Download Dilated heart failure in transgenic mice expressing the Epstein

Journal of General Virology (1993), 74, 1381-1391. Printed in Great Britain
1381
Dilated heart failure in transgenic mice expressing the Epstein-Barr virus
nuclear antigen-leader protein
David S. Huen,' Andrew Fox, ~ Prem Kumar 2 and Peter F. Searle 1.
Cancer Research Campaign Laboratories, Department o f Cancer Studies and 2 Department of" Physiology, University
o f Birmingham Medical School, Birmingham B15 2 T J, U.K.
The Epstein-Barr virus nuclear antigen-leader protein
(EBNA-LP) is required for high efficiency B lymphocyte
growth transformation by the virus. To test the potential
contribution of EBNA-LP to tumorigenesis in vivo, we
produced transgenic mice carrying an EBNA-LP eDNA
construct, using the widely expressed metallothionein
promoter. Expression of EBNA-LP was detected in
liver, kidney, heart, lung and spleen. There were no
apparent oncogenic consequences of EBNA-LP expression. Unexpectedly however, at ages ranging from
about 4 months to over a year, transgenic mice
developed symptoms of congestive heart failure, in-
cluding left ventricular dilatation, right ventricular
hypertrophy, left atrial thrombosis, pulmonary oedema
and hydrothorax. Fibrillation was not apparent in the
electrocardiograph; however a reduction in T-wave
amplitude suggested that the development of an abnormality of ventricular repolarization may precede the
manifestation of overt symptoms. The highly predictable
development of dilated heart failure in these transgenic
mice suggests they may be a useful model for the
pathophysiological changes associated with human
dilated cardiomyopathy.
Introduction
and cellular promoters (Abbot et al., 1990; Fahraeus et
al., 1990; Wang et al., 1990). Expression of EBNA-2 in
The Epstein-Barr virus (EBV) is a common human
herpesvirus with a complex life cycle involving chronic
replication in epithelial tissues and virus persistence as a
non-productive or 'latent' infection in B lymphocytes
(Fields & Knipe, 1990). Infection usually occurs asymptomatically in early childhood, whereas primary infection
during adolescence can cause infectious mononucleosis
(glandular fever). The subsequent lifelong persistence of
the virus is generally asymptomatic, and kept in check by
a cytotoxic T cell response (Rickinson et al., 1992).
However EBV is also associated with several major
human malignancies including Burkitt's lymphoma,
Hodgkin's lymphoma and nasopharyngeal carcinoma.
Immortalized lymphoblastoid cell lines (LCLs) can be
derived from B lymphocytes infected with EBV, and
these express a limited set of nine viral proteins
comprising the six EBV nuclear antigens [EBNA-1,
EBNA-2, EBNA-3A/B/C, EBNA-leader protein
(EBNA-LP)], three latent membrane proteins (LMP-1,
LMP-2a and LMP-2b) as well as two small nuclear
RNAs (EBERs). Although the minimal subset of viral
proteins needed for immortalization has yet to be
determined, both EBNA-2 and LMP-1 have major roles
in growth transformation. The EBNA-2 protein is a
trans-activator that upregulates transcription from EBV
rodent fibroblasts reduces their serum requirement for
growth (Dambaugh et al., 1986), and mutant viruses
lacking the EBNA-2 gene are unable to transform B
lymphocytes (Cohen et al., 1989; Hammerschmidt &
Sugden, 1989; Skate et al., 1985). Expression of the
LMP-1 protein induces expression of cell surface markers
associated with B lymphocyte activation in Burkitt's
lymphoma lines and reduces their sensitivity to apoptosis
through the upregulation of the bcl-2 proto-oncogene
(Henderson et aL, 1991; Wang et al., 1988). LMP-I
expression also confers anchorage independence and
tumorigenicity in some rodent fibroblast lines (Wang et
al., 1985) and inhibits differentiation in human epithelial
cell lines (Dawson et al., 1990; Fahraeus et al., 1990).
Consistent with the latter role, expression of LMP-1 in
the skin of transgenic mice produced a phenotype of
hyperplastic dermatosis (Wilson et al., 1990), more
widespread expression resulting in death.
A third viral protein, EBNA-LP, has an important
auxiliary role in growth transformation by EBV. Although its biochemical function is unknown, the protein
was recently reported to co-localize with a biochemically
distinct fraction of the retinoblastoma gene protein to
large granular structures within the nucleus, as also
observed for the simian virus 40 (SV40) T antigen (Jiang
0001-1421 © 1993SGM
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D. S. H u e n and others
et al., 1991). Viruses with mutations in the EBNA-LP
gene are greatly impaired in their ability to transform B
lymphocytes (Hammerschmidt & Sugden, 1989; Mannick et al., 1991). EBNA-LP is a polymorphic protein
consisting of a varying number of amino-terminal repeats
of a motif rich in basic residues, and a short unique
carboxy-terminal domain with some similarity to a
region of the adenovirus 5 E1A protein (Bodescot et al.,
1984; Huen et al., 1988; Sample et al., 1986). The repeat
motifs in EBNA-LP are each encoded by a pair of exons
within a series of 3.4 kb direct repeats in the viral
genome. The EBNA-LP open reading frame (ORF)
occurs within the leader region of mRNAs encoding each
of the other EBNAs, derived by alternative processing of
large primary transcripts from the C and W promoters.
