CuZnSOD and nNOSlocalization in erythrocytes from Duchenne-Becker patients

Abstract

Aside of sarcolemma dystrophin deficiency associated with Duchenne-Becker muscular dystrophy (DMBD), higher oxidative pressure also contributes to muscle fiber degeneration and disease progression. The increased activity of catalase, CuZn superoxide dismutase (CuZnSOD) and glutathione transferase (GST) in order to protect both muscle and non-muscle cells from oxidative damage has been shown, but their cellular localization remains unsolved. Also, the role of neuronal nitric oxide synthase (nNOS) situated near dystrophin in sarcolemma has not been investigated in non-muscle cells. We have previously shown significant differences in the erythrocytesʼ catalase localization in DMBD patients comparing to control and this study aimed to further examine presence and localization of CuZnSOD and nNOS in erythrocytes of DMBD patients. In the present study immunogold labeling was used to compare CuZnSOD and nNOS localizations in erythrocytes from patients with Duchenne-Becker muscular dystrophy and from age-matched normal boys. Immunogold labeling was performed on ultrathin Araldite sections (70 nm), using antibodies against CuZnSOD and nNOS, examined under the transmission electron microscope and quantified by Image J. Significantly higher amount of CuZnSOD and reduced amount of nNOS is present in erythrocytes of DMBD patients compared to control. Ultrastructural localization of CuZnSOD and nNOS by gold immunocytochemistry showed that the label is mostly localized in erythrocytes’ cytoplasm, with sporadic decoration of the cell membrane. In addition, clustering of nNOS-gold complexes was observed in DBMD samples. The lesser presence of nNOS is observed in acquired and inherited neuromuscular disorders including Duchenne muscular dystrophy while the increase of CuZnSOD is noticed in vascular dysfunction in various diseases. In the light of disturbed erythrocyte structure, deficiency of nNOS may lead to and may contribute to reduced blood flow, e.g. tissue ischemia and thus degeneration of muscle fibers in DMBD as well. The timely treatment of muscular dystrophy patients, based on reduction of oxidative damage, could diminish muscle injury, and possibly change progression of the disease.