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 2

D96V-CaM impairs INa inactivation in murine and human iPSC-CMs.

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D96V-CaM impairs INa inactivation in murine and human iPSC-CMs. (A) Repr...
(A) Representative late INa traces recorded in WT murine cardiomyocytes dialyzed with 6.5 μM WT CaM (black), 6.5 μM D96V-CaM in the absence (red) or presence (blue) of 300 nM of 4,9ahTTX, and 5.4 μM WT CaM (83%) plus 1.1 μM D96V-CaM (17%; orange). The voltage protocol is illustrated above the traces. In these experiments, the recombinant CaMs were not FLAG tagged. (B) Summary: Late INa integral. For WT CaM, n = 21 cells from 9 mice (n = 4 females, n = 5 males, 6–26 weeks old); D96V-CaM n = 23 cells from 11 mice (n = 6 males, n = 5 females, 6–15 weeks old); D96V-CaM plus 4.9ahTTX, n = 9 cells from 6 mice (n = 3 males, n = 3 females, 6–15 weeks old); 5.4 μM WT CaM (83%) plus 1.1 μM D96V-CaM (17%), n = 17 cells from 8 mice (n = 4 males, n = 4 females, 12–24 weeks old). **q < 0.01 and ****q < 0.0001, by ordinary 1-way ANOVA test with the original FDR method of Benjamini and Hochberg for multiple comparisons. (C) Steady-state inactivation curves and (D) the corresponding half-maximal voltage (V1/2) of inactivation. For WT CaM, n = 12 cells from 6 mice (n = 2 females, n = 4 males, 6–26 weeks old); D96V-CaM, n = 19 cells from 8 mice (n = 5 males, n = 3 females, 6–25 weeks old); D96V-CaM plus 4.9ahTTX (300 nM), n = 10 cells from 5 mice (n = 2 males, n = 3 females, 6–25 weeks old), 5.4 μM WT CaM (83%) plus 1.1 μM D96V-CaM (17%), n = 10 cells from 7 mice (n = 4 males, n = 3 females, 11–24 weeks old). *q < 0.05, **q < 0.01, and ***q < 0.001, by ordinary 1-way ANOVA with the original FDR method of Benjamini and Hochberg for multiple comparisons. (E) Peak INa I–V relationship and (F) normalized INa conductance with the corresponding V1/2 of activation (inset). For WT CaM, n = 13 cells from 7 mice (n = 4 males, n = 3 females, 6–26 weeks old); D96V-CaM n = 10, n = 7 (n = 5 males, n = 2 females, 6–25 weeks old); D96V-CaM plus 4.9ahTTX (300 nM) n = 9, n = 7 (n = 4 males, n = 3 females, 6–25 weeks old); 5.4 μM WT CaM (83%) plus 1.1 μM D96V-CaM (17%) n = 6, n = 3 (n = 2 males, n = 1 female, 12–18 weeks old). *q < 0.05 for peak INa of D96V-CaM plus 4.9ahTTX versus D96V-CaM at –45 mV. q > 0.05 (NS), by Kruskal-Wallis test with the original FDR method of Benjamini and Hochberg for multiple comparisons. (G) Representative late INa recorded from human iPSC-CMs dialyzed with 6.5 μM WT CaM (black) or D96V-CaM in the absence (red) or presence (blue) of 300 nM 4,9ahTTX. (H) Summary: Late INa integral. For WT CaM, n = 14; D96V-CaM, n = 17; D96V-CaM plus 4.9ahTTX (300 nM), n = 11. **q < 0.01, by Kruskal-Wallis test with the original FDR method of Benjamini and Hochberg for multiple comparisons. A∙s∙F–1, amperes seconds per Farad; G/Gmax, normalized membrane conductance.