The STAT3 Inhibitor Stattic Impairs Cardiomyocyte Mitochondrial Function Through Increased Reactive Oxygen Species Formation

The signal transducer and activator of transcription 3 (STAT3) transduces stress signals from the plasma membrane to the nucleus but has recently also been identified in mitochondria. Inhibition of cardiomyocyte mitochondrial STAT3 with the STAT3-specific inhibitor Stattic decreases ADP-stimulated respiration and enhances calcium-induced mitochondrial permeability transition pore (MPTP) opening. The aim of the present study was to analyze whether or not these effects of STAT3 inhibition by Stattic are mediated by the formation of reactive oxygen species (ROS). The H2O2 formation from isolated rat left ventricular mitochondria was measured continuously in the presence of the complex 1 substrates glutamate and malate using the H2O2 indicator Amplex UltraRed. Stattic dose-dependently increased mitochondrial ROS formation (slope of Amplex UltraRed fluorescence/ time; DMSO: 0.39 +/- 0.01; 1 mu M Stattic: 0.40 +/- 0.03; 10 mu M Stattic: 0.71 +/- 0.04; 25 mu M Stattic: 1.43 +/- 0.05; 50 mu M Stattic: 3.53 +/- 0.23; 100 mu M Stattic: 9.23 +/- 0.69, n= 5 mitochondrial preparations, p<0.05 for 10-100 mu M Stattic). The increase in the ROS signal by 100 mu M Stattic was abolished in the presence of the ROS scavenger N-acetylcysteine (Nac, 0.46 +/- 0.02, n=7, p<0.05). Mitochondria treated with 100 mu M Stattic produced less ATP than control mitochondria (86 +/- 3 arbitrary units (a.u.) vs. 128 +/- 7 a.u., n=9, p<0.05). Again, in the presence of Nac ATP production was similar between Stattic-treated and control mitochondria (142 +/- 4 a.u. vs. 147 +/- 12 a.u., n=5, p=ns). MPTP opening was induced by lower amounts of calcium in Stattic-treated than in control mitochondria (in nmol CaCl2/mg protein, Stattic: 507 +/- 57, n=7; control: 857 +/- 70, n=7, p<0.05). There was no difference in calcium-induced MPTP opening between Stattic-treated (833 +/- 57, n=6) and control mitochondria (921 +/- 75, n=7, p=ns) in the presence of Nac. Taken together, our data show that inhibition of mitochondrial STAT3 by Stattic impacts on mitochondrial ATP production and MPTP opening through enhanced ROS formation.