MnSOD activity protects mitochondrial morphology of quiescent fibroblasts from age associated abnormalities

Abstract

Previously, we have shown manganese superoxide dismutase (MnSOD) activity protects quiescent human normal skin fibroblasts (NHFs) from age associated loss in proliferative capacity. The loss in proliferative capacity of aged vs. young quiescent cells is often characterized as the chronological life span, which is clearly distinct from replicative senescence. We investigate the hypothesis that MnSOD activity protects the mitochondrial morphology from age associated damage and preserves the chronological life span of quiescent fibroblasts. Aged quiescent NHFs exhibited abnormalities in mitochondrial morphology including abnormal cristae formation and increased number of vacuoles. These results correlate with the levels of cellular reactive oxygen species (ROS) and mitochondrial morphology in MnSOD homozygous and heterozygous knockout mouse embryonic fibroblasts. The abnormalities in mitochondrial morphology in aged quiescent NHFs cultured in presence of 21% oxygen concentration were more severe than NHFs cultured in 4% oxygen environment. The alteration in mitochondrial morphology was associated with a significant increase in cell population doubling: 54h in 21% compared to 44h in 4% oxygen environment. Overexpression of MnSOD decreased ROS levels, and preserved mitochondrial morphology in aged quiescent NHFs. These results demonstrate that MnSOD activity protects mitochondrial morphology and preserves the proliferative capacities of quiescent NHFs from age associated loss.

Figure 1. MnSOD overexpression protects mitochondrial morphology of quiescent NHFs from age associated abnormalities

A. Transmission electron microscopy pictures of mitochondria in young (2 days) and old (50 days) contact inhibited quiescent NHFs cultured in 21% oxygen environment (left panels). Two day old quiescent NHFs were infected with 30 MOI of AdBgl II and AdMnSOD adenoviruses, and cultured for a total of 50 days. Cells were collected by scraping and processed for visualization of mitochondrial morphology using transmission electron microscopy (right panels); scale ranges from 100 nm to 0.2 µm. Representative of 2 or more experiments. B. Total cellular protein extracts prepared from 7 days control and adenovirus infected quiescent NHFs were analyzed for MnSOD protein levels by immunoblotting (Left panel), and MnSOD activity using native gel-electrophoresis assay (Right panel). C. Electron resonance spectroscopy measurements of cellular ROS levels in control, AdBgl II, and AdMnSOD infected quiescent NHFs; Left panel: representative ESR spectra, Right panel: ESR peak height per million cells. Asterisk indicates significant difference between MnSOD overexpressing NHFs compared to control, and AdBgl II infected cells. n=3, p < 0.05.

Figure 2. MnSOD activity protects mitochondrial morphology and suppresses cellular ROS levels

A. MnSOD genotype MEFs were cultured in 4% oxygen environment and harvested for transmission electron microscopy visualization of mitochondria morphology; scales range from 100 nm to 0.2 µm. Representative of 2 or more experiments. B. MnSOD genotype MEFs cultured in 4% oxygen environment were incubated with MitoTracker (left panel), and MitoSox (right panel). Fluorescence was measured by flow cytometry. Asterisks (left panel) indicate significant difference in MitoTracker fluorescence in MnSOD (−/−) compared to (+/+) and (+/−) MEFs; asterisks in right panel represent significant difference in MitoSOX fluorescence in MnSOD (+/+) and (+/−) compared to MnSOD (−/−) MEFs; n=3, p<0.05.

Figure 3. Lower oxygen environment protects quiescent NHFs from age associated abnormalities in mitochondrial morphology and loss in proliferative capacity

A. Transmission electron microscopy visualization of mitochondrial morphology in quiescent NHFs cultured in 1, 4, and 21% oxygen environment. Scales range from 100 nm to 0.2 µm. Representative of 2 or more experiments. B. Upper panel: total cellular protein extracts were analyzed for MnSOD and CuZnSOD protein levels by immunoblotting. Actin levels were used for loading correction. Quantitation was performed using Alpha Imager and Image J software. Band intensities were normalized to actin in individual samples and fold change calculated relative to control. Lower panel: Relative MnSOD mRNA levels in 12 days quiescent NHFs in 4% vs. 21% oxygen tension using real-time PCR as described in Materials and Methods. Relative MnSOD mRNA levels were calculated relative to 2-day quiescent NHFs. C. Growth characteristics of NHFs replated from 60 and 80 days quiescent NHFs cultured in 1, 4, and 21% oxygen environment. Replated cells were cultured in the same oxygen environment and cell numbers counted at indicated times. Asterisks indicate significant difference among different oxygen concentrations; # indicates significant difference in 80 days compared to 60 days quiescence; n=3, p < 0.05.