| Mitochondria are a major cellular source of oxygen free radicals. Under normal conditions, free radical scavengers and antioxidant molecules protect cellular components from damage. However, during the progression of numerous degenerative conditions such as myocardial ischemia-reperfusion injury, mitochondria are a source of increased free radical generation and exhibit declines in respiratory function. It has therefore been suggested that oxidative damage to mitochondrial components plays a critical role in the pathology of these processes. Polyunsaturated fatty acids are the most susceptible cellular component to oxidation. Fatty acid oxidation (lipid peroxidation) results in the formation of aldehydes, alkenals, and hydroxyalkenals. 4-Hydroxy-2-nonenal (HNE), the major hydroxyalkenal produced, is highly cytotoxic by virtue of its reactivity with protein. Furthermore, degenerative diseases associated with increased mitochondrial free radical production are often paralleled by increases in HNE formation. It is therefore hypothesized that HNE represents an important mediator of free radical induced mitochondrial dysfunction. In this study, the effects of HNE on intact cardiac mitochondria were investigated to gain insight into potential mechanisms by which free radicals mediate mitochondrial dysfunction. Exposure of mitochondria to micromolar concentrations of HNE for brief periods resulted in loss of NADH-dependent state 3 respiration. HNE exerted its effects on intact mitochondria by decreasing the availability of NADH by inhibiting α-ketoglutarate dehydrogenase (KGDH). Because of the central role of KGDH in mitochondrial metabolism, inhibition of this enzyme would have profound effects on cellular homeostasis. It was therefore necessary to determine the molecular mechanism of inhibition to evaluate the role of this process during physiological conditions of oxidative stress. Mechanistic studies revealed that lipoic acid, a cofactor covalently bound to the E2 subunit of oxo-acid dehydrogenases and required for activity, was highly susceptible to HNE modification. To assess the occurrence of this modification during reperfusion of the ischemic heart, a physiological condition of oxidative stress associated with mitochondrial dysfunction and loss of KGDH activity, antibodies were raised against the lipoic acid-HNE adduct. These antibodies identified the presence of HNE modified lipoic acid during reperfusion, demonstrating for the first time a mechanism whereby free radicals induce mitochondrial dysfunction during oxidative stress. |
