Abstract
In large mammals, many SR Ca2+ channels (RyRs) are uncoupled from sarcolemma or T-tubules (TTs). Tools for exploring RyR regulation in relation to T-tubule proximity are limited. Here we present a method to measure TT distance and investigate release from coupled and uncoupled RyRs. In isolated pig ventricular myocytes consecutive vertical confocal images were taken after labeling with wheat germ agglutinin-Alexa594. After image processing, the 3-D TT geometry was assessed using Euclidean distance mapping. Whole-cell voltage clamp was subsequently used to elicit Ca2+ release at 0.5, 1 and 2 Hz and Ca2+ was recorded in a confocal line scan (0.3 micron pixel-1, 649 Hz). The temporal mid-point of the local Ca2+ transient upstroke (TF50) was used to assess latency of release. Correlation of the TF50 for each site with distance to TT in 3-D yielded a linear relationship for distance between 0.5 and 3 μm from T-tubules. This allowed the use of TF50 to map subcellular Ca2+ release regions as coupled ( 2 μm). In coupled regions, TF50 occurred within 18 ms, 17 ms and 12 ms, respectively for 0.5, 1 and 2 Hz. This resulted in 56% (0.5 Hz), 57% (1 Hz) and 49% (2 Hz) of coupled regions. Spark frequency increased with stimulation frequency in coupled release sites but significantly less so in uncoupled ones. In the presence of the F-actin disrupter Cytochalasin D, this increase in spark frequency was abolished and there was no difference between coupled and uncoupled sites. Therefore, the regional discrimination of RyR reveals a preferential activation of coupled RyRs with frequency, which is dependent on a cytoskeletal interaction.
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