Imaging Ca2+ nanosparks in heart with a new targeted biosensor

Abstract

RATIONALE:: In cardiac dyads, junctional Ca directly controls the gating of the ryanodine receptors (RyRs), and is itself dominated by RyR-mediated Ca release from the sarcoplasmic reticulum. Existing probes do not report such local Ca signals because of probe diffusion, so a junction-targeted Ca sensor should reveal new information on cardiac excitation-contraction coupling and its modification in disease states. OBJECTIVE:: To investigate Ca signaling in the nanoscopic space of cardiac dyads by targeting a new sensitive Ca 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 Ca transients (Ca nanosparks) that were ≈50× smaller in volume than conventional Ca sparks (measured with diffusible indicators). The presence of the biosensor did not disrupt normal Ca 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 Ca release events. CONCLUSIONS:: This new, targeted biosensor allows selective visualization and measurement of nanodomain Ca 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. © 2013 American Heart Association, Inc.