Figure 3. LCR rats show increased mitochondrial oxidative stress, mtDNA damage, circulating mitochondrial DAMPs and dysfunction in old age. Representative fluorescence images of aortic cross sections stained with MitoSOX Red (MSR) (A, top panel, scale 50 μm) and VSMCs stained with MitoTracker Green (MTG) and MitoSOX Red (B, top panel, scale 10 μm) from young and old age HCR and LCR rats. MitoSOX Red fluorescence was presented as integrated density (mean ± SEM, n = 4) (A and B bottom panels). Representative long PCR amplicon of 13.4 kb mitochondrial DNA from aortas (C, top panel) from old age HCR and LCR rats. PCR product band intensities were normalized to a short amplicon of 0.22 kb GAPDH (mean ± SEM, n = 3) (C, bottom panel). Western blot analysis and densitometric quantification of TFAM in VSMC lysates (D, top panel). Data are presented as fold change ± SEM where n = 4 (D, bottom panel). The amount of mtDNA was estimated by qPCR of cell-free DNA isolated from a fixed volume of VSMC cell-free culture medium (E). Data presented as cycle number ± SEM, n = 3-4. Mitochondrial membrane potential was measured by flow cytometry and the data presented as percentage of cells scored positive for high tetramethylrhodamine ethyl ester (TMRE) fluorescence. Data are mean ± SEM, n = 3-4 (F). ATP levels were measured using a colorimetric assay kit and the data presented are arbitrary units (mean ± SEM, n = 4 (G). Oxygen consumption rate (OCR) of aortic VSMCs isolated from young and old age rats. VSMCs were plated at a density of 15000 cells/well in the XF96 culture plates and OCR was measured at the basal level and in the presence of 2 μM oligomycin, 500 nM trifluoromethoxy carbonylcyanide phenylhydrazone (FCCP), and 0.5 μM antimycin A + 500 nM rotenone in VSMCs from young (H, left panel) and old age (I, left panel) LCR and HCR rats. Mitochondrial bioenergetic parameters were derived from VSMC OCR measurements (H and I, right panels). Data are mean ± SEM, n = 4. *P < 0.05; **P < 0.01; ****P < 0.0001.
