1 Department of Genetics, Harvard Medical School and Howard Hughes Medical
Institute, Boston, MA 02115, USA
2 Department of Pathology and Cardiac Registry, Children's Hospital and Harvard
Medical School, Boston, MA 02115, USA
3 Molecular Cardiology Research Center and Section of Cardiac Electrophysiology,
University of Pennsylvania, Philadelphia, PA 19104, USA
4 Department of Cardiology, Children's Hospital and Department of Pediatrics,
Harvard Medical School, Boston, MA 02115, USA
5 Programs in Cardiovascular Research and Developmental Biology, The Hospital
for Sick Children, Toronto, ON M5G 1X8, Canada
6 Department of Molecular and Medical Genetics, University of Toronto, Toronto,
ON M5S 1A8, Canada
7 Departments of Anesthesiology and Pharmacology, Vanderbilt University School
of Medicine, Nashville, TN 37232-6602, USA
8 Departments of Medicine and Pharmacology, Vanderbilt University School of
Medicine, Nashville, TN 37232-6602, USA
9 Division of Cardiology, Brigham and Women's Hospital, and Howard Hughes
Medical Institute, Boston, MA 02115,USA
* Author for correspondence (e-mail: seidman{at}genetics.med.Harvard.edu)
Accepted 4 May 2004
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SUMMARY |
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Key words: Cardiac, Conduction, Tbx5, Mouse
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Introduction |
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A molecular marker of the conduction system has been reported, in a study
in which selective ß-galactosidase expression in the cardiac conduction
system was established by placing the lacZ gene downstream of the
potassium channel minK promoter
(Kupershmidt et al., 1999;
Kondo et al., 2003
). In adult
mice, ß-galactosidase expression from the minK:lacZ allele
(minKlacZ/+) demarcates nuclei of cells in the mature
central conduction system, including the sinoatrial node, the atrioventricular
node, the atrioventricular bundle and the right and left bundle branches, from
the working myocardium. In concert with in-vivo electrophysiologic techniques
(Gehrmann and Berul, 2000
),
minKlacZ/+ mice enable detailed morphological and
functional analyses of the mammalian conduction system.
We employed these tools to study the role of the transcription factor Tbx5
in the development of the cardiac conduction system. Tbx5 belongs to
the T-box gene family, the members of which share a highly conserved
180-amino-acid domain required for DNA binding
(Herrmann et al., 1990;
Muller and Herrmann 1997
)
(reviewed by Papaioannou and Silver,
1998
). Mutations in several human T-box genes cause dominant
disorders with a variety of developmental malformations
(Bamshad et al., 1997
;
Merscher et al., 2001
). In
humans, haploinsufficiency of functionally null TBX5 mutations causes
HoltOram syndrome (Basson et al.,
1997
; Li et al.,
1997
; Basson et al.,
1999
), manifest by congenital heart defects, conduction-system
abnormalities and upper-limb deformities. Morphologic cardiac defects are most
commonly atrial septal defects of the secundum type, although a range of
structural abnormalities has been reported
(Basson et al., 1997
;
Li et al., 1997
,
Basson et al., 1999
). Common
electrophysiologic abnormalities found in HoltOram syndrome include
progressive atrioventricular block, bundle-branch block and sick sinus
syndrome (Basson et al., 1994
).
Some HoltOram syndrome patients have electrophysiologic defects in the
absence of structural heart defects (Basson
et al., 1994
; Newbury-Ecob et
al., 1996
), thereby suggesting a direct role for TBX5 in
the conduction system that is independent of this transcription factor's
function in cardiac septation.
Mice lacking a functional Tbx5 allele
(Tbx5del/+) were constructed to understand how
Tbx5 haploinsufficiency disturbs limb and cardiac development
(Bruneau et al., 2001). Like
HoltOram patients, adult Tbx5del/+ mice were found
to have atrial septal defects (ASDs), including secundum or primum ASDs, and
conduction system abnormalities including atrioventricular conduction delay.
