Occurrence of Spontaneous Sparks in Ventricular Myocytes From Junctional and Non-junctional Ryr Clusters

Abstract

In isolated rabbit ventricular myocytes, we found a significant number of ryanodine receptor (RyR) clusters that are not associated with the sarcolemma (non-junctional RyRs). The contribution of non-junctional RyR clusters to calcium transients is unclear. Here, we investigated if these non-junctional RyRs are able to produce spontaneous local calcium release events (sparks), and compared the probability of non-junctional versus junctional sparks. We imaged spontaneous sparks in cells loaded with fluo-4 and bathed in Tyrode solution with dextran (Molecular Weight: 10 kDa) linked to Texas Red dye. We evoked spontaneous sparks using field stimulation in the presence of 1 μM isoproterenol and 4 mM calcium. After 5 stimuli applied with a frequency of 0.5 Hz, we simultaneously imaged the sarcolemma and spontaneous sparks using a line scan confocal microscope (Biorad MRC-1024). Furthermore, a 3D image stack of the Texas Red associated signal was acquired to identify the sarcolemma including the transverse tubular system. We classified sparks as non-junctional if their distance to the sarcolemma is larger than 1 μm. All other sparks were assumed to be junctional. In measurements on 12 isolated cells, 38 sparks (51%) were identified as non-junctional, 36 (49%) as junctional. Our measurements clearly demonstrate that non-junctional RyR clusters are able to release calcium and produce spontaneous sparks. We expect that our approach for distinguishing between non-junctional and junctional sparks underestimates the number of non-junctional sparks. If this is true, the probabilities of the types of sparks are similar to probabilities of the two types of RyR clusters identified in related immunolabeling and microscopic studies. This would suggests that spark generation probability of RyR clusters does not depend on their type.