File S1 - Mitochondrial Ca2+-Handling in Fast Skeletal Muscle Fibers from Wild Type and Calsequestrin-Null Mice

Supporting text and figures. Figure S1. In situ determination of Rmin (left) and Rmax (right) of the cameleon (4mtD3cpv). The 4mtD3cpv responses (YFP, red; CFP, blue; ratio YFP/CFP, green) are shown. Aliquots of ionomycin (Iono) were added up to a final concentration of 5 μM (left) and 10 μM (right); 50 μM N-benzyl-ptoluene sulphonamide (BTS) was added in order to reduce movement artifacts during the Rmax determination. The minimum YFP/CFP value reached (Rmin) amounted to 1.5. Addition of 5 mM CaCl2 caused an abrupt increase in the YFP/CFP ratio up to a maximum value of 6.3. Upon the addition of 5 mM CaCl2, the fiber started to contract despite of the presence of BTS (for 7 minutes), resulting in an increase in the CFP (and YFP) intensity and moved out of the focal plane of the microscope near the end of the recording (resulting in a decline in the YFP and CFP intensity). Figure S2. Correction for bleaching. Top: during a train of stimuli at 0.1 Hz, the amplitude of the increase in the YFP/CFP ratio declined as a result of bleaching of the probe. Bottom: a linear relation was observed between peak amplitude and the concurrent baseline value, yielding a straightforward way to correct the ΔR values for bleaching of the probe (see Results). Figure S3. Recording of the change in [Ca2+]mito during and after a single twitch. The baseline corrected 4mtD3cpv response (YFP, red; CFP, blue; ratio YFP/CFP, green) in a WT fiber electrically stimulated by a single pulse in the presence of 1 mM Ca2+. The YFP/CFP ratio shown was obtained by using a 10-points running average. The final part of the decay phase could be well fitted to a single exponential (bright green) with a rate constant of 0.21 s−1. Figure S4. Comparison of the time course of the 4mtD3cpv responses and the Fura-2 responses. In each panel, the upper figure shows the 4mtD3cpv response and the lower figure shows the Fura-2 response in WT at 1, 5 and 60 Hz stimulation (A, B and C) and in dCASQ-null at 60 Hz (D). Note that the decline in the 4mtD3cpv transients at 1 and 5 Hz occurs more slowly than in the Fura-2 transients. As a result there is -during the stimulation period- a gradual rise in the baseline free Ca2+ concentration inside the mitochondria, whereas the cytosolic Ca2+ concentration in between stimuli remains rather constant (Panels A and B). The comparison between WT and dCASQ-null at 60 Hz illustrates that in dCASQ-null fibers the initial peak value is similar to WT but there is a gradual decline in the cytosolic free Ca2+ concentration. At 60 Hz stimulation, the free [Ca2+]mito after an early rapid increase accumulates gradually in WT but remains rather constant in dCASQ-null fibers (Panels C and D). (DOC)