Download Upregulation of the cardiac homeobox gene Nkx2–5 (CSX) in feline

Upregulation of the cardiac homeobox gene Nkx2–5
(CSX) in feline right ventricular pressure overload
JERRY T. THOMPSON, MARY S. RACKLEY, AND TERRENCE X. O’BRIEN
Office of Research and Development, Ralph H. Johnson Department of Veterans Affairs Medical
Center, and Cardiology Division, Department of Medicine, Medical University of South Carolina
and the Gazes Cardiac Research Institute, Charleston, South Carolina 29425
cardiac hypertrophy; pulmonary artery banding
THE NK CLASS OF HOMEODOMAIN proteins is essential in
myogenic lineage development (19). In Drosophila, the
NK2 homeodomain protein tinman is a transcription
factor required for insect cardiac mesodermal determination (3, 5). In vertebrates, tinman is represented by a
closely related family of NK2 genes including the
murine cardiac homolog Nkx2–5 (21) (also called CSX,
Ref. 20), the Xenopus homolog XNkx2.3 (15), and the
human homolog CSX1, which has three isoforms (27).
Nkx2–5 is detectable before both cardiac myogenic
differentiation and the expression of cardiac-specific
genes such as a-cardiac actin (21) and atrial natriuretic
factor (ANF) (14). Nkx2–5 is then continually expressed throughout cardiogenesis as well as in the
adult heart of mice (20) and humans (27). The presence
of Nkx2–5 in the adult myocardium suggests a role in
maintaining the cardiac phenotype. Mice with an homologous knockout of Nkx2–5, like that of tinman in
Drosophila (15), result in an embryonic lethal phenotype that occurs before cardiac looping (22). Nkx2–5
has been characterized predominantly as a cardiac
transcription factor, and its target DNA binding element, NKE, resembles the serum response element
(6, 7). Nkx2–5 has been recently found to provide
specific transcriptional activation of two cardiac genes
important in both the adult and embryonic heart:
a-cardiac actin (6, 8) and ANF (14).
Adult cardiac myocytes are terminally differentiated
and respond to growth stimuli by hypertrophying (10).
Likewise, the adult heart compensates for increased
hemodynamic pressure load with a hypertrophic response. The degree of increase in cardiac mass is
dependent on the severity and type of ventricular wall
stress imposed (12). Sustained hemodynamic overload
of the heart often leads clinically to congestive heart
failure. Genes induced or upregulated during hypertrophic induction secondary to hemodynamic load include,
but are not limited to, cardiac contractile proteins (10),
certain protooncogenes (18), and the reexpression (or
marked increase in expression) of a set of transcripts
normally quiescent in the adult ventricle but that are
predominantly expressed during embryonic life. Examples include ANF (28), skeletal a-actin (26), b-myosin
heavy chain (17, 23, 25), and atrial light chain-1 (16).
Because the exact mechanism(s) for transducing hemodynamic load into the cardiac hypertrophic response is
unknown, it becomes important to examine genes that
are potentially involved in the regulation of gene
transcription during cardiac growth.
Because Nkx2–5 is expressed in the adult heart and
is known to be capable of contributing to the activation
of ANF (14) and a-cardiac actin (6, 8) in vitro, the
question is raised whether such a cardiac homeobox
gene critical to the heart during development might
also be capable of transcriptional activation in response
to pressure overload in adult myocardium. A first step
would be to examine Nkx2–5 transcript levels during
hypertrophic stimulation. To address this, a feline right
ventricular (RV) pressure overload (RVPO) model utilizing pulmonary artery banding was employed. This
model allows study of the physiological effect of a
doubling of RV hemodynamic load as an initiating and
continuing stimulus. The effect of hemodynamic load
on the RV is separated from systemic variables that
would affect both ventricles. RV hypertrophy occurs
over the first several days after pulmonary artery
banding, and the RV wall growth response is largely
completed by 14 days (29). Such an in vivo model offers
several advantages including hemodynamic changes
that resemble human disease (13), the ability to consistently examine different durations of pressure overload, and the use of the unloaded left ventricle (LV) as a
same animal control (24). As such, specific cDNA probes
for Nkx2–5 were used to compare changes in transcript
levels during RVPO and to correlate with changes in
ANF and a-cardiac actin.
