title: Two-photon molecular excitation imaging of Ca2+ transients in Langendorff-perfused mouse hearts.
abstract: The ability to image calcium signals at subcellular levels within the intact depolarizing heart could provide valuable information toward a more integrated understanding of cardiac function. Accordingly, a system combining two-photon excitation with laser-scanning microscopy was developed to monitor electrically evoked [Ca(2+)](i) transients in individual cardiomyocytes within noncontracting Langendorff-perfused mouse hearts. [Ca(2+)](i) transients were recorded at depths </=100 microm from the epicardial surface with the fluorescent indicators rhod-2 or fura-2 in the presence of the excitation-contraction uncoupler cytochalasin D. Evoked [Ca(2+)](i) transients were highly synchronized among neighboring cardiomyocytes. At 1 Hz, the times from 90 to 50% (t(90-50%)) and from 50 to 10% (t(50-10%)) of the peak [Ca(2+)](i) were (means +/- SE) 73 +/- 4 and 126 +/- 10 ms, respectively, and at 2 Hz, 62 +/- 3 and 94 +/- 6 ms (n = 19, P < 0.05 vs. 1 Hz) in rhod-2-loaded cardiomyocytes. [Ca(2+)](i) decay was markedly slower in fura-2-loaded hearts (t(90-50%) at 1 Hz, 128 +/- 9 ms and at 2 Hz, 88 +/- 5 ms; t(50-10%) at 1 Hz, 214 +/- 18 ms and at 2 Hz, 163 +/- 7 ms; n = 19, P < 0.05 vs. rhod-2). Fura-2-induced deceleration of [Ca(2+)](i) decline resulted from increased cytosolic Ca(2+) buffering, because the kinetics of rhod-2 decay resembled those obtained with fura-2 after incorporation of the Ca(2+) chelator BAPTA. Propagating calcium waves and [Ca(2+)](i) amplitude alternans were readily detected in paced hearts. This approach should be of general utility to monitor the consequences of genetic and/or functional heterogeneity in cellular calcium signaling within whole mouse hearts at tissue depths that have been inaccessible to single-photon imaging.
authors.affiliation: Wells Center for Pediatric Research, Indianapolis, Indiana 46202, USA. mrubartv@iupui.edu
authors.forename: Michael
authors.initials: M
authors.lastname: Rubart
authors.forename: Exing
authors.initials: E
authors.lastname: Wang
authors.forename: Kenneth W
authors.initials: KW
authors.lastname: Dunn
authors.forename: Loren J
authors.initials: LJ
authors.lastname: Field
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism*; D020013:Calcium Signaling / Q000502:physiology*; D002614:Chelating Agents / Q000378:metabolism; D015638:Cytochalasin D / Q000378:metabolism; D003931:Diacetyl / Q000031:analogs & derivatives* / Q000378:metabolism; D003952:Diagnostic Imaging / Q000379:methods*; D004533:Egtazic Acid / Q000031:analogs & derivatives* / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000378:metabolism; D005456:Fluorescent Dyes / Q000378:metabolism; D016257:Fura-2 / Q000378:metabolism; D006321:Heart / Q000502:physiology*; D006575:Heterocyclic Compounds, 3-Ring; D066298:In Vitro Techniques; D051379:Mice; D008815:Mice, Inbred Strains; D008822:Mice, Transgenic; D032342:Muscle Cells / Q000166:cytology / Q000378:metabolism; D009119:Muscle Contraction / Q000502:physiology; D019384:Nucleic Acid Synthesis Inhibitors / Q000378:metabolism; D010477:Perfusion; D017785:Photons; D016212:Transforming Growth Factor beta / Q000235:genetics / Q000378:metabolism; D053773:Transforming Growth Factor beta1
chemical_list: D002614:Chelating Agents; D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D006575:Heterocyclic Compounds, 3-Ring; D019384:Nucleic Acid Synthesis Inhibitors; C508961:Tgfb1 protein, mouse; D016212:Transforming Growth Factor beta; D053773:Transforming Growth Factor beta1; C068483:rhod-2; C004717:diacetylmonoxime; D015638:Cytochalasin D; D004533:Egtazic Acid; C025603:1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; D003931:Diacetyl; D002118:Calcium; D016257:Fura-2