The initiation codon of EBNA-LP is assembled by only
two of four alternative splices at the 5' end of this
transcript (Rogers et al., 1990; Sample et al., 1986).
As an approach to investigating the roles of the EBV
latent gene products in cell transformation and tumorigenesis we attempted the expression of several of the
proteins, including EBNA-LP, in transgenic mice. Initially we produced three lines of transgenic mice in which
the EBNA-LP cDNA was coupled to the immunoglobulin heavy chain enhancer with an SV40 promoter,
in an attempt to achieve B cell-specific expression;
however mice in these lines did not express the transgene
(D. S. Huen et al., unpublished results). We describe here
several lines of transgenic mice expressing EBNA-LP
from a metallothionein promoter, in a variety of tissues.
There were no apparent oncogenic consequences of
EBNA-LP expression. Unexpectedly however, after
several months of apparent good health, transgenic mice
in multiple independently derived lineages became ill and
died prematurely of congestive (or dilated) heart failure.
The highly predictable development of this condition in
these transgenic mice suggests that they may be a
valuable model for the pathophysiological changes
occurring in human dilated cardiomyopathy.
Methods
Construction of MT-LP-hGH fusion gene. Plasmid pUC-LP contains
the EBNA-LP-encoding portion of the T65 cDNA cloned into the
SmaI site of pUC18 (Sample et al., 1986; gift of Dr F. Wang, Boston,
Mass., U.S.A.). A 1.8 kb BamHI-HindIII fragment from the human
growth hormone (hGH) gene was inserted between the BamHI and
HindIII sites within the polylinker downstream of the EBNA-LP ORF
to form p(DH55)LP-hGH. A 1"7 kb EcoRI-BglII fragment containing
the mouse metallothionein-I (MT) promoter and transcription initiation site was then inserted upstream of the LP-hGH fusion gene
between the EcoRI and KpnI sites of the pUC18 polylinker to give
p(DH61)MT-LP-hGH. The injected fragment was excised from the
polylinker as a 4.5 kb EcoRI HindIII fragment (shown schematically
in Fig. l b) and separated from vector sequences by agarose gel
electrophoresis, electroelution and recovery using a QIAGEN tip-20
(Diagen).
Production of transgenic mice. Procedures were essentially as
described (Hogan et al., 1986). Briefly, the purified DNA fragment
(MT-LP-hGH) was microinjected into fertilized mouse eggs derived
from C 5 7 B L / 6 J x C B A / C a F a hybrid parents. The manipulated
embryos were cultured overnight and then re-implanted into pseudopregnant female mice. This resulted in the birth of 35 pups, of which 30
survived to weaning age. Transgenic progeny were identified by
hybridizing dot blots of tail DNA with a probe spanning the LP-hGH
portion of the construct (indicated in Fig. 1b). All procedures using
animals were carried out with the authority of appropriate Home Office
Project and Personal Licenses.
Transgene analysis of genomic DNA. Genomic DNA samples from
the tail (5 ~tg) were digested with the appropriate restriction enzyme as
recommended by the supplier in a total volume of 40 ~tl. Samples were
then electrophoresed in a Tris-acetate-EDTA-buffered agarose gel and
vacuum-transferred to Hybond-N+ nylon membranes (Amersham).
Hybridization was performed as described by Sambrook et al. (1989)
using a 32P-labelled random-primed probe prepared by using an
ofigolabelling kit (Pharmacia).
Detection of EBNA-LP in transgenic mouse tissues. Tissue samples
were homogenized in chilled RIPA buffer (20 mM-Tri~HC1 pH 7"5,
5mM-EDTA, 400mM-NaC1, 0.5% Triton X-100, 0"5% sodium
deoxycholate, 0"1% SDS) using an Ultra-Turrax homogenizer (Janhke
& Kunkel). Contaminating serum immunoglobulins were removed by
preclearing the samples with anti-mouse immunoglobulin-Sepharose
beads. These were prepared by binding a goat anti-mouse immunoglobulin serum (Sigma) to CNBr-activated Sepharose 4B beads (Sigma)
as indicated in the manufacturer's instructions. Protein concentrations
were determined using the phenol-tannic acid method (MejbaumKatzenellenbogen & Dobryszycka, 1959). Protein samples (100 ~tg)
were separated on SDS~olyacrylamide gels and electrophoretically
transferred onto nitrocellulose membranes (Gelman) essentially as
described (Towbin et al., 1979). Transferred protein was visualized
using Ponceau S (Sigma) and the positions o f M r markers (Sigma) were
noted. The filter was then blocked with BLOTTO, a 5 % suspension of
non-fat dried milk in PBS supplemented with 0.1% Tween-20
(PBS-Tween) for 1 h. A mouse monoclonal antibody (MAb) specific
for EBNA-LP (JF186, kind gift of Dr M. Rowe; Finke et al., 1987),
was then applied to the filter as a 10-fold dilution of hybridoma
supernatant in BLOTTO and incubated at 4 °C overnight, then washed
with three changes of PBS-Tween. The filter was then incubated with
a 1000-fold dilution of rabbit anti-mouse IgG (Dakopatts) for 1 h and
washed again with three changes of PBS-Tween. Finally, the filter was
incubated with 20 pCi of radioiodinated Protein A (Amersham) in
BLOTTO for 3 h, washed as before with PBS-Tween and exposed to
X-ray film (Kodak X-Omat S).
