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Circ Res. 2014 Jan 31;114(3):412-20. doi: 10.1161/CIRCRESAHA.114.302938. Epub 2013 Nov 20.

Imaging Ca2+ nanosparks in heart with a new targeted biosensor.

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From the State Key Laboratory of Biomembrane and Membrane Biotechnology, Beijing Key Laboratory of Cardiometabolic Molecular Medicine, Institute of Molecular Medicine, Peking-Tsinghua Center for Life Sciences (W.S., F.L., T.S., J.X., Y.W., G.W., L.C., X.W., H.C.) and State Key Laboratory of Biomembrane and Membrane Biotechnology, College of Life Science (L.-L.L., S.-Q.W.), Peking University, Beijing, China; and Department of Physiology and Pharmacology, University of Bristol, City of Bristol, United Kingdom (M.B.C.).



In cardiac dyads, junctional Ca2+ directly controls the gating of the ryanodine receptors (RyRs), and is itself dominated by RyR-mediated Ca2+ release from the sarcoplasmic reticulum. Existing probes do not report such local Ca2+ signals because of probe diffusion, so a junction-targeted Ca2+ sensor should reveal new information on cardiac excitation-contraction coupling and its modification in disease states.


To investigate Ca2+ signaling in the nanoscopic space of cardiac dyads by targeting a new sensitive Ca2+ biosensor (GCaMP6f) to the junctional space.


By fusing GCaMP6f to the N terminus of triadin 1 or junctin, GCaMP6f-triadin 1/junctin was targeted to dyadic junctions, where it colocalized with t-tubules and RyRs after adenovirus-mediated gene transfer. This membrane protein-tagged biosensor displayed ≈4× faster kinetics than native GCaMP6f. Confocal imaging revealed junctional Ca2+ transients (Ca2+ nanosparks) that were ≈50× smaller in volume than conventional Ca2+ sparks (measured with diffusible indicators). The presence of the biosensor did not disrupt normal Ca2+ signaling. Because no indicator diffusion occurred, the amplitude and timing of release measurements were improved, despite the small recording volume. We could also visualize coactivation of subclusters of RyRs within a single junctional region, as well as quarky Ca2+ release events.


This new, targeted biosensor allows selective visualization and measurement of nanodomain Ca2+ dynamics in intact cells and can be used to give mechanistic insights into dyad RyR operation in health and in disease states such as when RyRs become orphaned.


biosensing techniques; calcium signaling; excitation-contraction coupling; junctin; ryanodine receptor calcium release channel; triadin

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