title: Measuring local gradients of intramitochondrial [Ca(2+)] in cardiac myocytes during sarcoplasmic reticulum Ca(2+) release.
abstract: BACKGROUND
  Mitochondrial [Ca(2+)] ([Ca(2+)](mito)) regulates mitochondrial energy production, provides transient Ca(2+) buffering under stress, and can be involved in cell death. Mitochondria are near the sarcoplasmic reticulum (SR) in cardiac myocytes, and evidence for crosstalk exists. However, quantitative measurements of [Ca(2+)](mito) are limited, and spatial [Ca(2+)](mito) gradients have not been directly measured.
  
  
  OBJECTIVE
  To directly measure local [Ca(2+)](mito) during normal SR Ca release in intact myocytes, and evaluate potential subsarcomeric spatial [Ca(2+)](mito) gradients.
  
  
  RESULTS
  Using the mitochondrially targeted inverse pericam indicator Mitycam, calibrated in situ, we directly measured [Ca(2+)](mito) during SR Ca(2+) release in intact rabbit ventricular myocytes by confocal microscopy. During steady state pacing, Δ[Ca(2+)](mito) amplitude was 29±3 nmol/L, rising rapidly (similar to cytosolic free [Ca(2+)]) but declining much more slowly. Taking advantage of the structural periodicity of cardiac sarcomeres, we found that [Ca(2+)](mito) near SR Ca(2+) release sites (Z-line) versus mid-sarcomere (M-line) reached a high peak amplitude (37±4 versus 26±4 nmol/L, respectively P<0.05) which occurred earlier in time. This difference was attributed to ends of mitochondria being physically closer to SR Ca(2+) release sites, because the mitochondrial Ca(2+) uniporter was homogeneously distributed, and elevated [Ca(2+)] applied laterally did not produce longitudinal [Ca(2+)](mito) gradients.
  
  
  CONCLUSIONS
  We developed methods to measure spatiotemporal [Ca(2+)](mito) gradients quantitatively during excitation-contraction coupling. The amplitude and kinetics of [Ca(2+)](mito) transients differ significantly from those in the cytosol and are respectively higher and faster near the Z-line versus M-line. This approach will help clarify SR-mitochondrial Ca(2+) signaling.
authors.affiliation: Department of Pharmacology, University of California, Davis, CA 95616, USA.
authors.forename: Xiyuan
authors.initials: X
authors.lastname: Lu
authors.forename: Kenneth S
authors.initials: KS
authors.lastname: Ginsburg
authors.forename: Sarah
authors.initials: S
authors.lastname: Kettlewell
authors.forename: Julie
authors.initials: J
authors.lastname: Bossuyt
authors.forename: Godfrey L
authors.initials: GL
authors.lastname: Smith
authors.forename: Donald M
authors.initials: DM
authors.lastname: Bers
mesh_terms: D000818:Animals; D015374:Biosensing Techniques / Q000592:standards; D002118:Calcium / Q000378:metabolism*; D020013:Calcium Signaling; D002138:Calibration; D002304:Cardiac Pacing, Artificial; D002478:Cells, Cultured; D003600:Cytosol / Q000378:metabolism; D056966:Excitation Contraction Coupling; D005456:Fluorescent Dyes / Q000378:metabolism; D007700:Kinetics; D018613:Microscopy, Confocal / Q000592:standards; D008856:Microscopy, Fluorescence / Q000592:standards; D008929:Mitochondria, Heart / Q000378:metabolism*; D009200:Myocardial Contraction; D032383:Myocytes, Cardiac / Q000378:metabolism*; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000378:metabolism*; D014162:Transfection
chemical_list: D005456:Fluorescent Dyes; D002118:Calcium