High-Resolution Imaging of GFP-Tagged Cardiac Ryanodine Receptor in Intact Heart and Brain

Abstract

The cardiac ryanodine receptor (RyR2) is most abundantly expressed in the heart and brain. RyR2 is located in the sarco(endo)plasmic reticulum (ER/SR) membrane and functions as an intracellular calcium release channel, important for a number of fundamental processes, such as muscle contraction, secretion, neurotransmitter release, learning and memory. Proper function of RyR2 critically depends on its subcellular distribution. In ventricular myocyte of the heart, RyR2 is organized in highly-ordered arrays of clusters, which is thought to be important for synchronous, stable calcium release during excitation-contraction coupling. However, little is known about RyR2 distribution in other cardiac cells. In the brain, RyR2 is thought to play a central role in learning and memory, but the cellular/subcellular distribution of RyR2 in the brain remains largely undefined. Recently, we have generated a knock-in mouse model that expresses a green fluorescence protein (GFP)-tagged RyR2. This GFP-RyR2 mouse model allows us to directly and specifically monitor the cellular/subcellular distribution and expression of RyR2 in various cells and tissues. To improve the detection of GFP, we have also developed novel GFP-specific probes based on anti-GFP single domain antibodies (i.e. nanobodies). High-resolution confocal imaging of intact GFP-RyR2 heart sections and brain slices revealed highly ordered arrays of GFP-RyR2 in various regions of the heart, but disperse distribution of GFP-RyR2 in hippocampus and other regions of the brain. Using Alexa-Fluor 647 (AF647)-conjugated GFP-specific probes, we will perform super-resolution imaging to further define the subcellular distribution of GFP-RyR2 in the heart and brain (Supported by NSERC, CFI, CIHR, and LCIA).