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Title: Characterisation of a novel interaction between the cardiac myosin binding protein-C and the ryanodine receptor/calcium release channel
Author: Stanczyk, Paulina J.
ISNI:       0000 0004 6349 1935
Awarding Body: Cardiff University
Current Institution: Cardiff University
Date of Award: 2016
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Muscle excitation-contraction coupling (ECC) is a fundamental physiological process by which electrically-evoked sarcoplasmic reticulum (SR) Ca2+ release triggers cardiomyocyte contraction. Human ryanodine receptor 2 (RyR2) is an ion channel present in the SR membrane of cardiac myocytes, responsible for mediating calcium efflux. Cardiac myosin binding protein-C (cMyBP-C) is a modular protein anchored to the thick filament through its C-terminal region, whereas the N-terminal region of cMyBP-C is thought to regulate myocardial contractility by modifying actin-myosin association. Here, we present several lines of evidence indicating potential RyR2:cMyBP-C interaction, which could provide a novel retrograde regulation of SR Ca2+ release by the sarcomere. Firstly, co-immunoprecipitation (co-IP) experiments indicated that the detected putative association between RyR2 and cMyBP-C can be applicable to large mammalian species as well as to recombinant human proteins heterologously expressed in a mammalian cell line, with RyR2 N-terminus and cMyBP-C C-terminus being responsible for the binding. A 3D co-localisation immunocytochemistry study supported the possibility for the proteins to be readily available for protein-protein interaction, with recombinant human RyR2 and cMyBP-C shown to co-localise in HEK293 cells. Secondly, with the aid of two complementary techniques, namely co-IP and yeast 2-hybrid (Y2H), we showed that RyR2 residues 1-906 comprise the main cMyBP-C site, with residues 1-346 (corresponding to subdomain A and B of RyR2 NTD) and 654-906 (domain SPRY1 and P1) being important determinants for the binding with cMyBP-C C-terminus. Further mapping studies implied that cMyBP-C residues 820-972 and 1061-1274 are involved in the interaction with RyR2 N-terminus, with cMyBP-C Fn domain/s proposed to be directly responsible for the binding. Additionally, parallel experiments with the skeletal muscle RyR1 demonstrated that the corresponding N-terminus region (RyR2 1-906 amino acids) of mammalian isoform RyR1 can also interact with MyBP-C, which could indicate that the detected association can also be relevant in the regulation of ECC in skeletal muscle. Thirdly, we pursued the possible functional significance of RyR2:cMyBP-C interaction. The results from single cell Ca2+ imaging indicated that cMyBP-C interaction with RyR2 may have an inhibitory effect on Ca2+ release channel function, with cMyBP-C binding diminishing RyR2 channel activity. In particular, HEK293 cells co-expressing RyR2 and cMyBP-C had a reduced frequency of spontaneous intracellular calcium oscillations compared to cells expressing only RyR2. Lastly, further co-IP data demonstrated that cMyBP-C:RyR2 binding is not affected by four different RyR2 single point mutation variants (S166C, R176Q, R420Q and L433P) associated with different pathological phenotypes. Thus, while a physical and functional association between RyR2 and cMyBP-C was shown to possibly regulate normal cardiac physiology, it is unlikely to be involved in RyR2-related cardiac disease. In order to unravel the pathological significance of the detected cMyBP-C:RyR2 association, more studies are needed to understand what role cMyBP-C plays in RyR2 channel dysfunction that could potentially affect muscle contraction/relaxation.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available