Abstract 16971: CaMKinase II Modulation of Ryanodine Receptors during High Frequency Stimulation is Restricted to Junctional Microdomains
Background. In ventricular myocytes from large mammals, including humans, T-tubules (TTs) are more sparse and a significant fraction of ryanodine receptors (RyRs) are not coupled to sarcolemma and L-type Ca2+channels (LTCC). Non-coupled and coupled receptors may have different properties related to local signaling in the junctional microdomain. We tested this hypothesis during high frequency stimulation, known to lead to activation of CaMKII.
Methods. Ca2+ release from the sarcoplasmic reticulum (SR) was elicited during depolarizing steps under whole-cell voltage clamp and recorded using confocal line scan imaging with Fluo-4 as Ca2+ indicator in single myocytes from pig left ventricle. Subsequent Z-stack images of TTs and sarcolemma system (stained with WGA-Alexa595) allowed establishing for every pixel on the line scan the nearest distance to TTs in 3D. This 3D-distance map was correlated to the rise time of the local [Ca2+]i transients. Spontaneous RyR release events, sparks, were recorded during a 15 s period following stimulation at 0.5 or 2 Hz for 2 minutes. Based on the 3-D and [Ca2+]i transient analysis, sparks in sites > 2 micron away from TT were considered non-coupled, sparks less than 0.5 micron away from the TT were classified as coupled.
Results. Increasing the stimulation frequency from 0.5 Hz to 2 Hz resulted in an increase in spark frequency that was restricted to the RyR clusters in coupled sites (from 1.2+/-0.2 to 3.9+/-0.6 sparks/100micron/s, P<0.05, in coupled, vs. from 1.1+/-0.2 to 2.0+/-0.3, P=NS in non-coupled, n= 13). Selective inhibition of CaMKII with AIP (10 microM, incubation for 1h and inclusion in pipette solution, n=12) prevented this response. Immunostaining for RyR, CaMKII and NCX as reporter for sarcolemma and TT, showed equal colocalization of coupled and non-coupled RyR with CaMKII (n=21). However, disrupting this colocalization by incubation with cytochalasin D (10microM incubation for 1h , confirmed with immunostaining) inhibited the frequency response in coupled RyR (n=12).
Conclusions. Increased open probability of RyRs during high frequency stimulation is restricted to junctional microdomains where CaMKII is selectively activated.
- © 2012 by American Heart Association, Inc.