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

Rubart, Michael, Exing Wang, Kenneth W. Dunn, and Loren J. Field. Two-photon molecular excitation imaging of Ca2 transients in Langendorff-perfused mouse hearts. Am J Physiol Cell Physiol 284: C1654–C1668, 2003. First published February 12, 2003; 10.1152/ajpcell.00469. 2002.—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 m 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-2induced 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.