The Role of MAPRE2 and Microtubules in Maintaining Normal Ventricular Conduction

Abstract

Background: 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 EB2 (microtubule end-binding protein 2), implicating microtubule involvement in Brugada syndrome.

Methods: A mapre2 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 cdh2 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 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-CMs. 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-CMs 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.

Keywords: arrhythmias, cardiac; electrophysiology; microtubules; sodium channels; zebrafish.

Figure 1.. mapre2 loss-of-function leads to decreased ventricular conduction and Na_V function.

A-C. Voltage mapping of hearts isolated from WT vs. heterozygous (HET) and homozygous (HOMO) mapre2 KO zebrafish. In HOMO hearts, there was a nonsignificant decrease in ventricular conduction velocity (CV; A), a significant decrease in ventricular V_max (maximum action potential upstroke velocity, dV/dt; P=0.0121 vs. WT; B), and a nonsignificant increase in ventricular action potential duration (APD; C). There was also a significant decrease in ventricular V_max in HET hearts compared to WT (P=0.0363). Multiple comparisons were done using Dunnett’s test if one-way ANOVA was significant (P=0.0147 for V_max in B). D-F. Voltage mapping of hearts isolated from control (CTL) vs. mapre2 morpholino (MO) injected larvae. In MO hearts, there was a significant decrease in ventricular CV (unpaired t-test: P<0.0001; D) and V_max (unpaired t-test: P<0.0001; E), and a significant increase in ventricular APD (Mann-Whitney test: P=0.0021; F). All hearts (represented by dots) were isolated from 5 dpf zebrafish larvae. The dotted squares in A and D reflect the main ventricular area in the hearts from which the parameters were measured. APD was measured at 80% repolarization while the hearts were paced at 100 bpm.

Figure 2.. mapre2 loss-of-function leads to conduction slowing in adult fish.

Two-lead surface ECG was performed in anesthetized fish from the mapre2 KO line. A. Representative averaged ECG tracings demonstrating similarity to human ECG with the notable exception of the inverted T wave. B-G. In homozygous mapre2 KO, there is a nonsignificant increase in P wave duration and a significant increase in QRS duration, suggesting ventricular conduction slowing, with heterozygotes showing an intermediate phenotype (one-way ANOVA P<0.0001 followed by Tukey’s multiple comparisons test: P=0.0435 WT vs. HET; P=0.0314 HET vs. HOMO; P<0.0001 WT vs. HOMO; F). Each dot represents one fish.

Figure 3.. mapre2 loss-of-function leads to decreased Na⁺ current.

A. Voltage clamp protocol used to measure I_Na (top panel) and typical I_Na traces (bottom) of a freshly isolated WT and KO myocyte. As detailed in the Supplement, we collected ventricles from 4–5 adult fish which were pooled for each cell isolation. The ‘n’ in this figure indicates the number of cells measured from three isolations. B. Average current voltage (I-V) relationships (left panel) and dot plots of I_Na density at −20 mV (right panel) in WT and KO myocytes showing a decrease of I_Na density in KO myocytes. I-V relationships were compared with the two-way repeated measures ANOVA (P=0.036) followed by pairwise comparison using the Student–Newman–Keuls test. The I_Na densities at −20 mV were compared using the Student’s t-test. The numbers indicate the P values lower than 0.05. C. The time course of current inactivation at −20 mV was fitted by a double-exponential equation: I/I_max=A_f×exp(−t/τ_f)+A_s×exp(−t/τ_s), where A_f and A_s are the fractions of the fast and slow inactivation components, and τf and τs are the time constants of the fast and slow inactivating components, respectively. Data did not differ significantly (Student’s t-test). Neither the time constants nor the relative amplitudes were different between WT and KO myocytes. D. Voltage-dependency of activation (left panels) and V_1/2 and k of the Boltzmann fits of every cell measured (right panels). Solid lines are Boltzmann fits to the average data. Data did not differ significantly (Student’s t-test). E. Voltage clamp protocol used to measure voltage dependency of I_Na inactivation (top panel) and typical I_Na inactivating traces (bottom panels). F. Voltage-dependency of inactivation and V_1/2 and k of the Boltzmann fits of all cells (right panels). Solid lines are Boltzmann fits to the average data. Data did not differ significantly (Student’s t-test).

Figure 4.. mapre2 loss-of-function leads to disruption of adherens junctions.