Molecular studies of Tbx5del/+ mice identified atrial
natriuretic factor (ANF) and connexin 40 (Cx40;
Gja5 Mouse Genome Informatics) as gene targets of this
transcription factor: expression of ANF and Cx40 is
abrogated in Tbx5del/+ mice. Both ANF and
Cx40 are expressed in cells of the conduction system
(Houweling et al., 2002
;
Coppen et al., 2003
). Cx40 is
localized at cellular appositions called gap junctions, which function to
propagate electrical impulses between cells. Cx40 null mice
(Cx40/) have electrophysiologic defects
similar to Tbx5del/+ mice, including atrioventricular
block and bundle-branch block. Taken together, previous work suggested that
Cx40 deficiency could account for conduction system disease in
Tbx5del/+ mice and in HoltOram syndrome.
To study the role of Tbx5 in conduction system development, we examined the expression of this transcription factor during the anatomical and functional maturation of the cardiac electrophysiologic system. We investigated the consequences of Tbx5 haploinsufficiency on central conduction system function and morphology by analyzing wild-type, Tbx5del/+ and compound Tbx5del/+/minKlacZ/+ mice and Cx40//minKlacZ/+ mice. We report specific developmental and functional requirements for Tbx5 in the atrioventricular node, atrioventricular (His) bundle and bundle branches of the conduction system. These findings identify Tbx5 as a critical transcription factor for the morphologic patterning and electrophysiologic maturation of the mammalian conduction system.
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Materials and methods |
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ECGs
Surface ECGs were recorded from unanesthetized
Tbx5del/+ newborn mice (n=14) and compared with
those for littermate wild-type mice (n=22). Surface ECG recordings
and complete in-vivo electrophysiological studies (EPS) were recorded from
14-week-old adult Tbx5del/+ mice (n=21) and
compared with those for wild-type littermate mice (n=13). Surface ECG
recordings were also recorded from 14-week-old adult
Cx40//minKlacZ/+ and
Tbx5del/+/minKlacZ/+ mice
(n=19) and compared with those for age-matched wild-type mice
(n=14).
Protocols for the surface ECG and in-vivo mouse electrophysiology studies
are previously described (Berul et al.,
1996; Maguire et al.,
2000
). Briefly, 6-limb ECGs with a right precordial lead were
obtained using 25-gauge subcutaneous electrodes in unanesthetized newborn
mice. Adult mice were lightly anesthetized with pentobarbital (0.033 mg/kg IP)
and 6-limb lead ECGs with a right precordial lead were obtained using 25-gauge
subcutaneous electrodes. For in-vivo electrophysiology studies in adult mice,
a jugular vein cutdown was performed and an octapolar 2-French electrode
catheter (CIBer mouse-EP; NuMED, Inc.) was placed in the right atrium and
ventricle under electrogram guidance to confirm catheter position. Recording
of atrioventricular bundle potentials was confirmed by the presence of a
triphasic signal on one of the distal bipole electrograms, and was
accomplished using simultaneous multielectrodes and persistent catheter
manipulation (Maguire et al.,
2000
).
Electrophysiology study
In-vivo electrophysiologic studies were performed in all adult mice using
standard pacing protocols to assess atrial and ventricular conduction,
refractoriness and arrhythmia inducibility
(Maguire et al., 2000;
Berul et al., 2001
). ECG
channels were filtered between 0.5 and 250 Hz and intracardiac electrograms
filtered between 5 and 400 Hz. Signals were displayed on an oscilloscope and
simultaneously recorded through an A-D converter at a digitization rate of 2
kHz (MacLab Systems, Inc.) for offline analysis. ECG intervals were measured
in 6-limb leads and a right precordial lead by two independent observers,
blinded to genotype.
