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Am J Physiol Cell Physiol 284: C1654-C1668, 2003. First published February 12, 2003; doi:10.1152/ajpcell.00469.2002
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Vol. 284, Issue 6, C1654-C1668, June 2003

Two-photon molecular excitation imaging of Ca2+ transients in Langendorff-perfused mouse hearts

Michael Rubart1, Exing Wang2, Kenneth W. Dunn2, and Loren J. Field1

1 Wells Center for Pediatric Research and Krannert Institute of Cardiology, and 2 Department of Medicine, Division of Nephrology, Indiana University School of Medicine, Indianapolis, Indiana 46202

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 [Ca2+]i transients in individual cardiomyocytes within noncontracting Langendorff-perfused mouse hearts. [Ca2+]i transients were recorded at depths <= 100 µm from the epicardial surface with the fluorescent indicators rhod-2 or fura-2 in the presence of the excitation-contraction uncoupler cytochalasin D. Evoked [Ca2+]i transients were highly synchronized among neighboring cardiomyocytes. At 1 Hz, the times from 90 to 50% (t90-50%) and from 50 to 10% (t50-10%) of the peak [Ca2+]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. [Ca2+]i decay was markedly slower in fura-2-loaded hearts (t90-50% at 1 Hz, 128 ± 9 ms and at 2 Hz, 88 ± 5 ms; t50-10% 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 [Ca2+]i decline resulted from increased cytosolic Ca2+ buffering, because the kinetics of rhod-2 decay resembled those obtained with fura-2 after incorporation of the Ca2+ chelator BAPTA. Propagating calcium waves and [Ca2+]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.

rhod-2; fura-2; BAPTA; 2,3-butanedione monoxime; cytochalasin D


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