Activation of Calcium Release from RyR Clusters Depends on their Distance from the Sarcolemma

Abstract

Summation of microscopic release events from RyRs underlies the upstroke of the calcium transient in mammalian ventricular cells. These release events are initiated when RyRs are gated by transmembrane calcium influx. In rabbit cardiac myocytes the majority of RyR clusters are closely associated with the sarcolemma where they form couplons. However, significant numbers of RyR clusters are non-junctional and do not form couplons. It is unclear if or by what mechanism non-junctional RyRs are activated. We analyzed the activation of both types of RyRs. We studied calcium transients in rabbit isolated ventricular cells that exhibit a sparse t-system with Fluo-4 and high-speed two-dimensional confocal microscopy. We constructed distance maps indicating that some intracellular regions are up to 3.5 μm away from the sarcolemma. Image sequences showed that transients were spatially and temporally inhomogeneous. Transients originated in regions where we detected sarcolemma and spread with a significant delay to areas lacking t-system where non-junctional RyRs are present. We found an increasing relationship between sarcolemmal distances and local activation times with activation times of 13.0±6.6 ms at sarcolemmal sites, and 20.5±5.1 ms at distances 3-3.5 μm. The relationship between sarcolemmal distances and maximal upstroke of the transient was more complex. Maximal upstroke at sarcolemmal sites was 13.2±4.1 %/ms versus 8.8±1.6 ms at 3-3.5 μm. To interpret these results we used a one-dimensional model of calcium diffusion, which explains the spread of upstroke that we see experimentally. We conclude that non-junctional RyRs within 1.5 μm of the sarcolemma are activated. Beyond 1.5 μm the rise in calcium could result from either diffusion or slow activation. In both cases, activation of non-junctional RyRs would occur by a mechanism different than local control, i.e. a modified common pool model.