ß-galactosidase (lacZ) activity
Dissected hearts were fixed at 4°C in 4% paraformaldehyde in 0.1 mol/L
PBS (pH 7.4) for 1 hour. Following a PBS rinse, hearts were permeabilized in
0.01% sodium deoxycholate, 0.02% NP-40, and 2 mmol/L MgCl2 in 0.1
mol/L PBS for 30 minutes. Following a PBS rinse, hearts were stained in
permeabilization solution plus 1 mg/ml X-Gal, 5 mmol/L potassium ferrocyanide,
and 5 mmol/L ferrocyanide at 37°C overnight. Hearts were washed at least
three times with PBS and post-fixed and stored in 4% paraformaldehyde at
4°C.
In-situ hybridization
Whole-mount in-situ hybridization was performed as previously described
(Bruneau et al., 2001). To
insure probe access to the entire heart, the systemic and pulmonary veins were
opened and the ventricular apex was removed. In-situ hybridization was
performed on slide sections with the following modifications. Newborn hearts
were dissected, washed once in PBS, fixed overnight in 4% paraformaldehyde in
PBS and either stored at room temperature in ethanol 70% or immediately
paraffin embedded and sectioned. In-situ hybridization on paraffin wax tissue
sectioned at 5 µm was performed using radioactive Tbx5 probe
(Bruneau et al., 2001
) labeled
with 35S-UTP according to previously described protocol
(Sibony et al., 1995
).
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Results |
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To clarify the relationship between conduction and Tbx5-expressing
cells, Tbx5 expression was examined at higher resolution in
histologic sections. The conduction system was identified by visualizing
ß-galactosidase activity in sections from minKlacZ/+
mouse hearts (Fig. 1E) or
connexin 40 expression in sections from wild-type mouse hearts
(Fig. 1G). Connexin 40
expression was comparable to that previously described
(Coppen et al., 2003). In
newborn minKlacZ/+ mouse hearts, ß-galactosidase
activity was present in the atrioventricular bundle
(Fig. 1E, arrow) and bundle
branch (arrowhead) conduction. Tbx5 expression was observed in the
same structures of the centralized conduction system, including the
atrioventricular bundle in cross sections from wild-type hearts
(Fig. 1F, arrow), and bundle
branches (Fig. 1F, arrowheads).
Connexin 40 and Tbx5 expression were assessed by in-situ
hybridization on serial sections from the same newborn wild-type heart
(Fig. 1G,H, arrow). These RNAs
were observed in precisely the same locations in the atrioventricular bundle
(Fig. 1G,H, arrow), and left
bundle branch and right bundle branch (Fig.
1G,H, arrowheads). A higher level of Tbx5 expression was
observed in the structures of the conduction system than in the surrounding
myocardium.
Tbx5 haploinsufficiency prevents atrioventricular canal conduction system maturation
Given Tbx5 expression in the central conduction system of newborn
mice and ECG abnormalities in Tbx5del/+ mice
(Bruneau et al., 2001), we
hypothesized that Tbx5 haploinsufficiency might affect conduction
system development. Hearts from compound heterozygous
Tbx5del/+/minKlacZ/+ mice (see
Materials and methods) and minKlacZ/+ mice were compared
after X-gal staining. At the atrioventricular junction, hearts from newborn
minKlacZ/+ (Fig.
2A) and
Tbx5del/+/minKlacZ/+
(Fig. 2C) mice demonstrated
similar rings of specialized conduction tissue surrounding both the tricuspid
annulus and mitral annulus. This same pattern of ß-galactosidase activity
was observed in all minKlacZ/+ (n=25) and
Tbx5del/+/minKlacZ/+ (n=22)
newborn mouse hearts.
|
To ascertain whether Tbx5 had functional, as well as
morphological, roles in atrioventricular conduction system maturation, we
evaluated the consequences of Tbx5 haploinsufficiency using surface
ECG monitoring and in-vivo electrophysiology. Surface ECGs were recorded from
unanesthetized, newborn animals and lightly anesthetized adult animals. The PQ
interval, which detects the time for electrical conduction from the sinoatrial
node to the atrioventricular node, atrioventricular bundle, and proximal
bundle branches, was measured (Fig.
3A, Table 1)
(Marriott and Conover, 1998).
