The Role of <i>MAPRE2</i> and Microtubules in Maintaining Normal Ventricular Conduction.

Abstract

Brugada syndrome is associated with loss-of-function <i>SCN5A</i> variants, yet these account for only ≈20% of cases. A recent genome-wide association study identified a novel locus within <i>MAPRE2</i>, which encodes EB2 (microtubule end-binding protein 2), implicating microtubule involvement in Brugada syndrome. A <i>mapre2</i> 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 <i>cdh2</i> 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 <i>MAPRE2</i> knockdown and knockout, respectively. Voltage mapping of <i>mapre2</i> 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 <i>mapre2</i> loss of function, associated with a decrease of detyrosinated tubulin. <i>MAPRE2</i> knockdown in iPSC-CMs led to an increase in microtubule growth velocity and distance, indicating changes in microtubule dynamics. Finally, knockdown of <i>ttl</i> encoding tubulin tyrosine ligase in <i>mapre2</i> knockout larvae rescued tubulin detyrosination and ventricular maximum upstroke velocity of the action potential. Genetic ablation of <i>mapre2</i> 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 <i>ttl</i> knockdown led to rescue of voltage-gated sodium channel-related functional parameters in <i>mapre2</i> knockout hearts. Taken together, our study implicates microtubule dynamics in the modulation of ventricular conduction.