AU4485499A - Diagnostic method - Google Patents

Description

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AUSTRALIA

Patents Act 1990 Imperial College of Science, Technology Medicine

ORIGINAL

COMPLETE SPECIFICATION STANDARD PATENT Invention Title: Diagnostic method The following statement is a full description of this invention including the best method of performing it known to us:- 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 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 P-blockers) would involve up to 80% of patients being unnecessarily exposed to drug therapy, a means to o predict post-operative AF is considered highly desirable. Apart from intraoperative induction ofAF, 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 postoperatively 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) 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 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-junctional channels with distinctive properties unit channel conductances, permeabilities and sensitivity to transjunctional voltage) (Gros and 15 Jongsma, 1996; Veenstra, 1996; Jongsma, 1997).

Summary of the Invention \We hypothesised that distinctive patterns of connexin expression contribute to oo 20 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).

Typically the determination of the levels of connexin40 comprises measuring the levels of 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 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.

15 The present invention further provides a substance capable of down regulating .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 postoperative atrial fibrillation in a patient which method comprises administering to said patient an effective amount of a substance capable of down regulating expression or inhibiting connexin40 function.

Typically, the compound may have been identified by the assay methods of the invention.

Brief Description of the Figures 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 (A and for connexin45 and for connexin43 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.

Figure 4. A, Typical western blot analysis of heart protein homogenate for (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 Ih 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 el al., 1999), and because this part of the right atrium is always available during surgery and it s 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 cellular 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).

Northern blotting of purified total cellular RNA may be performed using standard techniques known in the art. Typically, hybridisation is performed at high stringency 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 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) p 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 mRNA levels. Results are typically calculated as a percentage of the results obtained for the positive control.

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 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 A preferred antibody is antibody Y21Y(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.

-7- Samples may also be tested using standard ELISA protocols. Samples may conveniently be tested in multi-well plate formats. ELISA techniques are well known in the art.

Whiichever 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, binding assay) encompassing the Y2lIY peptide (see Examples) can be used as a positive control in a range of concentrations which give densitometric values found in post-operative AF resistant patients. For ELISA, a positive control can be constituted either by the Y2 1Y 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 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 fuinction Our results demonstrate a link between pre-operative elevated levels of 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 expression or affects connexin40 function in cardiac tissue. An example of such a substance would be an antisense eonnexin40 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.

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 I.D.

Nos. 1 and 2 of W098/02150 and homologues thereof having an analogous biological function 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 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 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 -9batches 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 as cardiac myocytes which will be exposed to the inhibitor and tested for inhibition of connexin40 function and/or expression.

binding assays One type of assay for identifying substances that bind to connexin40, possibly interfering with its function, synthesis, trafficking and/or 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-agarose 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 nonimmobilised.

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 histidinetagged 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 enzymeantibody 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.

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 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 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 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 -11intercellular communication is genetically modified (by transfection) to express only 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 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 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 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.

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.

-12- 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 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 connexin4O expression to treat the resulting AF or prevent its occurrence. Such substances may include antisense 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.

-13- 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 phosphatebuffered 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 0.01 to 30 mg/kg body weight, preferably from 0.1 to 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 I naked nucleic acid construct. The polynucleotides/vectors may further comprise flanking S"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 pg to 10 mg, preferably from 100 4g 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 lipofectam M and transfectam). 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- -14buffered 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.

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 The antibodies recognise a 40 kDa bands in atrial tissues, some ventricular tissues and lung (lanes RA1, LA1, LV2 and Lung in panel but not in most ventricular tissues (known to lack connexin40 in working myocardium; lane RV1 and LV1). A spurious band at 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 (A and for connexin45 and for connexin43 Connexin staining is seen as red fluorescence, autofluorescence of elastic fibres as green, autofluorescence of lipofuscin granules as white-purple or white-red 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 (0 in A and Distribution is somewhat heterogeneous, as seen in 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 the much higher power of the laser setting necessary to record signal thus results in much brighter intensity of lipofuscin fluorescence in C compared to A, B and D. Connexin43 signal is prominent but, in contrast to connexin40, is absent from arteries (indicated by 10., 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 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 0" 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 (upper panel), connexin43 (middle panel) and coomassie blue staining of these samples *B (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 anticonnexin43 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 preoperative ECG. Forty five samples of the right atrial appendage matching these criteria were collected 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.

S. 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, 0 1998). Fragments of the PCR products were cloned into pT7/T3at-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 32 P (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 pg/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/ig 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 of the results was done by expressing the data as a percentage of the signal obtained from a chosen non-AF 6 6 patient (Table 1, patient no 1) run in all experiments.

