Mitochondrial disease in mouse results in increased oxidative stress

Abstract

It has been hypothesized that a major factor in the progression of mitochondrial disease resulting from defects in oxidative phosphorylation (OXPHOS) is the stimulation of the mitochondrial production of reactive oxygen species (ROS) and the resulting damage to the mtDNA. To test this hypothesis, we examined the mitochondria from mice lacking the heart/muscle isoform of the adenine nucleotide translocator (Ant1), designated Ant1(tm2Mgr) (-/-) mice. The absence of Ant1 blocks the exchange of ADP and ATP across the mitochondrial inner membrane, thus inhibiting OXPHOS. Consistent with Ant1 expression, mitochondria isolated from skeletal muscle, heart, and brain of the Ant1-deficient mice produced markedly increased amounts of the ROS hydrogen peroxide, whereas liver mitochondria, which express a different Ant isoform, produced normally low levels of hydrogen peroxide. The increased production of ROS by the skeletal muscle and heart was associated with a dramatic increase in the ROS detoxification enzyme manganese superoxide dismutase (Sod2, also known as MnSod) in muscle tissue and muscle mitochondria, a modest increase in Sod2 in heart tissue, and no increase in heart mitochondria. The level of glutathione peroxidase-1 (Gpx1), a second ROS detoxifying enzyme, was increased moderately in the mitochondria of both tissues. Consistent with the lower antioxidant defenses in heart, the heart mtDNAs of the Ant1-deficient mice showed a striking increase in the accumulation of mtDNA rearrangements, whereas skeletal muscle, with higher antioxidant defenses, had fewer mtDNA rearrangements. Hence, inhibition of OXPHOS does increase mitochondrial ROS production, eliciting antioxidant defenses. If the antioxidant defenses are insufficient to detoxify the ROS, then an increased mtDNA mutation rate can result.

Figure 1

Mitochondrial H₂O₂ production is maximized in Ant1^tm2Mgr (−/−) skeletal muscle and heart. The bars represent the mean ± SD level of H₂O₂ production for mitochondria from normal (+/+) and Ant1^tm2Mgr (−/−) animals with and without antimycin A. ∗∗, P < 0.01 for normal (+/+) as compared with (−/−) mitochondria for the same tissue; n = 5 for each group.

Figure 2

Mitochondrial H₂O₂ production is increased partially in Ant1^tm2Mgr (−/−) brain cortex and cerebellum. Data are presented as in Fig. 1. ∗, P < 0.05 for normal (+/+) as compared with (−/−) mitochondria from the same tissue without inhibitor; ∗∗, P < 0.01 for (−/−) without inhibitor as compared with (−/−) with inhibitor; n = 4–6 for each group.

Figure 3

Mitochondrial H₂O₂ production is normal in Ant1^tm2Mgr (−/−) livers. Data are presented as in Fig. 1. ∗, P < 0.05 for comparison of (+/+) or (−/−) mitochondria with or without inhibitor; n = 5 for each group.

Figure 4

Increased Sod2 and Gpx1 protein levels in Ant1^tm2Mgr (−/−) skeletal muscle and heart. Western blot analysis was performed on total tissue lysates (a and b) or isolated mitochondria (c and d). Each of the channels of the blots was loaded with equal amounts of protein from a different animal, and the blots were probed with polyclonal antibodies raised against Sod2 (Left) or Gpx1 (Right). The molecular mass of the Sod2 monomer is 24 kDa, and that of the Gpx1 monomer is 22 kDa.

Figure 5

Increased Sod2 mRNA in Ant1^tm2Mgr (−/−) skeletal muscle and heart as determined by Northern blot analysis of heart and skeletal muscle. The mean mRNA levels were calculated from a total of five determinations. For skeletal muscle, P < 0.01 for (+/+) as compared with (−/−) mice.

Figure 6

Increased mtDNA rearrangements in 16- to 20-month-old Ant1^tm2Mgr (−/−) heart. (a) Heart LX-PCR products: lanes 1–2, wild-type (+/+); lanes 3–4, Ant1^tm2Mgr (+/−); lanes 5–8, Ant1^tm2Mgr (−/−); lane 9, 32-month-old wild-type heart DNA; and lane 10, no template DNA. Each lane (–9) represents the products of a reaction that used template DNA from a different animal. The arrow indicates a 15.9-kb mtDNA product. (b) Heart minicircle PCR products. Sample order as in a. The “362-bp standard region” is the product encompassed between the two primers (14).