Note: Descriptions are shown in the official language in which they were submitted.
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DIAGNOSTIC METU~n
Field of the Invention
The present invention relates to the diagnosis of post-operative atrial
fibrillation (AF) by
determining the levels of connexin40 in a sample of cardiac tissue taken from
a patient. The
present invention further relates to a method for preventing or treating post-
operative atrial
fibrillation by administering to a patient a substance capable of down
regulating connexin40
expression or inhibiting connexin40 function. The present invention also
relates to assays for
identifying suitable substances.
Background to the Invention
Post-operative atrial fibrillation (AF) is a common complication after
cardiothoracic surgery,
occurring in up to 40% of patients. Although, in the absence of hemodynamic
compromise,
post-operative AF is considered relatively benign, it is associated with an
increased risk of
stroke and ventricular tachycardia, and results in higher costs due to
prolonged hospital stay.
As prophylactic drug treatment (especially (3-blockers) would involve up to
80% of patients
being unnecessarily exposed to drug therapy, a means to predict post-operative
AF is
considered highly desirable. Apart from intraoperative induction of AF, which
has a negative
predictive value of 93%, no practical intraoperative screening technique is
currently available.
There is thus considerable interest in the prevention and prediction of post-
operative AF, and
in understanding the mechanisms of its genesis.
The underlying cellular mechanism of AF involves re-entrant circuits resulting
from areas of
slow conduction and functional conduction block. However, why AF develops post-
operatively in some patients but not others with apparently identical heart
conditions is
unclear. Recent discussion has focussed on the idea that predisposition to AF
may reside in
the passive electrical properties of the atrial myocardium (Spach et al.,
1995) i.e., on the cell-
to-cell conduction properties determined by gap junctions (Rohr et al., 1997;
Shaw and Rudy,
1997). Gap junctions are clusters of channels which link neighbouring cells,
forming low
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resistance pathways which allow transmission of action potentials between each
and every
myocyte in the heart (Bruzzone et al., 1996; Severs, 1999; Gros and Jongsma,
1996). Each
channel consists of two hemichannels called connexons, and each connexon is a
hexamer of
connexin subunits (Severs,1999; Gros and Jongsma,1996). In the human heart,
myocyte gap
junctions may be constructed from up to three different connexin types,
connexin43,
connexin40 and connexin45, expressed in a distinctive tissue and chamber
related pattern, with
an additional isoform, connexin 37, in vascular endothelial cells (Severs,
1999; Vozzi et al.,
1999). Connexin43 and connexin45 are homogeneously distributed throughout the
atria at
high and low levels respectively (Vozzi et al. ,1999). Connexin40, however, is
heterogeneous
in distribution, expressed at levels 2-3 fold higher in the right atrium than
the left, reaching,
in the former, similar levels to connexin43 (Vozzi et al., 1999). When
expressed individually
in artificial systems, these different connexin types confer gap functional
channels with
distinctive properties (e.g., unit channel conductances, permeabilities and
sensitivity to
transjunctional voltage) (Gros and Jongsma, 1996; Veenstra, 1996; Jongsma,
1997).
Summary of the Invention
We hypothesised that distinctive patterns of connexin expression contribute to
predisposition
of surgical patients to post-operative AF. To address this hypothesis, we have
collected right
atrial appendages from ischemic patients undergoing coronary by-pass operation
for analysis
of connexin expression by immunoconfocal, northern and western blotting
techniques. As
expected, a number of patients subsequently developed AF, allowing
retrospective division
of the samples into two groups, control and AF-prone. Surprisingly we have
found that one
connexin type, connexin40, is expressed at significantly higher levels in the
group predisposed
to AF.
Accordingly, the present invention provides a method for determining the
susceptibility of a
patient to post-operative atrial fibrillation (AF) which method comprises
determining the
levels of connexin40 in a sample of cardiac tissue taken from said patient
wherein the presence
in said sample of elevated levels of connexin40 is indicative of a
susceptibility to post-
operative atrial fibrillation (AF).
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Typically the determination of the levels of connexin40 comprises measuring
the levels of
connexin40 mRNA and/or connexin40 protein in said sample.
The present invention also provides a method for identifying a substance
capable of down
regulating connexin40 expression which method comprises contacting a cell
which expresses
connexin40 with a candidate substance and determining whether connexin40
expression is
reduced.
In another embodiment, the present invention provides a method for identifying
a substance
capable of inhibiting connexin40 function which method comprises contacting a
connexin40
polypeptide with a candidate substance and determining whether said substance
binds to the
connexin40 polypeptide.
The present invention further provides a substance capable of down regulating
connexin40
expression or inhibiting connexin40 function identified by the assay methods
of the invention.
In addition, the present invention provides a method for preventing or
treating post-operative
atrial fibrillation in a patient which method comprises administering to said
patient an effective
amount of a substance capable of down regulating connexin40 expression or
inhibiting
connexin40 function.
Typically, the compound may have been identified by the assay methods of the
invention.
Brief Description of the Fib
Figure 1. Characterisation of anti-connexin 40 antibodies by western blots
(panel A) and by
colloidal immunogold electronmicroscopy of atrial tissues (panel B and C).
Figure 2. Low magnification of atrial samples immunofluorescently labelled for
connexin40
(A and B), for connexin45 (C) and for connexin43 (D).
Figure 3. A, Representative northern blot analysis of total RNA extracted from
patients with
no post-operative AF (patients 1, 2, 3, 4 and 5) and from patients with post-
operative AF
(patients 6 to 14). B, Graph showing quantification of A.
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Figure 4. A, Typical western blot analysis of heart protein homogenate for
connexin40 (upper
panel), connexin43 (middle panel) and coomassie blue staining of these samples
(lower panel).