H1569
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Thompson, Jerry T., Mary S. Rackley, and Terrence
X. O’Brien. Upregulation of the cardiac homeobox gene
Nkx2–5 (CSX) in feline right ventricular pressure overload.
Am. J. Physiol. 274 (Heart Circ. Physiol. 43): H1569–H1573,
1998.—The recent characterization of the cardiac-specific
homeobox gene Nkx2–5 (or CSX) and its detection in normal
adult heart tissue raises the possibility of a role in adult
hypertrophy. Using pressure overload as a primary stimulus,
we used a feline pulmonary artery banding model to produce
right ventricular hypertrophy (RVH). Total RNA was hybridized to a full-length murine Nkx2–5 cDNA probe that contained the NK family homeodomain. Nkx2–5 mRNA levels
increased 5.1-fold (P , 0.05) and 3.9-fold vs. the corresponding left ventricles at 2 and 7 days of RVH, respectively, during
the period of maximal myocardial growth. By 2 wk, when the
RVH response had been completed, Nkx2–5 mRNA levels
were returning toward baseline. Hybridization with an Nkx2–
5 probe not containing the NK homologous homeodomain
demonstrated that upregulation was specific for the Nkx2–5
gene. Atrial natriuretic factor and a-cardiac actin, both
activated in part by Nkx2–5 DNA binding elements, also
increased with RVH. These data suggest that a cardiac
homeobox gene may play a role in the induction of adult
cardiac hypertrophy.
H1570
NKX2–5 IN RIGHT VENTRICULAR HYPERTROPHY
MATERIALS AND METHODS
RESULTS
Nkx2–5 transcript levels are upregulated during cardiac hypertrophic growth. All PA-banded cats at death
had an ending RV pressure of .40 mmHg (a pressure
known to induce hypertrophic growth, Ref. 13) as well
as a significant increase in their RV free wall-to-body
weight ratios. These parameters for all the animals
used in these experiments are listed in Table 1. Note
that that although three animals were examined at
each RVPO time point for each cDNA probe, because of
tissue limitations not all animals were tested with
every probe. To determine the relative level of Nkx2–5
mRNA during hemodynamic pressure overload, total
RNA, and for one animal at each time point, poly(A)
RNA, was isolated from each PA-banded and control
animal’s RV and LV. GAPDH hybridization was used to
normalize the RNA loading between the RV and LV
samples, since there is a significant increase in ribosomal RNA during cardiac hypertrophy (24) and
GAPDH levels do not appreciably increase (32). Note
that GAPDH normalization was between each of the
RV and LV pairs for each animal, and as such, the
RV-to-LV ratio is unity for each time point. Northern
analysis utilizing the full-length murine Nkx2–5 probe
demonstrated mRNA upregulation in triplicate RVPO
samples after 2 days (mean, 5.1-fold; P , 0.05), 7 days
(mean, 3.9-fold; P 5 NS), and to a lesser degree, 14 days
(mean, 1.9 fold) of PA banding as compared with the
same animal’s LV as well as sham RV and LV controls
(Figs. 1 and 2). No differences were found between
results using total RNA and poly(A) RNA. Because the
full-length Nkx2–5 cDNA probe contained the NK
family homologous homeodomain, there is the possibil-
Table 1. Pulmonary artery banded cat hemodynamics
Time
Banded
Number
Banded
Mean RV Pressure
at Death, mmHg
Mean RV-to-Body
Weight Ratio
Control
2 Days
7 Days
14 Days
4
5
3
4
24/0.5 6 2.4/0.5
42/7 6 5.2/1.6*
44/2 6 7.0/1.1*
57/5 6 5.5/1.3*
0.72 6 0.04
1.24 6 0.15*
1.18 6 0.05*
1.19 6 0.10*
Values are means 6 SE. RV, right ventricle. * P , 0.05 by Dunnett’s
1-tailed mean greater than control test.
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RVPO models. Briefly, adult cats (Felis domesticus) between 2.5 and 4.1 kg were gently anesthetized with meperidine (10 mg/kg im), methohexital sodium (20 mg/kg ip), and
a-chloralose (60 mg/kg iv) followed by arterial cannulation for
blood pressure monitoring, thoracotomy, and pericardiotomy.
