Final Report Summary - CPAF (Calcium Propagation in Atrial Fibrillation) Research objectives1) Explore the involvement of site-to-site propagation in modulation of E-C coupling in atrial myocardium of sheep. How does this change in AF?2) Study the factors affecting RyR mediated Ca2+ leak in atrial myocytes; investigate the contribution of extra-dyadic RyRs and the changes occurring in AF.3) Find histological evidence for extra-dyadic RyRs in atrial myocytes; quantify the density and sise of these clusters, compare in control and AF animals.4) Design image analysis software for deconvolution and quantification of acquired microscopy images.The work performed since the beginning of the projectThe fellow has carried out experiments on intact and permeabilised cells form control and AF sheep, with a goal of meeting objective 1 and 2. He has also initiated a collaboration with the Hofkens group in the Department of Chemistry KU Leuven, utilising their super resolution microscopy techniques to answer objective 3. Computer software has been written utilising deconvolution algorithms which have improved detection of RyR clusters as detailed in objective 4. These are implemented in the Python programming language and are freely available. This has allowed the quantification of RyR clusters sise and geometry. This has been presented at several international meetings (see dissemination section) and a publication is in preparation.The main results achieved so farThe fellow has identified differential activation of dyadic and non-dyadic RyRs, which is dependent of CaMKII activation in the dyadic region. In AF, this dyadic activation is lost and extradyadic RyRs appear more sensitive to activation by CaMKII. In AF, this shift from dyadic to extra-dyadic activation leads to a two-fold higher probability of spontaneous release upon CaMKII stimulation. This spontaneous Ca2+ release, coupled with the increased electrogenic current, activated by the sodium / calcium exchanger (NCX) would predispose the atria to trigger extra-systolic beats, contributing to the induction and persistence of atrial fibrillation as is observed in this animal model.In terms of RyR density, it has been observed that RyR density is altered in AF, compared to control. Experimental evidence published by the fellow (MacQuaide et al., 2010) has reported that smaller RyR clusters are necessary for the initiation and propagation of spontaneous Ca2+ waves and modelling (Sobie et al., 2006) has predicted that these are more sensitive to phosphorylation. This could explain the shift in activation of extradyadic RyRs in AF, allying super resolution structural information with the functional experimental evidence.The expected final results and their potential impact and use (including the socio-economic impact and the wider societal implications of the project so far).It is expected that the final results will constitute a new paradigm in the induction of spontaneous Ca2+ release and how this can predispose to the fibrillating cardiac phenotype. The techniques described may be of use in the exploration of other arrhythmogenic phenotypes, such as the increased inducibility of ventricular fibrillation seen in many hypertrophic animal models and eventually in human tissue.These new insights into the mechanisms which underlie the spontaneous release of Ca2+, leading to the formation of ectopic foci may have important role in the generation of an arrhythmogenic substrate. Other ionic currents, such as INCX, IK1, Ito, Ica and ICaCl are likely to be remodeled to produce the pronounced shortening of the action potential. This complex system will need further integration into how these systems may interact in the intact heart. There is however a growing body of evidence that clear that the spontaneous Ca2+ release is capable of acting as a trigger for the induction of a fibrillating cardiac phenotype (Weiss et al., 2011).It is hoped the work from this study can pave the way for future studies, assisting in the development of future therapeutic options for patients of AF and other arrhythmogenic myopathies. The potential socio-economic benefit and the wider societal implications are evident as improved antiarrhythmic pharmacological treatments, reducing the need for surgical implantation of implantable cardioverter-defibrillator devices (ICDs) in patients susceptible to ventricular fibrillation (VF) and improvement in the susceptibility to stroke and general improvement in quality of life in sufferers of AF.Reference listMacQuaide N., Ramay H. R., Sobie E. A., and Smith G. L. (2010). Differential sensitivity of Ca(2)+ wave and Ca(2)+ spark events to ruthenium red in isolated permeabilised rabbit cardiomyocytes. J Physiol 588, 4731 - 4742.Sobie E. A., Guatimosim S., Gomez-Viquez L., Song L. S., Hartmann H., Saleet J. M., and Lederer W. J. (2006). The Ca2+ leak paradox and rogue ryanodine receptors: SR Ca2+ efflux theory and practice. Prog Biophys Mol Biol 90, 172 - 185.Weiss J. N., Nivala M., Garfinkel A., and Qu Z. (2011). Alternans and arrhythmias: from cell to heart. Circ Res 108, 98 - 112.