B. Graph showing quantification of A.
Detailed Description of the Invention
Although in general the techniques mentioned herein are well known in the art,
reference may be
made in particular to Sambrook et al., Molecular Cloning, A Laboratory Manual
(1989) and
Ausubel et al., Short Protocols in Molecular Biology (1999) 4'h Ed, John Wiley
& Sons, Inc.
A. Measuring-connexin40 levels in patients
Levels of connexin40 may be measured in samples of cardiac tissue using a
variety of standard
techniques. Cardiac tissue is typically obtained from surgical patients during
procedures
carried out on the patient's heart or associated tissue, such as for example
during a coronary
by-pass operation. Typically, tissue taken from the right atrial appendage is
preferred because
of the heterogeneity of connexin40 expression in atria (Vozzi et al., 1999),
and because this
part of the right atrium is always available during surgery and its ablation
does not pose any
hazard to patient health. Generally, samples are snap-frozen in liquid
nitrogen within a few
minutes after collection. This in particular preserves total cellular RNA for
subsequent
analysis by northern blotting or reverse transcriptase polymerase chain
reaction (RT-PCR) as
well as cellulax protein for subsequent analysis by western blotting and other
immunological
detection methods such as ELISA.
Measurement of connexin40 mRNA levels
Total cellular RNA is typically purified from frozen, pulverised tissues using
a modified
isothiocynanate/acid phenol extraction procedure (Kilarski et al., 1998;
Chomczynski et al.,
1987).
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Northern blotting of purified total cellular RNA may be performed using
standard techniques
known in the art. Typically, hybridisation is performed at high stringency
(65°C 5 x SSC)
using a random primed DNA probe.
RT-PCR is performed on total RNA preparations under standard conditions.
Suitable cycle
parameters include denaturation at 94°C for 5 mins, annealing at 45 to
65 °C (depending on the
primer set) for 1 min, and extension at 72°C for 1.5 mins. Persons
skilled in the art will have
no difficulty in selecting other suitable parameters.
The nucleotide sequence of connexin40 is set out in W098/02150 and Kanter et
al., 1994,
both of which are incorporated herein by reference. This sequence and other
connexin40
sequences may be used to design suitable PCR primers and/or probes for RT-PCR
and/or
northern blotting. Fragments obtained by PCR from, for example, genomic DNA,
may be
used as probes for northern blotting.
An example of a suitable PCR primer pair is:
5'-ATGGGCGATTGGAGCTTCCTGGGA-3' (forward primer)
5'-CACTGATAGGTCTACTGACCTTGC-3' (reverse primer)
Northern blotting and RT-PCR are generally performed on samples from surgical
patients
together with a suitable negative control (such as an in vitro transcribed RNA
that can be
accurately measured and used at a range of concentrations which would mirror
the range found
in patients resistant to post-operative AF) and a suitable positive control
(such as a sample
from a healthy patient with normal levels of connexin40 expression).
Results are typically measured in a quantitative or semi-quantitative manner,
such as by
scanning densitometry of bands corresponding to hybridising fragments and/or
amplification
products. Alternatively, results may be gauged by eye. Competitive (using an
internal
standard), quantitative RT-PCR is a preferred method for measuring connexin40
mRNA
levels. Results are typically calculated as a percentage of the results
obtained for the positive
control.
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For a positive diagnosis, a susceptible patient may typically have at least
30% higher levels
of connexin40 mRNA in total cellular RNA taken from cardiac tissue during or
shortly after
surgical procedures as compared with a control standard range of in vitro
transcribed RNA.
Preferably the connexin40 mRNA levels are at least 50% higher than the
control, more
preferably at least 60, 70 or 80%.
A sample from a particular patient is preferably tested more than once and the
mean of the
determinations used.
Measurement of connexin40 protein levels
For determination of connexin40 protein levels, tissue samples are typically
lysed in a solution
comprising a detergent such as SDS. Other methods for lysing whole cells are
known in the
art and include mechanical and/or chemical means.
For western blotting, lysed samples are resolved by electrophoresis and
transferred onto a
suitable membrane such as nitrocellulose or PVDF using standard techniques.
The membrane
is then blocked with standard buffers and incubated with an antibody to
connexin40. A
preferred antibody is antibody Y21 Y(R968) described in the Examples.
After washing and incubation with, for example, enzyme conjugated secondary
antibody, the
presence of connexin40 protein is determined by standard means and typically
quantitated
using densitometric scanning. It may be desirable to also probe blots with a
control antibody
- such as anti-myosin antibody - to normalise for lane loading and adjust the
readings to
myocardial volume. Alternatively, or in addition, parallel samples may be
stained with, for
example, coomassie blue and densitometrically scanned.
Samples may also be tested using standard ELISA protocols. Samples may
conveniently be
tested in mufti-well plate formats. ELISA techniques are well known in the
art.
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Whichever method is used, results are typically calculated as a percentage of
the results
obtained for the positive control. For western blotting, a GST fusion protein
(see below,
connexin40 binding assay) encompassing the Y21 Y peptide (see Examples) can be
used as a
positive control in a range of concentrations which give densitometric values
found in post-
s operative AF resistant patients. For ELISA, a positive control can be
constituted either by the
Y21 Y peptide or the GST fusion protein used in the range of concentrations
mimicking the
readings determined from AF resistant patients
For a positive diagnosis, a susceptible patient may typically have at least
30% higher levels
of connexin40 protein in cell extracts taken from cardiac tissue before,
during or shortly after
surgical procedures as compared with a control sample taken from a healthy
patient.
Preferably the connexin40 protein levels are at least 50% higher than the
positive control value
determined above, more preferably at least 60, 70 or 80%.
A sample from a particular patient is preferably tested more than once and the
mean of the
determinations used.
