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

Cardiac-specific expression of D96V-CaM promotes NaV1.6-mediated cellular and in vivo arrhythmias.

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Cardiac-specific expression of D96V-CaM promotes NaV1.6-mediated cellula...
(A) Late INa measurements. For WT, n = 13 cells from 6 mice (n = 3 males, n = 3 females, 6–19 weeks old); FLAG-tagged cD96V, n = 9 cells from 4 mice (n = 2 males, n = 2 females, 20–25 weeks old); and FLAG-tagged cD96V cNaV1.6-KO, n = 15 cells from 5 mice (n = 2 males, n = 3 females, 7–25 weeks old). ****q < 0.0001, by ordinary 1-way ANOVA with the original FDR method of Benjamini and Hochberg for multiple comparisons. (B) Ca2+ waves from WT (top), cD96V (middle), and cD96V cNaV1.6-KO (bottom) cardiomyocytes paced at 0.3 kHz. Black lines above the image denote stimuli. For WT, n = 96 cells from 13 mice (n = 7 males, n = 6 females, 9–27 weeks old); cD96V, n = 106 cells from 10 mice (n = 6 males, n = 4 females, 9–31 weeks old); and cD96V cNaV1.6-KO, n = 92 cells from 8 mice (n = 6 males, n = 2 females, 6–31 weeks old). ***q < 0.001 and ****q < 0.0001, by Kruskal-Wallis test with the original FDR method of Benjamini and Hochberg for multiple comparisons. (C) Surface ECGs and QTc intervals from WT, cD96V, and cD96V cNaV1.6-KO mice. For WT, n = 9 mice (n = 4 males, n = 5 females, 12–25 weeks old); cD96V, n = 12 mice (n = 7 males, n = 5 females, 6–18 weeks old); and cD96V cNaV1.6-KO, n = 13 mice (n = 9 males, n = 4 females, 6–26 weeks old). ***q < 0.001, by Kruskal-Wallis test with the original FDR method of Benjamini and Hochberg for multiple comparisons. (D) ECGs recorded from mice after carbachol challenge (0.5 mg/kg i.p. injection), which resulted in sinus bradycardia (SB) (heart rate <200 beats per minute). VT was only observed in 4 of 12 cD96V mice. *P < 0.05, by χ2 test.