Ablation of Tbx20 in adult mouse myocardium results in dilation of cardiac
chambers and lethality within 15 days.
To address whether Tbx20 may be playing a role in adult heart, we first determined
the expression profile of Tbx20 in hearts of adult mice. Tbx20
protein is present at the highest levels in the LV free wall and ventricular septum,
at intermediate levels in the RV, and at the lowest levels in the left and right
atria (Figure A).
Cardiomyocyte-specific ablation of Tbx20 in adult mice
results in cardiac chamber dilation and lethality.
To directly evaluate the role of Tbx20 in adult cardiomyocytes, we
generated adult cardiomyocyte-specific Tbx20-KO mice by injecting
mice with tamoxifen intraperitoneally daily for 5 consecutive days (referred to as
Tbx20 conditional KO mice in the remainder of the manuscript).
Following tamoxifen injections, X-gal staining to monitor
efficiency demonstrated Cre-mediated excision within a substantial number of
ventricular myocytes, with little to no excision in atria (Figure B). Tbx20 mRNA levels were
determined by quantitative RT-PCR (qRT-PCR) (not shown), and protein levels were
analyzed by Western blot analyses of total protein isolated from cardiac ventricles
(Figure C). Relative to levels in control
littermates, Tbx20 mRNA and protein expression in mutant ventricles
was substantially downregulated by day 3 following tamoxifen injection. Together,
these results demonstrated the efficacy of the conditional KO system.
Mice with Tbx20
ablated in cardiomyocytes exhibited a severe
phenotype 3 to 4 days after initiation of tamoxifen treatment, with decreased motor
activity and death occurring within 5–16 days (Figure D). Control littermates exhibited no adverse
effects of tamoxifen treatment, with similar results in control mice carrying the
transgene compared with mice without the transgene. Histological analysis with
H&E staining of mutant hearts revealed enlarged right and left cardiac
ventricles with thinner walls (Figure E).
Ratios of heart weight to body weight and heart weight to tibia length were
significantly increased in Tbx20
conditional KOs (heart weight:body
weight, 0.0075 vs. 0.0047, P
< 0.01; heart weight:tibia
length, 0.091 vs. 0.064, P
< 0.001). Increased cell death was
observed in mutant hearts relative to controls by TUNEL staining 5 days after
tamoxifen injection (Figure F). Early signs of
fibrosis detected by trichrome staining were observed in mutant hearts 7 days after
tamoxifen injection (Supplemental Figure 1A; supplemental material available online
with this article; doi:
). Extensive fibrosis observed within a short time
frame in our adult myocyte-specific KOs of Tbx20 was usually detected during acute
ischemic episodes. The cause of the fibrosis is not yet clear, but will be a subject
of future investigation.
Adult cardiomyocyte-specific Tbx20 conditional KO mice exhibit a severe
cardiomyopathy with heart failure and arrhythmias.
Our histological analysis suggested a dilated cardiomyopathy in mutant hearts. We
therefore assessed heart function in vivo by echocardiography on mutant and control
mice. Tbx20 conditional KO mice showed enlarged LVs and reduced
thickness of the LV walls, quantified by M-mode measurements 6 days after tamoxifen
induction (Figure , A and B, and Supplemental
Table 1). Changes in cardiac dimensions in mutant mice were accompanied by a dramatic
decrease in LV systolic function, observed by a significant decrease in fractional
shortening (%FS) and velocity of circumferential fiber shortening (VCF) compared with
littermate controls. Reduction of cardiac function could be detected as early as 3
days after tamoxifen injection and was severe at day 4 after injection (Supplemental
Data Files 1 and 2 [videos]).
Tbx20 ablation in adult mouse myocardium results in
cardiomyopathy and arrhythmia.