B Assays for substances capable of affecting connexin40 expression or function
Our results demonstrate a link between pre-operative elevated levels of
connexin40 expression
and post-operative acute AF. Consequently, one possible means of treating or
preventing AF,
in particular post-operative AF would be to administer to a patient in need of
such treatment
an effective amount of a substance that downregulates connexin40 expression or
affects
connexin40 function in cardiac tissue. An example of such a substance would be
an antisense
connexin40 construct.
Other suitable substances may be identified by screening methods of the
invention. For
example, a candidate substance whose activity it is desired to test may be
administered to a
cell which expresses connexin40 in the absence of the candidate substance.
Such screening
methods may also be performed in vivo on intact multicellular animals, such as
mammals, for
example mice, rats or hamsters.
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The present invention also provides assays that are suitable for identifying
substances that bind
to connexin40 polypeptides. Such assays are typically in vitro. Assays are
also provided that
test the effects of candidate substances identified in preliminary in vitro
assays on intact cells
in whole cell assays and/or in intact multicellular animals.
Reference to connexin40 is taken to include human connexin40 as set out in SEQ
LD. Nos. 1
and 2 of W098/02150 and homologues thereof having an analogous biological
function (i.e.
as a constituent of gap junctions in cardiac tissue). For example, the
nucleotide and
polypeptide sequences of human, rat, mouse and dog connexin40 are available in
the Genbank
database as Accession Nos. L34954, M83092, X61675 and M81347, respectively. In
binding
assays, it may not be necessary to use the full length polypeptide: fragments
of the full length
sequence may also be used. Such fragments comprise at least 20, 30, 40 or 50
amino acids,
more preferably at least 100 amino acids of the full length sequence and
preferably encompass
either the intracellular loop or the cytoplasmic C tail which are different
between each
connexin and are readily accessible sequences (as determined by membrane
topology
analysis).
Candidate substances
Suitable candidate substances include peptides, especially of from about 5 to
30 or 10 to 25
amino acids in size, based on the sequence of the various domains of
connexin40, or variants
of such peptides in which one or more residues have been substituted. Peptides
from panels
of peptides comprising random sequences or sequences which have been varied
consistently
to provide a maximally diverse panel of peptides may be used.
Combinatorial libraries, peptide and peptide mimetics, defined chemical
entities,
oligonucleotides, and natural product libraries may be screened for activity
as inhibitors of
connexin40 expression and/or function. The candidate substances may be used in
an initial
screen in batches of, for example 10 substances per reaction, and the
substances of those
batches which show inhibition tested individually. Candidate substances which
show activity
in in vitro screens such as those described below can then be tested in whole
cell systems, such
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as cardiac myocytes which will be exposed to the inhibitor and tested for
inhibition of
connexin40 function and/or expression.
Connexin40 bindin assays
One type of assay for identifying substances that bind to connexin40, possibly
interfering with
its function, synthesis, trafficking andlor degradation in vivo, involves
contacting a connexin40
polypeptide, which is immobilised on a solid support, with a non-immobilised
candidate
substance determining whether and/or to what extent the connexin40 polypeptide
and
candidate substance bind to each other. Alternatively, the candidate substance
may be
immobilised and the connexin40 polypeptide non-immobilised.
In a preferred assay method, the connexin40 polypeptide is immobilised on
beads such as
agarose beads. Typically this is achieved by expressing the component as a GST-
fusion
protein in bacteria, yeast or higher eukaryotic cell lines and purifying the
GST-fusion protein
from crude cell extracts using glutathione-agarose beads. As a control,
binding of the
candidate substance, which is not a GST-fusion protein, to glutathione-agaxose
beads (and/or
a GST only control) is determined in the absence of the connexin40
polypeptide. The binding
of the candidate substance to the immobilised connexin40 polypeptide is then
determined.
This type of assay is known in the art as a GST pulldown assay. Again, the
candidate
substance may be immobilised and the connexin40 polypeptide non-immobilised.
It is also possible to perform this type of assay using different affinity
purification systems for
immobilising one of the components, for example Ni-NTA agarose and histidine-
tagged
components.
Binding of the connexin40 polypeptide to the candidate substance may be
determined by a
variety of methods well-known in the art. For example, the non-immobilised
component may
be labelled (with for example, a radioactive label, an epitope tag or an
enzyme-antibody
conjugate). Alternatively, binding may be determined by immunological
detection techniques.
For example, the reaction mixture can be Western blotted and the blot probed
with an antibody
that detects the non-immobilised component. ELISA techniques may also be used.
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Candidate substances are typically added to a final concentration of from 1 to
1000 nmol/ml,
more preferably from 1 to 100 nmol/ml.
Whole cell assays - including whole animal studies
Candidate substances may also be tested on whole cells for their effect on
connexin40
expression and/or function. Preferably the candidate substances have been
identified by the
above-described in vitro methods. Alternatively, rapid throughput screens for
substances
capable of inhibiting connexin40 function may be used as a preliminary screen
and then used
in the in vitro binding assay described above to confirm that the affect is on
connexin40.
The candidate substance, i.e. the test compound, may be administered to the
cell in several
ways. For example, it may be added directly to the cell culture medium or
injected into the
cell. Alternatively, in the case of polypeptide candidate substances, the cell
may be transfected
with a nucleic acid construct which directs expression of the polypeptide in
the cell.
Preferably, the expression of the polypeptide is under the control of a
regulatable promoter.
Suitable whole cells for testing inhibition, are those which express
connexin40. Since
connexin40 expression is generally confined to cardiac tissue and endothelium,
suitable cells
may include immortalised cell lines derived from cardiac atrial myocytes
(Claycomb et al.,
1998) and endothelial cells, primary cardiac cells and samples of cardiac
tissue obtained from
test animals. Typically cardiac cells are preferred to avoid cell type-
specific regulation of
connexin40 (such as found in endothelial cells for example). Since cardiac
cells usually
express more than one connexin they are generally not suitable for a
functional investigation.
