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Am J Physiol Cell Physiol (February 12, 2003). doi:10.1152/ajpcell.00469.2002
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Submitted on October 4, 2002
Accepted on February 10, 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 Medicine, Wells Center for Pediatric Research and Krannert Institute of Cardiology, Indianapolis, IN, USA
2 Nephrology, Indiana University School of Medicine, Indianapolis, IN, USA

* To whom correspondence should be addressed. E-mail: mrubartv{at}iupui.edu.

The ability to image calcium signals at sub-cellular levels within the intact depolarizing heart could provide valuable information towards 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 non-contracting 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 (mean ± SEM) 73±4 ms and 126±10 ms, respectively, and at 2 Hz, 62±3 ms 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, since 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.




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