Proceedings of The Physiological Society
University of Oxford (2011) Proc Physiol Soc 23, PC158
A novel method to localize subcellular Ca2+ release using 3-D distance mapping
E. Dries1, G. Antoons1, V. Bito1, K. Sipido1, N. Macquaide1
1. Katholieke Universiteit Leuven, Leuven, Belgium.
In ventricular myocytes, L-type Ca2+ channels (LTCC) on the sarcolemma (SL) and clusters of ryanodine receptors (RyRs) on the sarcoplasmic reticulum (SR) come in close proximity. Invaginations of the SL permeate the cell, called T-tubules (TT). These allow functional coupling of the SL and SR, allowing a rapid global increase in cytosolic [Ca2+] for the activation of contraction. However, not all RyR clusters are coupled, leading to temporal inhomogeneity of systolic Ca2+ release, which is more pronounced in larger mammals (Heinzel et al., 2002). Investigation of release, assigning events to coupled and uncoupled regions is currently limited by the temporal resolution of confocal microscopy to simultaneously monitor the 3D configuration of release sites and SL. Here we present a new method to investigate the subcellular activation of release of Ca2+ with proximity to TTs in pig ventricular myocytes. Whole-cell voltage clamp was used to elicit Ca2+ release by depolarizing steps at different frequencies (0.5 and 2 Hz) at 37°C. Cytochalasin D was used to inhibit contraction. Confocal line scanning of fluo-4 fluorescence was used to monitor Ca2+ transients during depolarizing steps from -70 mV to +10 mV at different frequencies. The temporal mid-point of the rising phase (TF50) was used to assess latency of release. SL membranes were fluorescently labeled with wheat germ agglutinin-Alexa594 and vertical image stacks were recorded for each cell. The 3-D TT geometry was assessed using the Elucidean distance mapping method. This method allowed the correlation of Ca2+ TF50 for each site in the line scan recording with distance to TT in 3D. A linear relationship was found for release between 0.5 and 3 µm from T-tubules at 2 frequencies: 0.5 Hz (R2 = 0.88; ncells=7, Npigs=5) and 2 Hz (R2 = 0.80; ncells=7, Npigs=5). This allowed the use of TF50 to map subcellular regions of Ca2+ release defined as coupled (< 0.5 µm) and uncoupled (> 1 µm). Regions of coupled release were found to reach TF50 within 19 ms and 14 ms, respectively for 0.5 Hz and 2 Hz stimulation. This resulted in 55% (0.5 Hz) and 49% (2 Hz) of coupled regions. Non-coupled release regions were found to reach TF50 within 22 ms and 17 ms, for 0.5 Hz and 2 Hz respectively. Hence 38% of regions were classified as non-coupled for both frequencies. This novel method shows 3-D distance mapping is a useful technique to localize subcellular Ca2+ release. This will be extended to allow investigation of spontaneous diastolic Ca2+ release events (sparks) from RyRs in different subcellular domains.
Where applicable, experiments conform with Society ethical requirements