Two-photon molecular excitation imaging successfully monitored Ca2+ transients in intact mouse hearts, revealing slower decay kinetics with fura-2 compared to rhod-2 (P<0.05).
Two-photon excitation microscopy enables subcellular imaging of calcium transients in intact mouse hearts, demonstrating that fura-2 increases cytosolic Ca2+ buffering compared to rhod-2.
p-value: p=<0.05
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.
Rubart et al. (Sun,) conducted a other in Langendorff-perfused mouse hearts (n=19). Two-photon molecular excitation imaging was evaluated on Ca2+ transient decay times (p=<0.05). Two-photon molecular excitation imaging successfully monitored Ca2+ transients in intact mouse hearts, revealing slower decay kinetics with fura-2 compared to rhod-2 (P<0.05).