Abstract
Ryanodine receptors (RyR2s) are ion channels in the sarcoplasmic reticulum
(SR) that are responsible for Ca$^2+$ release in rat ventricular
myocytes. Localization of RyR2s is therefore crucial for our understanding
of contraction and other Ca$^2+$-dependent intracellular processes.
Recent results (e.g. circular waves and Ca$^2+$ sparks in perinuclear
area) raised questions about the classical views of RyR2 distribution
and organization within ventricular cells. A Ca$^2+$ spark is
a fluorescent signal reflecting the activation of a small group of
RyR2s. Frequency and spatio-temporal characteristics of Ca$^2+$
sparks depend on the state of cytoplasmic and intraluminal macromolecular
complexes regulating cardiac RyR2 function. We employed electron
microscopy, confocal imaging of spontaneous Ca$^2+$ 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 Ca$^2+$
sparks showed the existence of functional groups of RyR2s in these
intracellular compartments. We found that within the sarcomere about
20\% of Ca$^2+$ 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 Ca$^2+$
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 Ca$^2+$-dependent
gene regulation, but their existence raises many additional questions.
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