FIG. 1.
α1-adrenoceptor (α1-AR) stimulation accelerates mitochondrial Ca2+ uptake in H9c2 cells. (A) Expression of mitochondria-targeted Ca2+-sensitive probe, Mitycam. Mitochondrial location was determined using mt-RFP. A cell stably transfected with only mt-RFP is also shown in the top row to confirm almost no background fluorescence in the GFP channel. Mitycam localized to mitochondria. Scale bars, 20 μm. (B) Color scatter plotting (right) and frequency scatter plotting (left) obtained from representative pictures in (A). (C) Time course of Mitycam fluorescence intensity during 3 μM thapsigargin (TG) treatment. Scale bar, 10 μm. (D) Left: Representative time courses of [Ca2+]mt (top) and [Ca2+]c (bottom) in cells stimulated by TG in control cells (CTR: nontransfected cells) (black), MCU (red), and MCUB (blue)-overexpressed cells. Arrow indicates TG application. [Ca2+]mt and [Ca2+]c were measured using Mitycam and Fura-Red, respectively. Mitycam fluorescence (F) was converted into 1 − (ΔF/F0), which shows the changes in [Ca2+]mt, where Fo stands for initial fluorescence levels. Averaged data are shown in Supplementary Fig. S2. Right: summary data of left panels. [Ca2+]mt was measured at 60 s after stimulation. For [Ca2+]c, peak [Ca2+]c within 120 s was measured. *p<0.05, compared to CTR. (E) Effect of Phe pretreatment on [Ca2+]mt and [Ca2+]c. Representative time course of [Ca2+]mt (top) and [Ca2+]c (bottom) after TG stimulation (arrow) in cells with (red) or without (black) pretreatment with Phe (100 μM, 15 min). [Ca2+]mt and [Ca2+]c were simultaneously measured using Mitycam and Fura-red, respectively, in a single cell (see Supplementary Materials and Methods section). Summary data are shown in Supplementary Fig. S5. (F) Left: Averaged time course of [Ca2+]mt after TG stimulation (arrow) in cells with (red) or without (black) pretreatment with Phe (100 μM, 15 min). [Ca2+]mt in cells overexpressing MCU-DN (blue) or MCUB (sky blue) is also shown for comparison. Right: Averaged time course of [Ca2+]mt after TG stimulation (arrow) in MCU-transfected cells pretreated with (red) or without (black) Phe. (G) Summary data of (F) at 60 s after stimulation. [Ca2+]mt in cell pretreatment with Phe and prazosin (Praz, 1 μM) (purple) is also shown (see also Supplementary Fig. S5). *p<0.05, compared to CTR without Phe treatment. #p<0.05, N.S., not significant. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars