Fig. 3.

New steady-state characteristics emerge from pharmacological perturbation of a functionally coupled cardiomyocyte syncytium. A: HL-1 cardiomyocytes (25) were cultured into a spontaneously oscillating monolayer, and Ca²⁺-dependent Fluo4 fluorescence was recorded using confocal laser scanning microscopy (46, 48, 50). The boundaries of seven cells are shown. Bar represents 10 μm. B: basal Ca²⁺ oscillations and the responses to a single bolus of caffeine (5 mM, added at 30 s) of four cells in this cluster (1, red; 2, blue; 3, green; 4, black) are shown. This experiment can be viewed in full via the Supplemental Movie. C: expanded sections of the basal and caffeine-stimulated states from cell nos. 1–4 (horizontal bars = 5 s; B) were overlaid. D: caffeine elicited a marked reduction in the intercellular synchronization of Ca²⁺ oscillations. E: basal Ca²⁺-dependent fluorescence was unchanged in cell nos. 1–7 following caffeine-stimulation [73.9 ± 6.5 vs. 64.4 ± 4.3 fluorescence units (FU)]. F: despite the near-identical oscillatory behavior of all cells in the basal state, consistent with a high-degree of intercellular synchronization, cell no. 3 (green square) is functionally isolated from its neighbors in the post-caffeine state and exhibited a reduced oscillatory frequency. Cell-to-cell contacts and cell boundaries remained unaltered following caffeine addition (see Supplemental Movie). G: caffeine reduced the steady-state Ca²⁺ transient amplitude in a uniform manner. The standard error expressed as a percentage of the mean value (SE % mean) was 6.0% post-caffeine compared with 4.0% in the basal nonstimulated state. H: the duration of Ca²⁺-transient decay following caffeine addition exhibited a level of cell-to-cell variability that was >16 times greater than that determined in the basal state [SE (% mean) = 6.0% (caffeine) vs. 0.36% (basal)].