The Role of and Microtubules in Maintaining Normal Ventricular Conduction.

BACKGROUND

Brugada syndrome is associated with loss-of-function variants, yet these account for only ≈20% of cases. A recent genome-wide association study identified a novel locus within , which encodes EB2 (microtubule end-binding protein 2), implicating microtubule involvement in Brugada syndrome.

METHODS

A knockout zebrafish model was generated using CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeat-associated protein 9) and validated by Western blot. Larval hearts at 5 days post-fertilization were isolated for voltage mapping and immunocytochemistry. Adult fish hearts were used for ECG, patch clamping, and immunocytochemistry. Morpholinos were injected into embryos at 1-cell stage for knockdown experiments. A transgenic zebrafish line with tandem fluorescent timer was used to study adherens junctions. Microtubule plus-end tracking and patch clamping were performed in human induced pluripotent stem cell derived cardiomyocytes (iPSC-CMs) with knockdown and knockout, respectively.

RESULTS

Voltage mapping of knockout hearts showed a decrease in ventricular maximum upstroke velocity of the action potential and conduction velocity, suggesting loss of cardiac voltage-gated sodium channel function. ECG showed QRS prolongation in adult knockout fish, and patch clamping showed decreased sodium current density in knockout ventricular myocytes and arrhythmias in knockout iPSC-CMs. Confocal imaging showed disorganized adherens junctions and mislocalization of mature Ncad (N-cadherin) with loss of function, associated with a decrease of detyrosinated tubulin. knockdown in iPSC-CMs led to an increase in microtubule growth velocity and distance, indicating changes in microtubule dynamics. Finally, knockdown of encoding tubulin tyrosine ligase in knockout larvae rescued tubulin detyrosination and ventricular maximum upstroke velocity of the action potential.

CONCLUSIONS

Genetic ablation of led to a decrease in voltage-gated sodium channel function, a hallmark of Brugada syndrome, associated with disruption of adherens junctions, decrease of detyrosinated tubulin as a marker of microtubule stability, and changes in microtubule dynamics. Restoration of the detyrosinated tubulin fraction with knockdown led to rescue of voltage-gated sodium channel-related functional parameters in knockout hearts. Taken together, our study implicates microtubule dynamics in the modulation of ventricular conduction.