Chen, S. R. WayneHiess, Florian2018-01-262018-01-262018-01-23Hiess, F. (2018). Subcellular Distribution and Function of Cardiac Ryanodine Receptor in Ventricular Myocytes and Hippocampal Neurons. (Doctoral thesis, University of Calgary, Calgary, Canada). Retrieved from https://prism.ucalgary.ca. doi:10.11575/PRISM/5439http://hdl.handle.net/1880/106358Cardiac ryanodine receptors (RyR2s) are intracellular calcium (Ca2+) release channels most abundantly expressed in the heart and brain. They are clustered in the endo/sarcoplasmic reticulum (ER/SR) membrane to form elementary units for Ca2+ release. The distribution of these units determines the spatiotemporal profile and stability of ER/SR Ca2+ release. Thus, RyR2 distribution is believed to be essential in cellular processes, such as excitation-contraction coupling and learning and memory. The distribution of RyR2s has been extensively studied in cells/tissues using anti-RyR2 antibody immunostaining. However, sample preparation required for immunostaining may affect cellular structures, besides rendering the cells/tissues non-functional. Hence, the functional relevance of the distribution of RyR2 clusters in live cells/tissue is unclear. We have generated a knock-in mouse model that expresses green fluorescence protein (GFP)-tagged RyR2s. These mice allow us to monitor cellular/subcellular distribution of RyR2 in live cells/tissues by virtue of GFP fluorescence. To improve the detection of GFP-RyR2, we developed a novel GFP-specific probe based on anti-GFP single domain antibodies (nanobodies). Fluorescence imaging was employed to study Ca2+ release and the distribution of GFP-RyR2 in the interior and periphery of live ventricular myocytes and in intact hearts isolated from GFP-RyR2 expressing mice. We found highly-ordered arrays of stationary GFP-RyR2 clusters in the interior of cardiomyocytes in the z-line zone. In contrast, irregular and dynamic distribution of GFP-RyR2 clusters was observed in the periphery of cardiomyocytes. Imaging of intact GFP-RyR2 brain sections revealed a widespread distribution of RyR2 in various brain regions, most prominently in regions involved in spatial learning and memory, such as the hippocampus. To investigate the functional role of RyR2 in this region, we performed electrophysiological studies using hippocampal slices prepared from knock-in mice harboring a cardiac arrhythmia-associated human RyR2 mutation (R4496C) with enhanced channel activity. We found that enhanced RyR2 function reduces long-term potentiation (LTP) in Schaffer collateral inputs to CA1 pyramidal cells. Thus, RyR2 plays a critical role in LTP at these synapses. Behavioral studies on RyR2 mutant mice further supported the role of RyR2 in learning and memory. Overall, these results reveal, the distribution of RyR2 clusters and its functional significance in living ventricular myocytes and hippocampal neurons.engUniversity of Calgary graduate students retain copyright ownership and moral rights for their thesis. You may use this material in any way that is permitted by the Copyright Act or through licensing that has been assigned to the document. For uses that are not allowable under copyright legislation or licensing, you are required to seek permission.Cardiac Ryanodine ReceptorEducation--Sciencesdoctoral thesis10.11575/PRISM/5439