ECG analyses showed a variety of abnormalities in Tbx20 conditional
KOs, including slower heart rate, changes in AV conduction, and altered ventricular
depolarization and repolarization as well as tachyarrhythmias and bradyarrhythmias
(Figure , C–F). To eliminate the
effect of anesthesia, which is required for ECG analysis, we confirmed these findings
by telemetric recordings in awake mice (Figure C) and on isolated hearts (Figure D)
to further minimize effects caused by vagal innervation or circulating
catecholamines. Mutant mice exhibited a widening of QRS complexes compared with
controls (QRS duration 11.07 ± 0.38 ms vs. 24.92 ± 2.41 ms;
P < 0.0001) and an elevation or depression of the ST
segment (Figure , E and F). Mutant mice further
showed sinus bradycardia and progressive AV block, resulting in complete heart block
with a typically slow and in some cases polymorphic ventricular escape rhythm as a
preterminal rhythm (Figure , C and D).
Additionally, telemetry tracing revealed self-limiting polymorphic wide-QRS
tachycardias in some of the mice (Figure C),
suggesting a severe derangement of normal electrophysiology in mutant hearts.
Together, these results reveal an ongoing requirement for Tbx20 in adult heart
function and show that loss of Tbx20 results in heart failure. Supporting this
notion, we observed a reduction in Tbx20 mRNA and protein levels in
other murine models of heart failure (Supplemental Figure 2).
Loss of Tbx20 in adult cardiomyocytes leads to changes in expression of genes
required for transcriptional regulation, ion transport, and contractility and
disrupts critical gene expression gradients in myocardium.
To evaluate molecular mechanisms underlying the fulminant disease in
Tbx20 conditional KOs, we analyzed expression of a number of
candidate genes expressed in adult myocardium that might be involved in the
development of the phenotype. Expression levels of candidates including transcription
factors, ion channels, gap junction proteins, and proteins involved in calcium
cycling as well as cytoskeletal and sarcomeric proteins were assayed by qRT-PCR
Loss of Tbx20 in adult myocardium leads to changes in
expression of important cardiac genes.
At 3 days after tamoxifen induction, we found marked decreases in mRNA expression of
cardiac transcription factors Mef2c, Tbx5,
Gata4, Irx4, and Irx5 in hearts
of Tbx20 conditional KO mice compared with controls, while the
transcription factor Irx1 was substantially upregulated in mutant mice. Furthermore,
we found downregulation in expression of numerous genes encoding proteins with
critical functions in cardiomyocytes. These included genes encoding the gap junction
protein Gja1 (connexin 43); potassium channel genes Kcnd2,
Kcnd3, Kcnj2, Kcnj3,
Kcnip2, and Kcnh2; calcium channel genes
Cacna1c, Cacna1g, Cacna2d1,
Cacnb2, and Cachd1; genes encoding calcium
cycling or regulatory proteins Serca2 (Atp2a2),
phospholamban (Pln), ryanodine receptor 2 (Ryr2),
and calmodulin-dependent protein kinase type II delta chain
(Camk2d); and those encoding cytoskeletal proteins cypher
(Ldb3) and desmin (Des). In contrast, genes for
β-tubulin (Tubb2b, not shown) and atrial natriuretic
factor (Nppa) were significantly upregulated (Figure A). Expression of other candidate genes,
Gata6, Tbx3, Irx2,
Irx3, Nkx2-5, or Kcne1, was not
significantly altered in Tbx20 conditional KO mice (not shown).
Several genes whose expression was downregulated in Tbx20
conditional KO mice were further evaluated at the protein level by Western blot
analysis of cardiac protein extracts obtained 5 days following tamoxifen injection
(Supplemental Figure 1, B and C). Consistent with decreases in mRNA levels, most of
these proteins were downregulated. Only 2 of the tested proteins,
Girk1 and Serca2, appeared unchanged despite a
marked downregulation of the cognate Kcnj3 and
Atp2a2 mRNAs. These differences may indicate a slower turnover of
some proteins and/or compensatory alterations in posttranscriptional gene regulation.
Immunohistochemical analysis of the main ventricular gap junction protein
α 1 (connexin 43) revealed a striking loss of cell-cell connections
between neighboring cardiomyocytes compared with that in control hearts (Figure B). Staining with antibodies for the L-type
calcium channel Cav1.2 and the potassium voltage-gated channel
Kv4.2 likewise demonstrated reduced expression in mutant hearts.
