Increased oxidative stress in <i>Sod1</i>^−/− mice leads to early onset limb muscle atrophy and weakness.

<p>(<i>A</i>) Gastrocnemius and extensor digitorum longus muscle mass expressed as % of body mass from in wild-type (WT, N = 47, 7 months old, white) and <i>Sod1</i>^−/− (N = 32, 7 months old, gray) mice. (<i>B, left</i>) Chart showing the average (10 trials) grip strength in WT (N = 14, 5–6 months old, white) and <i>Sod1</i>^−/− (N = 13, 5 months old, gray) mice. (<i>B, right</i>) Chart showing time to fall in the wire hanging assay for WT (N = 10, 5–6 months old, white) and <i>Sod1</i>^−/− (N = 8, 4–5 months old, gray) mice. All mice are between 4 to 10 months of age. Data are plotted as mean with error bars representing standard error. Statistics: <i>t-test</i>, *p<0.05, **p<0.01.</p

Quantification of neuromuscular junction (NMJ) denervation in wild-type and <i>Sod1</i>^−/− muscles.

<p>N depicts the number of animals used, and n depicts the total number of NMJs sampled.</p

Morphology-synaptic function relationship in wild-type and <i>Sod1</i>^−/− EDL neuromuscular junctions (NMJs).

<p>(<i>A</i>) Scatter plot showing the relationship between AChRs total area and its occupancy by axon shown as % of overlap over the AChRs total area in wild-type (WT, white) and <i>Sod1</i>^−/− (gray) mice. NMJs labeled as “#” and “1, 2 and 3” are examples of individual NMJs in WT and <i>Sod1</i>^−/− mice which were recorded for electrophysiology shown in (<i>C</i>). (<i>B</i>) Quantification of average AChR density (<i>F_int/Area</i>) in EDL muscle NMJs from WT (n = 30, white) and <i>Sod1</i>^−/− (n = 30, gray) mice. Data are plotted as mean with error bars representing standard error. (<i>C</i>) Representative images of individual EDL NMJs showing the overlap (yellow) between axonal (green) and AChRs (red) fluorescence and their respective spontaneous mEPP and evoked EPP electrophysiological responses in WT and <i>Sod1</i>^−/− mice. The areas and corresponding occupancies for the NMJs are shown by numbered circles in (<i>A</i>).</p

Aberrant electromyographic characteristics in <i>Sod1</i>^−/− muscle imply deficits in neurotransmission and denervation.

<p>(<i>A</i>) Representative traces of gastrocnemius compound muscle action potential (CMAP) when stimulated at 0.2 Hz and 10 Hz. Dashed line shows the amplitude of the initial peak. Traces were arbitrarily spaced 1ms apart for easy visualization. (<i>B</i>) Quantitative result of CMAP decrement in WT (N = 17, 7 months old, white) and <i>Sod1</i>^−/− (N = 11, 7 months old, gray) mice in response to repetitive nerve stimulation. Data are plotted as mean with error bars representing standard error. All mice are between 4 to 10 months of age. Statistics: <i>t-test</i>, *p<0.05, **p<0.01.</p

Degenerative morphological alterations in neuromuscular junctions (NMJs) in wild-type and <i>Sod1</i>^−/− muscles.

<p>NMJs in EDL and gastrocnemius muscles were visualized via Thy1-YFP fluorescence in the presynaptic axon and nerve terminal (green), as described in detail in Materials and Methods, and by staining acetylcholine receptors (nAChRs) with fluorophore conjugated α-bungarotoxin (red). Partially innervated and completely denervated NMJs in <i>Sod1</i>^−/− muscle were indicated (arrows). Axon thinning was also frequently observed in <i>Sod1</i>^−/− mice (arrow heads). Scale bars: 50µm.</p

Spontaneous and evoked synaptic release are impaired at <i>Sod1</i>^−/− EDL neuromuscular junctions (NMJs).

