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

Calmodulin (CaM) plays critical roles in cardiomyocytes, regulating Na+
(NaV) and L-type Ca2+ channels (LTCC). 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 induces
pro-arrhythmic 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. 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 (VT)
in vivo. Cardiac-specific NaV1.6 knockout protected cD96V mice from
increased T-tubular late NaV activity, and its arrhythmogenic
consequences. In summary, we demonstrate that D96V-CaM promotes
arrhythmias by dysregulating LTCC and NaV1.6 within T-tubules and thereby,
facilitating aberrant Ca2+ release.

钙调蛋白（Calmodulin, CaM）在心肌细胞中发挥关键调控作用，可调节钠离子通道（NaV）与L型钙离子通道（L-type Ca²⁺ channels, LTCC）的功能。突变型钙调蛋白引发的LTCC功能失调，已被证实与动作电位时程（action potential duration, APD）延长及致心律失常性长QT综合征（arrhythmogenic long QT syndrome, LQT）密切相关。值得注意的是，尽管D96V突变型钙调蛋白（D96V-CaM）诱导的LTCC功能障碍水平与其他LQT相关钙调蛋白相当，但其引发的APD延长程度却更为显著，这提示其还可通过调控其他去极化通道引发功能异常。本研究为横小管（transverse tubules, T-小管）内的NaV功能失调参与D96V-CaM相关心律失常提供了实验证据。研究发现，D96V-CaM可通过损伤钠通道亚型1.6（NaV1.6）的失活过程，诱导促心律失常的晚钠电流（late Na⁺ current, INa），但对心肌细胞主要表达的钠通道亚型NaV1.5并无此作用。我们借助心肌特异性表达D96V-CaM的小鼠模型（cD96V）探究心律失常的致病机制。超分辨率显微镜技术（super-resolution microscopy）显示，T-小管内NaV1.6与兰尼碱受体2（Ryanodine receptor 2, RyR2）存在紧密邻近关系；与野生型（WT）小鼠相比，cD96V小鼠T-小管内的NaV1.6密度显著升高。结合D96V-CaM对该区域内NaV1.6的功能失调作用，我们观测到T-小管内的晚钠电流活性显著增强。由此产生的晚钠电流可诱发心肌细胞内异常钙离子释放，并延长APD，最终在活体水平引发LQT与室性心动过速（ventricular tachycardia, VT）。心肌特异性敲除NaV1.6可使cD96V小鼠免受T-小管晚钠电流活性升高及其致心律失常后果的影响。综上，本研究证实D96V-CaM可通过失调T-小管内的LTCC与NaV1.6功能，诱导异常钙离子释放，进而促进心律失常的发生。