized in the grooves. (D) Percentage of Na_v1.5 cluster expression at the grooves calculated per each individual cell. n represents the number of cells measured. WT: N = 3 mice, mdx: N = 3 mice. *P < 0.05, Mann–Whitney test.

Figure 7

EpoY treatment decreases MT detyrosination and increases I_Na density in mdx cardiomyocytes. (A) Typical examples of confocal images showing immunolabelled total α-tubulin (red), detyrosinated α-tubulin (green), and overlay (yellow) in mdx cardiomyocytes (CMs) after 3–5 h incubation with either DMSO or EpoY (scale bar, 20  μm). (B) Quantification of total α-tubulin, detyrosinated α-tubulin, and ratio of detyrosinated α-tubulin on total α-tubulin in mdx CMs after 3–5 h incubation with either DMSO or EpoY (20 µM). n represents the number of cells measured. mdx: N = 3 mice. *P < 0.05, ***P < 0.001, unpaired Student’s t-test or Mann–Whitney test. (C) Representative I_Na traces (left panel), average current–voltage (IV) relationships (middle panel) and average voltage dependence of (in)activation (right panel) in mdx CMs after 3–5 h incubation with either DMSO or EpoY (20  μM). Insets: voltage-clamp protocols. n represents the number of cells measured. mdx: N = 5–7 mice. *P < 0.05, two-way ANOVA repeated measures followed by Holm–Sidak test for post hoc analysis.