Reversible redox-dependent modulation of mitochondrial aconitase and proteolytic activity during in vivo cardiac ischemia/reperfusion

Abstract

Prooxidents can induce reversible inhibition or irreversible inactivation and degradation of the mitochondrial enzyme aconitase. Cardiac ischemia/reperfusion is associated with an increase in mitochondrial free radical production. In the current study, the effects of reperfusion-induced production of prooxidants on mitochondrial aconitase and proteolytic activity were determined to assess whether alterations represented a regulated response to changes in redox status or oxidative damage. Evidence is provided that ATP-dependent proteolytic activity increased during early reperfusion followed by a time-dependent reduction in activity to control levels. These alterations in proteolytic activity paralleled an increase and subsequent decrease in the level of oxidatively modified protein. In vitro data supports a role for prooxidants in the activation of ATP-dependent proteolytic activity. Despite inhibition during early periods of reperfusion, aconitase was not degraded under the conditions of these experiments. Aconitase activity exhibited a decline in activity followed by reactivation during cardiac reperfusion. Loss and regain in activity involved reversible sulfhydryl modification. Aconitase was found to associate with the iron binding protein frataxin exclusively during reperfusion. In vitro, frataxin has been shown to protect aconitase from [4Fe-4S](2+) cluster disassembly, irreversible inactivation, and, potentially, degradation. Thus, the response of mitochondrial aconitase and ATP-dependent proteolytic activity to reperfusion-induced prooxidant production appears to be a regulated event that would be expected to reduce irreparable damage to the mitochondria.

Fig. 1.

Alterations in mitochondrial carbonylated proteins and proteolytic activity during coronary ischemia/reperfusion. Mitochondria were prepared from left ventricular free wall after 90 min of sham-operated perfusion (P), 30 min of occlusion (I₃₀), or 30 min of occlusion followed by 5, 15, or 60 min of reperfusion (R₅,R₁₅, and R₆₀, respectively). (A) Oxidatively modified proteins were detected upon treatment of mitochondrial extracts (5.0 μg of protein per lane) with 2,4-dinitrophenylhydrazine to derivatize protein carbonyl groups followed by Western blot analysis using polyclonal antibodies to the 2,4-dinitrophenyl moiety. (B) Mitochondrial proteolytic activity was measured as the rate of degradation of FITC-casein in the presence of 8.0 mM ATP. Values represent the mean ± SEM for five separate experiments. (C) The level of Lon protease was detected by Western blot analysis of mitochondrial protein (10.0 μg per lane) using a polyclonal antibody against Lon protease.

Fig. 2.

Effect of coronary ischemia/reperfusion on mitochondrial aconitase content and activity. Mitochondria were prepared from left ventricular free wall after 90 min of sham-operated perfusion (P), 30 min of occlusion (I₃₀), or 30 min of occlusion followed by 5, 15, or 60 min of reperfusion (R₅,R₁₅, and R₆₀, respectively). (A) Mitochondrial protein (10.0 μg per lane) was subjected to Western blot analysis using a polyclonal antibody against aconitase. Densitometric analysis was performed (National Institutes of Health image software) on Western blots of mitochondrial protein isolated from four separate rats for each experimental protocol, and results are represented as mean ± SD with the mean for P intensity assigned a value of 100. Using a two-tailed t test, no statistically significant differences were observed between any of the experimental conditions indicated. (B) After each experimental protocol, mitochondria were disrupted and aconitase activity was determined as described in Materials and Methods. Values represent the mean ± SEM for five separate experiments.

Fig. 3.

Alterations in aconitase association with frataxin, EPR detectable [3Fe-4S]⁺¹ cluster, and oxidant-derived sulhydryl modifications during coronary ischemia/reperfusion. (A) Mitochondria were prepared from left ventricular free wall after 90 min of sham-operated perfusion (P), 30 min of occlusion (I₃₀), or 30 min of occlusion followed by 5, 15, or 60 min of reperfusion (R₅,R₁₅, and R₆₀, respectively). Mitochondria were disrupted in 0.05% Triton X-100, and immunoprecipitation was performed by using a specific antiserum raised against rat aconitase. Immunoprecipitated protein was then evaluated by Western blot analysis using polyclonal antibody specific to frataxin. (B) Mitochondria (30 mg/ml) (P, solid black line; I_30, dashed black line; and R₅, R₁₅, and R_60, solid black lines) were analyzed by EPR spectroscopy as described in Materials and Methods. To obtain a known EPR spectrum for the [3Fe-4S]⁺¹ cluster of aconitase, intact mitochondria (30 mg of protein per ml) were incubated with 2.0 mM H₂O₂ in the presence of 2.0 mM citrate for 40 s (solid gray line). (C) Mitochondria were prepared from left ventricular free wall after 90 min of sham-operated perfusion or 30 min of occlusion followed by 5 min of reperfusion. Mitochondria were then solubilized in 0.05% Triton X-100 and incubated with 5.0 mM DTT for 1.0 min before measurement of aconitase activity. Values represent the mean ± SEM for five separate experiments.