GENERAL METHODOLOGY AND INITIAL STUDIES

The principles and methods o f acquiring and processing the myocardial specimens investigated were common to all o f this series o f studies, and are detailed in this chapter.

2.1 Patient selection

Patients for inclusion were selected from those satisfying the study criteria (specified in the relevant chapters) during the period o f tissue collection for each study, on the operating lists o f the surgeons who had agreed to take part; Graeme Bennett, John Pepper, Asghar Khagani and Sir Magdi Yacoub for the adult and transplant tissue, and Christopher Lincoln, Daryl Shore and Sir Magdi Yacoub for paediatric tissue. There was no interference with, or modification of, patient preparation for surgery, anaesthesia or operative procedures, which were carried out according to routine surgical practice. When the planned surgical procedure itself required resection or biopsy o f tissue appropriate for these studies, this tissue was handed over for processing as soon as it was excised. The specimens from cardiac transplantation hearts fell into this category. Approval o f the Ethics Committee o f the Royal Brompton National Heart and Lung Hospital was obtained to biopsy patients specifically for the studies undertaken, either by scalpel excision o f up to three small ventricular samples, or by use o f a needle bioptome (Trucut) to obtain transmural samples. Potentially appropriate patients from this category were seen preoperatively, and the details o f the study were explained to them both verbally and in writing. All questions were answered and if patients had any remaining concerns about the work they were advised not to give consent. When consent was obtained, if the operative findings were as expected, and at the surgeon’s discretion, tissue for study was resected at the earliest opportunity after establishment o f routine cardiopulmonary bypass and hypothermic cardiac arrest.

2.2 Acquisition of tissue

To maximise tissue preservation, all biopsies required immediate fixation on removal, and the specimen was therefore divided as required in the operating theatre, keeping note o f tissue orientation to enable matching o f areas analysed by the different microscopical techniques used. Perfusion fixation o f fresh human specimens is obviously not possible.

and the methods o f immersion fixation o f whole myocardium used in this series o f experiments were based on standard published techniques (Gourdie et al. 1991), some o f which had been assessed by other members o f the laboratory. Modifications o f some o f these techniques, particularly for application to very small tissue samples and isolated myocytes, were developed in the course o f the series o f studies included in this thesis, as were the optimal conditions for preservation o f specimens for electron microscopic analysis (see below - section 2.5).

2.3 Tissue preparation for immunohistochemistry

Tissue was put in Zamboni’s fixative (2% paraformaldehyde, 0.2% picric acid, O.IM phosphate-buffered saline, pH 7.4) (Toshimori et al. 1987) for 2-6 hours. After fixation, all samples were washed in tap water, dehydrated in alcohol, placed in chloroform and embedded in wax following standard histological procedures (Gourdie et al. 1991).

2.3.1 Antibodies

The primary antiserum used to localise cardiac gap-junctional connexin43 was raised (by N J Severs and C R Green) against a synthetic peptide matching residues 131-142 o f the cytoplasmiccilly-exposed segment o f the connexin43 molecule (Gourdie et al. 1990b; Harfst et al. 1990). The peptide, supplied by N. B. Gilula (Research Institute o f the Scripps Clinic, La Jolla, California) was prepared using the simultaneous multiple peptide synthesis procedure o f Houghten (1985), and was coupled using glutaraldehyde to the carrier protein, keyhole limpet haemocyanin, for subcutaneous injection into Sandy half­ lop rabbits. A summary o f the derivation o f this antibody (designated "HJ") is illustrated in Figure 2 .1 , and full details o f the production and characterisation o f the polyclonal antiserum are reported by other members o f the laboratory (Gourdie et al. 1990b; Harfst et al. 1990; Green et al. 1993; Severs et al. 1993).

