Transcriptional Patterning of the Ventricular Cardiac Conduction System - PubMed (original) (raw)

. 2020 Jul 17;127(3):e94-e106.

doi: 10.1161/CIRCRESAHA.118.314460. Epub 2020 Apr 15.

Ozanna Burnicka-Turek 1 2 3, Jeffrey D Steimle 1 2 3, Bastiaan J Boukens 5 6, Nataliya B Petrenko 7, Kohta Ikegami 1 2 3, Rangarajan D Nadadur 1 2 3, Yun Qiao 5, David E Arnolds 1 2 3, Xinan H Yang 1 2 3, Vickas V Patel 8, Marcelo A Nobrega 3, Igor R Efimov 5, Ivan P Moskowitz 1 2 3

Affiliations

Transcriptional Patterning of the Ventricular Cardiac Conduction System

Ozanna Burnicka-Turek et al. Circ Res. 2020.

Abstract

Rationale: The heartbeat is organized by the cardiac conduction system (CCS), a specialized network of cardiomyocytes. Patterning of the CCS into atrial node versus ventricular conduction system (VCS) components with distinct physiology is essential for the normal heartbeat. Distinct node versus VCS physiology has been recognized for more than a century, but the molecular basis of this regional patterning is not well understood.

Objective: To study the genetic and genomic mechanisms underlying node versus VCS distinction and investigate rhythm consequences of failed VCS patterning.

Methods and results: Using mouse genetics, we found that the balance between T-box transcriptional activator, Tbx5, and T-box transcriptional repressor, Tbx3, determined the molecular and functional output of VCS myocytes. Adult VCS-specific removal of Tbx5 or overexpression of Tbx3 re-patterned the fast VCS into slow, nodal-like cells based on molecular and functional criteria. In these cases, gene expression profiling showed diminished expression of genes required for VCS-specific fast conduction but maintenance of expression of genes required for nodal slow conduction physiology. Action potentials of _Tbx5_-deficient VCS myocytes adopted nodal-specific characteristics, including increased action potential duration and cellular automaticity. Removal of Tbx5 in vivo precipitated inappropriate depolarizations in the atrioventricular (His)-bundle associated with lethal ventricular arrhythmias. TBX5 bound and directly activated _cis_-regulatory elements at fast conduction channel genes required for fast physiological characteristics of the VCS action potential, defining the identity of the adult VCS.

Conclusions: The CCS is patterned entirely as a slow, nodal ground state, with a T-box dependent, physiologically dominant, fast conduction network driven specifically in the VCS. Disruption of the fast VCS gene regulatory network allowed nodal physiology to emerge, providing a plausible molecular mechanism for some lethal ventricular arrhythmias.

Keywords: His bundle/atrioventricular bundle; Tbx3; Tbx5; arrhythmia; cardiac conduction system; heart rhythm; ventricular conduction.

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Figures

Figure 1.

Figure 1.. Tbx5/Tbx3 balance determines the regional functional output within the specialized mature CCS.

