Evoked Centripetal Ca2+ Activation in Cardiac Purkinje Cells: CICR or Ca2+ Diffusion?

In large mammals, cardiac Purkinje cells (Pcells) lack transverse tubules (T-tubules). However, Pcells respond to stimulations by massive Ca-releases from the sarcoplasmic reticulum (SR) similar to those of ventricular myocytes (VMs) with T-tubules. Ca-imaging revealed a wave of elevated Ca-concentration (Cai) propagating from the sarcolemma (SL) toward the centre of Pcells upon stimulation. Ca-diffusion was proposed to explain this centripetal propagation. A thin layer of SR expressing RyR3 was found ∼5µm below the SL while other SR-regions expressed RyR2. Ca-Induced Ca-Release (CICR) from RyR3-SR-region could relay Ca-signal from Ca-entry to RyR2-Ca-release sites in the core, playing equivalent role as T-tubules in VMs. Our objective wasto examine whether centripetal Ca-signaling results exclusively from Ca-diffusion or involved intermediate RyR3-CICR. Methods:Pcells were prepared from Yucatan swine (30-40kg) and incubated with Fluo4-AM. Ca-dynamics was assessed by 2D-confocal microscopy (30-100fps); pH7.3,35oC. 26 cells were field stimulated at various external Ca-concentrations (Cao). Ca-data were compared with those from a model of Ca-propagation across 3 virtual adjacent SR-Ca-release regions. Results: In stimulated Pcells, Ca-increase first happened under SL with amplitude increasing from 0.1 to 0.5µmol/L with Cao from 1 to 4mmol/L. A Ca-wave then propagated uniformly from the SL toward cell center at 100-250µm/s. An overall Ca-transient appeared when Ca-wave reached the core region. When the model included intermediate CICR-region, predicted data were consistent with Pcells observations: minimal amplitude (minAmp) of peripheral Ca-increase mediating a central Ca-transient similar to that of Pcells, was 130nmol/L; this generated Ca-propagation with velocity (propVel) of 160µm/s; propVel was independent on Cao. In contrast, minAmp and propVel were 220nmol/L and 40µm/s respectively, and propVel increased with Cao when only diffusion was considered. Conclusion: Our data were consistent with an intermediate CICR-mechanism which “boosts” Ca-propagation between SL and core of Pcells.