For functional assays, as described below, a cell line devoid of intercellular
communication
is genetically modified (by transfection) to express only connexin40 under the
control of an
inducible promoter to allow for varied levels of expression (for example the
Tet off system
supplied by Promega).
Typically, an assay to determine the effect of a candidate substance
identified by the method
of the invention on connexin40 comprises administering the candidate substance
to a cell and
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determining whether the substance inhibits or reduces connexin40 expression
and/or
connexin40 function.
Connexin40 expression may be determined by measuring mRNA and/or protein
levels as
described above. A candidate substance is typically considered to be an
inhibitor of
connexin40 expression if connexin40 expression is reduced to below 50%,
preferably below
40, 30, 20 or 10% of that observed in untreated control cells. However, it is
also preferred that
inhibition of expression in cells from patients or experimental animals with
upregulated
connexin40 expression does not reduce the levels of connexin40 expression
significantly
below normal levels. In other words, preferably the inhibitor of connexin40
expression
reduces connexin40 levels in the cardiac tissue of patients suffering from AF
to no less than
70, 80 or 90% of normal levels found in patients that are not susceptible to
AF. In practice,
however, the level of inhibition of expression in a patient may preferably be
modulated
through changing substance dosage.
Connexin40 function may be measured by, for example, determining whether gap
junctions
which comprise connexin40 are functional - such as by taking electrical
measurements of unit
channel conductances, permeabilities and/or sensitivity to transjunctional
voltage, for example
as described in Gros and Jongsma, 1996; Veenstra, 1996 and Jongsma, 1997. The
properties
of connexin40-containing gap junctions in treated cells is compared with the
properties of
connexin40-containing gap junctions in an untreated control cell population to
determine the
degree of inhibition, if any.
The concentration of candidate substances used will typically be such that the
final
concentration in the cells is similar to that described above for the in vitro
assays.
The types of assays performed on whole cells described above may also be
performed on
intact multicellular animals, typically mammals such as rodents, pigs or non-
human primates.
Typically, the inhibitor of connexin40 expression reduces its expression to no
less than 50%
of the normal level in these animals which corresponds approximately to the
difference found
between the AF and the control group (see example). Candidate substances may
be
administered to the animal as described below in section D. In one type of
assay, AF is
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induced by surgical procedures such as opening the thoracic cage and
pericardium and
stopping the heart by cryocardioplegia, preferably on bigger animals (for
example pigs dogs
and non-human primates since AF is not inducible in small laboratory animals).
The candidate
substance is administered and the effect determined in terms of reduction of
connexin40
expression and incidence of post-operative AF.
C. Diagnostic and Therapeutic Applications
The measurement of connexin40 levels as described in section A above may be
used in
methods of diagnosis for susceptibility to AF, typically acute AF brought on
by surgical
procedures, which generally occurs within 2 to 3 days after surgery.
Measurements may be
taken before, during and/or after surgical procedures, preferably immediately
after, and the
results used by clinicians to decide on the necessary course of clinical
treatment such as
prophylaxis using pharmacological agents known to prevent and/or revert
arrhythmias (for
example beta-blockers).
Substances capable of reducing connexin40 expression and or function may be
administered
to a patient diagnosed as having elevated levels of connexin40 expression to
treat the resulting
AF or prevent its occurrence. Such substances may include antisense connexin40
constructs
and/or substances identified by the assay methods of the present invention.
D. Administration
Substances identified or identifiable by the assay methods of the invention
may preferably be
combined with various components to produce compositions of the invention.
Preferably the
compositions are combined with a pharmaceutically acceptable carrier or
diluent to produce
a pharmaceutical composition (which may be for human or animal use). Suitable
carriers and
diluents include isotonic saline solutions, for example phosphate-buffered
saline. The
composition of the invention may be administered by direct injection. The
composition may
be formulated for parenteral, intramuscular, intravenous, subcutaneous,
intraocular or
transdermal administration. Typically, each substance may be administered at a
dose of from
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0.01 to 30 mg/kg body weight, preferably from 0.1 to 10 mg/kg, more preferably
from 0.1 to
1 mg/kg body weight.
Polynucleotides/vectors encoding polypeptide components (or antisense
constructs) for use
in inhibiting connexin40 expression and/or function may be administered
directly as a naked
nucleic acid construct. The polynucleotides/vectors may further comprise
flanking sequences
homologous to the host cell genome.
When the polynucleotides/vectors are administered as a naked nucleic acid, the
amount of
nucleic acid administered may typically be in the range of from 1 ~g to 10 mg,
preferably from
100 ~g to 1 mg. It is particularly preferred to use polynucleotides/ vectors
that target
specifically cardiac cells, such as atrial myocytes, for example by virtue of
suitable regulatory
constructs or by the use of targeted viral vectors. The delivery of genetic
material to specific
locations in cardiac tissue is described in W098/02150 and reference therein.
Uptake of naked nucleic acid constructs by mammalian cells is enhanced by
several known
transfection techniques for example those including the use of transfection
agents. Example
of these agents include cationic agents (for example calcium phosphate and
DEAE-dextran)
and lipofectants (for example lipofectamTM and transfectamTM). Typically,
nucleic acid
constructs are mixed with the transfection agent to produce a composition.
Preferably the polynucleotide or vector according to the invention is combined
with a
pharmaceutically acceptable carrier or diluent to produce a pharmaceutical
composition.
Suitable carriers and diluents include isotonic saline solutions, for example
phosphate-buffered
saline. The composition may be formulated for parenteral, intramuscular,
intravenous,
subcutaneous or transdermal administration.