Tbx20–/– cardiomyocytes exhibit altered ion
currents, decreased calcium transient amplitudes, and reduced cell shortening during
The combination of phenotypes and gene expression analysis in adult
Tbx20 myocardial mutant mice suggests that Tbx20
conditional KO mice may be developing dilated cardiomyopathy in part due to altered
ion flux regulation. To directly test this hypothesis, we performed
electrophysiological analyses of isolated cardiomyocytes.
In accordance with the finding that voltage-gated, L-type calcium channels are
significantly downregulated in mutant hearts, single-cell patch clamp analysis showed
a significant reduction of L-type calcium current in
compared with controls (Figure A).
Additionally, also in line with the aforementioned experiments, the peak inactivating
outward potassium current was significantly reduced (Figure A), while the slow component of the outward potassium current was
not significantly altered (Figure A).
Single-cell electrophysiology analysis reveals altered ion flux regulation in
adult Tbx20-null cardiomyocytes.
Expression analysis revealed downregulation of various other genes encoding proteins
involved in ion flux and regulation of intracellular calcium levels. In keeping with
these findings, calcium measurements by line scan confocal microscopy showed
significantly reduced calcium transient peaks and significantly reduced cell
shortening (Figure , B and C) in cardiomyocytes
from Tbx20 conditional KO mice.
ChIP-seq analysis in adult mouse hearts identifies direct downstream targets of
Tbx20 with critical functions in cardiomyocytes.
To improve our understanding of molecular mechanisms underlying the severe cardiac
phenotypes observed in Tbx20 conditional KO mice, we sought to
determine which cardiac genes with altered expression in mutant mice were directly
regulated by Tbx20. Given the data presented previously, we hypothesized that Tbx20
is an upstream transcription regulator of many critical ion transport genes. To
address this question, we generated a genome-wide map of Tbx20-binding regions in
adult mouse heart and direct Tbx20 downstream gene targets, using ChIP followed by
massive parallel sequencing (ChIP-seq).
As none of the commercially available Tbx20 antibodies that we tested were ChIP
grade, we decided to use a tagging strategy. We identified a BAC encompassing
Tbx20 and its long-range cardiac transcriptional enhancers (M.A.
Nobrega, personal communication). Utilizing BAC recombineering, we fused a GFP cDNA
to the C terminus end of Tbx20 and generated transgenic mice
carrying this engineered BAC transgene. Because we used a BAC containing the
Tbx20 cardiac enhancers, the fused Tbx20-GFP
protein recapitulated the endogenous cardiac expression pattern of
Tbx20. This was confirmed both by RT-PCR and by
immunohistochemistry (Supplemental Figure 3). We also determined, by qRT-PCR, that
the fusion Tbx20-GFP mRNA is expressed at the same level as
endogenous Tbx20 in 6-week-old hearts, effectively doubling the
level of Tbx20 in the heart. Together, these results illustrate that
the Tbx20-GFP fusion cassette is expressed in embryonic and adult
hearts in a pattern recapitulating the endogenous Tbx20 expression,
and at quasiphysiological levels. We then employed a well-established, ChIP-grade
polyclonal anti-GFP antibody for ChIP.
Our ChIP analysis resulted in 4,012 peaks of Tbx20 binding at 2% false discovery
rate. Around 23% of these peaks were within 6 kb of the transcription start site
(TSS) of RefSeq genes (P = 10–40), indicating
that although Tbx20 directly binds the promoter regions of several genes, the
majority of its regulation relies on binding to longer range DNA elements.
Tbx20-binding regions within ±6 kb of the TSS were clustered at the
center of this region, close to the TSS, which is the typical binding position for
PolII and other transcription factors, supporting the role of Tbx20 as a core
transcriptional coregulator. We observed a higher degree of evolutionary conservation
of Tbx20-binding regions compared with the rest of the genome, suggesting that the
peaks are functional binding sites (Figure A).
Validation of Tbx20 ChIP peaks as long distance enhancers.
