MiP2005: Session 7Mitochondrial Physiology Network 10.9: 94 (2005) - download pdf
Evidence for increased superoxide production in atrial fibrillation detected by ESR and HPLC based assay.
Bruno Fink, N Fink
Noxygen Science Transfer & Diagnostics GmbH, Elzach, Germany. - info@noxygen.de
Atrial fibrillation is the most common cardiac arrhythmia. It is associated with a 5-6 fold increase in the incidence of a stroke, due to almost exclusively to thrombus formation in the left atrial appendage. We hypothesized that this decrease in NO· may be due to increased superoxide (O2·-) production and oxidative destruction of NO·. To address this hypothesis, we induced AF in pigs using rapid atrial pacing.
We studied the reaction of O2·- with a new spin probe, 1-hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine (CMH), for analysis of O2·- production in suspension of cardiomyocytes and in isolated heart tissue. Parallel to ESR we investigated the superoxide formation using dihydroethidium and a newly developed HPLC method [1]. After 10 min of incubation, the intracellular concentration of CMH in cells reached 18.1 %. For loading the cells with DHE we incubated them for 20 min. Intracellular O2·- production was measured from PEG-SOD inhibited formation of 3-methoxy-carbonyl radical (CM·) or from selective DHE interaction product oxyethidium (OxyEt). A 1.8-fold increase in LAA O2·- from 80 to 140 a.u./mg tissue/10 min was confirmed using ESR and using formation of OxyEt. Treatment of cardiomyocytes with lactate leads to 3-fold increase in O2·- production in LAA.
We conclude that atrial fibrillation is associated with increased O2·- and decreased NO• production in the left atrial appendage. Using new synthesized cyclic hydroxylamine CMH and DHE we detected intracellular O2·- production from mitochondria. High cell permeability and high reactivity with O2·- of CMH allow effective detection of low amounts of intra- and extracellular O2·-. These findings we confirmed using dihydroethidium and new developed HPLC based assay.
1. Fink B, Laude K, McCann L, Doughan A, Harrison DG, Dikalov S (2004) Detection of intracellular superoxide formation in endothelial cells and intact tissues using dihydroethidium and an HPLC-based assay. Am. J. Physiol. Cell. Physiol. 287: C895-C902.