The routes of administration and dosages described are intended only as a
guide since a skilled
practitioner will be able to determine readily the optimum route of
administration and dosage
for any particular patient and condition.
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The invention will now be further described by way of Examples, which are
meant to serve
to assist one of ordinary skill in the art in carrying out the invention and
are not intended in
any way to limit the scope of the invention. The Examples refer to the
Figures.
Detailed Description of the Figures
Figure 1. Characterisation of the anti-connexin 40 antibodies by western blots
(panel A) and
by colloidal immunogold electronmicroscopy of atrial tissues (panel B and C).
The antibodies
recognise a ~ 40 kDa bands in atrial tissues, some ventricular tissues and
lung (lanes RA1,
LA1, LV2 and Lung in panel A), but not in most ventricular tissues (known to
lack
connexin40 in working myocardium; lane RV l and LV 1 ). A spurious band at ~
65 kDa is
present in all cardiac tissues but absent in lung. Panel B and C show that the
colloidal gold
labelling is exclusively confined to structures recognised as gap junctions by
their typical
pentalaminar appearance.
Figure 2. Low magnification of atrial samples immunofluorescently labelled for
connexin40
(A and B), for connexin45 (C) and for connexin43 (D). Connexin staining is
seen as red
fluorescence, autofluorescence of elastic fibres as green, autofluorescence of
lipofuscin
granules as white-purple or white-red (L, lipofuscin). Connexin40 is present
at the intercalated
disks seen in various orientations from transverse to en face (insert in A),
and in the
endothelial cells of intramural arteries ( ~ in A and B). Distribution is
somewhat
heterogeneous, as seen in (B) where the right part of the field shows almost
no positive
staining while the left part displays prominent labelling. Connexin45 is
present only in low
amounts (C); the much higher power of the laser setting necessary to record
connexin45 signal
thus results in much brighter intensity of lipofuscin fluorescence in C
compared to A, B and
D. Connexin43 signal is prominent (D) but, in contrast to connexin40, is
absent from arteries
(indicated by , identified by the ring of green fluorescence of medial elastic
fibres).
Figure 3. A, Representative northern blot analysis of total RNA extracted from
patients with
no post-operative AF (patients 1, 2, 3, 4 and 5) and from patients with post-
operative AF
(patients 6 to 14). The same membranes have been hybridised with the cDNA for
the connexin
indicated on the left side of the picture. The position of the wells, the 28S
rRNA and the 18S
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rRNA are indicated on the right side of the panels. Each probe labels a single
band at the
expected molecular weight. The lower panel is a hybridisation with an
oligonucleotide specific
for the 18S ribosomal RNA used to assess equivalent loading and to normalise
the values
obtained with the specific probes. B, Quantification of each connexin. All
values are expressed
as a percentage of the value for patient 1 to allow comparison of different
experiments. Error
bars correspond to the SEM. Only comparison of connexin40 gave significant p
values (<
0.05).
Figure 4. A, Typical western blot analysis of heart protein homogenate for
connexin40 (upper
panel), connexin43 (middle panel) and coomassie blue staining of these samples
(lower panel;
used to assess gel loading and normalise the data with the values obtained by
densitometric
scanning for myosin). Patient number and post-operative diagnosis are
indicated on top of the
gels. The position of the separating gel (arrow) and molecular weight
standards are indicated
to the left. Connexin40 migrates at ~40 kDa. The anti-connexin43 antibody
labels a single
band at ~44kDa representing the highly phosphorylated form of this protein;
some lower
molecular weight bands just below are also weakly labelled and represent low
amounts of the
dephosphorylated or unphosphorylated forms. Quantification of connexin40 and
connexin43
protein is shown in B. As for northern blot quantification, signals were
normalised with the
values from the coomassie blue stained myosin and are expressed as a
percentage of the value
for patient 1. Error bars represent the SEM. Only comparison of connexin40
returned
significant p values (< 0.05).
EXAMPLES
MATERIALS AND METHODS
Collection of diseased human samples
Patients were selected using three criteria to obtain a clinically homogenous
group; i) they all
had ischemic heart disease, ii) they were undergoing coronary by-pass graft
operation, and iii)
they had no previous arrhythmic disorder as assessed by medical history and
pre-operative
ECG. Forty five samples of the right atrial appendage matching these criteria
were collected
CA 02281010 1999-08-27
P007306 CTH _ 16-
consecutively over a period of three months at the Harefield and Royal
Brompton Hospital
NHS Trust. All samples were snap frozen in liquid nitrogen within a few
minutes after
collection. This approach to sample collection permitted total RNA
purification for analysis
by northern blotting, extraction of protein in SDS buffer for western
blotting, and frozen
sectioning of intact tissue blocks for immunoconfocal analysis.
Production of probes for the detection of connexin mRNAs
To obtain DNA molecular probes, we used PCR amplification of human genomic DNA
with
primers specific for connexin37, connexin40, connexin43 and connexin45
(Kilarski, 1998).
Fragments of the PCR products were cloned into pT7/T3a-18 (GIBCO-BRL). To be
used as
probe, the inserts were released from the vector using the appropriate
restriction enzymes,
purified by electrophoresis in low melting point agarose, and radiolabelled
with 32P (dCTP)
by random primer labelling (supplied as a kit by Boehringer Mannheim).