RVPO to 40–50 mmHg was induced with a partially occluding
3.5-mm pulmonary artery (PA) band. This is a level that
produces RV hypertrophy that becomes maximal in terms of
wall thickness within a 14-day period without evidence of
ischemia or infarction (13). Controls consisted of shamoperated animals. At the time of death, animals received
identical anesthesia with RV, and systemic pressure measurements were obtained before the isolation of the heart and
perfusion with ice-cold heparinized saline via the coronary
arteries. The RV and LV free walls were dissected and stored
in liquid N2 (24). An adequate response consisted of either a
doubling of baseline RV systolic pressure, measurements
above 42 mmHg (average baseline was 24 6 1.2 mmHg), or a
marked increase in RV-to-body weight ratio. By 14 days,
RVPO hypertrophy reaches its maximum gross response as
measured by serial echocardiography (29) and histology (13).
All procedures and the care of the animals were in accordance
with institutional guidelines and National Institutes of Health
‘‘Guide for the Care and Use of Laboratory Animals’’ [Department of Health and Human Services Publication No. (NIH)
85–23, Revised 1985].
RNA isolation and hybridization. Total RNA was isolated
from frozen tissue pieces homogenized in a polytron in 4.0 M
guanidinium thiocyanate using standard techniques (2).
Poly(A)-enriched RNA was prepared using a Fast Track
mRNA Isolation kit (Invitrogen). RNA samples were denatured, size separated on 1.0% agarose-formaldehyde gels, and
transferred to Duralon nylon membranes (Stratagene). 32Plabeled specific probes were hybridized in 50% formamide, 53
standard saline citrate (SSC), 23 Denhart’s solution, 0.1%
SDS, 0.5% dextran sulfate, and 100 µg/ml tRNA at 42°C
overnight. After samples were serial washed to 0.23 SSC and
0.1% SDS at 42°C, signals were visualized with autoradiography at 270°C for up to 72 h. RNA loading was normalized for
equivalency between each animal’s RV and LV based on
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) signal.
All autoradiography images were digitized and quantitated
using National Institutes of Health image software. Statistical analysis was performed on all data with Super ANOVA
software, and statistical significance was defined as a P value
,0.05 by Dunnett’s one-tailed (sample mean larger than
control mean) test.
Feline a-cardiac actin 38-UTR cloning. The 38-untranslated
region (UTR) for the feline a-cardiac actin gene was cloned
using standard methods for rapid amplification of cDNA ends
(38-RACE). Briefly, first-strand cDNA synthesis was performed from feline heart total RNA using a Superscript
preamplification system for first-strand cDNA synthesis kit
(GIBCO-BRL). This served as a template for polymerase
chain reaction (PCR) using a gene-specific 58-primer, 58CTGTCCACCTTCCAGCA-38, and an oligo(dT)-specific 38primer, 58-GACTC GAGTCGACATCG(T)17-38. The PCR reaction products were cloned into pCR2.1 (Invitrogen) according
to the manufacturer’s instructions. Several clones were sequenced and shown to be a-actin. Comparison with the
published feline a-skeletal actin (24) and other species’ acardiac actin sequences led to the identification of a feline
a-cardiac actin clone.
32P probe generation. A DNA fragment containing the
GAPDH coding region from amino acids 142 to 207 (1) was
PCR amplified from pBluescript using M13-forward and
M13-reverse primers. A feline ANF-specific DNA fragment
was PCR amplified using the 58-primer 58-GACGCCAGCATGAGCTCCTTC-38 and the 38-primer 58-CTCCAATCCTGTCCATCCTGC-38 from a feline cardiac myocyte library. A
1,300-bp DNA fragment containing the entire murine coding
region for Nkx2–5 was obtained from the expression plasmid
pCGNCSX (a generous gift of Dr. Timothy McQuinn, Ref. 6)
by digestion with EcoR I. A 341-bp DNA fragment of Nkx2–5
containing the sequence between bases 299 and 640 (21) was
PCR amplified using the 58-primer 58-GCCCACGCCYTTCTCAGTCA-38 and the 38-primer 58-TCCAGCTCCACYGCCTTCTG-38 and pCGNCSX as template DNA to generate an
Nkx2–5-specific probe [58-341(Nkx2–5)] that did not contain
the NK family homeodomain. These cDNAs were isolated on
1.25% low-melting-point agarose gel by electrophoresis, purified using standard techniques, and labeled using 32P (Du
Pont-New England Nuclear) and a nick translation kit
(GIBCO-BRL).