As previously described (Bruneau et al.,
2001
), adult Tbx5del/+ mice have a prolonged
PQ interval compared with that of wild-type mice (49.1±4.6 ms vs.
41.9±1.0 ms; P<0.01)
(Fig. 3B, Table 1). However, in newborn
mice, the PQ intervals of wild-type and Tbx5del/+ mice
were not significantly different (48.0±7.6 ms vs. 50.7±5.8 ms;
P<0.38) (Fig. 3B, Table 1). Neither the presence
of the minKlacZ allele nor the type of atrial septal
defect (primum or secundum) altered the PQ interval of the wild-type or
Tbx5del/+ newborn mice (ASD primum 50.7±5.6
ms vs. ASD secundum 52.5±5.3 ms; P-value not significant).
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In-vivo electrophysiology studies were performed in adult animals to localize the functional deficit in Tbx5del/+ mice to the atrium, atrioventricular node, or the atrioventricular bundle and proximal bundle branches. The AH interval (representing conduction propagation through the atrium and the atrioventricular node) and the HV interval (representing conduction propagation through the atrioventricular bundle and proximal bundle branches) were measured separately (Fig. 3A). The HV interval of adult Tbx5del/+ mice and wild-type mice were not significantly different (10.3±2.0 ms vs. 11.5±1.5 ms; P<0.69; n=6). However, the AH interval of adult Tbx5del/+ mice was significantly longer than the AH interval of adult wild-type mice (42.3±6.0 ms vs. 37.0±1.5 ms; P<0.001; n=6), demonstrating a functional deficit of atrial or atrioventricular node conduction in adult Tbx5del/+ mice. To distinguish between these, propagation of the electrical signal in the atria (atrial depolarization or P-wave) was directly measured by analyzing P-wave duration. P-wave duration was not significantly different between adult Tbx5del/+ mice and wild-type mice (15.9±2.5 ms vs. 15.0±1.1 ms; P<0.36; n=12). Taken together, the normal P-wave duration and AH interval prolongation suggests that the PQ prolongation in Tbx5del/+ mice is the result of a defect of the atrioventricular node or its connection with the atria or atrioventricular bundle.
Tbx5 haploinsufficiency causes atrioventricular bundle and bundle branch conduction system patterning defects
We used ß-galactosidase activity in minKlacZ/+
mice to characterize the morphology of the postnatal ventricular conduction
system, including the atrioventricular bundle and bundle branches. The left
bundle branch, lying on the left side of the interventricular septum, was
identified in all newborn minKlacZ/+ mouse hearts
(n=25), as a broad sheet of cells with ß-galactosidase activity
(Fig. 4B, arrowhead). This
pattern was consistent with classic histological descriptions of the left
bundle branch in young mouse hearts (Fig.
4B) (Lev and Thaemert,
1973). In all adult minKlacZ/+ mouse hearts
(n=18), ß-galactosidase activity was more discretely
concentrated into a bundle branch fascicle. In all adult
minKlacZ/+ mouse hearts (n=18),
ß-galactosidase activity also demarcated a well-defined atrioventricular
bundle, located on the crest of the interventricular septum
(Fig. 4C, red arrowhead).
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The right bundle branch, present on the surface of the right side of the
interventricular septum, had a different structure than that of the left
bundle branch (Fig. 4F-H). In
newborn minKlacZ/+ mouse hearts (n=25),
ß-galactosidase activity identified a loose bundle of cells adjacent to
the septal band and anterior papillary muscle of the right ventricle,
consistent with classic histological descriptions
(Lev and Thaemert, 1973) of
the right bundle branch (Fig.
4G). The right bundle branch in adult
minKlacZ/+ mouse hearts
(Fig. 4H) was more well-defined
than that in newborn mice, displaying a highly stereotyped pattern in all 18
hearts examined that was remarkably analogous to that of the adult human right
bundle branch.
