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 [less than or equal to] 100 [micro]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 [[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 [+ or -] SE) 73 [+ or -] 4 and 126 [+ or -] 10 ms, respectively, and at 2 Hz, 62 [+ or -] 3 and 94 [+ or -] 6 ms (n = 19, P < 0.05 vs. 1 Hz) in rhod-2-loaded cardiomyocytes. [[Ca.sup.2+]].sub.i] decay was markedly slower in fura-2-loaded hearts ([t.sub.90-50%] at 1 Hz, 128 [+ or -] 9 ms and at 2 Hz, 88 [+ or -] 5 ms; [t.sub.50-10%] at 1 Hz, 214 [+ or -] 18 ms and at 2 Hz, 163 [+ or -] 7 ms; n = 19, P < 0.05 vs. rhod-2). Fura-2-induced 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 genera] 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. rhod-2; fura-2; BAPTA; 2,3-butanedione monoxime; cytochalasin D