In situ Ca2+ dynamics of Purkinje fibers and its interconnection with subjacent ventricular myocytes

Purkinje fibers play essential roles in impulse propagation to the ventricles, and their functional impairment can become arrhythmogenic. However, little is known about precise spatiotemporal pattern(s) of interconnection between Purkinje-fiber network and the underlying ventricular myocardium within the heart. To address this issue, we simultaneously visualized intracellular Ca2+ dynamics at Purkinje fibers and subjacent ventricular myocytes in Langendorff-perfused rat hearts using multi-pinhole type, rapid-scanning confocal microscopy. Under recording of electrocardiogram at room temperature spatiotemporal changes in fluo3-fluorescence intensity were visualized on the subendocardial region of the right-ventricular septum. Staining of the heart with either fluo3, acetylthiocholine iodide (ATCHI), or di-4-ANEPPS revealed characteristic structures of Purkinje fibers. During sinus rhythm (about 60 bpm) or atrial pacing (up to 3 Hz) each Purkinje-fiber exhibited spatiotemporally synchronous Ca2+ transients nearly simultaneously to ventricular excitation. Ca2+ transients in individual fibers were still synchronized within the Purkinje-fiber network not only under high-K+ (8 mM) perfusion-induced Purkinje-to-ventricular (P–V) conduction delay, but also under unidirectional, orthodromic P–V block produced by 10-mM K+ perfusion. While spontaneous, asynchronous intracellular Ca2+ waves were identified in injured fibers of Purkinje network locally, surrounding fibers still exhibited Ca2+ transients synchronously to ventricular excitation. In summary, these results are the first demonstration of intracellular Ca2+ dynamics in the Purkinje-fiber network in situ. The synchronous Ca2+ transients, preserved even under P–V conduction disturbances or under emergence of Ca2+ waves, imply a syncytial role of Purkinje fibers as a specialized conduction system, whereas unidirectional block at P–V junctions indicates a substrate for reentrant arrhythmias.