TBX5 drives Scn5a expression to regulate cardiac conduction system function
David E. Arnolds, … , Vickas V. Patel, Ivan P. Moskowitz
David E. Arnolds, … , Vickas V. Patel, Ivan P. Moskowitz
Published June 25, 2012
Citation Information: J Clin Invest. 2012;122(7):2509-2518. https://doi.org/10.1172/JCI62617.
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Research Article

TBX5 drives Scn5a expression to regulate cardiac conduction system function

Abstract

Cardiac conduction system (CCS) disease, which results in disrupted conduction and impaired cardiac rhythm, is common with significant morbidity and mortality. Current treatment options are limited, and rational efforts to develop cell-based and regenerative therapies require knowledge of the molecular networks that establish and maintain CCS function. Recent genome-wide association studies (GWAS) have identified numerous loci associated with adult human CCS function, including TBX5 and SCN5A. We hypothesized that TBX5, a critical developmental transcription factor, regulates transcriptional networks required for mature CCS function. We found that deletion of Tbx5 from the mature murine ventricular conduction system (VCS), including the AV bundle and bundle branches, resulted in severe VCS functional consequences, including loss of fast conduction, arrhythmias, and sudden death. Ventricular contractile function and the VCS fate map remained unchanged in VCS-specific Tbx5 knockouts. However, key mediators of fast conduction, including Nav1.5, which is encoded by Scn5a, and connexin 40 (Cx40), demonstrated Tbx5-dependent expression in the VCS. We identified a TBX5-responsive enhancer downstream of Scn5a sufficient to drive VCS expression in vivo, dependent on canonical T-box binding sites. Our results establish a direct molecular link between Tbx5 and Scn5a and elucidate a hierarchy between human GWAS loci that affects function of the mature VCS, establishing a paradigm for understanding the molecular pathology of CCS disease.

Authors

David E. Arnolds, Fang Liu, John P. Fahrenbach, Gene H. Kim, Kurt J. Schillinger, Scott Smemo, Elizabeth M. McNally, Marcelo A. Nobrega, Vickas V. Patel, Ivan P. Moskowitz

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Figure 6

TBX5 directly regulates an enhancer downstream of Scn5a.

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TBX5 directly regulates an enhancer downstream of Scn5a. (A) Bioinform...
(A) Bioinformatic identification of a candidate enhancer downstream of Scn5a. We used previously reported data sets to identify potential TBX-responsive enhancers: bioinformatic predictions of cardiac enhancers (24); p300 ChIP-seq peaks to mark active enhancers in the E11.5 heart (23); ChIP-seq studies identifying both p300 and TBX5 binding sites in the atrial cardiomyocyte HL-1 cell line (22); and evolutionary conservation, as assessed by genomic evolutionary rate profiling score (21). A region demonstrating overlap in all 4 data sets, approximately 15 kb downstream of Scn5a, is shaded yellow. (B) The WT candidate enhancer demonstrated robust TBX5-mediated activation in dual luciferase reporter assays in HEK-293T cells. Luciferase activity was blunted by single mutation of any of 3 conserved T-box elements (TBE1 mut, TBE2 mut, or TBE3 mut) and eliminated by mutation of all 3 T-box elements (TBE123 mut). *P < 0.05 versus all other groups; n ≥ 3; mean ± SEM. (C–H) VCS of transient transgenic embryos, analyzed at E13.5. Whereas the WT enhancer reproducibly drove lacZ expression from a minimal promoter (C–E), mutation of the T-box elements in the enhancer resulted in blunted and regionally variable expression (F–H). Shown are representative posterior (C and F) and sagittal section (D and G) views of X-Gal staining. Higher-magnification views of boxed regions in D and G demonstrated X-gal expression (E) or its absence (H) in the developing AV bundle (arrowhead) and bundle branches (arrow). Note that, in contrast to the weak, non-CCS lacZ expression in F, the more robustly stained heart in G demonstrated ectopic expression in the endocardial cushions and compact myocardium. (I) Model for the role of TBX5 in driving fast conduction in the VCS via direct regulation of Scn5a and Gja5. Original magnification, ×4 (D and G); ×40 (E and H).