Figure 2. Ca²⁺ hot spots observed following depolarization in UBSM cells from RyR2^+/+ and RyR2^+/−.

Membrane currents and Ca²⁺ events were simultaneously monitored in single UBSM cells isolated from RyR2^+/+ and RyR2^+/− under voltage clamp. Ca²⁺ images were obtained using fluo-4 and laser scanning confocal microscopy. A, representative Ca²⁺ images during and following depolarization from −60 to 0 mV for 50 ms in RyR2^+/+ and RyR2^+/− UBSM cells. Voltage clamp depolarization started at 0 ms. The edge of cells is indicated by the white line. Arrows indicated hot spots. The Ca²⁺ hot spots were identified and measured in the images as follows (Ohi et al. 2001). (1) The averaged fluorescence intensity ratio (F/F₀) in a cluster of pixels, which includes neighbouring 4 pixels or more, was larger than 2.0. (2) The F/F₀ in a hot spot was measured as the average at pixels in a circular spot of 1.3 μm in diameter. (3) The area of the hot spot spreads and the F/F₀ in it increased or maintained the high level over 100 ms. B, changes in F/F₀ in the small area of hot spots ‘a’ (red) and ‘b’ (green) were measured from two Ca²⁺ hot spots indicated by red and green arrowheads in A, respectively. The data are plotted against time. F₀ was the average fluorescence intensity before depolarization. F/F₀ ratios measured as the average from whole cell area (blue) were also plotted. The black dots show membrane currents under whole cell voltage clamp. C, summarized data of [Ca²⁺]_i increases detected as peak F/F₀ at Ca²⁺ hot spots and in whole cell areas. Data were obtained from experiments shown in A. The numerals in parentheses indicate number of cells examined. D, number of Ca²⁺ hot spots per cell. The Ca²⁺ hot spots were measured in one confocal plane obtained 18.7 ms after the start of depolarization in the experiments shown in A. The numerals in parentheses indicate number of cells examined. E, peak amplitude of outward currents activated by depolarization from −60 to 0 mV for 50 ms as shown in A. The numerals in parentheses indicate number of cells examined. *P < 0.05, **P < 0.01 versus RyR2^+/+ in C, D and E.