Early and delayed afterdepolarizations in rabbit heart Purkinje cells viewed by confocal microscopy

We investigated action potentials and Ca(2+) transients in rabbit Purkinje myocytes using whole cell patch clamp recordings and a confocal microscope. Purkinje cells were loaded with 5 microM Fluo-3/AM for 30min. Action potentials were elicited by application of a stimulus delivered through the recording pipettes. When Purkinje cells were stimulated in 2.0mM Ca(2+), transverse XT line scans revealed a symmetrical 'U'-shaped Ca(2+) transient demonstrating that the transient was initiated at the cell periphery. When Purkinje cells were superfused with 1 microM isoprenaline, both early and delayed afterdepolarizations were induced. XT line scans of cells exhibiting early afterdepolarizations showed a second symmetrical 'U'-shaped transient. This Ca(2+) transient was initiated at the cell periphery suggesting reactivation of the Ca(2+) current. In contrast, in Purkinje cells exhibiting delayed afterdepolarizations and a corresponding transient inward current, XT line scans revealed a heterogenous rise in Ca(2+) at both peripheral and central regions of the cell. Immunofluorescence staining of Purkinje cells with an antibody to ryanodine receptors (RyRs) revealed that RyRs are located at regularly spaced intervals throughout the interior of Purkinje cells. These results suggest that, although RyRs are located throughout Purkinje cells, only peripheral RyRs are activated to produce transients, sparks and early afterdepolarizations. During delayed afterdepolarizations, we observed a heterogenous rise in Ca(2+) at both peripheral and central regions of the cell as well as large central increases in Ca(2+). Although the latter may result from central release, we cannot exclude the possibility that it reflects Ca(2+) diffusion from subsarcolemmal sites.