Cardiac expression of ryanodine receptor subtype 3; a strategic component in the intracellular Ca2+ release system of Purkinje fibers in large mammalian heart

Autor: Bruno D. Stuyvers, Rebecca E. Daniels, John J. McGuire, Vincenzo Sorrentino, Elizabeth W. Chia, Kazi T. Haq, Masahito Miura, Lawson Miller
Rok vydání: 2017
Předmět:
Zdroj: Journal of Molecular and Cellular Cardiology. 104:31-42
ISSN: 0022-2828
Popis: Background Three distinct Ca 2 + 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 Ca 2 + release channel ( InsP3R ) were found in the cell periphery. How each of these channels contributes to the Ca 2 + cycling of P-cells is unclear. Recent modeling of Ca 2 + mobilization in P-cells suggested that Ca 2 + sensitivity of Ca 2 + induced Ca 2 + release ( CICR ) was larger at the P-cell periphery. Our study examined whether this numerically predicted region of Ca 2 + release exists in live P-cells. We compared the regional Ca 2 + dynamics with the arrangement of intracellular Ca 2 + 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 Ca 2 + activity and electrically-evoked Ca 2 + mobilization were imaged by 2D spinning disk confocal microscopy. Functional regions of P-cell were differentiated by the characteristics of local Ca 2 + events. We used the Ca 2 + propagation velocities as indicators of channel Ca 2 + sensitivity. Results RyR2 gene expression was identical in Purkinje fibers and myocardium (6 hearts) while RyR3 and InsP 3 R gene expressions were, respectively, 100 and 16 times larger in the Purkinje fibers. Specific fluorescent immuno-staining of Ca 2 + 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 Ca 2 + -transients: (1) large asymmetrical Ca 2 + sparks under the membrane, and (2) typical Ca 2 + -wavelets propagating exclusively around the core of the cell. Larger cell-wide Ca 2 + waves ( CWW s) 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 Ca 2 + 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 2 + concentration under the membrane which preceded the propagation of Ca 2 + into the interior of the cell. Elevated Ca i 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 Ca 2 + release channels in the periphery of Pcell: an outer layer of InsP 3 Rs under the membrane and an inner layer of RyR3s . The propagation of Ca 2 + events in these layers revealed that Ca 2 + sensitivity of Ca 2 + 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.
Databáze: OpenAIRE
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