Functional groups of ryanodine receptors in rat ventricular cells

Abstract

Ryanodine receptors (RyR2s) are ion channels in the sarcoplasmic reticulum (SR) that are responsible for Ca2+ release in rat ventricular myocytes. Localization of RyR2s is therefore crucial for our understanding of contraction and other Ca2+-dependent intracellular processes. Recent results (e.g. circular waves and Ca2+ sparks in perinuclear area) raised questions about the classical views of RyR2 distribution and organization within ventricular cells. A Ca2+ spark is a fluorescent signal reflecting the activation of a small group of RyR2s. Frequency and spatio-temporal characteristics of Ca2+ sparks depend on the state of cytoplasmic and intraluminal macromolecular complexes regulating cardiac RyR2 function. We employed electron microscopy, confocal imaging of spontaneous Ca2+ sparks and immunofluorescence to visualize the distribution of RyR2s in ventricular myocytes and to evaluate the local involvement of the macromolecular complexes in regulation of functional activity of the RyR2 group. An electron microscopy study revealed that the axial tubules of the transverse-axial tubular system probably do not have junctions with the network SR (nSR). The nSR was found to be wrapped around intermyofibrillar mitochondria and contained structures similar to feet of the junctional cleft. Treatment of ventricular myocytes with antibodies against RyR2 showed that in addition to the junctional SR, a small number of RyR2s can be localized at the middle of the sarcomere and in the zone of perinuclear mitochondria. Recordings of spontaneous Ca2+ sparks showed the existence of functional groups of RyR2s in these intracellular compartments. We found that within the sarcomere about 20% of Ca2+ sparks were not colocalized with the zone of the junctional or corbular SR (Z-line zone). The spatio-temporal characteristics of sparks found in the Z-line and A-band zones were very similar, whereas sparks from the zone of the perinuclear mitochondria were about 25% longer. Analysis of the initiation sites of Ca2+ sparks within the same junctional SR cluster suggested that 18-25 RyR2s are in the functional group producing a spark. Because of the similarity of the spatio-temporal characteristics of sarcomeric sparks and ultrastructural characteristics of nSR, we suggest that the functional groups of RyR2s in the middle of the sarcomere are macromolecular complexes of approximately 20 RyR2s with regulatory proteins. Our data allowed us to conclude that a significant number of functional RyR2s is located in the middle of the sarcomere and in the zone of perinuclear mitochondria. These RyR2s could contribute to excitation-contraction coupling, mitochondrial and nuclear signalling, and Ca2+-dependent gene regulation, but their existence raises many additional questions.