Figure 1.
Effect of proximity of TTs to RyR on spontaneous Ca2+ sparks.
A. Typical example of a line scan image during and after 1 Hz stimulation. After loading the SR with conditioning pulses from −70 to +10 mV at 1 Hz, stimulation was stopped and 15 seconds of diastole were recorded for Ca2+ sparks. Sparks were assigned to early (blue) and delayed (red) release areas corresponding to their position on the scan line. B. Spark frequency and morphology in early vs. delayed release areas in CTRL pigs (Npigs = 12, ncells = 41). * denotes P<0.05.
Figure 2.
Effect of sarcolemmal fluxes on spark frequency and duration.
A. Effect of NCX block by 5 mM nickel on spark frequency and duration in early (left) and delayed (right) release areas in CTRL cardiomyocytes (Npigs = 3, ncells = 7). B. Effect of ICaL block by 50 µM cadmium on spark frequency and duration in early (left) and delayed (right) release areas in CTRL cardiomyocytes (Npigs = 2, ncells = 9). * denotes P<0.05.
Figure 3.
Effect of remodeling in the area adjacent to MI on Ca2+ sparks.
A. Representative line scan image of Ca2+ spark recording in MI. The types of release areas are marked in blue and red for early and delayed release areas respectively. The artifact region (marked by –) was excluded for analysis. B. Whole-cell spark frequency and morphology in CTRL (Npigs = 12, ncells = 41) and MI (Npigs = 7, ncells = 33). C. Spark mass, calculated by amplitude*width*duration, and spark-mediated Ca2+ leak, calculated by spark frequency*spark mass, in CTRL (Npigs = 12, ncells = 41) and MI (Npigs = 7, ncells = 33). D. SR Ca2+ content as µmoles/L accessible cytosol, in CTRL (Npigs = 4, ncells = 15) and MI (Npigs = 4, ncells = 12). * denotes P<0.05.
Figure 4.
Subcellular spark properties in myocytes from the area adjacent to MI.
A. Spark frequency, and, B, duration in early vs. delayed release areas in MI, with the MI-dependent change in each area (CTRL Npigs = 12, ncells = 41 vs. MI Npigs = 7, ncells = 33). C. Example of long spark in 3D and fraction of cells showing long-lasting sparks (>47.6 ms) in CTRL (24/43 cells with long sparks) and MI (22/31 cells with long sparks). * denotes P<0.05.
Figure 5.
NCX in myocytes from the area adjacent to MI.
A. NCX function was measured as the tail current upon repolarization to −70 mV (arrow) after a step to +10 mV in CTRL and MI. B. Averaged NCX current density in CTRL (Npigs = 9, ncells = 30) and MI (Npigs = 7, ncells = 19), with [Ca2+]i measured simultaneously. * denotes P<0.05.
Figure 6.
Modulation of Ca2+ sparks with TT loss during culture.
A. Representative confocal images of TT staining with di-8-ANEPPS (left) and RyR staining (right) after 48 hours of culture. B. Representative line scan image of Ca2+ spark protocol in CULT. The type of release areas is marked in blue and red for early and delayed release areas respectively. C. Comparison of spark duration in early (left) and delayed (right) release areas in CTRL (Npigs = 7, ncells = 29) vs. CULT (Npigs = 7, ncells = 14). D. Fraction of cells showing long-lasting sparks (>47.6 ms) in CTRL (17/35 cells with long sparks) and CULT (10/15 cells with long sparks). E. NCX current density in CTRL (Npigs = 4, ncells = 14) and CULT (Npigs = 4, ncells = 7). * denotes P<0.05.
Figure 7.
Ca2+ removal by NCX during caffeine application.
A. Example of current and [Ca2+]i transient recording obtained during the last conditioning pulse from -70 to +10 mV and caffeine application (left). The decay of the current was fit by a 1- or 2-exponential according to the goodness of fit (R2>0.95) and with the 2 amplitudes being negative. The right panel is a typical example of a 2-exponential decay in a CTRL myocyte. B. Example of monophasic INCX decay (left) and mean data on incidence of biphasic decay (middle). The percentage of cells better fit by a biphasic exponential was significantly higher in CTRL than in MI (CTRL, Npigs = 4, ncells = 17; MI, Npigs = 4, ncells = 12, P<0.05). In addition, Tau of fast component of INCX decay tended to be faster in CTRL than in MI (right, CTRL, Npigs = 4, ncells = 8, vs. MI, Npigs = 2, ncells = 4, P = NS). * denotes P<0.05.