NaV1.6 dysregulation within myocardial T-tubules by D96V calmodulin enhances proarrhythmic sodium and calcium mishandling
Mikhail Tarasov, … , Rengasayee Veeraraghavan, Przemysław B. Radwański
Mikhail Tarasov, … , Rengasayee Veeraraghavan, Przemysław B. Radwański
Published February 23, 2023
Citation Information: J Clin Invest. 2023;133(7):e152071. https://doi.org/10.1172/JCI152071.
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Research Article Cardiology Cell biology

NaV1.6 dysregulation within myocardial T-tubules by D96V calmodulin enhances proarrhythmic sodium and calcium mishandling

Abstract

Calmodulin (CaM) plays critical roles in cardiomyocytes, regulating Na+ (NaV) and L-type Ca2+ channels (LTCCs). LTCC dysregulation by mutant CaMs has been implicated in action potential duration (APD) prolongation and arrhythmogenic long QT (LQT) syndrome. Intriguingly, D96V-CaM prolongs APD more than other LQT-associated CaMs despite inducing comparable levels of LTCC dysfunction, suggesting dysregulation of other depolarizing channels. Here, we provide evidence implicating NaV dysregulation within transverse (T) tubules in D96V-CaM–associated arrhythmias. D96V-CaM induced a proarrhythmic late Na+ current (INa) by impairing inactivation of NaV1.6, but not the predominant cardiac NaV isoform NaV1.5. We investigated arrhythmia mechanisms using mice with cardiac-specific expression of D96V-CaM (cD96V). Super-resolution microscopy revealed close proximity of NaV1.6 and RyR2 within T-tubules. NaV1.6 density within these regions increased in cD96V relative to WT mice. Consistent with NaV1.6 dysregulation by D96V-CaM in these regions, we observed increased late NaV activity in T-tubules. The resulting late INa promoted aberrant Ca2+ release and prolonged APD in myocytes, leading to LQT and ventricular tachycardia in vivo. Cardiac-specific NaV1.6 KO protected cD96V mice from increased T-tubular late NaV activity and its arrhythmogenic consequences. In summary, we demonstrate that D96V-CaM promoted arrhythmias by dysregulating LTCCs and NaV1.6 within T-tubules and thereby facilitating aberrant Ca2+ release.

Authors

Mikhail Tarasov, Heather L. Struckman, Yusuf Olgar, Alec Miller, Mustafa Demirtas, Vladimir Bogdanov, Radmila Terentyeva, Andrew M. Soltisz, Xiaolei Meng, Dennison Min, Galina Sakuta, Izabella Dunlap, Antonia D. Duran, Mark P. Foster, Jonathan P. Davis, Dmitry Terentyev, Sándor Györke, Rengasayee Veeraraghavan, Przemysław B. Radwański

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Figure 5

Cardiac-specific expression of D96V-CaM promotes T-tubular late NaV1.6 activity and aberrant Ca2+ release.

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Cardiac-specific expression of D96V-CaM promotes T-tubular late NaV1.6 a...
(A) 3D SICM image of a cardiomyocyte lateral membrane from a WT cardiomyocyte. Arrows indicate T-tubule openings. (B) Confocal image of a section of myocardial tissue from a WT mouse (independent sample from A) immunolabeled for NaV1.6 (blue) and RyR2 (red). Scale bar: 1 μm (A and B). (C) Fluorescence intensity profiles of NaV1.6 (blue) and RyR2 (red) from B overlaid with a topology profile from A correlate NaV1.6 and RyR2 signal intensity with T-tubules (arrows). (D) SICM-guided “smart” patch INa recordings from T-tubule openings of WT (left, black), FLAG-tagged cD96V (middle, red), and FLAG-tagged cD96V cNaV1.6-KO (right, blue) cardiomyocytes. Uppermost traces show full current recordings obtained during the voltage-step protocol; lower traces show late INa recordings only (50 ms after the test potential onset). Insets: Dashed rectangles from region 1 (top trace), enlarged. (E) Late INa recordings enlarged from dashed rectangles 2–5 from D. (F) Histograms of NaV openings recorded from T-tubules (relative to the total number of attempts). For WT n = 52 cells from 16 mice (n = 10 males, n = 6 females, 7–13 weeks old); cD96V, n = 57 cells from 19 mice (n = 10 males, n = 9 females, 11–26 weeks old); and cD96V cNaV1.6-KO, n = 36 cells from 17 mice (n = 7 males, n = 10 females, 9–25 weeks old). ***P < 0.001, **P < 0.01, and *P < 0.05, by χ2 test. (G) Frequency of burst NaV openings (normalized to the number of NaVs in membrane patches and the cumulative durations of current registrations). For WT, n = 7 cells form 4 mice (n = 3 males, n = 1 female, 7–10 weeks old); cD96V, n = 19 cells from 11 mice (n = 4 males, n = 7 females, 11–26 weeks old); and cD96V cNaV1.6-KO, n = 8 cells from 6 mice (n = 4 males, n = 2 females, 9–25 weeks old). **q < 0.01 and *q < 0.05, by Kruskal-Wallis test with the original FDR method of Benjamini and Hochberg for multiple comparisons. (H) Confocal Ca2+ sparks recorded in linescan mode from WT (left), cD96V (middle), and cD96V cNaV1.6-KO (right) cardiomyocytes paced at 0.3 Hz. (I) Ca2+ spark frequencies. For WT, n = 96 cells from 13 mice (n = 7 males, n = 6 females, 8–23 weeks old); cD96V, n = 106 cells from 10 mice (n = 4 males, n = 6 females, 10–26 weeks old); and cD96V cNaV1.6-KO, n = 74 cells from 8 mice (n = 5 males, n = 3 females, 6–26 weeks old). *q < 0.05, by Kruskal-Wallis test with the original FDR method of Benjamini and Hochberg for multiple comparisons. (J) SR Ca2+ load measured as Ca2+ transient amplitude elicited with 20 mM caffeine (caffeine-induced Ca2+ transient). For WT, n = 34 cells from 8 mice (n = 6 males, n = 2 females, 9–23 weeks old); cD96V, n = 30 cells from 12 mice (n = 4 males, n = 8 females, 8–25 weeks old); and cD96V cNaV1.6-KO, n = 27 cells from 6 mice (n = 3 males, n = 3 females, 14–26 weeks old). **q < 0.01 and ****q < 0.0001, by ordinary 1-way ANOVA with the original FDR method of Benjamini and Hochberg for multiple comparisons.