<p>(<i>A</i>, <i>top</i>) Representative traces of spontaneous mEPPs in wild-type (WT) and <i>Sod1</i>^−/− EDL muscles. (<i>A</i>, <i>bottom</i>) Amplitude-frequency histogram of spontaneous mEPPs for WT (white) and <i>Sod1</i>^−/− (gray). Data were analyzed from pooled results from 22 individual records (∼1000 mEPPs) and binned in 0.1 mV intervals. (<i>B</i>) Representative traces of evoked EPPs recorded in WT (black) and <i>Sod1</i>^−/− (gray) EDL muscle fibers. (<i>C</i>) EPP rundown analysis over 5s at 10 Hz and 40 Hz (EPP amplitude, <i>top</i>; normalized result, <i>bottom</i>) for WT (n = 34∼36 NMJs from N = 5 mice, 5 months old, triangles) and <i>Sod1</i>^−/− (n = 22∼28 NMJs from N = 5 mice, 5–6 months old, diamonds).</p

Summary of EDL muscle electrophysiological properties in wild-type (WT) and <i>Sod1</i>^−/− mice.

<p>Values are mean ± standard errors. N depicts the number of animals used, and n depicts the total number of NMJs sampled. Statistics: <i>t-test</i>, *p<0.05 and **p<0.01.</p

Determination of PMP22 protein carbonylation, changes in hydrophobicity, and aggregation in <i>Dbdb</i> mice.

<p>(<b>A</b>) A Kyte-Doolittle Hydropathy plot was determined for PMP22 hydrophobicity. FTC labeled (<b>B</b>) cytosolic and (<b>C</b>) detergent-soluble protein fractions were immunoprecipitated with anti-PMP22 polyclonal antibody, loaded onto SDS-PAGE gels and analyzed for protein carbonylation. (<b>D</b>) The detergent soluble fraction was analyzed by Western blot against PMP22. The higher order PMP22 aggregates are indicated by *. Results are expressed as mean ± SEM (n = 6; *<i>p</i><0.05 by two-tailed <i>t-test</i>).</p

p53 contributes to DOX-induced cardiac injury.

<p>A. Representative electron micrographs demonstrating cardiomyocyte injury and morphometric quantification of subcellular injury in cardiomyocytes in WT and p53^(−/−) mice. WT (a) and p53^(−/−) (b) mice treated with saline demonstrated normal ultrastructure of heart muscle with cardiomyocytes showing numerous mitochondria (M), prominent myofilaments (Myo) and lipids (Lip). WT (c) and p53^(−/−) (d) mice treated with DOX demonstrated the following pathologic changes: lysosomal degradation of mitochondria (asterisk), mitochondria with loss of cristae (arrow), peri-mitochondrial swelling (double arrow), disruption of mitochondrial membranes (arrowhead), and mitochondrial degeneration (d); scale bar, 1 µm. B. Pathologic changes were quantified for saline and DOX treated mice in the mitochondria and total cellular area (excluding nuclei). Quantification of damage for each specific compartment is expressed in the ratio of damaged area versus the total area. All graphs represent the Mean ± SEM for each group. *p<0.05 when compared with saline treated mice of the same genotype, and ^#p<0.05 when compared with WT mice treated with DOX. n = 6 or 7. C & D. Left ventricular function, assessed by percentage of ejection fraction (LV%EF) (C) and fractional shortening (LV%FS) (D), is expressed in percentage of change from basal levels caused by saline and DOX three days after injections for WT and p53^(−/−) mice. All bar graphs represent the Mean ± SEM for each group. ^#p<0.05 when compared to all three other groups. n = 6–11.</p

<i>In vitro</i> assessment of oxidative stress-induced aggregation of PMP22 protein.

<p>Purified PMP22 protein was subjected to <i>in vitro</i> oxidation with increasing concentrations of tert-butyl hydroperoxide. SDS-PAGE was performed on oxidized PMP22 protein from both the soluble and detergent-soluble protein fractions. Results are expressed as mean ± SEM (n = 6; *<i>p</i><0.05 by two-tailed <i>t-test</i>).</p