Declines in mitochondrial respiration during cardiac reperfusion: Age-dependent inactivation of α-ketoglutarate dehydrogenase

Abstract

We previously reported that cardiac reperfusion results in declines in mitochondrial NADH-linked respiration. The degree of inactivation increased with age and was paralleled by modification of protein by the lipid peroxidation product 4-hydroxy-2-nonenal. To gain insight into potential sites of oxidative damage, the present study was undertaken to identify specific mitochondrial protein(s) inactivated during ischemia and reperfusion and to determine which of these losses in activity are responsible for observed declines in mitochondrial respiration. Using a Langendorff rat heart perfusion protocol, we observed age-dependent inactivation of complex I during ischemia and complex IV and ␣-ketoglutarate dehydrogenase during reperfusion. Although losses in complex I and IV activities were found not to be of sufficient magnitude to cause declines in mitochondrial respiration, an age-related decrease in complex I activity during ischemia may predispose old animals to more severe oxidative damage during reperfusion. It was determined that inactivation of ␣-ketoglutarate dehydrogenase is responsible, in large part, for observed reperfusion-induced declines in NADHlinked respiration. ␣-Ketoglutarate dehydrogenase is highly susceptible to 4-hydroxy-2-nonenal inactivation in vitro. Thus, our results suggest a plausible mechanism for age-dependent, reperfusion-induced declines in mitochondrial function and identify ␣-ketoglutarate dehydrogenase as a likely site of free radical-mediated damage.Cardiac mitochondria exhibit declines in the rate of NADHlinked respiration and ATP synthesis as a result of myocardial ischemia and reperfusion (1-5). Loss in mitochondrial respiratory function because of reperfusion is more pronounced in senescent relative to adult heart (1). Because mitochondria are a site of free radical production and oxidative damage during reperfusion (6-9), it is likely that free radical events contribute to reperfusion-induced declines in mitochondrial function. The goals of this study were to (i) identify and distinguish between specific mitochondrial protein(s) inactivated as a result of ischemia and͞or reperfusion; (ii) determine which of these losses in activity are responsible for observed declines in NADH-linked respiration; and (iii) evaluate the effects of age on these processes. Identification of enzymes inactivated during ischemia and reperfusion is necessary for rigorous assessment, at the molecular level, of events responsible for enzyme inactivation. Evaluation of which losses in activity are responsible for declines in the rate of overall mitochondrial respiration indicates the physiological consequences of enzyme inactivation. Distinguishing between ischemia-and reperfusioninduced declines in function will provide insight into factors contributing to free radical production and mitochondrial dysfunction. Thus, these studies are critical to elucidating mechanism(s) by which free radicals mediate declines in mitochondrial function during cardiac reperfusion.We have previously demonstrated ...

Two subpopulations of mitochondria in the aging rat heart display heterogenous levels of oxidative stress

“…In vitro, exposures to 4-HNE deactivates a number of mitochondrial proteins, such as glucose-6-phosphate, pyruvate, and α-ketoglutarate dehydrogenases and the adenine nucleotide exchanger [5,[40][41][42]. Similarly, multiple subunits on Complex IV (subunits II, IV, Vb, VIIa, VIIc, and VIII) are readily modified by 4-HNE and lead to its inactivation [22].…”

“…In addition, there is an age-related decline in the capacity to withstand stress, such as ischemia/reperfusion [3][4][5]. In its most severe form, cardiac decay results in congestive heart failure, one of the leading causes of death in people over the age of 65.…”

Mitochondria: A mirror into cellular dysfunction in heart disease

“…As a result, the OxPhos pathway of the mitochondria has been the subject of numerous investigations. Complex 1 of the OxPhos pathway has been shown to be affected by I/R [20,[90][91][92][93], whereby the activity of the complex is significantly reduced, with concurrent changes in the abundance of several complex 1 subunits, as identified by proteomic techniques including 2-DE [76,94]. To investigate the discrete compositional alterations with I/R, isolated mitochondria treated with elevated levels of ROS and/or NO resulted in the inactivation of complex 1 [95][96][97][98][99][100][101], with in vitro preparations showing a similar inactivation of complex 1 by increased concentration of both ROS and Ca 21 [102].…”

Dissociation of Cytochrome c from the Inner Mitochondrial Membrane during Cardiac Ischemia

“…Immediate consequences of oxygen deprivation are a reduction in the rate of oxidative phosphorylation, incomplete oxidation of glycolytic metabolites, depletion of creatine phosphate and ATP, and metabolic acidosis (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11). Declines in both pH and ATP concentration adversely affect the maintenance of ionic gradients and are in part responsible for intracellular Ca 2ϩ overload (4,5).…”