3D dSTORM imaging reveals novel detail of ryanodine receptor localization in rat cardiac myocytes

Shen, X. et al. (2019) 3D dSTORM imaging reveals novel detail of ryanodine receptor localization in rat cardiac myocytes. Journal of Physiology, 597(2), pp. 399-418. (doi: 10.1113/jp277360) (PMID:30412283) (PMCID:PMC6332759)

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Cardiomyocyte contraction is dependent on Ca2+ release from ryanodine receptors (RyRs). However, the precise localization of RyRs remains unknown, due to shortcomings of imaging techniques which are diffraction limited or restricted to 2D. We aimed to determine the 3D nanoscale organization of RyRs in rat cardiomyocytes by employing direct stochastic optical reconstruction microscopy (dSTORM) with phase ramp technology. Initial observations at the cell surface showed an undulating organization of RyR clusters, resulting in their frequent overlap in the z‐axis and obscured detection by 2D techniques. Non‐overlapping clusters were imaged to create a calibration curve for estimating RyR number based on recorded fluorescence blinks. Employing this method at the cell surface and interior revealed smaller RyR clusters than 2D estimates, as erroneous merging of axially aligned RyRs was circumvented. Functional groupings of RyR clusters (Ca2+ release units, CRUs), contained an average of 18 and 23 RyRs at the surface and interior, respectively, although half of all CRUs contained only a single ‘rogue’ RyR. Internal CRUs were more tightly packed along z‐lines than surface CRUs, contained larger and more numerous RyR clusters, and constituted ∼75% of the roughly 1 million RyRs present in an average cardiomyocyte. This complex internal 3D geometry was underscored by correlative imaging of RyRs and t‐tubules, which enabled quantification of dyadic and non‐dyadic RyR populations. Mirroring differences in CRU size and complexity, Ca2+ sparks originating from internal CRUs were of longer duration than those at the surface. These data provide novel, nanoscale insight into RyR organization and function across cardiomyocytes.

Item Type:Articles
Additional Information:This work was supported by the European Union's Horizon 2020 research and innovation programme (Consolidator grant, W.E.L.) under grant agreement No. 647714. Additional support was provided by The South‐Eastern Norway Regional Health Authority, Anders Jahre's Fund for the Promotion of Science, The Norwegian Institute of Public Health, Oslo University Hospital Ullevål, and the University of Oslo. Research reported in this publication was supported by the Maximizing Investigators' Research Award (MIRA) (R35) from the National Institute of General Medical Sciences (NIGMS) of the National Institutes of Health (NIH) under grant number R35GM124977 (to P.K.H.).
Glasgow Author(s) Enlighten ID:MacQuaide, Dr Niall
Authors: Shen, X., van den Brink, J., Hou, Y., Colli, D., Le, C., Kolstad, T. R., MacQuaide, N., Carlson, C. R., Kekenes-Huskey, P. M., Edwards, A. G., Soeller, C., and Louch, W. E.
College/School:College of Medical Veterinary and Life Sciences > Institute of Cardiovascular and Medical Sciences
Journal Name:Journal of Physiology
ISSN (Online):1469-7793
Published Online:09 November 2018
Copyright Holders:Copyright © 2018 The Authors
First Published:First published in Journal of Physiology 597(2):399-418
Publisher Policy:Reproduced under a Creative Commons License

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