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Skeletal Muscle Atrophy in Sedentary Zucker Obese Rats Is Not Caused by Calpain-Mediated Muscle Damage or Lipid Peroxidation Induced by Oxidative Stress

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Skeletal Muscle Atrophy in Sedentary Zucker Obese Rats Is Not Caused by Calpain-Mediated Muscle Damage or Lipid Peroxidation Induced by Oxidative Stress

Nancy Pompeani et al. J Negat Results Biomed.

Abstract

Background: Skeletal muscle undergoes significant atrophy in Type 2 diabetic patients and animal models. We aimed to determine if atrophy of Zucker rat skeletal muscle was due to the activation of intracellular damage pathways induced by excess reactive oxygen species production (specifically those associated with the peroxidation of lipid membranes) and calpain activity. 14 week old obese Zucker rats and littermate lean controls were injected with 1% Evan's Blue Dye. Animals were anaesthetised and extensor digitorum longus and soleus muscles were dissected, snap frozen and analysed for ROS-mediated F2-isoprostane production and calpain activation/autolysis. Contralateral muscles were histologically analysed for markers of muscle membrane permeability and atrophy.

Results: Muscle mass was lower in extensor digitorum longus and soleus of obese compared with lean animals, concomitant with reduced fibre area. Muscles from obese rats had a higher proportional area of Evan's Blue Dye fluorescence, albeit this was localised to the interstitium/external sarcolemma. There were no differences in F2-isoprostane production when expressed relative to arachidonic acid content, which was lower in the obese EDL and soleus muscles. There were no differences in the activation of either μ-calpain or calpain-3.

Conclusions: This study highlights that atrophy of Zucker rat skeletal muscle is not related to sarcolemmal damage, sustained hyperactivation of the calpain proteases or excessive lipid peroxidation. As such, establishing the correct pathways involved in atrophy is highly important so as to develop more specific treatment options that target the underlying cause. This study has eliminated two of the potential pathways theorised to be responsible.

Figures

Figure 1
Figure 1
Plasma insulin and glucose concentration (normalised to body weight) and relative insulin sensitivity as determined by the quantitative insulin sensitivity check index (QUICKI) in lean versus obese Zucker rats (n = 7). ***p < 0.001; **p < 0.005; *p < 0.05 Lean versus Obese Zucker.
Figure 2
Figure 2
Skeletal muscle histology. (A) Average fibre area of skeletal muscle tissues (µm2). H & E stained skeletal muscle sections of EDL (B) and soleus (C) of obese and lean rats. Magnification ×100. *p < 0.01 vs obese. #p < 0.01 vs EDL. n = 14 for each group.
Figure 3
Figure 3
EBD staining in muscle fibres of Zucker rats. Proportion (%) of cross-sectional area that is EBD+ (A). Average Fluorescence intensity (arbitrary units) of EBD+ areas (B). Mean strength of EBD signal in myofibres and the interstitium as scored by a semi-quantitative Likert scale (C). Muscle sections of EDL (D), and Soleus (E) of obese and lean rats showing EBD accumulation around the skeletal muscle membranes of obese rats (solid arrows), with minimal penetration of the dye in lean rats. Magnification ×100. *p < 0.05 vs obese. #p < 0.05 vs EDL. n = 14 for each animal group.
Figure 4
Figure 4
Isoprostane and arachidonic acid content of Zucker skeletal muscle. (A) Total muscle F2-isoprostane production per md tissue. (B) Total muscle arachidonic acid content. (C) F2-isoprostane production corrected for arachidonic acid content. *p < 0.05 vs obese. **p < 0.01 vs obese. #p < 0.05 vs EDL. ##p < 0.01 vs EDL. n = 13 for each group.
Figure 5
Figure 5
Calpain activity of Zucker skeletal muscle. Percentage of activated Calpain-3 (A) and µ-Calpain (B). Western blots show either μ-Calpain or Calpain-3 in obese and lean rats (C), with molecular mass markers, taken as a white light image prior to chemiluminescence and then images superimposed, indicated on the left. n = 6 for all groups. *indicates the non-specific band seen in rodent skeletal muscle with this antibody.

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