Figure 3. Behavior of ECC couplons at variable stimulation frequencies and during β-adrenergic stimulation.

Rat ventricular myocytes were electrically paced into steady state at the stimulation intervals given. (A) The resulting CaCLEAN ECC couplon maps at 600 ms (a) and 150 ms (b) stimulation intervals under control conditions (Ctrl). Subcellular regions highlighted with white boxes were replotted at a high magnification (c) alongside with segmented ECC couplon sites (c, red). (B) Data similar to those in (A) but in a ventricular myocyte following β-adrenergic stimulation (10 nM isoproterenol, 5 min) (Iso). (C) (a) The negative Ca²⁺ transient amplitude-stimulation frequency relationship for control conditions and following stimulation with 10 nM isoproterenol, here, global refers to the average of the confocal section (b) The density of active ECC couplons. (c) The released Ca²⁺ amplitudes from individual ECC couplons and (d) the size of these ECC couplons (indicated as full width at half maximum, FWHM) decreases with increasing stimulation frequencies for control conditions (filled symbols) and following isoproterenol stimulation (open symbols); (e) the coefficient of variance (CV) of active ECC couplons increases between consecutive inter-CaT couplons and (f) within individual Ca²⁺ transients (intra-CaT couplon CV) with increasing stimulation frequencies under control conditions and during β-adrenergic stimulation. Significant differences are indicated by the black lines in the lower parts of the panels. (D) Fast Fourier Transform (FFT) analysis of CaCLEAN maps at increasing stimulation frequencies under control conditions and following β-adrenergic stimulation. Panel (a) exemplifies the power spectra of the FFT analysis; panel (b) displays the normalized amplitudes of the characteristic power peaks (marked with an arrow in a) and panel (c) shows the characteristic spatial distances of those power peaks. For all conditions, at least 23 cells from three different animals were analyzed. Error bars smaller than their symbols were omitted. For statistical details see Figure 3—source data 1.

Figure 3—figure supplement 1. CaCLEAN map (left part of each panel) and segmented ECC couplons (right part of each panel) for a typical rat ventricular myocyte under control conditions (A) and during simulation with 10 nM isoproterenol (B) at the given stimulation intervals.

These are the complete data for the two cells exemplified in Figure 3.

Figure 3—figure supplement 2. Comparison of CaCLEAN couplon map and anti-RYR immunolabeling with Fast Fourier Transform (FFT) analysis.

(A) A typical CaCLEAN couplon map taken from a rat ventricular myocyte (top) and its 2D-FFT transform (bottom). (B) A typical distribution of RyR proteins after immunolabeling with a RYR-specific primary antibody in a rat ventricular myocyte (top) and its 2D-FFT transform (bottom). (C) Line traces of the normalized FFT power taken along the longitudinal axis of the FFT power maps. Blue highlights the trace from the CaCLEAN map while the RyR trace is in red. Apart from the height of the frequency peaks, the two traces are superimposable. (D) Compares the height of the spatial frequency peak marked with an asterisk in (C) for the anti-RYR staining (red) and CaCLEAN (blue). The cells (n = 26 and 48 for immunolabeling and CaCLEAN maps, respectively) were analyzed from three different rats.