Ventricular weights. Ventricular weights were measured using hearts
fixed for 24 h in formal saline. The atria were removed, then the free
right ventricular wall was excised along its margin with the interventricular septum. The left ventricle was then cut open longitudinally,
both ventricles were washed free of blood and clots and patted dry with
paper tissue. The free wall of the right ventricle, and the left ventricle
with interventricular septum were then weighed (see Table 1).
Electrocardiography. Electrocardiograms (ECGs) were recorded
from subcutaneous needle electrodes placed above the right shoulder
and lower left ribcage of the dorsal surface of mice under Hypnodil
(metomidate hydrochloride) anaesthesia. Signal amplification was
achieved with Neurolog equipment (Digitimer) incorporating a 0-5 Hz
to 300 Hz bandpass and 50 Hz notch filter. Data capture was achieved
by the use of a pulse code modulator (Sony 501-ES) sampling at
44.1 kHz and recorded on a VHS video recorder. ECG profiles were
obtained by playback of recorded material and examination on a
digital oscilloscope equipped with hardcopy facilities (Gould 420).
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Heart failure in E B N A - L P transgenic mice
Results
Production of E B N A - L P transgenic mice
To produce transgenic mice expressing EBNA-LP,
mouse zygotes were injected with the M T - L P - h G H gene
construct shown diagrammatically in Fig. 1 (b), in which
a c D N A fragment encoding E B N A - L P was placed
downstream of the mouse M T p r o m o t e r region. Sequences encompassing most of the h G H gene transcription unit and 3' sequences were included downstream of the E B N A - L P c D N A to provide splice and
poly(A) signals. Three founder transgenic mice (designated 53-2, 53-18 and 53-26) were identified by dot blot
analysis of tail D N A . On subsequent breeding, the
variation a m o n g progeny in signal intensity on dot blots,
and in the case of 53-2 a high proportion of transgenic
progeny (16 out of 20), suggested that each of the
founder mice had incorporated transgene copies in at
least two segregating sites in the genome, and therefore,
multiple sublines were established by further breeding of
several offspring from each of the original transgenic
founders.
(a)
7 8
123456
-*---MT-LP-hGH
(b)
K
I
s
B
I
I
[.....
LP
Fig. l(a) shows a Southern blot of BamHI-digested
D N A from transgenic mice in three sublines derived
from 53-2 and 53-18, probed with the E B N A - L P - h G H
fragment indicated in Fig. 1 (b). The transgene contains
a single internal BamHI site, and so the unit-length
4.5 kb band in each of the lanes indicates the presence of
a tandem array of transgenes in each of the sublines.
Subline 53-2A clearly differs from 53-2B in the intensity
of this band, indicating a difference in transgene copy
number, and in the pattern of minor bands which include
junction fragments with mouse D N A and additional
variable bands believed to result from internal transgene
rearrangements (see below). The transgene loci in these
two sublines are therefore considered to result from
independent integration events. In contrast, the pattern
obtained with subline 53-2C D N A is similar to that
obtained with subline 53-2B. Although there are subtle
differences such as the faint band of about 5.8 kb
observed in 53-2B and not in 53-2C, the similarities
suggest that these two sublines carry derivatives of the
same transgene locus which has subsequently undergone
additional minor rearrangements in at least one of the
lineages. By similar reasoning, the transgene loci in
sublines 53-18B and -C m a y be related whereas that in
subline 53-18D probably results from an independent
integration event. Two sublines were also established
from transgenic founder 53-26; however, one of these,
53-26X, was lost at an early stage owing to the disease
phenotype described below. The absence of this phenotype in the surviving subline, 53-26A, suggests that these
two lineages must also have received distinct transgene
loci. As listed in Table 1, the number of copies of the
M T - L P - h G H gene in the different sublines ranged from
about 2 to 100.
s
I
\\\\\\\\\\\\x
MT
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Expression of E B N A - L P
I
hGH
Probe
1 kb
Fig. 1. (a) Southern blot ofBamHI-digested DNA from transgenicmice
in each of three sublines derived from founder animals 53-2 and 53-18
(lanes 1 to 3, 53-2A, -B and -C, respectively;lanes 4 to 6, 53-18B, -C and
-D, respectively). Lane 7 contained DNA from a non-transgenic
littermate; lane 8 contained 30 pg of the microinjected DNA fragment
and non-transgenic carrier DNA. The positions of DNA size markers
are indicated. (b) Schematic diagram of the MT-LP-hGH fusion gene
construct used to produce transgenic mice.The open, solid and hatched
areas represent the MT promoter, EBNA-LP ORF frame and the hGH
gene sequences respectively. Selected restriction endonuclease sites are
indicated as follows: K, KpnI ; S, SstI; B, BamHI. The thick line below
the construct shows the region of homology between the LP-hGH
fragment used as probe and the transgene construct. The short thin
extension represents a SV40 promoter-derived region not contained in
the transgene.
