Electrophysiological characteristics of individual atrioventricular nodal myocytes in the rat

Kathryn Yuill, Kim Dora, Christopher Garland
University of Bath, Bath, Somerset, United Kingdom

The atrioventricular node (AVN) is the primary conduction pathway from the atria to the ventricles, and is essential for normal cardiac function. This region displays some interesting and unique properties, acting to slow conduction from atria to ventricles, thus timing ventricular filling. The AVN has therefore long been a target for pharmacological intervention, and is of primary importance in a variety of cardiac arrhythmias and conduction abnormalities. The elucidation of AVN function has been primarily derived from single cell studies in rabbit, and more recently guinea-pig (Yuill & Hancox 2002) and mouse (Marger et al., 2007). The present study explores the feasibility of characterising individual myocytes isolated from the rat AVN as a means to further explore the cellular electrophysiology of this region, and help determine whether the cellular properties are preserved across species.

Adult male Wistar rats (200-250g) were killed humanely by cervical dislocation. Hearts were rapidly removed and retrogradely perfused with an enzymatic solution (containing protease, collagenase and BSA). The AVN region was excised and cells isolated using a combination of mechanical and enzymatic dispersion (Hancox et al., 1993; Yuill & Hancox 2002). All recordings were carried out at 37°C, using the perforated patch mode of the voltage clamp technique, and standard physiological K+-based solutions. Electrophysiological recordings were only conducted on healthy AVN myocytes exhibiting regular spontaneous activity. Cells were stored before experimentation at 4°C, in a K+ rich, low-Ca2+ solution. Individual AVN myocytes displayed a spindle-type morphology, and the following dimensions, length = 87.5 ± 2.7μm and width = 8.72 ± 0.45μm (n=54). The mean cell capacitance measured was 31.1 ± 1.8pF (n=23).

AVN function is modulated largely by autonomic transmitters. Here, the responses to acetylcholine and isoprenaline were used as a measure of cell viability, demonstrating that muscarinic and beta receptor function was retained, post enzymatic dispersion. Bath application of 1μM acetylcholine induced a concomitant cessation of spontaneous action potential activity, coupled to hyperpolarisation, which was readily reversible on washout. Conversely, an increase in firing rate was observed upon external application of 1μM isoprenaline. Recordings of membrane current suggest subtle differences compared to previous findings in guinea-pig and rabbit AVN myocytes. L-type calcium current was present in all cells, with a current density similar to previous findings in rabbit AVN myocytes (Hancox & Levi 1996). However, transient outward K+ current was present, in contrast to findings in guinea-pig AVN (Yuill & Hancox 2002). The time-dependent If, and IK were also present, and an INa was observed in a small proportion of cells. The present study indicates that these cells can be isolated in a viable state. As such they will enable direct comparisons of AVN function in health and disease, as many disease models are widely available in the rat.

Hancox et al., (1993) Am. J. Physiol. 265: H755-766; Hancox & Levi (1996) Br J. Pharmacol. 118 6: 1447-54; Marger et al (2007) Proc. Physiol. Soc. (University of Manchester) PC34; Yuill & Hancox (2002) Pflügers Arch. 445 3:311-20.

Supported by the British Heart Foundation