6* *666 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 of human 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.

S

Complete, detailed characterisation of the anti-connexin45 (Q14E(GP42))antibody is described in Coppen et al. (1998).

The new human connexin40 antibody developed for this study (designated Y21Y(R968)) was characterised by western blot (FigurelA) and by immunolabelling of ultrathin section of Lowicryl-embedded atrial tissues (Figure 1B and 1C). 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 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 -18aggregated form of connexin40. The 65 kDa band probably represents a spurious crossreactivity 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 1B and 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.

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 .tg/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 for each mg of frozen powder) (Dupont et al., 1988). Four Lg 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: 150 mM NaC1) for the mouse monoclonal anti-connexin43 and the same buffer supplemented with 4% non fat dry milk (Marvel) for the rabbit polyclonal 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 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 pg 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 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 15 anti-connexin43 and CY3 conjugated goat anti-guinea-pig IgG to detect the anticonnexin45; 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 (CY5, CY3 and FITC fluorescence) and were transformed 20 into projection views of optical sections taken at 0.5 pm 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=very 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).

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 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.

-21- 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 Mmode echocardiography or ventriculography) did not produce significant p values Immunofluorescence analysis 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 15 (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.

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). Connexin43 was also homogeneously present in large amounts between myocytes (Figure 2D) at fluorescence intensities similar to those observed for 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, 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, 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).

20 Western blot analysis o and connexin43 were detected as single bands at ~40 and -43 kDa respectively (Figure 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=0.0 2 2 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.

-23- 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 r 2 =0.5291, p=0.0009; for connexin43, r 2 =0.26 and p=0.0364, indicating that the amounts of both connexins are closely related to their corresponding transcript steady state level.

DISCUSSION

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 reentrant 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 -24expression of each connexin can be identified, then the possibility exists for development of specific therapeutic intervention.

The discovery of significantly higher levels of connexin4O in patients susceptible to postoperative 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 connexin.40 distribution, our data show that measurement of overall mRNA and protein quantities would have predicted AF with a positive and negative rate of at least 75%, even with the small number of samples used in this study. By comparison, other clinical means of prediction based on electrocardiography 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 15 detrimental effects on patient health.

How might the difference in conmexin40 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 postoperative 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 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.

*0 .00 0 000 S 0@ 00 0 0000 00 0000 0** 0 0 000 0 0 0 000 0 000 0 OS 00 0 00 @0 0 0 0 000 0 00 0 0 0 0 00 S Table 1: Patient details n A Q ICAA ThIM OI arii e ex ~virv code Grafti lamIp ture By-pa~ss time LN J min min mEq/1 I' LA Size LVEF Post-op AF wave sec mmn Y/N Pre-op Post-op M H-T,MI M H-TMI M HT, MI M MJ, HC M HT M HiC M NIDDM M MI, HC F HT M MI, NIDDM M HT F HT M MI, HC

M

M

M MI M MI, HC M HT, MI F MI 3.7 4.6 0.1 4.3 4.4 0.04 4 4.8 0.08 4.4 4.2 0.1 4.3 4.5 0.04 4.7 4.1 0.1 4.4 5.8 0.08 4.7 4.8 0.08 3.5 4.4 0.04 4.2 5 0.08 4.2 5.2 0.1 4.4 4.6 0.08 4 4.3 0.08 4.3 4.5 0.1 3.7 4.7 0.06 4.3 3.8 0.08 4.1 4.7 0.1 3.9 4 0.12 4.7 4.4 0.08 39 N/A 30-49 27.5. 30-49 27.6. 30.1 30-49 N/A >50 36 >50 32.7 30-49 31.4 >50 38 30-49 38.5 <30 27.8 >50 N/A <30 28 30-49 30.5 >50 N/A 30-49 28.8 30-49 36.3 >50 N/A 30-49 PMH: previous medical history, HT: hypertension MI: myocardial infarction HC: hypercholesteralemia, NIDDM: non-insulin dependant diabetis meltus LA: left atrium LVEF: left ventricular ejection fraction CP: cardioplegia, N/A: not available.

References Bruzzone R, White TW, Paul DL: Connections with connexins: The molecular basis of direct intercellular signaling. Eur. J Biochem. 1996;238:1-27 Chomczynski P, Sacchi N: Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 1987; 162:156-159 Claycomb WC, Lanson NA Jr, Stallworth BS, Egeland DB, Delcarpio JB, Bahinski A, Izzo NJ Jr: HL-1 cells: a cardiac muscle cell line that contracts and retains phenotypic characteristics of the adult cardiomyocyte. Proc Natl Acad Sci USA 1998; Mar 17;95(6): 2979-84 Coppen SR, Dupont E, Rothery S, Severs NJ: Connexin45 expression is preferentially associated with the ventricular conduction system in mouse and rat heart. Circ. Res.