Severe patterning defects of the right bundle branch were observed in newborn and adult Tbx5del/+/minKlacZ/+ mice. In all newborn Tbx5del/+/minKlacZ/+ mouse hearts studied (n=22), there was a paucity of cells with ß-galactosidase activity in the right ventricle. The most severe cases (10/22 hearts) had complete absence of a discrete right bundle branch on the right ventricular septal surface (Fig. 4I). In less severe cases (12/22 hearts), the right bundle branch was foreshortened and failed to associate with the anterior papillary muscle (data not shown).
As in neonates, adult Tbx5del/+/minKlacZ/+ mouse hearts exhibited markedly abnormal right bundle branches. In 15 hearts examined, there was a marked paucity of ß-galactosidase-expressing cells on the right ventricular septal surface. In 8 of 15 hearts, the right bundle branch was entirely missing, and only a few dispersed cells with ß-galactosidase activity could be identified on the right ventricular septal surface (Fig. 4J). In the remaining mutant hearts (n=7), a foreshortened right bundle branch was present (not shown).
To verify the severe bundle-branch-patterning defects observed in Tbx5del/+/minKlacZ/+ mouse hearts, conduction system morphology in Tbx5del/+ newborn mouse hearts was also evaluated by in-situ hybridization of connexin 40 expression on sagital sections. In wild-type newborn mouse hearts (n=6), connexin 40 expression uniformly marked the atrioventricular bundle and left and right bundle branches (Fig. 4L). In Tbx5del/+ newborn mouse hearts (n=6), connexin 40 was also observed in the atrioventricular bundle and left bundle branch (Fig. 4M). However, no right bundle branch could be identified in 3/5 Tbx5del/+ newborn hearts (Fig. 4M). In these cases, a right bundle branch was absent even from the region adjacent to the membranous septum, where the right bundle branch normally exits the atrioventricular bundle to enter the right ventricle (Fig. 4M).
To assess the functional consequences of the profound bundle branch
morphologic abnormalities in Tbx5del/+ mice, ventricular
conduction was evaluated using surface ECG analysis. Previous studies using
single lead Holter monitoring revealed no ventricular conduction differences
between wild-type and Tbx5del/+ mice
(Bruneau et al., 2001). Using
6-lead ECGs, we found that the QRS interval, produced by depolarization and
activation of the ventricular myocardium
(Surawicz and Knilans, 2001a
),
was significantly longer in Tbx5del/+ mice than that in
wild-type mice. QRS prolongation occurred both in neonates (12.7±2.6 ms
vs. 9.8±1.5 ms; P<0.001) and in adults (16.5±1.0 ms
vs. 13.7±1.3 ms; P<0.005)
(Fig. 3B; Table 1B). Neither the
minKlacZ/+ allele nor the type of septal defect (primum or
secundum) had a significant effect on the QRS interval of the wild-type or
Tbx5del/+ neonatal or adult mice (ASD primum
16.8±0.9 ms versus ASD secundum 16.3±3.2 ms; P-value
not significant).
Delay or loss of conduction through one of the bundle branches, termed
`bundle-branch block', results in lengthening of the QRS interval due to
delayed and asynchronous activation of the ventricular myocardium
contralateral to the affected bundle branch
(Surawicz and Knilans, 2001b)
(Fig. 3A). To ascertain whether
QRS prolongation in Tbx5del/+ mice was due to a
bundle-branch block, surface 6-limb ECGs with a right precordial lead were
recorded. The QRS complex recorded from the right precordial lead has a
distinct morphology in the case of either right or left bundle branch,
allowing their unique identification
(Surawicz and Knilans, 2001b
).
An RSr' morphology indicative of right-bundle-branch block was observed
in 9/11 Tbx5del/+ mice versus 3/27 adult wild-type mice
(P<0.001) (Fig.
3C). RSr' is the standard notation for a notched QRS
upstroke (an initial upward deflection (R) followed by a downward deflection
(S) followed by another upward deflection (r')). RSr' morphology
on ECG combined with prolongation of QRS duration is indicative of
right-bundle-branch block. The type of atrial septal defect in the heart
(secundum or primum) had no significant effect on the likelihood of
right-bundle-branch block in Tbx5del/+ adult mice
(ASD primum 4/4 vs. ASD secundum 5/7; P-value not
significant).
