PRUEBA DE HIPÓTESIS

IN GUINEA PIGS.

10.1 Introduction

Ventricular hypertrophy is associated with alterations in myocardial electrophysiology and mechanical function that cannot be explained entirely by changes in the action potential or histology o f the myocardium. Despite the abundant literature concerning the importance o f ventricular arrhythmias in patients with cardiac hypertrophy and failure, there is a lack o f consensus as to the nature and relevance o f the basic electrophysiology underlying these pathological conditions. A number o f studies over the last 20 years have reported a variety o f discrepant cellular electrophysiological measurements made on hypertrophied myocytes and whole tissue, and although reduction o f the upstroke velocity and prolongation o f the plateau phase o f the action potential are now generally accepted alterations in hypertrophy (Bassett and Gelband, 1973; Tritthart et al. 1975; Kowey et al. 1991; Pye and Cobbe, 1992), there is no direct evidence linking these changes to altered myocardial function or arrhythmogenesis, particularly in the early stages o f the hypertrophic response (Bassett and Gelband, 1973; Tritthart et al. 1975; Kowey et al.

1991; Pye and Cobbe, 1992). Cardiac failure and hypertrophy are associated with abnormalities o f ventricular wall motion, and studies o f the underlying contractile disturbance have also yielded conflicting results (Gulch, 1980; TenEick et al. 1989; Harding et al. 1991). Although the use o f different models o f hypertrophy and a lack o f standardisation, particularly o f the loading conditions o f the tissue, are partly to blame for inconsistent results (TenEick et al. 1989; Kowey et al. 1991), it is now known that there are rapid changes in the physiology o f cardiac myocytes in early hypertrophy (Bassett and Gelband, 1973; Tritthart et al. 1975; Swynghedauw, 1991), which may make the timing o f study critical. One factor that is relevant to both the coordination o f contraction and the arrhythmic tendency o f myocardium is conduction velocity, o f which gap-junctional cellular coupling is, as discussed earlier, the major determinant. However, few studies have investigated either conduction velocity or cellular coupling in hypertrophied or failing hearts. With the alterations o f myocardial architecture and the consequent changes in cellular contacts that must occur with myocyte enlargement and

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either elongation or so-called slippage o f myocytes in the failing heart (Gerdes et al. 1992), changes in cellular coupling would be expected to occur, even in the absence o f interstitial fibrosis that may contribute further to alterations o f myocyte interaction in longstanding hypertrophy. Although myocardial conduction velocity has been reported as slowing in hypertrophy, which would be consistent with, but not necessarily entirely attributable to, the changes in the myocyte action potential, conduction velocity in whole myocardium that has these action potential changes has been reported as being paradoxically increased during the active phase o f hypertrophy (Tritthart et al. 1975). This increase in conduction velocity, which may only occur transiently in the hypertrophic process, cannot be accounted for by the increasing myocyte diameter alone (Tritthart et al. 1975), and indicates that alterations in cellular coupling may indeed occur, and may be variable, in early hypertrophy.

The purpose o f this study was to investigate alterations o f gap-junctional organisation in left ventricular myocardium in response to renovascular hypertension. The guinea pig was considered an appropriate model to investigate this since the active membrane-channel properties in guinea pigs are more similar to those in man than are those o f other laboratory small mammals, with similar alterations o f the action potential under pathophysiological conditions. The cellular coupling o f guinea pig ventricular tissue, known to have the typical mammalian gap-junctional distribution (Gourdie et al. 1991), might also be expected to parallel alterations in man in response to induced hypertension.

10.2 M aterials and Methods

10.2.1 Renal artery clipping

Hypertension was induced by a modified Goldblatt 1-clip 2-kidney model (Smith et al. 1988). Male Dunkin-Hartley guinea pigs weighing between 2(X) and 3(X)g were anaesthetised using 6.5mg/kg intraperitoneal Hypnovel (midazolam, Roche) and 2.4mg/kg intramuscular Hypnorm (Janssen). Under sterile conditions. Dr K T MacLeod performed surgery in which the left renal artery was located via a dorsal incision, and a clip shaped from 0.1mm thick silver foil was placed around it and tightened to constrict, but not occlude, the lumen. The animal was allowed to recover and kept for a period o f 3 weeks, feeding ad libitum, before sacrifice for study. Weight-matched guinea pigs had

sham surgery, with exposure o f the renal artery but no clip placement.

10.2.2 Blood pressure measurement

Immediately before sacrifice, the animal was anaesthetised as above, and the arterial blood pressure measured by carotid artery cannulation.

10.2.3 Myocyte isolation and immunolabelling

Immediately after sacrifice, the heart was removed and a small deep subepicardial left ventricular wall sample was put in Zamboni’s solution. The heart was then mounted and perfused as detailed in Chapter 2, by the Langendorff method, and treated to isolate the myocytes o f the remaining left ventricle.

After fixation in Zamboni’s solution, the left ventricular whole-tissue specimens, and the isolated myocyte suspensions were processed for connexin43-immunolabelling using the standard procedures detailed in Chapter 2, and the labelled whole tissue was examined in the confocal microscope to determine the pattern o f connexin43 gap- junctional distribution.

10.2.4 Quantitative analysis o f the isolated myocytes

Immunolabelled cells were examined by conventional epifluorescence and confocal laser scanning microscopy. Phase contrast microscopy was used to confirm intact, regular sarcomeric striation o f the myocytes.

From each preparation, 6 myocytes that had retained a rod-like shape were randomly selected from under the confocal microscope for quantitative analysis. Planimetric measurement o f the volume o f the myocyte and the total surface area o f connexin43 gap-junctional present were determined by the following method. An optical section series was taken at 1.5 fim intervals in the vertical (z) axis through the entire volume o f the myocyte, by the same method as detailed in section 6.3.2 o f Chapter 6. The aperture control on the microscope was withdrawn by 1.5 mm, producing a degree o f confocality that provided minimal overlap between adjacent sections. A "black level" o f 4 and a gain level was set so that the cell cross sectional outline was just visible and the image data spanned the 255-point grey scale. Each image o f each series was binarised, processed and analysed by the same PC IMAGE program as described in

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Chapter 6 to determine the total cross-sectional area o f the cell. An additional analysis