A. Representative immunostaining of hearts from WT versus homozygous mapre2 KO larvae shows a general disorganization of ventricular N-cadherin (Ncad). Representative of 2 WT vs. 2 KO hearts. B. Immunocytochemistry of hearts from WT versus homozygous mapre2 KO larvae on the transgenic background with cdh2 tandem fluorescent timer (tFT). Immunostaining of zonula occludens-1 (ZO-1) was used to mark cell borders. Signal from GFP which takes 5 min to fold marks nascent Ncad whereas signal from RFP which takes 100 min to fold marks stable Ncad. Quantification of GFP and RFP signals using ZO-1 signal as a mask shows no significant change in nascent Ncad localization (C) but decreased stable Ncad localization at cell borders (unpaired t-tests: P=0.0020 in D and P=0.0278 in E), suggesting disruption of adherens junctions. Each dot represents one heart. Representative images were chosen based on closeness to group mean and image quality.

Figure 5.. mapre2 loss-of-function leads to decreased detyrosinated tubulin.

A. Representative immunostaining of hearts from WT (n=8) and homozygous KO (n=16) larvae showing a decrease in ventricular detyrosinated tubulin (Glu-tubulin) relative to total α-tubulin. B. Quantification of ventricular Glu-tubulin signal using α-tubulin signal as a mask (unpaired t-test: P=0.0072). C. Representative immunostaining of ventricular myocytes isolated from adult WT (21 cells from 2 fish) and homozygous KO fish (20 cells from 3 fish) also showing a decrease in ventricular detyrosinated tubulin (Glu-tubulin) relative to total α-tubulin. D. Quantification of ventricular Glu-tubulin signal using α-tubulin signal as a mask (unpaired t-test: P=0.0232). Representative images were chosen based on closeness to group mean and image quality.

Figure 6.. mapre2 loss-of-function leads to changes in microtubule dynamics.

A. Representative still images of microtubule plus-end tracking experiments in iPSC-derived cardiomyocytes with MAPRE2 knockdown (KD) using 2 different siRNA versus control siRNA. Scale bars = 10 μm. Please refer to the Supplement for representative videos (Video S1 is Control, Video S2 is siRNA#1, Video S3 is siRNA#2). B. Representative kymographs obtained from the live-cell imaging, which were used for measurement of microtubule parameters. Vertical scale bars = 10 seconds; horizontal scale bars = 5 μm. Compared to control, MAPRE2 knockdown with siRNA #1 and #2 resulted in 1.11-fold (P=0.0079) and 1.26-fold (P<0.0001) increases in microtubule growth velocity (C-D), and in 1.22-fold (P<0.0001) and 1.34-fold (P<0.0001) increases in microtubule growth distance (E-F), respectively. Dunn’s multiple comparisons tests were used following significant Kruskal-Wallis tests (P<0.0001 for both D and F). Data extracted from 368 Control microtubules, 314 siRNA #1 microtubules, and 194 siRNA #2 microtubules, in 5 sets of cells. Representative videos and images were chosen based on closeness to group mean and quality.

Figure 7.. Knockdown of ttl restores fraction of detyrosinated tubulin and stable to nascent Ncad in mapre2 KO hearts.

A. Representative immunostaining of hearts from homozygous mapre2 KO larvae injected with control (ctl) versus ttl morpholinos showing a restoration of ventricular detyrosinated tubulin (Glu-tubulin) signal relative to total α-tubulin signal. B. Quantification of ventricular Glu-tubulin signal using α-tubulin signal as a mask (unpaired t-test: P=0.0017). C. Immunocytochemistry of hearts from homozygous mapre2 KO larvae on the transgenic background with cdh2 tandem fluorescent timer (tFT). Immunostaining of zonula occludens-1 (ZO-1) was used to mark cell borders. D. Quantification of GFP and RFP signals using ZO-1 signal as mask shows a significant increase in stable to nascent Ncad localization at cell borders (unpaired t-test: P=0.0028), suggesting a restoration of Ncad balance at adherens junctions. Representative images were chosen based on closeness to group mean and image quality. Each dot represents one heart.

Figure 8.. Knockdown of ttl restores ventricular conduction in mapre2 KO hearts.

Voltage mapping of hearts isolated from homozygous mapre2 KO larvae injected with control (ctl) versus ttl morpholinos (MO). ttl knockdown resulted in a significant increase of ventricular conduction velocity (CV; Mann-Whitney test: P=0.0177; A) and V_max (maximum action potential upstroke velocity, dV/dt; unpaired t-test: P<0.0001; B), as well as a significant decrease in ventricular action potential duration (APD; unpaired t-test: P=0.0401; C). All hearts (represented by dots) were isolated from 5 dpf zebrafish larvae. The dotted squares in (A) reflect the main ventricular area from which the parameters were measured. APD was measured at 80% repolarization while the hearts were paced at 100 bpm.