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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online February 5, 2003 as doi:10.1096/fj.02-0729fje. |
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* Department of Biochemistry and Molecular Biology and CNR Unit for the Study of Mitochondria and Bioenergetics, and
Department of Emergency and Transplantation, University of Bari, Bari, Italy
2Correspondence: Department of Biochemistry and Molecular Biology, via E, Orabona, 4, 70126 Bari, Italy. E-mail: g.paradies{at}biologia.uniba.it
SPECIFIC AIMS
Reactive oxygen species (ROS) have been implicated in the heart tissue injury that follows ischemia and reperfusion. However, the mechanism for the enhanced ROS generation as well as subcellular targets of ROS attack remains still unclear. Cardiolipin has come to play an important role in mitochondrial bioenergetics. In this study, we tested the possibility that ROS generation during heart ischemia (30 min) and ischemia followed by reperfusion (15 min) may affect the activity of mitochondrial complex III via cardiolipin oxidative damage, leading to mitochondrial dysfunction.
PRINCIPAL FINDINGS
1. Enhanced capacity of mitochondria from ischemic and reperfused rat heart to produce H2O2
Mitochondria from control, ischemic, and reperfused rat heart were investigated for their capacity to generate oxygen radicals at the level of the respiratory chain. It has been shown that addition of succinate to aerobic mitochondria in the presence of rotenone (state 4) causes detectable H2O2 production, which arises from superoxide anion formed at the level of ubiquinone site of complex III. The amount of H2O2 generated in the presence of succinate and rotenone in these three types of mitochondria was measured. The production of H2O2 was enhanced by 16% and by 32% in mitochondria isolated from ischemic and reperfused rat hearts, respectively, compared with control hearts.
2. Impairment of complex III activity in mitochondria from ischemic and reperfused rat hearts
The activity of complex III was measured in mitochondria isolated from control ischemic, and reperfused rat hearts. In ischemic heart, mitochondrial complex III activity was reduced by 22% compared with normal hearts. In ischemic-reperfused hearts, the loss of complex III activity was much more pronounced, reaching 46%.
3. Loss of cardiolipin content in mitochondria from ischemic and reperfused rat hearts
The site of the ischemia and ischemia-reperfusion damage to complex III was approached in subsequent experiments. Mammalian complex III contains bound cardiolipin molecules that are essential for enzyme activity. Thus, ROS-induced oxidative damage to mitochondrial cardiolipin may be responsible for the observed defect of complex III activity. The composition of the major classes of phospholipids was analyzed in the mitochondrial preparations from normal ischemic, and ischemic-reperfused rat hearts. Alterations were found in the mitochondrial content of various phospholipid classes, the most pronounced change occurring in cardiolipin, the level of which decreased by 28% and by 50% as a function of ischemia and ischemia/reperfusion, respectively (Table 1
).
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4. Involvement of cardiolipin in the defect of complex III activity during ischemia-reperfusion
More firm evidence for the involvement of cardiolipin in the loss of complex III activity observed in mitochondria isolated from ischemic-reperfused hearts comes from the results of the experiment reported in Fig. 1
. Here, we investigated whether addition of exogenous cardiolipin to mitochondria isolated from ischemic-reperfused hearts was able to reverse the defect in complex III activity. As cardiolipin is poorly permeable to mitochondrial membrane, we used a method of fusion of vesicular lipids with mitochondrial membrane to enrich the inner mitochondrial membrane with cardiolipin. Fusion of mitochondria from ischemic-reperfused hearts with cardiolipin-liposomes resulted in an almost full restoration of complex III activity. No restoration was obtained with liposomes composed of other phospholipids or with peroxidized cardiolipin liposomes, suggesting that integral molecules of cardiolipin are required for the activation of complex III.
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CONCLUSIONS
A large body of experimental evidence indicates that reactive oxygen species play an important role in producing lethal cell injury associated with cardiac ischemia and reperfusion. The cellular sources and mechanism for the enhanced ROS generation, as well as subcellular targets of ROS attack and metabolic consequences of the resulting alterations to heart during ischemia-reperfusion injury, have not been established. Mitochondrial respiration is an important source of ROS production and hence a potential contributor to cardiac reperfusion injury. Reactive oxygen species lead to primary reaction and damage in the immediate surrounding where they are produced. Therefore, their effects should be greatest at the level of mitochondrial membrane constituents, including the complexes of the respiratory chain and phospholipids, particularly cardiolipin.
Due to its high content of unsaturated fatty acids and because of its location in the inner mitochondrial membrane near to the site of ROS production, cardiolipin appears particularly susceptible to ROS attack. This phospholipid has come to play an important role in mitochondrial bioenergetics. It would therefore be expected that changes that increase its susceptibility to oxidative damage would be deleterious to normal mitochondrial function. This condition is likely to be encountered on reperfusion of ischemic cardiac tissue. In this regard, recent results from this laboratory have shown that the activity of complex IV is decreased in mitochondria isolated from rat hearts subjected to ischemia-reperfusion. This decrease was attributed to ROS-induced oxidative damage of cardiolipin, which is required for full activity of this enzyme complex.
Very recently, it was reported that complex III contains tightly bound cardiolipin, which is essential for the catalytic function. We have reported that mitochondrial-mediated ROS production affects the activity of complex III via cardiolipin peroxidation in beef heart submitochondrial particles. Thus, it is possible that oxidative damage of cardiolipin may affect the activity of complex III in ischemic-reperfused rat hearts. This possibility was explored in the present investigation. Our results demonstrate that the activity of complex III was reduced by 22% and by 46% in mitochondria from ischemic and ischemic-reperfused rat hearts, respectively, compared with control hearts. These changes in complex III activity are quantitatively related to changes in the cardiolipin content. Thus, it is reasonable to assume that the molecular basis for the defect in the mitochondrial complex III activity associated with heart ischemia-reperfusion can be mainly ascribed to a specific loss in mitochondrial content of cardiolipin, which is essential for the functioning of this enzyme complex. This conclusion is further supported by the results of the experiment of Fig. 1
, demonstrating that exogenous added cardiolipin is able to fully restore the complex III activity in mitochondria from reperfused rat heart.
Complex III is considered an important source of oxygen radicals in mitochondria. The impairment of complex III activity due to the ROS induced cardiolipin oxidative damage, may increase the electron leak from the electron transport chain, generating more superoxide radicals and perpetuating a cycle of oxygen radical-induced damage, which ultimately leads to heart failure after ischemia-reperfusion. This hypothesis is summarized in Fig. 2
. The pattern of results presented here may prove useful in probing the molecular mechanism of ROS-induced oxidative damage to mitochondrial membrane constituents, which might contribute to mitochondrial dysfunction and contractile defects in ischemic heart upon reperfusion and in the development of effective antioxidants strategies.
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FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0729fje; to cite this article, use FASEB J. (February 5, 2003) 10.1096/fj.02-0729fje 
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