APs are triggered by spontaneous calcium oscillations. (A) Representative traces from simultaneous V_m and [Ca²⁺]_i measurements during I_f inhibition by Zeneca (n = 6; top) and SR inhibition by ryanodine (n = 5; bottom) from an E10 cardiomyocyte, demonstrating that inhibition of SR calcium release blocks the generation of APs, whereas inhibition of I_f does not. (B) Simultaneous V_m and [Ca²⁺]_i measurements from a spontaneously active E10-isolated cardiomyocyte showing the time difference between SR calcium release (location indicated in the phase contrast image with a red dot) and AP upstroke. The insert (gray background) shows both graphs in an expanded timescale with an 18-ms delay between the beginning of the release and initiation of AP upstroke. (C) Caffeine-induced calcium release (top) with corresponding inward current (bottom) in the absence and presence of 10 mM Ni²⁺, a blocker of the NCX. Right-side graph shows the relationship between cytosolic calcium and NCX current density at variable [Ca²⁺]_i levels pooled from 22 measurements during caffeine-induced calcium releases. Linear regression fit (solid line) with 95% confidence bands (dotted lines) shows a strong correlation between F/F₀ and NCX current density with a regression coefficient (r²) of 0.72 (P < 0.0001). In the presence of Ni²⁺, calcium signals fail to trigger an inward current (red dots).