Research Paper Volume 1, Issue 4 pp 425—437
Mitochondrial dysfunction and oxidative stress mediate the physiological impairment induced by the disruption of autophagy
Figure 1. Impaired mitochondrial function in Atg7 deficient skeletal muscle. (A) Western blot analysis of protein lysate obtained from mice with a conditional deletion of Atg7 within skeletal muscle (Atg7F/F:MCK-Cre) or control animals (Atg7F/+:MCK-Cre). Relative levels of Atg7, p62 and actin (loading control) were assessed. (B) Representative stained histological sections from soleus muscle. (C) Electron micrographs of skeletal muscle from 8 week old Atg7F/+:MCK-Cre or Atg7F/F:MCK-Cre mice. Electron micrographs demonstrate the accumulation of dysmorphic mitochondria within the Atg7F/F:MCK-Cre muscle. (D) Western blot analysis of purified mitochondria obtained from Atg7F/F:MCK-Cre or control animals Atg7+/+:MCK-Cre demonstrating an apparent similar level of electron transfer subunit composition. Mitochondrial protein lysates were probed for the Complex I component protein NDFS3 (NADH-ubiquinone oxidoreductase 30 kDa subunit), the Complex II component FP (flavoprotein subunit of complex II ), the Complex III protein Core 1 (Ubiquinol-cytochrome-c reductase complex core protein 1) and the mitochondrial membrane protein VDAC. (E) Blue Native gel electrophoresis using mitochondrial extracts isolated from control and Atg7-/- tissues demonstrating similar stoichiometry and assembly of electron transfer complex components. (F) Representative oxygen consumption tracings of mitochondria isolated from the skeletal muscle of 12 month old Atg7+/+:MCK-Cre or Atg7F/F:MCK-Cre mice. Atg7-deficient skeletal muscle demonstrated a pronounced reduction in respiration when assessed in the presence of the Complex II dependent substrate succinate. Rotenone was routinely added to prevent reverse electron transport. Measurements were made in the absence (State IV) and presence (State III) of ADP and following the Complex III inhibitor antimycin A (AA). (G) Composite determinations of mitochondrial respiration in the presence of succinate and rotenone. Graph represents the mean +/- SEM from Atg7+/+:MCK-Cre (n=3) or Atg7F/F:MCK-Cre mice (n=3). * p≤0.05; ** p≤0.01.