Background Three distinct Ca2 + release channels were identified in dog P-cells: the ryanodine receptor subtype 2 (RyR2) was detected throughout the cell, while the ryanodine receptor subtype 3 (RyR3) and inositol phosphate sensitive Ca2 + release channel (InsP3R) were found in the cell periphery. How each of these channels contributes to the Ca2 + cycling of P-cells is unclear. Recent modeling of Ca2 + mobilization in P-cells suggested that Ca2 + sensitivity of Ca2 + induced Ca2 + release (CICR) was larger at the P-cell periphery. Our study examined whether this numerically predicted region of Ca2 + release exists in live P-cells. We compared the regional Ca2 + dynamics with the arrangement of intracellular Ca2 + release (CR) channels. Methods Gene expression of CR channels was measured by qPCR in Purkinje fibers and myocardium of adult Yucatan pig hearts. We characterized the CR channels protein expression in isolated P-cells by immuno-fluorescence, laser scanning confocal microscopy, and 3D reconstruction. The spontaneous Ca2 + activity and electrically-evoked Ca2 + mobilization were imaged by 2D spinning disk confocal microscopy. Functional regions of P-cell were differentiated by the characteristics of local Ca2 + events. We used the Ca2 + propagation velocities as indicators of channel Ca2 + sensitivity. Results RyR2 gene expression was identical in Purkinje fibers and myocardium (6 hearts) while RyR3 and InsP3R gene expressions were, respectively, 100 and 16 times larger in the Purkinje fibers. Specific fluorescent immuno-staining of Ca2 + release channels revealed an intermediate layer of RyR3 expression between a near-membrane InsP3R-region and a central RyR2-region. We found that cell periphery produced two distinct forms of spontaneous Ca2 +-transients: (1) large asymmetrical Ca2 + sparks under the membrane, and (2) typical Ca2 +-wavelets propagating exclusively around the core of the cell. Larger cell-wide Ca2 + waves (CWWs) appeared occasionally traveling in the longitudinal direction through the core of Pcells. Large sparks arose in a micrometric space overlapping the InsP3R expression. The InsP3R antagonists 2-aminoethoxydiphenyl borate (2-APB; 3 μM) and xestospongin C (XeC; 50 μM) dramatically reduced their frequency. The Ca2 + wavelets propagated in a 5–10 μm thick layered space which matched the intermediate zone of RyR3 expression. The wavelet incidence was unchanged by 2-APB or XeC, but was reduced by 60% in presence of the RyR3 antagonist dantrolene (10 μM). The velocity of wavelets was two times larger (86 ± 16 μm/s; n = 14) compared to CWWs' (46 ± 10 μm/s; n = 11; P < 0.05). Electric stimulation triggered a uniform and large elevation of Ca2 + concentration under the membrane which preceded the propagation of Ca2 + into the interior of the cell. Elevated Cai propagated at 150 μm/s (147 ± 34 μm/s; n = 5) through the region equivalent to the zone of RyR3 expression. This velocity dropped by 50% (75 ± 24 μm/s; n = 5) in the central region wherein predominant RyR2 expression was detected. Conclusion We identified two layers of distinct Ca2 + release channels in the periphery of Pcell: an outer layer of InsP3Rs under the membrane and an inner layer of RyR3s. The propagation of Ca2 + events in these layers revealed that Ca2 + sensitivity of Ca2 + release was larger in the RyR3 layer compared to that of other sub-cellular regions. We propose that RyR3 expression in P-cells plays a role in the stability of electric function of Purkinje fibers.
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|Titolo:||Cardiac expression of ryanodine receptor subtype 3; a strategic component in the intracellular Ca2 + release system of Purkinje fibers in large mammalian heart|
|Citazione:||Cardiac expression of ryanodine receptor subtype 3; a strategic component in the intracellular Ca2 + release system of Purkinje fibers in large mammalian heart / Daniels, Rebecca E.; Haq, Kazi T.; Miller, Lawson S.; Chia, Elizabeth W.; Miura, Masahito; Sorrentino, Vincenzo; Mcguire, John J.; Stuyvers, Bruno D.. - 104(2017), pp. 31-42.|
|Appare nelle tipologie:||1.1 Articolo in rivista|
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