1998;82:232-243 Dupont E, El Aoumari A, Roustiau-Severe S, Briand JP, Gros D: Immunological 15 characterization of rat cardiac gap junctions: presence of common antigenic determinants in heart of other vertebrate species and in various organs. J Membr.

Biol. 1988;104:119-128 Gros DB, Jongsma HJ: Connexins in mammalian heart function. BioEssays 1996;18:719- 730 Fishman G, Spray D, Leinwand L. Molecular characterization and functional expression of the human cardiac gap junction channel. J. Cell Biol., 1990; 111:589-598.

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1992;117:1299-1310 Jongsma HJ: Gap junction channels and cardiac conduction, in Spooner PM, Joyner RW, Jalife J (eds): Discontinuous conduction in the heart. New York, Futura Publishing Company, Inc., 1997, pp 53-66 Kanter HL, Saffitz JE, Beyer EC: Molecular cloning of two human cardiac gap junction proteins, connexin40 and connexin45. J Mol. Cell. Cardiol.1994 Jul;26(7):861-8 Kilarski WM, Dupont E, Coppen SR, Yeh H-I, Vozzi C, Gourdie RG, Rezapour M, Ulmsten U, Roomans GM, Severs NJ: Identification of two further gap-junctional -28proteins, connexin40 and connexin45, in human myometrial smooth muscle cells at term. Eur. J. Cell Biol. 1998;75:1-8 Ko Y-S, Yeh H-I, Rothery S, Dupont E, Coppen SR, Severs NJ: Connexin make-up of endothelial gap junctions in the rat pulmonary artery as revealed by immunoconfocal microscopy and triple-label immunogold electron microscopy. J. Histochem.

Cytochem. 1999;47:683-692 Mendez RE, Pfeffer JM, Ortola FV, Bloch KD, Anderson S, Seidman JG, Brenner BM: Atrial natriuretic peptide transcription, storage and release in rats with myocardial infarction. Am.JPhysiol. 1987;253:H1449-H1455 Rohr S, Kucera JP, Fast VG, Kleber AG: Paradoxical improvement of impulse conduction in cardiac tissue by partial cellular uncoupling. Science 1997;275:841-844 Severs NJ: Cardiovascular disease, in Cardew G Gap Junction-Mediated Intercellular Signalling in Health and Disease. New York, John Wiley Sons Ltd., 1999, pp 188- 206 Shaw RM, Rudy Y: Ionic mechanisms of propagation in cardiac tissue Roles of the sodium and L-type calcium currents during reduced excitability and decreased gap junction coupling. Circ. Res. 1997;81:727-741 Spach MS, Starmer CF: Altering the topology of gap junctions a major therapeutic target for atrial fibrillation. Cardiovasc. Res. 1995;30:337-344 Veenstra RD: Size and selectivity of gap junction channels formed from different connexins. J Bioenerg. Biomembr. 1996;28:327-337 Vozzi C, Dupont E, Coppen SR, Yeh H-I, Severs NJ: Chamber-related differences in connexin expression in the human heart. J. Mol. Cell. Cardiol. 1999;31:991-1003 Yeh H-I, Dupont E, Rothery S, Coppen SR, Severs NJ: Individual gap junction plaques contain multiple connexins in arterial endothelium. Circ. Res. 1998;83:1248-1263

Claims (8)

1. A method for determining the susceptibility of a patient to 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 is indicative of a susceptibility to atrial fibrillation (AF).

2. The method of claim 1 wherein the determination of the levels of comprises measuring the levels of connexin40 mRNA.

3. The method of claim 1 wherein the determination of the levels of comprises measuring the levels of connexin40 protein.

4. A method for identifying a substance capable of down regulating expression which method comprises contacting a cell which expresses connexin40 with a candidate substance and determining whether connexin40 expression is reduced.

5. 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.

6. A substance capable of down regulating connexin40 expression or inhibiting function identified by the method of claim 4 or claim

7. 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.

8. The method of claim 7 wherein said compound has been identified by the method of claim 4 or claim DATED THIS 30 DAY OF AUGUST 1999 IMPERIAL COLLEGE OF SCIENCE, TECHNOLOGY AND MEDICINE Patent Attorneys for the Applicant:- F.B.RICE CO