Expression of E B N A - L P in tissues of transgenic mice
from each of the surviving sublines was detected by
probing Western blots with an a n t i - E B N A - L P M A b
(Fig. 2). The 66K band present in the negative control as
well as the transgenics is attributed to mouse serum
albumin in the samples, detected with the rabbit antimouse immunoglobulin second-step antibody. The
E B N A - L P c D N A incorporated into the transgene
construct contained five copies of the repeated exon pair,
and encoded a 42K EBNA-LP. A band of this size was
seen in several tissues from mice in each subline. The top
panel in Fig. 2 shows expression in the heart, and it is
apparent that in addition to the 42K band seen in each
of these transgenic sublines, EBNA-LPs both larger and
smaller than the main band are expressed at significant
levels in several sublines. Different isolates of EBV
contain a variable number of copies of the genomic
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D. S. Huen and others
Table 1. Summary of MT-LP-hGH transgenic mouse lineages
Founder
Estimated
transgene
Subline* copy no.t
Age (days) at presentation
with heart diseaseJ;
Mean
s.D.
No.
of mice§
Incidence of
heart
disease(%)[[
EBNA-LP
expression
in heart¶
53-2
53-2A
53-2B
53-2C
3
20-30
20-30
500
155
158
92
28
31
14
40
89
50
100
100
++++
+++++
+++++
53-18
53-18B
53-18C
53-18D
50-100
50-100
5 10
260
281
343
94
118
74
55
26
36
100
100
100
++
++++
++++
53-26
53-26A
53-26X
2
5-10
146
15
-7
None
100
+
NA
* Sublines derived from each of the three founder animals; those which appear on the basis of Southern
blotting to contain derivatives of the same initial transgene integration event are grouped.
t Copy number was estimated from intensity of Southern blot signal.
:~ Age at which mice died with evidenceof congestive heart failure or were found with symptoms and humanely
killed.
§ Number of mice included in statistics of previous columns.
I] See text.
¶ Indication of relative levels of expression based on Western blot of heart tissue; NA, not available.
3.4 kb repeats including the repeated exons of E B N A - L P
m R N A ; even during infection of h u m a n B cells with a
single EBV isolate, the E B N A - L P produced is heterogeneous in size. This is caused by splicing of a variable
number of the repeated exons into the m R N A , as seen in
the positive control sample from the L C L X50-7 in which
the 42K E B N A - L P is a minor species. However as a
c D N A was used in the transgene construct, such variable
splicing cannot be the cause of E B N A - L P size heterogeneity in the transgenic mice. Southern blotting of
genomic D N A using restriction enzyme sites that flank
the repeats, resulting in smaller fragments and hence
greater resolution than seen in the blot shown in Fig.
1 (a), indicated that the size variation of E B N A - L P in the
transgenic mice correlated with differences in the number
of 198 bp exon pair repeats in the genomic transgene
copies (data not shown). These m a y have arisen by
unequal recombination and, as the patterns appear to be
stably inherited, probably occurred during transgene
integration. A wide variation of E B N A - L P size was also
observed with both vaccinia virus and baculovirus
recombinants incorporating the same c D N A , which
suggests that the E N B A - L P repeats are strongly recombinogenic (M. Rowe, A. H. Davies & D. S. Huen, unpublished observations).
The lower panels of Fig. 2 show only the 42K band of
E B N A - L P detected in liver, kidney, lung and spleen.
Each of these other tissues gave the same characteristic
pattern of additional E B N A - L P bands as seen in the
heart (top panel). The heart blot shown was overexposed
in order to show the weaker E B N A - L P bands; in the 532B and -C sublines for example, similar levels of E B N A LP were present in the heart and liver samples. The
relative levels of expression in the different tissues varied
between different lineages. For example, in subline 5326A, expression in the heart was the lowest observed
whereas expression in the liver was relatively high,
exceeded only by that in subline 53-18D. Expression in
the lung and spleen was detectable only in the 53-18
sublines, at levels appreciably lower than in kidney, liver
or heart. Subline 53-26X was shown to express E B N A LP strongly in liver and kidney; other tissues were not
tested before the subline was lost. Immunohistochemical
localization of E B N A - L P in sublines of 53-2 and 53-18
showed the protein to be localized to the nucleus of
hepatocytes in liver sections and cardiomyocytes in the
heart (data not shown).
