title: Imaging Ca2+ nanosparks in heart with a new targeted biosensor.
abstract: BACKGROUND
  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.
  
  
  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.
authors.affiliation: From the State Key Laboratory of Biomembrane and Membrane Biotechnology, Beijing Key Laboratory of Cardiometabolic Molecular Medicine, Institute of Molecular Medicine, Peking-Tsinghua Center for Life Sciences (W.S., F.L., T.S., J.X., Y.W., G.W., L.C., X.W., H.C.) and State Key Laboratory of Biomembrane and Membrane Biotechnology, College of Life Science (L.-L.L., S.-Q.W.), Peking University, Beijing, China; and Department of Physiology and Pharmacology, University of Bristol, City of Bristol, United Kingdom (M.B.C.).
authors.forename: Wei
authors.initials: W
authors.lastname: Shang
authors.forename: Fujian
authors.initials: F
authors.lastname: Lu
authors.forename: Tao
authors.initials: T
authors.lastname: Sun
authors.forename: Jiejia
authors.initials: J
authors.lastname: Xu
authors.forename: Lin-Lin
authors.initials: LL
authors.lastname: Li
authors.forename: Yanru
authors.initials: Y
authors.lastname: Wang
authors.forename: Gang
authors.initials: G
authors.lastname: Wang
authors.forename: Liangyi
authors.initials: L
authors.lastname: Chen
authors.forename: Xianhua
authors.initials: X
authors.lastname: Wang
authors.forename: Mark B
authors.initials: MB
authors.lastname: Cannell
authors.forename: Shi-Qiang
authors.initials: SQ
authors.lastname: Wang
authors.forename: Heping
authors.initials: H
authors.lastname: Cheng
mesh_terms: D000818:Animals; D015374:Biosensing Techniques / Q000379:methods / Q000639:trends*; D002118:Calcium / Q000502:physiology*; D020013:Calcium Signaling / Q000235:genetics*; D002478:Cells, Cultured; D006321:Heart; D008297:Male; D056004:Molecular Dynamics Simulation / Q000639:trends*; D032383:Myocytes, Cardiac / Q000737:chemistry / Q000502:physiology*; D036103:Nanotechnology / Q000379:methods / Q000639:trends*; D051381:Rats; D017207:Rats, Sprague-Dawley
chemical_list: D002118:Calcium
keywords: biosensing techniques; calcium signaling; excitation-contraction coupling; junctin; ryanodine receptor calcium release channel; triadin