To determine whether the developmental failure of a morphologic right bundle branch correlated with the ECG finding of right-bundle-branch block, structure and function were assessed in 10 Tbx5del/+ mice. All (n=4) mice with morphologic absence of the right bundle branch demonstrated right-bundle-branch block by precordial ECG analysis. By contrast, only 2/6 mice with a morphologically visible right bundle branch demonstrated a right-bundle-branch block on ECG.
Normal conduction system patterning in mice lacking Cx40, a Tbx5-target gene
Previous studies demonstrated that Cx40, which encodes a gap
junction protein required for normal conduction system function, is a gene
target of Tbx5. Adult Tbx5del/+ mice express only
10% of normal levels of Cx40 transcripts, and Tbx5 directly binds and
activates the Cx40 promoter in primary cultures of cardiomyocytes
(Bruneau et al., 2001).
Cx40 null mice (Cx40/) have
prolonged PQ intervals, prolonged QRS intervals, and right-bundle-branch block
(Kirchhoff et al., 1998
;
Simon et al., 1998
;
Bevilacqua et al., 2000
;
Saffitz and Schuessler, 2000
;
Tamaddon et al., 2000
;
van Rijen et al., 2001
).
To test whether Cx40 insufficiency accounted for the morphologic
and electrophysiologic defects in the conduction system of
Tbx5del/+ mice, we studied
Cx40/ mice
(Simon et al., 1998). The
morphology of the conduction system was analyzed in compound
Cx40//minKlacZ/+ mice by
evaluating ß-galactosidase activity in adult hearts
(Fig. 5). Precordial ECG
analyses confirmed previously described electrophysiologic abnormalities:
19/19 Cx40/ mice had a prolonged PR interval
and 15/19 had a right-bundle-branch block (data not shown). Morphologic
analyses were performed in mice with the most severe ECG abnormalities, those
with prolonged PR interval and right-bundle-branch block. The atrioventricular
node, atrioventricular bundle, left bundle branch and right bundle branch
identified by ß-galactosidase activity were well-formed and normally
patterned in all
Cx40//minKlacZ/+ mice
(n=10) (Fig. 5A-F).
Both the amount and the distribution of ß-galactosidase activity were
indistinguishable from that observed in littermate
minKlacZ/+ mice (Fig.
5).
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Discussion |
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Tbx5 expression and specification of central conduction system cells
Early in embryogenesis, Tbx5 is expressed throughout the cardiac
crescent, but becomes restricted during formation of the linear heart tube.
High levels persist in the atria, but Tbx5 levels in the primordial
ventricles decrease throughout gestation, and prior to birth Tbx5
expression is largely restricted to the endocardial surface of the left
ventricle. By contrast, Tbx5 levels in the atrioventricular bundle and bundle
branches (Fig. 1) are
maintained at birth, and levels in these conduction system structures are
higher than in the surrounding ventricular myocardium
(Fig. 1). Tbx5 is also
known to activate the promoters of genes encoding the gap junction proteins
Cx40 and ANF (Bruneau et al.,
2001), two molecules that distinguish adult electrophysiologic
cells from working myocardial cells of the ventricle
(Houweling et al., 2002
;
Coppen et al., 2003
). The
temporal and spatial pattern of Tbx5 expression and transcriptional
activity is consistent with a role for this transcription factor in
specification of the conduction system cells.
Tbx5 is required for maturation of the atrioventricular canal conduction system
Development of the mature conduction system requires both specification of
cells with electrophysiologic functions and morphologic patterning of the
central and peripheral electrophysiologic components. Organization of
specialized electrophysiologic cells into distinct components of the mature
conduction system appears to occur in distinct temporal steps. Some cells in
the central conduction system appear to be specified early in development,
since node-like pacemaker activity is evident by the linear-heart-tube stage
(Kamino et al., 1981).