Heart disease in EBNA-LP transgenic mice
After several months of apparent good health, transgenic
mice in all sublines derived from transgenic founders 532 and 53-18, and one of the two 53-26 sublines, became
ill, showing symptoms of respiratory distress. I f not
killed at the appearance of symptoms, such animals died
typically within 1 to 3 days. Mice which had died or were
killed when symptoms became apparent were found to
have fluid in the lung alveoli (pulmonary oedema) and
pleural cavity (hydrothorax). Abnormalities of the heart
were also invariably observed, including distention o f the
left atrium by a prominent thrombus as shown in Fig.
3(b). Microscopic examination of the atrial thrombi
revealed large numbers of erythrocytes enmeshed between strands of fibrin, indicative of formation under
conditions of circulatory stasis. The right atrium was
also often enlarged to a lesser extent, without the
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Heart failure in EBNA-LP transgenic mice
120K -97K --
123
45
6789
66K --
45K --
29K --
Liver
Kidney
Lung
:/~;:;!:
Spleen
Fig. 2. Western blots showing EBNA-LP expression in tissues of
transgenic mice. The main panel shows heart samples from transgenic
mice in the available sublines (lanes 1 to 3, 53-2A, -B and -C,
respectively; lanes 4 to 6, 53-18B, -C and -D, respectively;lane 7, 5326A). Lane 8, non-transgenic mouse. Lane 9, positive control sample
that contains protein from 2 million X50-7 cells, which produce a
predominant EBNA-LP larger than that encoded by the injected
transgene; a 42K EBNA-LP equivalent to that expected in the mice is
present as a minor species (as indicated). Values of Mr markers (right)
are shown on the left. The top panel shows heart samples; this
autoradiograph was exposed for 1 week to show the fainter signals of
some sublines and the variant EBNA-LPs. Liver and kidney samples
gave similar patterns of variant EBNA-LPs; only the 42K regions of
the blots are shown. These were exposed for 1 day to avoid
overexposure. The blots of lung and spleen were also exposed for 1
week to show the low level expression of the 42K EBNA-LPs; variant
EBNA-LPs were below the limit of detection.
presence of a thrombus. The ventricles appeared to be
dilated with a larger cavity and thinner walls than
control hearts, as shown in Fig. 3(b, d). Equivalent
sections of normal hearts are shown in (a) and (c).
Transgenic mice examined prior to the development of
respiratory distress did not show these abnormalities
(data not shown).
To determine whether ventricular hypertrophy contributed to the pathology, hearts from normal nontransgenic mice, presymptomatic and symptomatic transgenic mice were dissected and the right and left ventricles
were weighed. As shown in Table 2, there was no
significant difference in the mass of the ventricles in
presymptomatic transgenic mice compared to nontransgenic controls. In symptomatic transgenic animals,
a 40 to 80 % increase in the mass of the right ventricle
was apparent, whereas there was no change in the left
ventricular weight. Light microscopic examination of the
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myocardium did not reveal obvious fibrosis, lymphocytic
infiltration, myocyte hypertrophy or other histological
abnormality; however in electron micrographs, regions
of myofibrillar disarray were apparent particularly in the
left atrium of affected animals. Fig. 4(b) shows the
typical ordered arrangement of myofibrils running in the
plane of the section in atrial myocytes from a nontransgenic control mouse. In contrast, atrial myocytes
from an affected transgenic mouse contain myofibrils
running in several directions, both in the plane of the
section and perpendicular to the section (Fig. 4a).
Similar changes, though less extreme, were observed also
in some transgenic mouse ventricular myocytes, including some from presymptomatic mice.
The disease was limited to transgenic mice within
susceptible sublines; non-transgenic littermates were
unaffected. Fig. 5 shows typical cumulative mortality
curves (for sublines 53-2C and 53-18D). N o transgenic
animals in subline 53-2C survived beyond 296 days, and
the majority developed heart disease between 125 and
200 days of age (black area of chart). Some transgenic
mice were killed for experimental purposes prior to the
development of symptoms; these are shown in the crosshatched area. Although it is obvious that only surviving
animals could be used, these were chosen essentially at
r a n d o m within the age range at which heart disease
develops, and so it is unlikely that we inadvertently
selected animals which would not have developed heart
disease. We conclude that in this subline the incidence of
heart disease is essentially 100%. Mice in subline 5318D developed heart disease over a greater range of ages,
i.e. from 227 to 556 days (Fig. 5b), although it again
appears that all transgenic mice in this subline would
ultimately develop the same pathology.
A summary of the mortality data for each of the
sublines is shown in Table 1. All transgenic mice in
subline 53-26X died by 5 months of age, leading to the
loss of this line at an early stage. Subline 53-26A is the
only lineage in which heart disease was not observed
a m o n g 139 transgenic animals, of which 30 were
monitored for over 400 days. In subline 53-2A the mean
age at which heart disease develops approaches the
normal lifespan of laboratory mice. Fewer mice in this
subline were monitored for the long period necessary,
but we estimate that around 50 % of animals develop
congestive heart failure before other causes of death
intervene.