(A-C) Relative Tbx3/Tbx5 expression ratio in wild-type AVN and VCS cardiomyocytes isolated from adult mouse hearts. The qRT-PCR (A) and western blot (B) demonstrated that Tbx3 expression predominates over Tbx5 in the slow AV-nodal cardiomyocytes and that Tbx5 predominates over Tbx3 expression in the fast VCS cardiomyocytes. The AVN cardiomyocytes used in these studies were marked by EGFP expression and isolated from Tbx3BAC-Egfp mice (27) at 10 weeks of age. VCS cardiomyocytes, used in these studies were marked by EYFP expression using a VCS-specific tamoxifen (TM) inducible Cre transgenic mouse line (MinK CreERT2) (14) in combination with conditional EYFP expression from the ROSA locus (R26 EYFP/+ ; MinK CreERT2/+) (7) and microdissected at 10 weeks of age. (C) Model of Tbx3/Tbx5 expression ratio in cardiac conduction system. (D-G) VCS-specific Tbx3 haploinsufficiency in Tbx3:Tbx5 compound heterozygous mice (Tbx3 fl/+ ;Tbx5 fl/+ ;R26 EYFP/+ ;MinKCre ERT2/+) rescues VCS defects caused by VCS-specific Tbx5 haploinsufficiency, in Tbx5 heterozygous mice (Tbx5 fl/+ ;R26 EYFP/+ ;MinK CreERT2/+). PR (D, left graph), QRS (D, right graph) and H-V (E, right graph) intervals prolongation, widening of the His duration (E, middle graph), as well as ventricular arrhythmias including reproducible pacing into ventricular tachycardia (VT) (F), observed in VCS-specific Tbx5 heterozygous mice (Tbx5 fl/+ ;R26 EYFP/+ ; MinK CreERT2/+), all got rescued in VCS-specific Tbx3:Tbx5 compound heterozygous mice (Tbx3 fl/+ ;Tbx5 fl/+ ;R26 EYFP/+ ; MinK CreERT2/+). Control VCS-specific Tbx3 heterozygous mice (Tbx3 fl/+ ;R26 EYFP/+ ;MinK CreERT2/+) showed neither conduction nor electrophysiological defects (D-F). Note, ventricular pacing at 100ms S1 drive train followed by 50ms S2 single extrastimulus induced VT in 5/9 Tbx5 fl/+ ;R26 EYFP/+ ; MinK _CreERT2/+_mice (F, upper panel, representative image), but failed to induced VT in all Tbx3/Tbx5 compound Tbx3 fl/+ ;Tbx5 fl/+ ;R26 EYFP/+ ; MinK CreERT2/+ mice (F, lower panel, representative image). (G) Summary of genetic interactions between Tbx5 and Tbx3 in the adult VCS. Data are presented as mean±SD. For (A), n=3 biological replicates/ analyzed tissue sample (AVN or VCS cardiomyocytes pooled from 30 mice per each biological replicate). (B), n=2 biological replicates/ analyzed tissue sample (AVN or VCS cardiomyocytes pooled from 50 mice per each biological replicate). For (D), n=4 animals/ genotype. For (E, F), n=4–9 animals/ genotype. For (A, B, D – QRP, F), Welch’s t-test; *P<0.05, ***P<0.01. For (D-PR, E), Mann-Whitney U test; *P<0.05. Abbreviations: PR, PR-interval duration; QRS, QRS complex, H-V, Hisio-Ventricular interval; Hd, His-duration.

Figure 2.

Figure 2.. Proper Tbx3/Tbx5 balance is required to maintain expression of VCS-specific fast conduction genes.

