PUBLICATION
The Role of MAPRE2 and Microtubules in Maintaining Normal Ventricular Conduction
- Authors
- Chiang, D.Y., Verkerk, A.O., Victorio, R., Shneyer, B.I., van der Vaart, B., Jouni, M., Narendran, N., Kc, A., Sampognaro, J.R., Vetrano-Olsen, F., Oh, J.S., Buys, E., de Jonge, B., Shah, D.A., Kiviniemi, T., Burridge, P.W., Bezzina, C.R., Akhmanova, A., MacRae, C.A.
- ID
- ZDB-PUB-231214-14
- Date
- 2023
- Source
- Circulation research 134(1): 46-59 (Journal)
- Registered Authors
- MacRae, Calum A.
- Keywords
- Brugada syndrome, action potentials, cadherins, immunohistochemistry, morpholinos
- MeSH Terms
-
- Action Potentials
- Adult
- Animals
- Brugada Syndrome*/genetics
- Brugada Syndrome*/metabolism
- Genome-Wide Association Study
- Humans
- Induced Pluripotent Stem Cells*/metabolism
- Microtubule-Associated Proteins/genetics
- Microtubules/metabolism
- Myocytes, Cardiac/metabolism
- NAV1.5 Voltage-Gated Sodium Channel/genetics
- NAV1.5 Voltage-Gated Sodium Channel/metabolism
- Tubulin/genetics
- Tubulin/metabolism
- Voltage-Gated Sodium Channels*/metabolism
- Zebrafish/genetics
- Zebrafish/metabolism
- PubMed
- 38095085 Full text @ Circ. Res.
Citation
Chiang, D.Y., Verkerk, A.O., Victorio, R., Shneyer, B.I., van der Vaart, B., Jouni, M., Narendran, N., Kc, A., Sampognaro, J.R., Vetrano-Olsen, F., Oh, J.S., Buys, E., de Jonge, B., Shah, D.A., Kiviniemi, T., Burridge, P.W., Bezzina, C.R., Akhmanova, A., MacRae, C.A. (2023) The Role of MAPRE2 and Microtubules in Maintaining Normal Ventricular Conduction. Circulation research. 134(1):46-59.
Abstract
Background The Brugada syndrome is associated with loss-of-function SCN5A variants, yet these account for only ≈20% of cases. A recent genome-wide association study identified a novel locus within MAPRE2, which encodes EB (microtubule end-binding protein) 2, implicating microtubule involvement in the Brugada syndrome.
Methods A mapre2 knockout zebra fish model was generated using CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeat-associated 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 zebra fish line with cdh2 tandem fluorescent timer was used to study adherens junctions. Microtubule plus-end tracking and patch clamping were performed in human iPSC-derived cardiomyocytes with MAPRE2 knockdown and knockout, respectively.
Results Voltage mapping of mapre2 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 cardiomyocytes. Confocal imaging showed disorganized adherens junctions and mislocalization of mature Ncad (N-cadherin) with mapre2 loss of function, associated with a decrease of detyrosinated tubulin. MAPRE2 knockdown in iPSC cardiomyocytes led to an increase in microtubule growth velocity and distance, indicating changes in microtubule dynamics. Finally, knockdown of ttl encoding tubulin tyrosine ligase in mapre2 knockout larvae rescued tubulin detyrosination and ventricular maximum upstroke velocity of the action potential.
Conclusions Genetic ablation of mapre2 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 ttl knockdown led to rescue of voltage-gated sodium channel-related functional parameters in mapre2 knockout hearts. Taken together, our study implicates microtubule dynamics in the modulation of ventricular conduction.
Genes / Markers
Expression
Phenotype
Mutations / Transgenics
Human Disease / Model
Sequence Targeting Reagents
Fish
Orthology
Engineered Foreign Genes
Mapping