Fig. 5.

HF reduced mitochondria and SR colocalization and disrupted mitochondria–SR microdomains. (A) Representative stimulated emission depletion microscopy images of sham and HF SANCs colabeled with RyR2 (purple) and COX IV (cyan). Two SANCs were seen in the sham group. (Scale bars, 5 μm [Upper] and 0.5 μm [enlarged images, Lower] for each group.) (B) PLA showing representative immunofluorescence confocal microscopy 3D rendered images, with PLA puncta (red) and DAPI (blue) from sham and HF SANCs, labeled with RyR2 + COX IV, RyR2 + Mfn2, and RyR2 + DRP-1. Positive cross-reactivity, which reflects an intermolecular distance of 40 nm or less, is detected as puncta, and the nuclei are depicted in blue. (C) Summary data of PLA fluorescent puncta per cell area (puncta per square micrometer) for the different combinations, including negative controls with one primary antibody. Data are represented as mean ± SEM. ****P < 0.0001. The symbols represent the number of cells within the bar graphs (n = 3 mice for each group).