Northern blot analysis
Total cellular RNA was purified from frozen, pulverised tissues using a
modified guanidinium
isothiocyanate/acid phenol extraction procedure (Kilarski, 1998; Chomczynski
and Sacchi,
1987). Equal amounts (5 ~g/lane) of each sample were run in formaldehyde
agarose gels and
capillary-transferred onto nylon membrane (Hybond N, Amersham). High
stringency
hybridisation was done (65 °C, 5 x SSC) with a random primed probe
generated from gel-
purified human connexin45, connexin43, connexin40 and connexin37 DNA inserts
(Kilarski,
1998). All probes used had specific activities between 1.9 to 2.1 dpm/~g of
DNA and were used
at concentrations between 2.2 and 2.5 ng/ml. Quantification of northern blots
was carried out by
densitometric scanning of the autoradiograms. Multiple exposures were obtained
to ensure
linearity of the film response. To take into account possible differences in
gel loading, a
hybridisation with a 5' end radiolabelled oligonucleotide specific for 18S
ribonucleotide RNA
was performed (Mendez et al. , 1987) and the densitometric values used to
normalise the
results obtained with the specific probes for the different connexins.
Standardised comparison
CA 02281010 1999-08-27
P007306 CTH _ 17_
of the results was done by expressing the data as a percentage of the signal
obtained from a
chosen non-AF patient (Table 1, patient no 1) run in all experiments.
Antibodies
For connexin43, a commercially available mouse monoclonal antibody against
residues 252
to 270 of rat connexin43 (Chemicon, Harrow, UK) was used. For antibodies
against connexins
45 and 40, peptides corresponding to residues 354 to 367 ofhuman connexin45
and to residues
316 to 336 of human connexin40 were used as immunogens in guinea pig and
rabbit
respectively to produce antisera through a customised service (Research
Genetics Inc.). Both
antisera were affinity purified against their respective peptide. Complete,
detailed
characterisation of the anti-connexin45 (Q 14E(GP42))antibody is described in
Coppen et al.
(1998).
The new human connexin40 antibody developed for this study (designated Y21
Y(R968)) was
characterised by western blot (Figure 1 A) and by immunolabelling of ultrathin
section of
Lowicryl-embedded atrial tissues (Figure 1 B and 1 C). Western blot analysis
showed positive
labelling of a distinct connexin40 band in tissues known to express high
levels of this
connexin (atria, lung); low or no connexin40 signal was seen in tissues
expressing little or no
connexin40 (ventricles) (Vozzi et al., 1999; Hennemann et al., 1992). A
prominent 65 kDa
band was present in all heart samples but absent in lung. There was no
relationship between
the intensity of the 40 and 65 kDa bands indicating that the latter was not an
aggregated form
of connexin40. The 65 kDa band probably represents a spurious cross-reactivity
with another
protein that is not present in lung. Peptide inhibition completely abolished
labelling of both
bands. That the antibody binds specifically and with high affinity to
morphologically defined
gap junctions of human atrial myocytes was demonstrated by immunogold
labelling and
electron microscopy (Figure 1 B and C). The labelling of the unknown 65 kDa
band in western
blot experiments was not associated with any labelling of structures other
than gap junctions
in immunoelectron or immunoconfocal microscopy.
CA 02281010 1999-08-27
P007306 CTH _ 1 g_
Controls in immunological tests (western blot, immunofluorescence and
immunogold) were
i) omission of the primary antibody and ii) preincubation of the diluted
antibody with 100
p,g/ml of the relevant peptide for one hour prior to use.
Protein extraction and western blotting
For western blotting, frozen, pulverised tissue was lysed in a solution
containing 20% SDS
(101 for each mg of frozen powder) (Dupont et al., 1988). Four ~,g of total
protein per lane
was run on 12.5% SDS polyacrylamide gels, electrophoretically transferred to
PVDF
membrane (Immobilon-P, Millipore) at constant voltage (60V) overnight.
Blocking and
dilution buffer (BDB) was 1% BSA, 0.1% Tween20 in TBS (TBS: 40mM Tris pH: 7.5,
150
mM NaCI) for the mouse monoclonal anti-connexin43 and the same buffer
supplemented with
4% non fat dry milk (Marvel) for the rabbit polyclonal anti-connexin40. All
incubations were
done at room temperature. The membrane was blocked, incubated with primary
antibody,
washed in BDB, incubated with appropriate alkaline phosphatase conjugated
secondary
antibodies diluted in BDB (goat anti mouse IgG for the anti connexin43, donkey
anti rabbit
IgG for the anti connexin40; Pierce), washed in BDB and in TBS. The enzymatic
activity was
revealed using NBT and BCIP substrate solution (Promega). Quantification of
western blots
was obtained by densitometric scanning (the 65 kDa band detected by the anti-
connexin40 was
excluded from analysis). Linearity range of optical density was verified by
loading a range of
total protein amounts and scanning the resulting immunolabelled membrane. In
order to relate
connexin to the myocytic compartment (bearing in mind that different samples
will contain
variable quantities of blood proteins), the same samples (8 ~g per lane) were
run in a parallel
gel, stained with coomassie blue and densitometrically scanned. The values
obtained for
myosin were used to normalise the values obtained with the anti-connexin43 and
the anti-
connexin40. Data are expressed as a percentage of the signal obtained from the
same patient
as in northern blots (No 1 ).
Immunofluorescent labelling
CA 02281010 1999-08-27
P007306 CTH _ 19-
All incubations were performed at room temperature. Frozen sections ( 10 pm)
were fixed by
immersion in methanol and washed with PBS. Blocking was carried out with 1 %
BSA in PBS
(PBS-BSA) before incubating the sections with the anti-connexin antibody of
choice in PBS-
BSA. The sections were washed with PBS and incubated with the appropriate
secondary
antibodies diluted in PBS-BSA (CY3 conjugated donkey anti-rabbit IgG to detect
the anti-
connexin40, FITC conjugated donkey anti-mouse IgG to detect the anti-
connexin43 and CY3
conjugated goat anti-guinea-pig IgG to detect the anti-connexin45; Chemicon).
The sections
were washed with PBS and mounted with Citifluor mounting medium (Agar
Scientific).
