Cytoplasmic Ca²⁺ signals in beating HL-1 cardiomyocytes expressing exogenous RyR2-WT and -K4750Q. (A) Cal520 (left) and mCherry (right) fluorescent images of HL-1 cells infected with WT-IRES-mCherry baculovirus. Bar, 100 µm. (B) Representative Ca²⁺ signals in mCherry-positive (a–c) and mCherry-negative cells (d). Thin blue lines indicate the onset of synchronized Ca²⁺ transients (a–d), induced by autogenic within clusters of HL-1 cells. The red arrow depicts the onset of a Ca²⁺ wave (c). Cells (a and b) displayed normal Ca²⁺ transients but no Ca²⁺ waves. (C) Cal520 (left) and mCherry (right) fluorescent images of HL-1 cells infected with K4750Q-IRES-mCherry baculovirus. (D) Representative Ca²⁺ signals in mCherry-positive (a–c) and mCherry-negative cells (d). Note that single Ca²⁺ waves (red arrows) occurred every three to four Ca²⁺ transients (blue lines), and the peak amplitudes of the Ca²⁺ waves are larger than those of the synchronized Ca²⁺ transients. The cell (a) displayed normal Ca²⁺ transients but no Ca²⁺ waves. (E) Mean Ca²⁺ wave frequency in mock-infected and RyR2-WT and -K4750Q–expressing cells (n = 24–30). ***, P < 0.001 versus mock. ^###, P < 0.001 versus WT. Values shown are mean ± SEM. (F) Typical Ca²⁺ signals at 2 mM (blue) and 5 mM (red) [Ca²⁺]_o. Note that the Ca²⁺ wave frequency was higher at 5 mM than at 2 mM [Ca²⁺]_o in both WT and K4750Q cells.