Fig. 2.

MiroEF suppresses and MiroV13 promotes the VP-induced Mito-motility inhibition. (A) Actual mitochondrial motility values (Left) and [Ca²⁺]_c (Right) during stimulation with VP (100 nM) in cells transfected with mitoYFP alone or mitoYFP+Miro1EF or mitoYFP+Miro2EF. For VP the lowest motility and highest [Ca²⁺]_c were calculated (n = 27–33). *, P < 0.01. For motility the baseline values were shown in Fig. 1B. (B) Simultaneous measurements of Mito-motility and [Ca²⁺]_c in H9c2 cells expressing mitoYFP (Upper) and coexpressing mitoYFP with MiroEF (Lower). Left images show mitoYFP fluorescence (grayscale); Middle and Right images show the sites of mitochondrial movement calculated by subtraction of sequential images (ΔF: change in fluorescence between the two time points; red for positive changes, green for negative changes) before and after application of 0.25 nM VP in each condition. Graphs show both [Ca²⁺]_c (Upper) and motility inhibition in cells expressing mitoYFP (Control; black) or coexpressing mitoYFP and MiroEF (red). (C) Dose-response relationships between Mito-motility and [Ca²⁺]_c. Vector (black, n = 58), MiroEF (pink, n = 24) and MiroV13 (cyan, n = 27) expressing cells. Mito-motility decrease during VP stimulation was normalized to baseline motility in each cell (% inhibition) and is plotted against the corresponding [Ca²⁺]_c elevations. The IC₅₀ and Hillslope values were in each condition: Control; 380 ± 10 nM and −4.6, V13; 300 ± 20 nM and −4.4, EF; 450 ± 20 nM and −4.5. (D) Mito-motility inhibition at the range of [Ca²⁺]_c = 300–400 nM were calculated in Control (n = 13), V13 (n = 10), and EF (n = 11).