(A-D) Characterization of _Tbx5_-dependent gene expression in the adult mouse VCS. Tbx5 was specifically removed from the mature VCS in Tbx5 fl/fl ;R26 EYFP/+ ;MinK CreERT2/+ mice (7), TM treated at 6-weeks of age and analyzed by qRT-PCR with two sets of CCS-specific markers at 10 weeks of age. (A) One set of markers comprised genes normally expressed in both the node and VCS (Pan-CCS) and important for the slow conducting nodal phenotype. None of these genes demonstrated _Tbx5_-dependent VCS expression by qRT-PCR. (B) The second set comprised genes normally expressed specifically in the VCS (excluded from the nodes) and essential for VCS function. Expression of all these genes was profoundly _Tbx5-_dependent by qRT-PCR. (C) Immunoblotting analysis confirmed _Tbx5_-dependent expression in the VCS. (D) Graphical summary of transcriptional changes observed in VCS of VCS-specific _Tbx5_-deficient mice. Genetic deletion of Tbx5 from the mature VCS uncovers its slow conduction, nodal molecular phenotype. (E-L) VCS-specific TBX3 overexpression in adult CT Tbx3/+ ;MinK CreERT2/+ mice. (E) Strategy to generate VCS-specific TBX3 overexpression in adult CT Tbx3/+ ;MinK CreERT2/+ mice. CT Tbx3 transgenic mice carrying a Cre-inducible TBX3 expression construct (CAG–CAT–Tbx3) (24) were crossed to VCS-specific tamoxifen inducible Cre transgenic mouse line (MinK CreERT2) (14) to generate CT Tbx3/+ ;MinK CreERT2/+ mice. The control (CT Tbx3/+ :MinK +/+) and mutant (CT Tbx3/+ :MinK CreERT2/+) mice were tamoxifen administrated at 6 week of age. The effects of VCS-specific TBX3 overexpression were assessed 4 weeks after tamoxifen administration (at 10 weeks of age). (F) Western blotting analysis demonstrates 3.8 fold induced overexpression of TBX3 in the mutant VCS cardiomyocytes compared to their control littermates. (G,H) VCS-specific TBX3 overexpression causes significant VCS conduction slowing in adult CT Tbx3/+ ;MinK CreERT2/+ mice. (G) Ambulatory telemetry ECG analysis showed increased PR and QRS intervals in CT Tbx3/+ :MinK CreERT2/+ mice compared to littermate CT Tbx3/+ ;MinK +/+ control mice. Moreover, intracardiac electrophysiology detected increased A-H, H-V, and His duration (Hd) intervals (H), all indicative of VCS conduction slowing, only in CT Tbx3/+ :MinK CreERT2/+ mutants never in CT Tbx3/+ ;MinK +/+ controls. (I,J) qRT-PCR analysis of molecular changes driven by Tbx3 overexpression in VCS of adult mice. VCS-specific Tbx3 overexpression did not affect expression of Pan-CCS-specific slow conduction genes (I), but caused a significant reduction in expression of VCS-specific fast conducting genes (J) in CT Tbx3/+ ;MinK CreERT2/+ mutants compared to littermate CT Tbx3/+ ;MinK +/+ controls. (K) Immunoblotting analysis confirmed a significant reduction in expression of VCS-specific fast conducting genes in CT Tbx3/+ ;MinK CreERT2/+ mutants compared to littermate CT Tbx3/+ ;MinK +/+ controls (L) Graphical summary of transcriptional changes observed in mice with induced VCS-specific Tbx3 overexpression. Data are presented as mean±SD. For (A, B, I, J), n=3 biological replicates/ genotype (VCS cardiomyocytes pooled from 30 mice per each biological replicate). For (C, F, K), n=2 biological replicates/ genotype (VCS cardiomyocytes pooled from 50 mice per each biological replicate). For (G), n=3 or 5 animals/ genotype. For (H), n=3 or 4 animals/ genotype. For (A, B, I, J), Welch’s t-test or Mann-Whitney U test, multiple testing correction using Benjamini & Hochberg procedure; *: FDR≤0.15. For (F-H), Welch’s t-test; *P<0.05. Abbreviations: OE, overexpression; A-H, Atrio-Hisian interval; H-V, Hisio-Ventricular interval; Hd, His-duration.

Figure 3.

Figure 3.. Electrophysiological characterization of mice with VCS-specific TBX5-deficiency.

(A-C) Action potentials (APs) from a control VCS (A), _Tbx5-_deficient VCS (B) and ventricular (C) cardiomyocyte by whole-cell patch–clamp. (A-B) AP of the _Tbx5_-deficient VCS cardiomyocytes demonstrate a slower upstroke (phase 0), longer plateau (phase 2), delayed repolarization (phase 3) and enhanced phase 4 depolarization compared to the control VCS cardiomyocyte. (C) AP of a ventricular cardiomyocyte from a VCS-specific, _Tbx5_-deficient mouse shows a rapid upstroke (phase 0), minimal plateau (phase 2), rapid repolarization (phase 3) and no phase 4 depolarization. The short horizontal bar in each panel represents the zero potential level. (D) Table showing detailed AP parameters recorded from mutant VCS, control VCS and ventricular cardiomyocytes. (E, F) VCS-specific removal of Tbx5 induces cellular automaticity in adult VCS cardiomyocytes. (E) No evidence of autonomous electrical activity, including phase 4 depolarization or autonomous beating, were observed in VCS cardiomyocytes isolated from control mice. (F) Phase 4 depolarization (arrows) and autonomous beating were observed in all VCS cardiomyocytes isolated from _Tbx5_-deficient mice. (G) Summary of cell autonomous defect observed in adult, _Tbx5_-deficient VCS cardiomyocytes. For (A-F), n=6 or 8 biological replicates/ genotype; Kruskal-Wallis H test; *P<0.05. Abbreviations: APD50, 90, AP duration at 50 and 90% of repolarization; APA, AP amplitude, RMP, resting membrane potential.

Figure 4.

Figure 4.. Spontaneous ventricular tachycardia (VT) in VCS-specific _Tbx5-_deficient adult mice originates in the VCS and is caused by inappropriate automaticity within the VCS.

