Two-photon molecular excitation imaging of Ca2+ transients in Langendorff-
perfused mouse hearts.
Rubart, Michael, Exing Wang, Kenneth W. Dunn, and Loren J. Field.
1Wells Center for Pediatric Research and Krannert Institute of Cardiology, and
2Department of Medicine, Division of Nephrology, Indiana University School of
Medicine, Indianapolis, Indiana 46202
APStracts 10:0073C, 2003.
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.
Received 3 October 2002; accepted in final form 10 February 2003
APS Manuscript Number C469-2.
Article publication pending Am J Physiol Cell Physiol
ISSN 1080-4757 Copyright 2003 The American Physiological Society.
Published in APStracts on 25 March 2003