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 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.
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-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.
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 and in disease states 
such as when RyRs become orphaned.