(A). Electrocardiograms recorded from adult VCS-specific_Tbx5_-deficient mice during Langendorff-perfusion showing sinus rhythm and ectopy (extra a-b). (B) Simultaneously recorded optical action potentials showed that ectopic beat originated in the ventricle. (C) Representative voltage activation maps from VCS-specific _Tbx5_–deficient mice have been reconstructed from sinus beat and two consecutive beats during ventricular ectopy. (D) ECG trace of episode of spontaneous VT (right) and reconstructed activation maps during two consecutive beats (left). Note, spontaneous VT was only detected in _Tbx5_-deficient mice, not in control mice. The activation patterns during sinus rhythm (SR) and VT are similar. (E) Graphic model of inappropriate activation pattern observed within the VCS of VCS-specific Tbx5 mutant mice that results in spontaneous VT. For (A-D), n=4 or 6 animals/ genotype.

Figure 5.

Figure 5.. TBX5 binds and directly activates cis-regulatory elements at VCS conduction loci, establishing a T-box-dependent gene regulatory network (GRN).

(A) Classification of the 12,436 peaks identified by TBX5 ChIP-seq from E14.5 whole heart based on distance to nearest transcription start site (TSS) and relevant histone data. (B) Central enrichment plots for the TBX5 ChIP-seq peaks**. (C,D)** Tbx5 directly regulates enhancer downstream of Ryr2 and upstream of Kcnk3. Genome browser views for the Ryr2 (C, upper panel) and Kcnk3 (D, upper panel) loci showing fold-enrichment tracks for TBX5, H3K27ac, and H3K27me3 ChIP-seq. Candidate cis-regulatory elements (CREs, highlighted red) were cloned into pGL4.23 vector to generate CRE-luciferase constructs. Both candidate enhancers demonstrated transcriptional activation in response to Tbx5 expression in dual luciferase reporter assays, in HEK-293T cells (lower panels of C and D, respectively), and in the HL-1 cardiomyocyte cell line with endogenous Tbx5 expression (lower panels of C and D, respectively). Enhancer activation was T-box dependent, as T-box binding element mutation neutralized enhancer activity in each case (lover panels of C and D, respectively). (E,F) Decreased RYR2 and KCNK3/TASK1 expression in the VCS following VCS-specific removal of Tbx5 in Tbx5 f/f :R26 EYFP/+ ;MinK CreERT2/+ mutant mice. Tbx5 +/+ :R26 EYFP/+ ;MinK CreERT2/+ control and Tbx5 f/f :R26 EYFP/+ ;MinK CreERT2/+ mutant mice were administered tamoxifen at 6 weeks of age. RYR2 (E) and KCNK3/TASK1 (F) protein expression was evaluated by immunohistochemistry (green signal) 4–5 weeks later. VCS components (AV bundle and bundle branches) were identified by positive contactin-2 expression on serial sections from control and Tbx5 mutant hearts (E and F, red signal). The contactin-2 positive AV bundle expressed high levels of RYR2 (E, green signal) and KCNK3 (F, green signal) in Tbx5 +/+ :R26 EYFP/+ ;MinK CreERT2/+ control hearts whereas RYR2 (E) and KCNK3/TASK1 (F) expression got drastically extinguished in the contactin-2 positive AV bundle of Tbx5 f/f :R26 EYFP/+ ;MinK CreERT2/+ mutant hearts. Nuclei were stained with DAPI. (G) TBX5 binds and directly activates cis-regulatory elements at fast conduction VCS-specific expressed genes, defining the identity of the adult VCS. In the absence of Tbx5 or mutation of T-box binding elements, the expression of the VCS fast conduction channel genes is suppressed. Data are presented as mean±SD normalized to blank vector control, Welch’s t-test; ***P≤0.01, original magnification, x20. For (C-F), n=3 biological replicates/ genotype.

Figure 6.

Figure 6.. Model of transcriptional architecture in specialized mature CCS.

(A) CCS regional specialization is driven by local expression of _Tbx5_-dependent fast conduction network in the VCS, which overlaps underlying Pan-CCS expression of nodal, slow conduction network. (B) VCS-specific removal of Tbx5 or overexpression of Tbx3 re-patterned the fast VCS into a slow, nodal-like system. (C) The balance of Tbx5, a T-box transcriptional activator, versus Tbx3, a T-box transcriptional repressor, determined the molecular and functional output of VCS myocytes. TBX5 binds and directly activates cis-regulatory elements at fast conduction loci, defining the identity of the adult VCS. In the absence of TBX5 or overexpression of TBX3, the expression of the VCS fast conduction channel genes is suppressed.

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