Imaging Ca²⁺ Nanosparks in Heart with a New Targeted Biosensor

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.