Differentiation of the fast-conducting atrioventricular bundle and bundle
branches occurs later in embryogenesis
(Delorme et al., 1995
;
Sedmera et al., 2003
).
Analysis of minKlacZ/+ mice
(Fig. 2A,B) extends the
temporal sequence for central conduction system development into the postnatal
period. At birth, rings of conduction tissue persist around both the tricuspid
annulus and mitral annulus. Consolidation of electrophysiologic cells into a
discrete atrioventricular node, atrioventricular bundle and bundle branches,
the pattern of a mature conduction system, is a postnatal process that in the
mouse is completed by week 14.
Although the rings of specialized electrophysiologic tissue are similar in newborn minKlacZ/+ mice and Tbx5del/+ mice, postnatal maturation of the atrioventricular canal conduction system fails to occur in Tbx5del/+ mice. Adult Tbx5del/+ mice maintain a neonatal pattern of specialized atrioventricular rings around both the tricuspid annulus and mitral annulus. The failure of morphologic atrioventricular canal maturation correlates with functional immaturity of the atrioventricular conduction system: absence of the normal age-dependent decrease of the PQ interval results in age-dependent atrioventricular block in Tbx5del/+ mice (Fig. 3).
In-vivo electrophysiology localized the anatomic source of the PQ
prolongation in adult Tbx5del/+ mice. The PQ interval
encompasses electrical conduction within the atrial musculature,
atrioventricular node, atrioventricular bundle and proximal bundle branches
(Fig. 3A). The normal HV
interval in Tbx5del/+ mice suggests that the
atrioventricular bundle has normal function, despite appearing physically
foreshortened (Table 1,
Fig. 4). The prolonged AH
interval in Tbx5del/+ mice placed the functional defect in
the atrial myocardium or atrioventricular node. Furthermore, a normal P-wave
duration, indicative of atrial depolarization, ruled out a functional deficit
within the atrial myocardium (Table
1). From these findings, we infer that the prolonged PQ interval
in Tbx5del/+ mice is the result of a maturation failure of
the atrioventricular node or its connection with the atria or atrioventricular
bundle. This finding could also reflect a deficit in one of the distinct
subpopulations of cells within the atrioventricular node
(Coppen et al., 2003).
Progressive atrioventricular block is found in human HoltOram syndrome. Conduction system disease is unusual at birth, but first or second-degree atrioventricular block occurs commonly in adult patients. The progressive onset of atrioventricular canal conduction dysfunction in humans and mice with Tbx5 haploinsufficiency suggests that the same postnatal Tbx5-dependent processes are required for the maturation of the atrioventricular canal conduction system in mice and humans.
Tbx5 and the ventricular conduction system
Tbx5 is also required for normal patterning and function of the
proximal ventricular conduction system, the atrioventricular bundle and the
left and right bundle branches. In adult Tbx5del/+ mouse
hearts, each of these components demonstrates a morphologic patterning defect
(Fig. 4). The fast-conducting
ventricular conduction system also demonstrates a functional deficit of the
right bundle branch but not the left bundle branch
(Table 1). We conclude that the
patterning of the right bundle branch is sufficiently disrupted to cause
malfunction, whereas patterning of the left bundle branch is not. Furthermore,
deficits in the right bundle branch are visible at birth
(Fig. 4). These findings define
a role for Tbx5 in the development of the right bundle branch and suggest a
substantial role in the differentiation of the components of the
fast-conducting ventricular conduction system, including the atrioventricular
bundle. Distinct differences in these components of the central conduction
system in newborn Tbx5del/+ mouse hearts suggest that
development of the atrioventricular node, atrioventricular bundle and bundle
branches have distinct molecular requirements. These data are consistent with
a model in which regional components of the conduction system are specified
independently and are then assembled into a continuous network.