Immunolabelled sections were examined by confocal laser scanning microscopy
using a Leica
TCS 4D system. The images recorded were taken using triple channel scanning
(CYS, CY3
and FITC fluorescence) and were transformed into projection views of optical
sections taken
at 0.5 p,m intervals.
To determine whether differences in connexin expression were detectable by
direct visual
inspection of immunofluorescent labelled specimens, coded samples (three
sections per
patient) were assessed by three investigators using conventional
epifluorescence microscopy,
and scored blind for the three connexins on a scale from 1 to 4 (1=no or
negligible labelling;
2=low to moderate labelling; 3=moderate to intense labelling; and 4~ery
intense labelling).
Each investigator sorted the samples into two groups, group 1 containing all
samples with
scores 1 and 2, and group 2 containing all samples with scores of 3 and 4. The
identities of the
samples were then decoded to determine whether distinctive patterns of
connexin expression
distinguished AF from non-AF groups.
Post-embedding immunogold thin-section transmission electron microscopy
Samples for post-embedding immunogold thin-section electron microscopy were
fixed (2%
paraformaldehyde in PBS), dehydrated in a series of ethanol, infiltrated and
embedded in
Lowicryl K4M (Agar Scientific) and polymerised with UV light in a Balzers FSU
010 low
temperature embedding unit (Yeh et al., 1998; Ko et al., 1999).
CA 02281010 1999-08-27
P007306 CTH _20-
Ultrathin sections on nickel grids were incubated at room temperature
successively in 1 % BSA
in PBS, 1% gelatin in PBS, 0.02M glycine in PBS, connexin40 antibody, PBS and
10 nm
gold-conjugated anti-rabbit antibodies (BioCell). After immunolabelling, the
sections were
washed with PBS, incubated in 1.25% glutaraldehyde, further washed with
distilled water,
dried and stained with uranyl acetate and lead citrate. All sections were
examined in either the
Philips EM301 or the Hitachi 900 electron microscope.
Statistical analysis
All analysis was done using GraphPad Prism 2.01 (GraphPad Software, Inc.).
Northern and
western data in histograms are expressed as mean ~ SEM. Data (experimental and
clinical) were
compared using unpaired Student's t test. Statistical differences were judged
significant at p ~ 0.05.
RESULTS
Patients
Details of the patients used in this study are summarised in Table 1. Of the
original forty five
patients, 22% developed symptomatic AF. Some of the samples were too small to
perform
western, northern and immunoconfocal analysis. We therefore used as many
samples as
possible in the AF group (nine samples for western, seven for northern) and
ten samples from
the control group, selected exclusively on the basis of tissue quantity.
The patients were a homogenous group with similar pathologies (ischemic heart
disease)
undergoing the same surgery (coronary artery by-pass graft). Statistical
analysis by unpaired
student t test for age, number of grafts, cross-clamp time, bypass time, pre-
and post-operative
serum potassium concentrations, P wave duration, left atrial size (assessed by
M-mode
echocardiography) and left ventricular ejection fraction (assessed by either M-
mode
echocardiography or ventriculography) did not produce significant p values
(>0.05).
Immunofluorescence analysis
CA 02281010 1999-08-27
P007306 CTH -21-
The human atrium is very rich in elastic fibres and lipofuscin which are
strongly
autofluorescent over a wide wavelength range and this impairs visualisation of
immunofluorescent signals when single channel recording is used. We therefore
used three
channel recording and combined them to generate the colour images presented.
In the images,
lipofuscin appears white (strong emission in all wavelengths), elastin green
(stronger emission
in the fluorescein channel) and CY3 red (stronger emission in the rhodamine
channel).
Connexin40 was consistently detected at the intercalated disks of atrial
myocytes and in
endothelial cells of intramural arteries (Figure 2A and 2B) (Vozzi et
al.,1999). Labelling was
heterogeneous, with large areas of myocardium displaying little staining
adjacent to other
areas that were heavily labelled (compare Figure 2A with Figure 2B, or the
right side with the
left side of Figure 2B). Visual inspection of the endothelial connexin40
labelling did not reveal
any detectable differences between or within samples. Connexin45 was present
exclusively
at myocyte intercalated disks but the fluorescence intensity was much lower
than that seen
using the anti-connexin40 or the anti-connexin43, as previously described
(Vozzi et al.,1999)
and its distribution was homogenous (Figure 2C). Conrlexin43 was also
homogeneously
present in large amounts between myocytes (Figure 2D) at fluorescence
intensities similar to
those observed for connexin40. Connexin37 was present exclusively in
endothelial cells (data
not shown).
Semi-quantitative analysis by scoring fluorescence label intensity for each
connexin type did
not reveal consistent or obvious differences between patients who developed AF
and those
who did not.
Northern blotting analysis
Figure 3 shows a typical northern analysis for connexin40, 43, 45 and 37. All
the probes for
the different connexins labelled a single mRNA band at the size expected from
previous
reports (Vozzi et al. , 1999; Kilarski et al. , 1998). Exposure times for the
gels in Figure 3 were
140 hours for connexin45 (first hybridisation), 15 hours for both connexin40
and connexin43
(second and third hybridisation, respectively) and 170 hours for connexin37
(fourth
hybridisation). From the exposure times using probes of similar sizes and
specific activities,
CA 02281010 1999-08-27
P007306 CTH _22-
connexin40 and connexin43 transcripts appear to be present in similar amounts.
Quantification
and comparison of the data from patients who developed AF with those who did
not are shown
in Figure 3B. Band intensities were normalised with the values for the 18S and
expressed as
a percentage of the value obtained for patient 1 in order to compare different
gels which all
contained this sample. Connexin40 mRNA was, on average, ~50% higher in atria
of patients
who subsequently developed post-operative AF than in those who did not
(p=0.001). On an
individual basis, by setting a threshold in the overlapping range between the
two groups, it
would have been possible to identify >75% of the patients prone to AF on the
basis of
connexin40 transcript content. The amounts of the other connexins were not
significantly
different between the groups (p>0.05).
