Location of the initiation site of calcium transients and sparks in rabbit heart Purkinje cells

Abstract

2+ ] i (calcium sparks) have been identified in cardiac, smooth and skeletal muscle (reviewed by Cheng et al. 1996b). A number of authors have suggested that Ca 2+ transients are due the spatio-temporal summation of many Ca 2+ sparks Cheng et al. 1995;Tanaka et al. 1998). These Ca 2+ sparks are the result of release of Ca 2+ from the SR and are believed to be the elementary events underlying E-C coupling in cardiac muscle (Cheng et al. 1993;Bridge et al. 1999). Shacklock et al. (1995) have shown that location of sparks coincides with the location of t-tubules in rat ventricular myocytes. Since L-type Ca 2+ channels and SR Ca 2+ release are co-localized at the t-tubule dyad junction, it has been suggested that a local accumulation of [Ca 2+ ] i near one or a cluster of SR Ca 2+ release channels gates their opening Cheng et al. 1996a). However, it is unclear whether Ca 2+ channels or junctions with the sarcolemma are absolutely necessary for the spontaneous occurrence of sparks.In contrast to the information available on sparks and Ca 2+ transients in ventricular muscle, relatively little is known about the spatio-temporal characteristics of Location of the initiation site of calcium transients and sparks in rabbit heart Purkinje cells ] i of ventricular cells showed a more uniform pattern of activation across the cell. Staining with di-8-ANEPPS revealed that Purkinje cells lack t-tubules, whereas ventricular cells have an extensive t-tubular system.3. When we superfused both cell types with a buffer containing 5 mM Ca 2+ -1 µM isoproterenol (isoprenaline) they produced Ca 2+ sparks spontaneously. Ca 2+ sparks occurred only at the periphery of Purkinje cells but occurred throughout ventricular cells. Sparks in both cell types could be completely abolished by addition of the SR inhibitor thapsigargin (500 nM). Brief exposure to nifedipine (10 µM) did not reduce the number of spontaneous sparks.4. Immunofluorescence staining of Purkinje cells with anti-ryanodine antibody revealed that ryanodine receptors (RyRs) are present at both peripheral and central locations.5. Computer simulations of experiments in which the calcium transient was evoked by voltage clamp depolarizations suggested that the increase in calcium observed in the centre of the cell could be explained by simple buffered diffusion of calcium. These computations suggested that the RyRs deep within the cell do not contribute significantly to the calcium transient.6. These results provide the first detailed, spatially resolved data describing Ca 2+ transients and Ca 2+ sparks in rabbit cardiac Purkinje cells. Both types of events are initiated only at subsarcolemmal SR Ca 2+ release sites suggesting that in Purkinje cells, Ca 2+ sparks only originate where the sarcolemma and sarcoplasmic reticulum form junctions. The role of the centrally located RyRs remains unclear. It is possible that because of the lack of t-tubules these RyRs do not experience a sufficiently large Ca 2+ trigger during excitation-contraction (E-C) coupling to become active.

2APB- and JTV519(K201)-sensitive micro Ca2+ waves in arrhythmogenic Purkinje cells that survive in infarcted canine heart

“…4). Similar to normal rabbit Purkinje cells with isoproterenol, 20 we found that in IZPCs (in the absence of isoproterenol), a cellwide wave is preceded by highly frequent μCaiTs (Fig. 1).…”

“…13 It also can restore coordination of Ca 2+ sparks in myocytes from an infarcted heart. 14 Others have suggested that isoproterenol has no effect on spark frequency or distribution, that it can or cannot increase spark amplitude but can accelerate its decay and increase the maximal rate of release 15 , 16 (but see references 17 -19 20 It is widely accepted that isoproterenol increases the rate of firing of canine Purkinje fibers. In this study, we report that isoproterenol increased both μCaiT and cell-wide Ca 2+ waves in normal Purkinje cell aggregates, consistent with observations that isoproterenol enhances membrane depolarization and the amplitude of delayed afterdepolarizations in normal canine Purkinje fibers.…”

Size Matters: Ryanodine Receptor Cluster Size Affects Arrhythmogenic Sarcoplasmic Reticulum Calcium Release

“…Individual RyR cluster size can vary over a continuous distribution (from <10 up to 250 RyRs), but some smaller clusters are quite close to others (eg, at the same junction) and, because of their proximity (within 100 nm), may function as a single junctional cluster. This proximity explains how a small local Ca 2+ influx can activate multiple RyRs via Ca 2+ ‐induced Ca 2+ ‐release (CICR) during the normal action potential–induced Ca 2+ transient and also during diastolic Ca 2+ sparks 7, 8, 9, 10, 11. Ca 2+ sparks are stochastic local CICR events that are typically restricted spatially to 1 cluster/junction without propagation to nearest‐neighbor junctions spaced 1.8 to 1.9 μm longitudinally or 0.4 to 0.8 μm transversely 8, 9, 10, 11, 12.…”

Ca 2+ Sparks and Waves in Canine Purkinje Cells

“…Most (82%) of these events (amplitudeϭ1.85Ϯ0.02 F/Fo; duration at half maximal amplitude [T 0.5 ]ϭ41Ϯ1 ms; full width at half maximal amplitude [FWHM]ϭ3.2Ϯ0.1 m; rateϭ0.6Ϯ0.2 events per s per 100 m; nϭ524) were similar to Ca 2ϩ sparks reported for rabbit Pcells. 6,15 The remaining nonpropagating Ca 2ϩ transients had amplitude above 3 F/Fo (amplitudeϭ4.9Ϯ0.2 F/Fo; T 0.5 ϭ43Ϯ2 ms; FWHM: 2.4Ϯ0.2 m; rateϭ0.13Ϯ0.04 events per s per 100 m scan; nϭ115; Figure 1A). Clusters of consecutive and/or simultaneous Ca 2ϩ sparks ( Figure 1B) were also detected predominantly in regions below the sarcolemma and were similar to compound sparks described previously.…”