ECG findings
The pathological findings in symptomatic M T - L P - h G H
transgenic mice described here indicated that heart
dysfunction was a major feature of the disease. In order
to investigate heart function non-invasively in live mice,
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D. S. Huen and others
(a)
(b)
Fig. 3. Photomicrographsof heart sectionsfrom normal miceand transgenicmicewith dilated heart failure. (a, b) Longitudinalsections
through hearts from (a) non-transgenicand (b) symptomatictransgenic mice; the arrows point to the left atria, which in the affected
transgenic heart contain a large thrombus. (c, d) Cross-sections through the ventricles of (c) non-transgenic and (d) symptomatic
transgenics; arrows indicate the cavity of the left ventricle, which is dilated in the affectedtransgenic. Bars represent 2 mm.
ECGs were obtained from control non-transgenic mice,
presymptomatic and symptomatic transgenic mice (see
Fig. 6). Murine ECGs differ from the usual human E C G
pattern in that the QRS complex (associated with
ventricular depolarization) and the T wave (associated
with ventricular repolarization) are not distinctly separated (Richards et al., 1953). In addition to the T wave,
which appeared within the QRS complex, two further
peaks of uncertain origin were noted to the right of the
QRS complex in our recordings (labelled U and V in Fig.
6). The non-transgenic controls and most transgenic
mice without visible disease symptoms showed essentially
identical ECGs (Fig. 6, top two traces). In transgenic
mice showing mild respiratory distress soon after the
onset of symptoms, the size of the T wave was reduced,
and the U wave increased in size (Fig. 6, third trace).
ECGs from a few animals not showing obvious symptoms also showed T wave reduction, suggesting that this
might be an early indicator of the onset of disease. In
more severely affected animals (bottom trace) the T wave
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Heart failure in EBNA-LP transgenic mice
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Table 2. Ventrieular weights
Mean weight (±S.D.) (mg)
Right ventricle*
Left ventricle*
No. of
animals
Male*
Control
Presymptomatic
Symptomatic
25.4_+3"5
22.4 ± 2.4
33.3 _+5"5t
109.1 + 12-9
93.7 ± 9.4
91.2 ± 12.3
9
8
7
Female*
Control
Presymptomatic
Symptomatic
18.7 ± 1.6
17.9 ± 1.8
32.3 ± 6"4t
79.1 ± 6.9
76.6 ± 3.5
79.3 ± 13.3
6
9
5
* Data for male and female mice are shown separately because the
hearts of males were significantly heavier than those of females.
Transgenic mice were classified as presymptomatic (i.e. normal
appearance) or symptomatic (showing signs of respiratory distress
when killed); controls were non-transgenic littermates.
~ For both males and females, right ventricles were significantly
heavier in symptomatic than either control or presymptomatic mice
(significant at 95 % level using Scheffe's F-test).
was further reduced and in some cases was almost
unrecognizable.
Atrial fibrillation was considered a possible reason for
the consistent distension and thrombus formation within
the left atrium, while ventricular fibrillation would
contribute to a reduced pumping efficiency and dilatation
of the ventricles. However the ECGs from transgenic
mice at all stages of disease progression showed a normal
rhythmicity indicating the absence of either atrial or
ventricular fibrillation. Also, as is apparent from the
alignment of ECGs in Fig. 6 (left panels), the time
interval between atrial and ventricular depolarization (P
to R) shows little change, indicating the absence of
conduction blocks at the atrial-ventricular junction;
similarly, despite the amplitude changes, the timing of
the R, T, U and V waves appeared unchanged. No
consistent changes in heart rate were noted, although
some variability between animals was observed, possibly
due to variation in the depth of anaesthesia.
D&cussion
We have established several transgenic mouse lineages
carrying the MT-LP-hGH gene construct designed to
express EBNA-LP from the mouse metallothionein
promoter, in a broad variety of tissues. We demonstrated
that EBNA-LP was expressed in each of these, at varying
levels in liver, kidney, spleen, lung and heart. Although
the MT promoter used in the construct is inducible by
heavy metal ions such as zinc and cadmium, all the
results described in this paper were due to the constitutive, non-induced activity of the promoter. Transgenic
mice in the majority of these lineages reproducibly
develop cardiopulmonary disease, within an age range
characteristic of the particular lineage. The appearance
Fig. 4. Transmission electron micrographs of left atrial tissue from (a)
a non-transgenic mouse and (b) a symptomatic transgenic mouse. The
thin arrows indicate myofibrils running in the plane of the section; the
thick arrow in (a) indicates myofibrils aligned perpendicular to the
plane of the section. Bar represents 10 gm.
of the same disease pathology in transgenic mice derived
from each of three original transgenic founder animals
indicates that the disease must be due to expression of
the transgene rather than to a chance mutation of the
host genome associated with transgene integration.