We then assigned all Tbx20 ChIP–binding regions to neighboring genes to
identify putative direct downstream gene targets. Peaks were assigned to a given gene
when mapping within the gene or within ±6 kb of the TSS. Peaks in distant
intergenic regions were assigned to both flanking genes. As a result, the 4,012 peaks
were assigned to 3,799 genes. Directly supporting our hypothesis that KO of
Tbx20 causes the observed cardiac phenotype by regulating
expression of ion transport genes, 26 of the 32 genes for which we tested mRNA
expression levels had a nearby ChIP peak. Among the 23 genes that were downregulated,
19 had a nearby ChIP peak, indicating that these genes are direct downstream targets
To interpret the large number of putative Tbx20 downstream targets in functional
terms, we performed a computational analysis for enrichment of Gene Ontology (GO)
). The top enriched GO terms were
related to heart muscle contraction, cardiac morphogenesis, muscle cell
proliferation, myofibril assembly, cell junction assembly, and adult heart
development (Figure B and Supplemental Data
File 3). Interestingly, we observed a significant overlap between our Tbx20-binding
regions and those recently reported for various cardiac transcription factors in
cardiac HL1 cells (31
). Supporting the notion
that Tbx20 physically interacts with Gata4, Nkx2-5, and Tbx5, we observed binding
events for Tbx20 and Gata4 to the same genomic coordinates 25-fold more frequently
than would be expected by chance. Cobinding of Tbx20 and Nkx2-5 to the same
coordinates occurred 14-fold more often than by chance, and that for Tbx20 and Tbx5
also occurred with 14-fold enrichment (Supplemental Table 2; also see Supplemental
Figure 4 for analysis of genes for which we measured mRNA expression). Finally, there
was an 80-fold enrichment in the cooccurrence of binding regions for Tbx20 and p300,
a signature for transcriptional enhancers (32
). The overrepresentation of these cooccurrences was strongly statistically
significant (Supplemental Table 2). We also observed a significant overrepresentation
of binding sites for Gata and Nkx in the Tbx20-binding regions (see Supplemental
Figure 4 and Supplemental Table 2), indicating that the high cooccurrence among
Tbx20, Gata4, Nkx2.5, and Tbx5 was detectable both at the DNA and the protein-DNA
interaction levels. Collectively, these results strongly validate our Tbx20 ChIP-seq
Computational analysis reveals a Tbx20-binding motif capable of predicting
binding sites not recognized by previously identified motifs.
As ChIP peaks putatively correspond to chromatin regions bound by Tbx20, we searched
these sequences for previously described Tbx20-binding motifs, that is, the motif
identified by selective evolution of ligands by exponential enrichment (33
), referred to here as the SELEX Tbx20 motif,
and the Eomes protein T-box motif (34
). To our
surprise, we found only a low number of peaks containing these motifs when compared
with random sequences generated with third-, fourth-, and fifth-order Markov models
(Eomes: 28% of the peaks, SELEX Tbx20: 4% of the peaks; Supplemental Table 3).
We then employed a de novo motif-finding program that recovered a motif that
resembles the known core T-box motif AGGTGTGA (33
) and matches JASPAR’s T (MA0009.1, P
), TRANSFAC’s Tbx5 (M01044,
= 6 × 10–5
UniProbe’s Eomes-primary (P
= 2 ×
) motifs (Supplemental Figure 5) (34
does not fully match any of these motifs (Figure C and Supplemental Figure 6). While clearly displaying most nucleotides
of the core T-box motif, our de novo motif contains one position with no nucleotide
preference, creating a gap at the center of the SELEX Tbx20 motif (the asterisk in
the consensus AGGTG*TGACAG). This de novo motif is present in 51% of the 4,012
Tbx20-binding regions at greater than 80% similarity to the consensus. Altogether,
SELEX Tbx20, Eomes, and our de novo Tbx20 motifs could be found in 69% of the peaks
(Figure D). The Tbx20 de novo motif was the
only one found by MEME, it resembles the SELEX Tbx20 and T-box motifs, and it is the
most overrepresented according to Clover. Therefore, we used the Tbx20 de novo motif
) in our subsequent analyses.
