Imaging Ca2+ Nanosparks in Heart with a New Targeted Biosensor

Wei Shang

Research output: Contribution to journalArticle (Academic Journal)

38 Citations (Scopus)

Abstract

Rationale: 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 due to probe diffusion, so a junction-targeted Ca2+ sensor should reveal new information on cardiac excitation-contraction coupling and its modification in disease states.
Objective: To investigate Ca2+ signaling in the nanoscopic space of cardiac dyads by targeting a new sensitive Ca2+ biosensor (GCaMP6f) to the junctional space.
Methods and Results: By fusing GCaMP6f to the N-terminus of triadin 1 or junctin, GCaMP6f-T/J 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-times faster kinetics than native GCaMP6f. Confocal imaging revealed junctional Ca2+ transients (“Ca2+ nanosparks”) that were ~50- times 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 co-activation of subclusters of RyRs within a single junctional region, as well as ‘quarky’ Ca2+ release events.
Conclusions: 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 as well as in disease states such as when RyRs become orphaned.
Original languageEnglish
Pages (from-to)412-420
Number of pages8
JournalCirculation Research
Volume114
Issue number3
Early online date20 Nov 2013
DOIs
Publication statusPublished - 31 Jan 2014

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