Selective Modulation of Coupled Ryanodine Receptors During Microdomain Activation of CaMKII in the Dyadic Cleft
Rationale: In ventricular myocytes of large mammals with low T-tubule (TT) density, a significant number of ryanodine receptors (RyR) are not coupled to the sarcolemma; cardiac remodeling increases non-coupled RyR.
Objective: To test the hypothesis that coupled and non-coupled RyRs have distinct microdomain-dependent modulation.
Methods and Results: We studied single myocytes from pig left ventricle. The TT network was analyzed in 3-D to measure distance to membrane of release sites. The rising phase of the Ca2+ transient was correlated with proximity to the membrane (confocal imaging, whole-cell voltage-clamp, K5fluo-4 as Ca2+ indicator). Ca2+ sparks following stimulation were thus identified as resulting from coupled or non-coupled RyRs. We used high frequency stimulation as a known activator of CaMKII. Spark frequency increased significantly more in coupled than in non-coupled RyRs. This specific modulation of coupled RyRs was abolished by the CaMKII-blockers AIP and KN-93, but not by KN-92. Colocalization of CaMKII and RyR was not detectably different for coupled and non-coupled sites but the F-actin disruptor cytochalasin D prevented the specific modulation of coupled RyRs. NOX2 inhibition by DPI or apocynin, or global ROS scavenging, also prevented coupled RyR modulation. During stimulated Ca2+ transients, frequency-dependent increase of the rate of Ca2+ rise was seen in coupled RyR regions only and abolished by AIP. After myocardial infarction, selective modulation of coupled RyR was lost.
Conclusions: Coupled RyRs have a distinct modulation by CaMKII and ROS, dependent on an intact cytoskeleton and consistent with a local Ca2+/ROS microdomain, and subject to modification with disease.
- ryanodine receptor
- calcium/calmodulin-dependent protein kinase II
- reactive oxygen species
- NAD(P)H oxidase
- Received May 28, 2013.
- Revision received September 26, 2013.
- Accepted September 30, 2013.