Our analyses of the remaining 31% of Tbx20-binding regions that did not contain an
identifiable Tbx20 motif (Tbx20–) suggest that they represent
functional binding sites of Tbx20. First, these regions display a significant degree
of evolutionary sequence conservation and were only slightly less conserved than the
binding regions containing recognizable Tbx20 motifs (Tbx20+) (Figure
E). Second, genes associated either with
Tbx20+ or Tbx20– regions show GO enrichment for
heart-related terms (Supplemental Data File 3). Furthermore, motif analyses in
Tbx20-binding regions revealed 29 other overrepresented transcription
factor–binding site (TFBS) motifs, 18 of which were also overrepresented
in Tbx20+ regions (3/29 expected by chance). Taken together, these data
suggest that at least a fraction of the peaks without a Tbx20 de novo motif are
functional. Whether these peaks reflect sites where Tbx20 is recruited to the locus
through protein-protein interactions or represent direct DNA-binding events using
motifs unrecognized by the various matrices we used is unclear.
Functional testing of identified Tbx20-binding sites.
To functionally test both Tbx20+
–binding regions for their cardiac
enhancer properties, we tested 41 individual binding regions in a zebrafish in vivo
reporter assay (37
). Because data from our murine adult
conditional KO model strongly suggested that a number of critical targets of Tbx20
were involved in ion transport, we prioritized sequences that were near cardiac ion
transport genes. Of 41 Tbx20-binding regions tested, 23 (56%) consistently drove GFP
expression in zebrafish embryonic hearts (Figure F and Supplemental Table 4). In these assays,
– and Tbx20–
sites were equally likely to behave as cardiac enhancers, further supporting the
notion that Tbx20–
regions represent functional sequences.
Transcription factor–binding motifs for Tbx20, Mef2a, Tead1, Esrr,
and Creb1 are required for coregulation of ion transport genes in adult
In addition to Tbx20
and T-box motifs, we searched the Tbx20-binding
region set for enrichment of all TFBS motifs from the JASPAR and UniProbe databases
) to uncover possible Tbx20 cofactors (Supplemental Data File 4).
Four transcription factor–binding motifs appeared particularly enriched
in binding regions associated with genes encoding for ion channel and transport
) Mef2a (MA0052.1),
Creb1 (MA0018.2), and Tead1 (MA0090.1) and UniProbe’s (34
) Esrr-primary (40
) were overrepresented in peaks associated with both Tbx20+
(Supplemental Data File 4). Genes in which Mef2A,
Tead1, Creb1, and Esrr motifs cooccur with Tbx20-binding regions were enriched for
functional GO terms connected to ion transport/contraction and heart
development/morphogenesis-related terms (Figure A), with more than 45% of enriched GO terms being ion
transport/contraction (see Supplemental Table 5 and Methods for a detailed
description of the analysis). This enrichment for the cooccurrence of the 5
transcription factor–binding motifs (those for Tbx20, Mef2A, Creb2,
Tead1, Essr) was statistically significant in that only 0.4% of 10,000 random groups
of any 5 TFBSs occurring in similar frequencies in the test motifs yielded comparable
Cooccurrence of transcription factor–binding motifs for
Tbx20, Mef2a, Tead1, Esrr, and Creb1 is important for gene
expression regulation of ion transport genes.
To test the functionality of TFBSs for Tbx20, Mef2A, Creb2, Tead1, and Essr in Tbx20
peaks containing binding sites for each of these factors, we selected 2
representative Tbx20 target enhancers, one adjacent to Cacna1c and
the other adjacent to Irx4, containing all 5 motifs and mutagenized
individual instances of each motif in these enhancers, evaluating the impact of each
mutation in the enhancer activity in our zebrafish in vivo reporter. Mutation of each
individual binding site led to a reduction in enhancer activity, reflected in the
decrease of the number of zebrafish expressing GFP in the heart by
50%–100% (Figure , B and C, and
Supplemental Table 6).
Taken together, these results suggest that Tbx20, in concert with a network that
includes Mef2, Tead, Creb, and Esrr, regulates calcium homeostasis and ion flux in