Downstream of the EBNA-LP coding sequence, the
transgene construct also contained most of the transcription unit from the hGH gene, included to allow
efficient nuclear processing of the RNA transcript; the
resulting mRNA is therefore expected to contain an
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1388
D. S. Huen and others
200
Q~S
180
Non-transgenic
160
~ P J i ~ - - t
-
~
~
~
140
120
100
transgemc
80
60
4O
Ons
20
.~
tof
symptoms
. . . .
i~,j
!
if
0
Terminal phase . . . . J/z,J "4'if..-/ ~ - ~ - - -
6 8O
t~'*
~-J~
Z
100 BV
60
l
100 gtV
-lOOms
l
,500ms
Fig. 6. Representative ECGs from non-transgenic mice and EBNA-LP
transgenics at various stages of disease progression. The traces on the
left show details of a single trace from the series shown on the right. The
P wave, QRS complex and T, U and V waves are indicated (see text).
Voltage and time scales are indicated below.
40
20
00
200
400
Age (days)
600
Fig. 5. Charts illustrating incidenceof dilated heart failure with age in
two representativetransgenicsublines. (a, 53-2C; b, 53-18D).The upper
line indicatesthe number of transgenicmicewhich have beenmonitored
to a particular age. The black area indicates the cumulative incidence
of dilated heart failure, and includes both animals which were found to
have died with congestive heart failure and those which were found
with symptomsand humanelykilled. The cross-hatchedarea represents
animals which were killed for experimental purposes prior to the
development of symptoms.
intact h G H O R F downstream of that encoding EBNALP. It appears unlikely that significant levels of h G H
would be translated from this hybrid mRNA, and in
accordance with this expectation the transgenic mice did
not grow larger than their transgenic littermates, as
observed with M T - h G H transgenic mice (Palmiter et al.,
1983). Furthermore, since M T - h G H transgenic mice did
not develop the pathology we observed (Brem et al.,
1989), we conclude that a possible low level of h G H
synthesis does not contribute to development of cardiopulmonary disease in M T - L P - h G H transgenic mice. The
latter do not develop turnouts at a rate detectably greater
than non-transgenic littermates, suggesting that EBNALP does not have an independent transforming ability.
Nevertheless, deletion mutants of EBV lacking the
EBNA-LP carboxy-terminal sequence were greatly impaired in their ability to transform B cells in vitro (Allan
et al., 1992; Hammerschmidt & Sugden, 1989; Mannick
et al., 1991). These two observations may be reconciled
only if EBNA-LP can facilitate growth transformation in
cooperation with some other EBV gene product. The
principle of oncogene cooperation to give a greater rate
of cell transformation in vitro or tumorigenesis in
transgenic mice than seen with either oncogene alone is
firmly established (Choi et al., 1988; van Lohuizen et al.,
1989; Hunter, 1991). It will be of interest to test the
ability of other EBV genes to cooperate with EBNA-LP
and induce tumours, using the M T - L P - h G H transgenic
mice that live longer. However it should be noted that
expression of EBNA-LP was detected in the spleen only
in the 53-18 sublines, and here the level was very low.
The possibility that higher levels of EBNA-LP may elicit
an observable phenotype in lymphocytes cannot therefore be excluded.
The pathological features o f the disease produced in
the M T - L P - h G H transgenic mice are indicative of
congestive heart failure. Heart failure in man can occur
secondary to hypertension (high blood pressure); however the increased resistance presented to the heart in this
condition induces growth of cardiomyocytes in the left
ventricle resulting in left ventricular hypertrophy, which
was not present at any stage of the disease in our
transgenic mice. Furthermore hypertensive transgenic
animals have been produced, yet these were not reported
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Heart failure in E B N A - L P transgenic mice
to develop the cardiopulmonary pathology described
here (Kimura et al., 1992; Mullins et al., 1990). The
consistent dilatation of the left ventricle in affected
transgenic mice without accompanying hypertrophy
suggests an underlying ventricular insufficiency. A
reduction in left ventricular output would impede
pulmonary drainage, increasing the pressure within the
pulmonary circulation. This could cause extravascular
fluid retention in the lungs and pleural cavity, giving rise
to oedema and hydrothorax. The hypertrophy observed
in the right ventricle of symptomatic transgenic mice is a
normal response to pulmonary hypertension, but may
further aggravate the problems. Increased pressure in the
pulmonary circulation may contribute to distension of
the left atrium, and the contraction of atrial myocytes
may also be compromised, as suggested by the observation of myofibrillar disarray. The combined effects
presumably cause thrombus formation, either because of
abnormal turbulence or, more probably as suggested by
the entrapment of erythrocytes within the clot, because
of stasis of the blood in the atrial appendage. Once
formed, the thrombus would further impair cardiac
function and pulmonary drainage, leading to escalation
of the effects and the appearance of visible symptoms.