Western blot analysis
Connexin40 and connexin43 were detected as single bands at ~40 and ~43 kDa
respectively
(Figure 4). Similar loading of myocytic protein was not feasible because all
the samples still
contained an unknown but large amount of blood and the actin/myosin ratio was
not constant.
Therefore, to normalise the western blot values, we used the values for myosin
(since actin is
a major component of the cytoskeleton in other cell types). As shown in Figure
4B,
connexin40 protein signal was significantly higher in the AF prone group than
in the control
group (p=0.022), whereas connexin43 was expressed at similar levels in both
groups. As with
the northern analysis, setting a threshold in the overlapping point between
the groups would
have led to detection of >75% of the patients prone to AF.
The correspondence of the results obtained by northern and western analysis
was examined
by plotting the individual mRNA values against the corresponding protein
values, followed
by linear regression analysis. For connexin40 this gave r2=0.5291, p=0.0009;
for connexin43,
rz=0.26 and p=0.0364, indicating that the amounts of both connexins are
closely related to
their corresponding transcript steady state level.
DISCUSSION
CA 02281010 1999-08-27
P007306 CTH -23-
This study demonstrates that one of the three connexins of atrial myocytes,
connexin40, is
expressed at significantly higher levels in patients who develop post-
operative AF than those
who do not. As connexin40 is expressed both in atrial myocytes and endothelial
cells, this
difference could theoretically be due to differential expression in either or
both cell types.
However, as the volume of endothelium is small compared with that of myocytes,
and
transcript for connexin37 (exclusively expressed in endothelium) does not
differ between the
groups, the difference in connexin40 is attributable largely if not
exclusively to myocytes
rather than endothelial cells.
The higher levels of connexin40 transcript found in AF patients is mirrored by
higher levels
of connexin40 protein demonstrated by quantitative western blotting, and hence
has the
potential to result in functional differences in conduction. Immunolabelling
revealed a
heterogeneity of connexin40 protein distribution, suggesting that spatially
adjacent regions of
atrial myocardium may have markedly different resistive properties and
conduction velocities.
These features could combine to enhance susceptibility to development of re-
entrant circuits
as the higher the overall level of connexin40, the more extreme would be the
differences
between the spatially adjacent regions. Our findings thus implicate a new
connexin-based
mechanism, in addition to the loss of side-to-side gap junctions associated
with increased
interstitial fibrosis, in the generation of atrial re-entrant circuits. The
linear regression results
suggest that connexin40 levels in the heart are regulated largely by the
transcript steady state
level, as previously reported for connexin43 (Vozzi et al., 1999). The finding
that different
connexin40 levels occur while connexin43 remains constant suggests that,
where, as in atria
and conductive tissues, the two connexins are co-expressed, they are regulated
independently.
If distinct "factors" or regulatory pathways that alter expression of each
connexin can be
identified, then the possibility exists for development of specific
therapeutic intervention.
The discovery of significantly higher levels of connexin40 in patients
susceptible to post-
operative AF raises the possibility that this difference could be exploited to
identify this group
of patients before symptoms develop. Although microscopical scoring of
immunofluorescently
stained specimens did not reliably discriminate between AF and non-AF groups
owing to the
heterogeneous pattern of connexin40 distribution, our data show that
measurement of overall
mRNA and protein quantities would have predicted AF with a positive and
negative rate of
CA 02281010 1999-08-27
P007306 CTH _24-
at least 75%, even with the small number of samples used in this study. By
comparison, other
clinical means of prediction based on electrocardiography (e.g. signal
averaged P wave)
involve continuous post-operative monitoring and have a lower positive
predictive value of
34%. Similarly, intraoperative induction of AF has a positive prediction rate
of ~50% but
involves longer operation time with possibly detrimental effects on patient
health.
How might the difference in connexin40 be used to develop a diagnostic test?
Both northern
and western blotting techniques, although giving accurate quantification of
mRNA and protein
respectively, are not well suited for this purpose because of the multiple,
time consuming steps
involved. To be practicable in a clinical situation, a predictive test would
need to be relatively
fast (a few hours), accurate and easy to set up in clinical laboratory, with
minimal requirement
for additional equipment. Two techniques potentially fulfil these
requirements: for mRNA,
quantitative RT-PCR using a simple RNA extraction method from a small sample
collected
at the time of surgery, and for protein, quantitative ELISA using similar
samples to relate
connexin40 and myosin expression, using our new specific human anti-connexin40
antibody.
Both these techniques are routine in many clinical laboratories and can be
performed within
in a few hours. A test based on these approaches could offer the possibility
of identifying
patients susceptible to post-operative AF before it actually occurs, and hence
the opportunity
for early initiation of preventative therapies.
In summary, we have identified a clear-cut difference in a key protein
responsible for cardiac
conduction properties that could offer potential in development of a
diagnostic tool to
detecting susceptibility to AF, and which could provide a potential target for
therapeutic
intervention.
All publications mentioned in the above specification are herein incorporated
by reference.
Various modifications and variations of the described methods and system of
the invention
will be apparent to those skilled in the art without departing from the scope
and spirit of the
invention. Although the invention has been described in connection with
specific preferred
embodiments, it should be understood that the invention as claimed should not
be unduly
limited to such specific embodiments. Indeed, various modifications of the
described modes
CA 02281010 1999-08-27
P007306 CTH _25-
for carrying out the invention which are obvious to those skilled in molecular
biology or
related fields are intended to be within the scope of the following claims.
CA 02281010 1999-08-27
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