Two-photon molecular excitation imaging of Ca[sup 2+] transients in Langendorff-perfused mouse hearts.

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[sup 2+]][sub i] transients in individual cardiomyocytes within noncontracting Langendorff-perfused mouse hearts. [Ca[sup 2+]][sub 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 [Ca[sup 2+]][sub i] transients were highly synchronized among neighboring cardiomyocytes. At 1 Hz, the times from 90 to 50% (t[sub 90-50%]) and from 50 to 10% (t[sub 50-10%]) of the peak [Ca[sup 2+]][sub i] were (means ± SE) 73 ± 4 and 126 ± 10 ms, respectively, and at 2Hz, 62 ± 3 and 94 ± 6 ms(n = 19, P<0.05vs. 1Hz) in rhod-2-1oaded cardiomyocytes. [Ca[sup 2+]][sub i] decay was markedly slower in fura-2-1oaded hearts (t[sub 90-50%] at I Hz, 128 ± 9 ms and at 2 Hz, 88 ± 5 ms; t[sub 50-l0%] 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 [Ca[sup 2+]][sub i] decline resulted from increased cytosolic Ca[sup 2+] buffering, because the kinetics of rhod-2 decay resembled those obtained with fura-2 after incorporation of the Ca[sup 2+] chelator BAPTA. Propagating calcium waves and [Ca[sup 2+]][sub 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. [ABSTRACT FROM AUTHOR]