2 .3 .2 Immunolocalisation

Ten-micrometer sections o f wax-embedded tissue were de-waxed and rehydrated, and incubated in a trypsin solution (containing 0.1% trypsin (Sigma T-8128), 0.1% CaCl2,

20mM Trizma base, pH 7.4) for 10 minutes at room temperature to re-expose antigenic sites altered by fixation and processing (Harlow and Lane, 1988). The sections were

5 6

B

CONNEXON - 6 C o n n e x in s

CONNEXIN - 4 3 KD

“COOH

H J = 01 u-l l#-Ly#-Ly #-P h#-Ly#-Tyr-Q ly-l l#-Q I u-GI u-HI #-(Cy #)

Rabbit Antl - HJ Antibody

Figure 2.1 . Summary o f the derivation o f the "HJ" antibody used for gap- junctional immunolocalisation. The antibody was raised in rabbits (C) to a 12-

residue segment (designated "HJ", with the sequence shown) o f the cytoplasmic loop o f the connexin43 molecule (B) o f the mammalian cardiac gap junction (A).

washed and treated with 0.1 M L-lysine (as blocking agent) in phosphate-buffered saline (PBS) containing 0.1 % Triton X-100 (as a wetting agent). Incubation with the primary antiserum (dilution 1:10 in PBS) was carried out for 1 hour at 37°C. After washing, secondary antibody treatment with swine anti-rabbit fluoroscein isothiocyanate (FITC: Dako, 1:20 dilution) was given for 1 hour in the dark at room temperature. After final washing in PBS, the slides were mounted using Citifluor mounting medium (Agar Scientific, Stansted, England). Under the conditions described, the immunolabelling procedure produces even and consistent staining through the depth of the sections. All immunolabelled tissue was stored at 4°C and when used for quantitative analysis, it was examined within 12 hours to avoid complications arising from significant fading of

fluorescence.

Immunolabelled sections were examined by conventional epifluorescence and confocal laser scanning microscopy. Phase contrast microscopy was used to obtain detail o f tissue structure, and adjacent sections, stained with haematoxylin and eosin, were routinely examined by standard light microscopy. Control specimens in which the antiserum was substituted by preimmune rabbit serum or buffered saline, or the second antibody by buffered saline, were routinely run in parallel in all experiments in this series o f studies. The specificity o f the antiserum for the peptide, for isolated cardiac gap- junctional protein and for ultrastructurally-deflned gap junctions was demonstrated as part

o f the characterisation o f the antiserum (Harfst et al. 1990; Severs et al. 1993).

2.4 Confocal laser scanning microscopy

For confocal microscopy, immunolabelled sections were examined using a Bio-Rad Lasersharp MRC-5(X), running on standard Bio-Rad software, with the digitised images stored on optical computer disks.

The principles behind confocal microscopy are illustrated diagrammatically in Figure 2.2. With a small aperture setting, the image acquired has a very narrow depth o f field, and can be considered as a single optical slice o f data with minimal thickness, at a particular depth with respect to the z-axis. The intensity o f scanning laser stimulation o f a specimen labelled cleanly with a high affinity antibody is such that optical slices can be acquired through a considerable depth o f tissue, without interference from signal or tissue outside the focal plane (Gourdie et al. 1990b). The motor-driven z-axis movement o f the specimen stage therefore permits a number o f optical slices to be obtained, through a known depth (ie. volume) o f tissue, and the Bio-Rad software enables an optical-section series so derived to be processed in a number o f ways. These include superimposition to create a single ("projection") image containing data from the entire volume o f tissue all in focus, creation o f a stereo pair o f images, and rotation o f the reconstituted volume o f data.

For the work conducted in the studies comprising this thesis, the microscope was used with the blue high-sensitivity filter block inserted for optimal excitation o f fluoroscein at its absorption peak wavelength o f 488 nm and detection o f the emission in the peak 520 nm range. A series o f objective lenses (xlO, x20, x60 oil) were used which, in conjunction with a series o f software zoom adjustments, permitted a range o f field sizes from 1080 fim x 720 ^m (xlO, zoom 1) to 45 /xm x 30 /xm (x60, zoom 4).

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