Myofibrils (Mr), which are fully competent of the contractile muscle apparatus and capable of calcium induced shortening in the presence of ATP, were used as a model to study the involvement of myoglobin in muscle impairment under oxidative stress. Mf were isolated from chicken legs and hearts. Incubation of Mf (1-2 mg/ml) in isotonic, pH 7.0 buffer at 37°C, with 30 #M myoglobin and 200/tM H202 but not each alone, resulted in crosslinking of the Mf within 90 minutes as appeared by formation of aggregates under light microscope. SDSPAGE gels revealed inter-molecular crosslinking of myosin monomers, but not actin, even after reducing S-S bonds. Appearance of a fluorescence emission typical to bi-tyrosines in the oxidized Mf (excitation wavelength of 325 nm and emission maximum at 403 nm), indicated the involvement of tyrosine residues in the inter-molecular crosslinked Mf. Globin-free heroin revealed the same peroxidative reactivity as that of myoglobin. Replacing of H202 bolus addition by glucose-oxidase/glucose mixture which yielded a low flow of H202 (0.03 #M/minute), resulted in similar protein crosslinking. Calcium ATPase activity of the crosslinked Mf was practically lost (residual of less than 10%). Because myoglobin is a vital component of muscles, our findings seem to be of primary importance in understanding the pathophysiology of cardiac and skeletal muscle tissues under ischemic conditions in which low flow of peroxidants is found.
|Published - 1997