A method for isolating rabbit atrioventricular node myocytes which retain normal morphology and function

Abstract

This report describes a method for isolating single rabbit atrioventricular (AV) node myocytes which retain their normal morphology when exposed to millimolar levels of calcium. Previous attempts to isolate cells from the AV node have produced myocytes that "round up" (i.e., go into contracture) when exposed to calcium. We show that the cells isolated with our technique possess properties similar to those described for intact AV nodal tissue. We find that single AV node myocytes are shorter and thinner (mean dimension = 103.5 +/- 2.3 by 7.8 +/- 0.2 microns; mean +/- SE, n = 90) than atrial or ventricular cells. Many of the cells produced by this isolation procedure generate spontaneous action potentials (188 +/- 9 beats/min; n = 6), which resemble action potentials recorded previously from the intact AV node. Voltage-clamp recordings from spontaneously active cells revealed similar membrane currents to those seen in intact tissue: fast sodium current and a L-type calcium current, followed by a delayed outward current. However, we found little evidence for the hyperpolarization-activated current (I(f)). Because the cells responded normally to concentrations of acetylcholine and isoproterenol within the physiological range, their cholinergic and adrenergic receptors appear to be well preserved by the isolation procedure. The ability to isolate morphologically and functionally normal AV myocytes may represent a significant advance for the investigation of nodal physiology at the cellular level.

Isolation and Characterization of Atrioventricular Nodal Cells From Neonate Rabbit Heart

“…Salient electrophysiological properties such as the action potential shape and ionic channel composition appear to vary with these different cellular morphologies. 17,21,25 Compared with the adult, neonatal AVNCs were observed to be relatively homogeneous in the phenotypic features, and only spindle and rod morphologies were found. Although spontaneous pacemaker activity was observed more frequently in spindled cells, both cell shapes had similar action potential configurations and, I f , I Ca , and I Na current densities (data not shown).…”

“…17 The extension of these techniques to the investigation of AV conducting mechanisms found in neonates has so far been extremely limited, due primarily to the technical difficulties associated with isolation and characterization of cells from this nodal area in the immature heart. For the present study, we developed an AVNC isolation technique for the 10-day-old (10-day) rabbit heart, which consistently produced a high-yield of spontaneously beating AVNCs.…”

“…17 Enzyme concentrations and perfusion rates were refined to ensure viable, Ca 2ϩ -tolerant AVNCs and SA nodal cells (SANCs) from the neonate heart. The complete procedure is described in online-only Data Supplement Table S2 and related material.…”

Novel Molecular Mechanism Involving α 1D (Cav1.3) L-Type Calcium Channel in Autoimmune-Associated Sinus Bradycardia

“…Rabbit SA and AV node tissues were prepared according to Denyer et al 17 and Hancox et al, 18 respectively. All animal protocols were approved by the Institutional Animal Care and Use Committee of VA New York Harbor Healthcare System.…”

Rate-dependency of Action Potential Duration and Refractoriness in Isolated Myocytes from the Rabbit AV Node and Atrium

“…Cells were isolated from the AV nodal region and the left atrium of rabbit hearts, using a modification of methods described previously. 10,24 Briefly, male New Zealand White rabbits (2.0-2.8kg) were administered a lethal dose of anaesthetic (sodium pentobarbitone, 80mg/kg) with added heparin (335U/kg), by intravenous injection. The heart was excised and retrogradely perfused via the aorta for 4min at 20ml/min, with a solution containing (mM): NaCl (130), KCl …”