FIGURE 4.

Alterations of mitochondria morphology induced by the age‐related decline of SIRT1 and SIRT3 under stress condition. (a) Representative images for H9c2 cells under normoxia and H/R stress conditions generated by applying the MiNA algorithm on the mitochondria stained with MitoTracker and DAPI. (b) Analysis of mitochondrial network morphology changes (mitochondria footprint, mitochondria individuals, and mitochondria networks) in H9c2 cells under normoxia and H/R stress condition using the MiNA macro for ImageJ. (c) Bar charts and relationship loop of mitochondria morphology parameters (mean network size and mean branch length) obtained from the analysis of 4–7 replicates for each group, with >10 cells counted for each replicate of H9c2 cells under normoxia and H/R stress conditions. *p < 0.05 vs. normoxia; ^† p < 0.05 vs. hypoxia condition. (d) Representative TEM images of in young (4–6 months)/aged (24–26 months) WT, SIRT1 ^f/f /icSIRT1^−/− (4–6 months), and SIRT3 ^f/f /cSIRT3^−/− (4–6 months) hearts under physiological and I/R stressed conditions. The images were analyzed using ImageJ to quantify the following morphological and shape descriptors: mitochondria surface area (µm²), perimeter (µm), Feret's diameter (µm), and aspect ratio. Values are means ± SEM. >50 mitochondria per group were measured. ^† p < 0.05 vs. sham, respectively; *p < 0.05 vs. young, SIRT1 ^f/f , SIRT3 ^f/f , respectively. (e) Representative microscopy images (200×) showing MitoSOX fluorescence in the myocardium in young/aged WT, SIRT1 ^f/f /icSIRT1^−/−, and SIRT3 ^f/f /cSIRT3^−/− hearts under physiological and I/R stressed conditions (upper panel). Quantification of MitoSOX oxidation from images. Values are means ± SEM. N = 5 donors, ^† p < 0.05 vs. sham, respectively; *p < 0.05 vs young, SIRT1 ^f/f , SIRT3 ^f/f , respectively