Complete absence or a diminutive right bundle branch was found in all Tbx5del/+ mice, indicating that normal levels of the transcription factor are essential for genesis of this structure. As in the atrioventricular canal, the morphologic deficits of the bundle-branch conduction system were mirrored by functional defects. In particular, there was a close correlation between the severity of the right-bundle-branch morphology defects and functional right-bundle-branch block in Tbx5del/+/minKlacZ/+ mice. All mice with morphologic absence of the right bundle branch had right-bundle-branch block by precordial ECG, whereas few mice with a visible right bundle branch demonstrated right-bundle-branch block. These data suggest that a functional electrophysiological deficit in Tbx5del/+ mice occurred as a consequence of an underlying primary, maldeveloped morphology in the conduction system.
Tbx5 haploinsufficiency directly disrupts the central conduction system
Several lines of evidence suggest that malformation of the central cardiac
conduction system in Tbx5del/+ mice occurs independent of
cardiac structural defects. First, despite an intact ventricular septum, all
Tbx5del/+ mice had malformations in the ventricular
conduction system, usually affecting both the right and left bundle branches.
Second, there was no relationship between the specific type of ASD in
Tbx5del/+ mice and conduction system abnormalities. The
presence of a secundum or primum ASD did not correlate with the severity of
morphologic defects in the central conduction system, and no statistically
significant differential effect was observed on the PQ interval, QRS interval
or likelihood of right-bundle-branch block
(Table 1). We conclude that
Tbx5 has a direct role in conduction system development independent
of its role in structural heart development. Furthermore, the finding that
Tbx5 is expressed at high levels in conduction system cells suggests
that its conduction system requirement may be cell-autonomous.
Cx40, a transcriptional target of Tbx5 that encodes a gap
junction protein required for normal electrophysiologic function of the heart,
was considered a potential cause for the patterning defects evident in the
central conduction system of Tbx5del/+ mice. Like
Tbx5del/+ mice, Cx40/
mice demonstrate prolonged PQ intervals, prolonged QRS intervals, and in some
cases right-bundle-branch block (data not shown)
(Kirchhoff et al., 1998;
Simon et al., 1998
;
Tamaddon et al., 2000
). The
degree to which the decrement in Cx40 transcription in
Tbx5del/+ mice accounts for the functional conduction
system abnormalities in Tbx5del/+ mice remains unclear.
Our recent findings demonstrate the critical importance of even limited
Cx40 expression in Tbx5del/+ mice: whereas
Tbx5del/+ mice usually live to adulthood,
Tbx5del/+/Cx40/ mice die
in utero (A.P., unpublished).
Cx40 deficiency does not, however, explain the morphologic abnormalities of the central conduction system found in Tbx5del/+ mice. Normal morphology of the atrioventricular node, atrioventricular bundle and bundle branches was present in all adult Cx40/ mice, indicating that this gap junction protein is not required for the morphologic maturation or patterning of the central conduction system. These findings implicate yet unidentified genes downstream of Tbx5 in the patterning of the conduction system.
Our results delineate several distinct roles for Tbx5 in conduction system development. Early in cardiac development, the temporal and spatial expression of Tbx5 is compatible with a role in specification of cells in the conduction system. Tbx5-dependent expression of Cx40, and presumably other molecules that are required for the critical electrophysiologic properties of these cells, supports this hypothesis. Tbx5 directs the expression of genes (e.g. Cx40) in the mature conduction system, after the primitive AV node, left bundle branch and right bundle branch have assumed their adult structures, which may account for why some Holt-Oram patients and Tbx5del/+ mice evolve conduction system disease with age. In addition to regulating gene transcription in the conduction system, Tbx5, but not Cx40, is critical for conduction system pattern formation. Normal morphology of the atrioventricular canal components and ventricular components of central conduction system are dependent on physiologic levels of this factor. Tbx5 is the first gene to be implicated in the pattern formation and the developmental maturation of the centralized cardiac conduction system. A link between a patterning abnormality of the developing conduction system and a functional abnormality of the mature conduction system is demonstrated.
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ACKNOWLEDGMENTS |
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