Calmodulin (CaM) prevents proarrhythmic late sodium current (INa) by facilitating normal inactivation of sodium channels (NaV). Since dysfunction of NaV1.6 has been implicated in late INa-mediated arrhythmias, we investigated its role in arrhythmias promoted by CaM mutant D96V. Super-resolution STED microscopy revealed enlarged NaV1.6 clusters in NaV1.6-expressing Chinese hamster ovary cells transfected with D96V-CaM relative to those transfected with WT-CaM. Therefore, we examined NaV1.6 clustering in transgenic mice with cardiac-specific expression of D96V-CaM (cD96V) with a C-terminal FLAG tag. Confocal microscopy confirmed expression of NaV1.6 and FLAG-tagged D96V-CaM in a striated pattern along with RYR2 in cD96V hearts, consistent with T-tubular localization. In both WT and cD96V hearts, STORM single molecule localization microscopy revealed that ∼50% of NaV1.6 clusters localized <100 nm from RYR2. However, NaV1.6 density within these regions was 67% greater in cD96V relative to WT. Consistent with this result, SICM-guided “smart” patch clamp recording of NaV activity from T-tubule openings revealed more frequent late-burst openings involving larger NaV clusters in cD96V myocytes relative to WT. Previous work identifies the sodium-calcium exchanger (NCX) as a key link between aberrant late NaV1.6 activity and proarrhythmic Ca2+ mishandling. Therefore, we explored the spatial organization of NaV1.6 and NCX using STORM. Consistent with their close association, 89% of NaV1.6 clusters localized <100 nm from NCX in cD96V hearts, compared with 77% in WT. Notably, density of both NaV1.6 and NCX was increased at these sites by 48% and 31%, respectively, in cD96V relative to WT. Consistent with these data, cD96V myocytes displayed larger, more frequent Ca2+ sparks relative to WT. These proarrhythmic functional effects were abrogated by cardiac-specific knockout of NaV1.6. To our knowledge, this is the first demonstration of proarrhythmic cardiac structural remodeling secondary to a defect in calmodulin, offering novel mechanistic insight into calmodulinopathy.

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