Abstract 15616: Inhibition of Mitochondrial Thioredoxin Reductase Leads to Increased Reactive Oxygen Species, Impaired Basal and β-Adrenergic Stimulated Cardiomyocyte Contractility in Mice
Chronic cardiac diseases are characterized by an overall oxidative milieu as evidenced by oxidation of two major thiol reductants glutathione (GSH) and thioredoxin (Trx). Release of diffusible H2O2 from mitochondria to the cytoplasm may lead to contractile dysfunction in these diseases. Mitochondrial Trx prevents the release of H2O2 from the mitochondrial compartment and is kept in its reduced (active) form by thioredoxin reductase (TrxR). Oxidative stress, where both GSH and Trx are oxidized, leads to reduced contractility, yet the effect of selective oxidation of Trx is unknown.
Hypothesis: TrxR activity protects against contractile dysfunction by improving mitochondrial Trx redox state, decreasing mitochondrial H2O2 and preventing oxidant-mediated decreases in contractility.
Methods: Freshly isolated mitochondria and ventricular myocytes were obtained from C57/BL6 mice. TrxR was inhibited by auronofin (AF), a gold compound effective against TrxR at nanomolar levels. Mitochondrial superoxide, NAD(P)H and H2O2 were assessed by two-photon microscopy. The redox state of Trx was examined via a redox western blot able to differentiate reduced versus oxidized forms. Cardiomyocyte function was assessed via sarcomere shortening and Ca2+ transients under basal and β-adrenergic stimulated conditions.
Results: Energizing mitochondria with substrate resulted in a 28±8% rise in the abundance of reduced (active) Trx (p<.05, n=3). Inhibiting TrxR with AF (100nM) prevented this effect and led to a 20-fold rise in H2O2 levels (from 50±4 to 1021±49 pmol H2O2/min/mg protein; p<.001). Mitochondrial membrane potential, NAD(P)H and GSH levels were unchanged, indicating a selective impact of AF on TrxR. When administered to cardiomyocytes (100 nM for 1hr), AF decreased basal and isoproterenol-stimulated (ISO, 10 nM) myocyte contractility. Fractional shortening was decreased by 26±0.04% (n=38, p<.001) under normal conditions and the ISO response was blunted by 22% (268±16% vs. 209±18% over baseline, p<.05). These changes were concomitant with a rise (16%±1) in cardiomyocyte H2O2 levels.
Conclusions: The Trx system is a critical antioxidant pathway in cardiomyocytes, and impairment of this pathway appears sufficient to trigger contractile dysfunction.
- Reactive oxygen intermediates
- Beta-adrenergic receptor agonists
- Oxidative stress
- © 2010 by American Heart Association, Inc.