Ca2+ Nanosparks: Junctional Ca2+ Dynamics Probed with a New Targeted Biosensor

Abstract

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. Being able to directly probe junctional Ca2+ dynamics should increase our understanding of cardiac excitation-contraction coupling and its modification in disease states. We developed a new targeted calcium biosensor based on the latest generation of fluorescent protein probes, GCaMP6f by fusing it to triadin and/or junctin. This probe targeted the dyad as shown by colocalization with RyRs after adenovirus-mediated transfection in single cardiac myocytes from adult rat. This biosensor had faster kinetics and slightly elevated Kd compared to native GCaMP6f. Confocal imaging revealed high-contrast biosensor fluorescent transients (“Ca2+ nanosparks”) that occupied a ∼50-times smaller volume than conventional Ca2+ spark records obtained with diffusible indicators. These spatially discrete signals showed no diffusive component outside the labeled region, allowing high-contrast recordings of dyad activation. The maintenance of high dyad-to-background contrast amidst whole cell Ca2+ transients allowed us to detect the latency and duration of individual dyad activation during normal excitation-contraction coupling. Furthermore, the high fidelity of the signal enabled a critical examination of non-spark Ca2+ leak that is currently thought to play a key role in heart diseases. In summay, this new, targeted biosensor permits the first selective detection of nanodomain Ca2+ dynamics and may be extended to whole tissues by transfection or transgenic approaches.