NKX2–5 IN RIGHT VENTRICULAR HYPERTROPHY
H1571
other than to express the mean RV to GAPDH signal for
each experimental group (Fig. 2).
DISCUSSION
ity that hybridization was not specific for Nkx2–5.
Additionally, all Nkx2–5 probes tested that contained
the homeodomain sequence bound to 28S rRNA, thereby
making the Northern analysis more technically difficult. Therefore, another cDNA probe was designed:
58-341(Nkx2–5), containing the Nk family specific TN
domain as well as the 58-Pro/Ala-rich sequence specific
for Nkx2–5 but not extending into the homologous
homeodomain. Specific hybridization of Nkx2–5 to
three animals at each time point showed statistically
significant increased transcript levels at 2 days (mean,
1.9-fold; P , 0.05) of RVPO with only a 1.5-fold increase
at 7 days (P 5 NS) and a return to baseline by 14 days
(Figs. 1 and 2). No correlation between the degree of
Nkx2–5 upregulation and any hemodynamic parameter
has been established.
Both a-cardiac actin and ANF transcript levels upregulate with Nkx2–5 during feline cardiac hypertrophic growth. Because Nkx2–5 activates the promoters
of both a-cardiac actin (8) and ANF (14), their respective transcript levels were investigated in the RVPO
animal model. The RNA membranes used to examine
for Nkx2–5 were hybridized with the feline a-cardiac
actin 38-UTR probe (Figs. 1 and 2), with mRNA levels
increasing at 2 days (3.2-fold), 7 days (3.9-fold), and 14
days (2.8-fold) of RVPO (all with statistically significant P values; P , 0.05) as compared with each
animal’s GAPDH normalized LV level. Similar results
were seen when these membranes were hybridized
with the ANF probe except that ANF was not detected
in the control RV or LV or in any of the PA-banded LV
samples (Figs. 1 and 2). The ANF signal for all the
RVPO samples was variable in intensity, which, with
no LV standardization, made formal statistics difficult
Fig. 2. Quantification of upregulation of Nkx2–5 full-length and
58-specific Nkx2–5 cDNA probes in RVPO-induced cardiac hypertrophy. Shown is Northern hybridization digital quantification with all
data points being averages of triplicate experiments with RVPO
durations of 0 (sham control), 2, 7, and 14 days (SE are shown). Each
RV and LV digitized hybridization value was normalized by dividing
it by digitized GAPDH signal. Then each RV-to-LV ratio was determined to quantitate Nkx2–5 upregulation (as such, GAPDH value
would be 1.0 for each time point). Both Nkx2–5 probes demonstrated
statistically significant increases in transcript levels with RVPO at 2
days, with a return toward baseline levels by 7 and 14 days. Because
ANF levels are below level of detection in all control and LV samples,
they are expressed as ratio of their RV signal normalized for GAPDH.
SE is not shown for ANF, since there is no detectable sham RV level
to normalize against and, once ANF becomes detectable, levels vary
widely between RVPO cats. a-Cardiac actin was significantly increased at all pulmonary artery-banded time points.
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Fig. 1. Upregulation of Nkx2–5 in right ventricular (RV) pressure
overload (RVPO)-induced cardiac hypertrophy relative to a-cardiac
actin and atrial natriuretic factor (ANF). Northern analysis of feline
heart total RNA isolated from RV and left ventricular (LV) tissue
after 0 (sham control), 2, 7, and 14 days of RVPO. All RV and LV
paired samples are from same animal, and same membrane was
hybridized to each of 5 cDNA probes shown. Hybridization with
full-length Nkx2–5 probe is seen at 1.5 kb (21) and is increased at 2
and 7 days and less so at 14 days in RV samples relative to control
and LV samples. Samples were normalized for glyceraldehyde-3phosphate dehydrogenase (GAPDH) mRNA hybridization as shown
[GAPDH does not change appreciably during development of cardiac
hypertrophy (32)]. 58-341(Nkx2–5) cDNA probe used was specific,
since it does not contain NK family homeodomain and demonstrated
increased hybridization at 2 and 7 days. Specific feline 38-untranslated region (UTR) a-cardiac actin and ANF cDNA hybridization
were also increased in RVPO samples as shown.