The molecular mechanism by which EBNA-LP causes
heart failure in the transgenic mice is not clear; however
the observation of a reduced T wave amplitude in ECGs
of affected animals and a proportion of the presymptomatic transgenic mice suggests that a defect ofventricular
repolarization develops in the transgenic mice and may
contribute to the end-stage disease. It appears most likely
that the cardiomyocytes themselves are the relevant
target cells for EBNA-LP expression and, with one
anomaly (subline 53-18B), there does appear to be a
correlation between the level of EBNA-LP expressed in
the heart and the severity of the disease, as judged by the
age of onset (see Table 1). However it is possible that the
effect could result from expression of EBNA-LP in a
particular region of the myocardium or even in a remote
tissue with an effect on heart function. The role of
EBNA-LP in the viral life cycle also remains unknown,
although the short region of identity with adenovirus 5
E1A protein and co-localization with the retinoblastoma
susceptibility gene product to granules within the nucleus
suggest that it may share cellular targets in common with
the oncogenes of other DNA tumour viruses, perhaps
affecting the transcriptional regulation of specific genes
(Huen et al., 1988; Jiang et al., 1991). A plausible
hypothesis to explain a role in the heart disease
pathogenesis is that EBNA-LP might affect the expression of genes involved in the normal function of the
heart, such as those encoding ion channels.
Heart disease has been described previously in only a
few lines of transgenic mice, and in none of these does the
1389
pathology appear identical to that described here.
Expression of the SV40 T antigen using the promoter of
the atrial natriuretic factor gene in transgenic mice
caused hyperplasia and a several hundredfold increase in
size of the right atrium (Field, 1988). The ECGs of such
mice were dominated by arrhythmias apparently due to
the development of multifocal pacemakers in the grossly
enlarged atrium. Cardiac expression of the c-myc gene
resulted in modest enlargement of the heart as a result of
myocyte hyperplasia, although in other respects the
myocardium appeared normal as judged by the absence
of fibrosis and expression of the appropriate actin
isoform (Jackson et al., 1990); these mice were not
reported to become ill with heart disease. Enlargement of
the heart was also a feature of v-fps transgenics in which
a form of congestive heart disease was observed; this was
accompanied by degeneration of cardiomyocytes and
marked fibrosis of the left ventricular wall, features
absent in the MT-LP-hGH transgenic mice (Yee et al.,
1989). The transgenic model most similar to that
described here is a single line of mice expressing
polyomavirus large T antigen (PVLT), also from the MT
promoter, reported while this work was in progress
(Chalifour et al., 1990). These MT-PVLT mice developed
congestive heart failure and were also found to develop
a left atrial thrombus. However the entire heart was
enlarged three- to 4.5-fold in the MT-PVLT transgenic
mice, and both vacuolar degeneration of myocytes and
interstitial fibrosis were apparent in the heart wall. This
contrasts with our MT-LP-hGH transgenic mice in
which there was no apparent underlying hypertrophy or
fibrosis. Nevertheless the causation of congestive heart
disease by both PVLT and EBNA-LP is consistent with
the hypothesis that these proteins may share cellular
targets. The differences in phenotype may relate to the
known ability of papovavirus large T antigens to interact
with a wide range of cellular growth-regulatory proteins
in contrast to the more limited selection expected for the
smaller, less complex EBNA-LP.
The disease produced by expression of EBNA-LP in
transgenic mice has some similarity to human congestive
heart failure or dilated cardiomyopathy (DCM), in
which dilatation of the ventricles occurs without a
proportionate increase in myocardial mass. In man
DCM has an estimated incidence of at least five to 10 per
100000 per year (Torp, 1980) and is associated with a
high mortality; end-stage disease can only be effectively
treated by heart transplantation (Brandenburg, 1985).
Several possible causes have been proposed for DCM in
man, including alcohol abuse, viral infection and
autoimmunity. Each of these may play a leading role in
some cases; however there remains a high proportion of
cases for which the cause is unknown (Olsen, 1985;
Wynne & Braunwald, 1988). The results presented here
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1390
D. S. Huen and others
suggest that the EBNA-LP of EBV can cause heart
failure in mice, indicating that individual viral proteins
can potentially precipitate heart failure, even without the
associated events of viral infection. Despite the widespread presence of EBV in the human population, it
appears unlikely that EBNA-LP plays a role in the
aetiology of human DCM as EBV is not known to infect
heart tissue. Although the primary insult may differ,
many features of disease progression may be similar in
MT-LP-hGH transgenic mice and human DCM patients,
and EBNA-LP may even mimic the effect of an initiating
infection by another virus. The highly predictable
development of dilated heart failure in transgenic mice
expressing EBNA-LP as described here should facilitate
investigation of the pathophysiological changes associated with ventricular dilatation, and may be valuable
in the development of preventative or therapeutic
approaches to human DCM.
Helpful advice was obtained from Dr W. Butler, Dr C. Edwards, Dr
M. D. Gammage, Professor W. A. Littler, Professor A. B. Rickinson
and Dr M. Rowe. The excellent technical assistance of K. Faulkner, V.
Nash, J. Pearson, P. Reeves and S. Williams is gratefully acknowledged. This project was funded by the Cancer Research Campaign.
D.H. was the recipient of an ORS Award from the CVCP and a Belt
Memorial Fellowship. This work forms a part of a body of work
included in a doctoral dissertation by D. S. H.
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