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

BACKGROUND

Three distinct Ca 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 release channel (InsP3R) were found in the cell periphery. How each of these channels contributes to the Ca cycling of P-cells is unclear. Recent modeling of Ca mobilization in P-cells suggested that Ca sensitivity of Cainduced Carelease (CICR) was larger at the P-cell periphery. Our study examined whether this numerically predicted region of Ca release exists in live P-cells. We compared the regional Ca dynamics with the arrangement of intracellular Ca 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 activity and electrically-evoked Ca mobilization were imaged by 2D spinning disk confocal microscopy. Functional regions of P-cell were differentiated by the characteristics of local Ca events. We used the Ca propagation velocities as indicators of channel Ca sensitivity.

RESULTS

RyR2 gene expression was identical in Purkinje fibers and myocardium (6 hearts) while RyR3 and InsPR gene expressions were, respectively, 100 and 16 times larger in the Purkinje fibers. Specific fluorescent immuno-staining of Ca 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-transients: (1) large asymmetrical Ca sparks under the membrane, and (2) typical Ca-wavelets propagating exclusively around the core of the cell. Larger cell-wide Ca 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; 3μM) and xestospongin C (XeC; 50μM) dramatically reduced their frequency. The Ca wavelets propagated in a 5-10μm 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 (10μM). The velocity of wavelets was two times larger (86±16μm/s; n=14) compared to CWWs' (46±10μm/s; n=11; P<0.05). Electric stimulation triggered a uniform and large elevation of Ca concentration under the membrane which preceded the propagation of Ca into the interior of the cell. Elevated Ca propagated at 150μm/s (147±34μm/s; n=5) through the region equivalent to the zone of RyR3 expression. This velocity dropped by 50% (75±24μm/s; n=5) in the central region wherein predominant RyR2 expression was detected.

CONCLUSION

We identified two layers of distinct Ca release channels in the periphery of Pcell: an outer layer of InsPRs under the membrane and an inner layer of RyR3s. The propagation of Ca events in these layers revealed that Ca sensitivity of Ca 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.