Poster Presentation 2014 International Biophysics Congress

Imaging Ca2+ nanosparks in heart with a new targeted biosensor (#524)

Fujian Lu 1 , Wei Shang 1 , Tao Sun 1 , Jiejia Xu 1 , Lin-Lin Li 2 , Yanru Wang 1 , Gang Wang 1 , Liangyi Chen 1 , Xianhua Wang 1 , Mark B. Cannell 3 , Shi-Qiang Wang 2 , Heping Cheng 1
  1. Institute of Molecular Medicine, Peking University, Beijing, China
  2. State Key Laboratory of Biomembrane and Membrane Biotechnology, College of Life Science, Peking University, Beijing, China
  3. Department of Physiology and Pharmacology, Medical Sciences Building, University of Bristol, Bristol, U.K.

    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. By fusing the new sensitive Ca2+ biosensor 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, and 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. Fast 2D imaging showed calcium propagation in a single cluster and between adjacent clusters. These results provide new evidence that RyR arrays in a single dyad do not necessarily activate in an all-or-none fashion, displaying complex gating behavior.