These studies demonstrate a link between the mechanical stimulus of hemodynamic load, which is a
primary regulator of cardiac mass (13), and the upregulation of Nkx2–5 transcript levels specifically in the
ventricle affected by the increased load. Nkx2–5 mRNA
levels increased markedly in the pressure-overloaded
RVs of PA-banded cats at 2 and 7 days, which was
during the period of maximal RV wall growth (13, 29).
Note that in this model 2 days is the earliest practical
time of death, since a full clinical recovery of the animal
from surgical effects may take up to 24 h. Because
Nkx2–5 is present constitutively in the normal adult
heart and transcript levels were found to increase
markedly during ventricular hypertrophic growth,
Nkx2–5 may be an important responder to adult ventricular wall stress. In support of this, overexpression
of (Xenopus) XNkx2–5 and XNkx2–3 in Xenopus embryos resulted in myocardial thickening in otherwise
morphologically normal hearts (11). Likewise, overexpression of Nkx2–5 in zebrafish produced disproportionately larger hearts in apparently otherwise normal
embryos (9). Whatever other covariables may be in-
H1572
NKX2–5 IN RIGHT VENTRICULAR HYPERTROPHY
Hemodynamic pressure overload in the heart has
been shown to evoke certain changes in cardiac transcription. Several gene products are reexpressed that
are only otherwise expressed during cardiac development. Examples include b-myosin heavy chain (17, 23,
25), ANF (28), a-skeletal actin (26), and atrial myosin
light chain-1 (16). This suggests that part of an otherwise embryonic cardiac transcriptional program might
reactivate in response to hemodynamic load (among
other possible stimuli). The upregulation of Nkx2–5
found in this study is compatible with this hypothesis.
Future studies will be required to determine if this
cardiac homeobox gene product previously thought
only active during cardiogenesis has a functional role in
adult cardiac growth regulation.
We thank Dr. Masayoshi Hamawoki for skillful animal surgery
and Drs. Paul McDermott, Tim McQuinn, and George Cooper for
critical review of this manuscript.
This work was supported by the Office of Research and Development, Medical Research Service, Ralph H. Johnson Department of
Veterans Affairs Medical Center (Charleston, SC), where T. X. O’Brien
is a Research Associate, and by National Heart, Lung, and Blood
Institute (NHLBI) Grant HL-55284 (to T. X. O’Brien) as well as
NHLBI Training Grant T32-HL-07260–19 (to J. T. Thompson).
Address for reprint requests: T. X. O’Brien, Cardiology Division,
816 CSB, 171 Ashley Ave., Charleston, SC 29425-2221.
Received 15 May 1997; accepted in final form 14 January 1998.
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Nkx2–5 binds to a specific DNA site, the NK element
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a-cardiac actin expression with Nkx2–5 in the RVPO
model. ANF and a-cardiac actin mRNA levels increased
in the RVPO cats during hypertrophic growth, paralleling early increases in Nkx2–5 (Figs. 1 and 2). It is
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increase after aortic banding (4). This may be a species
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Previous investigations have all included the NK
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21, 27); therefore, it was felt important to examine
specifically for Nkx2–5 independently of other NK
homeobox-containing genes. A cDNA probe consisting
of 341 nucleotides from the more divergent 58-end of the
coding region also demonstrated upregulation at 2 and
7 days of pressure overload (Figs. 1 and 2) in a similar
pattern to the full-length Nkx2–5 hybridization. The
possibility of other NK family members being present
and/or upregulated is suggested by the differences in
the increase between the full-length Nkx2–5 probe and
the 58-341(Nkx2–5) probe, but these cannot be directly
compared and this was not formally addressed in these
experiments. Because the full-length probe had greater
specific activity, this may contribute to differences in
signal levels. Also, the changes in Nkx2–5 transcript
levels observed may be from either differences in
transcription, mRNA stability, or some combination of
the two.
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