Adenosine- and 2-chloro-adenosine-induced cytopathic effects on myoblasticcells and myotubes: involvement of different intracellular mechanisms

We recently suggested that, in muscular dystrophies, the excessive accumula tion of adenosine as a result of an altered purine metabolism may contribut e to progressive functional deterioration and muscle cell death. To verify this hypothesis, we have taken advantage of C2C12 myoblastic cells, which c an be differentiated in vitro into multinucleated cells (myotubes). Exposur e of both proliferating myoblasts and differentiated myotubes to adenosine or its metabolically-stable analog, 2-chloro-adenosine, resulted in apoptot ic cell death and myotube disruption. Cytotoxicity by either nucleoside did not depend upon extracellular adenosine receptors, but, at least in part, by entry into cells via the membrane nitro-benzyl-thio-inosine-sensitive tr ansporter. The adenosine kinase inhibitor, 5-iodotubercidin, prevented 2-ch loro-adenosine-induced (but not adenosine-induced) effects, suggesting that an intracellular phosphorylation/activation reaction plays a key role in 2 -chloro-adenosine-mediated cytotoxicity. Conversely, adenosine cytotoxicity was aggravated by the addition of homocysteine, suggesting that adenosine effects may be due to the accumulation of S-adenosyl-homocysteine, which bl ocks intracellular methylation-dependent reactions. Both nucleosides marked ly disrupted the myotube structure via an effect on the actin cytoskeleton; however, also for myotubes, there were marked differences in the morpholog ical alterations induced by these two nucleosides. These results show that adenosine and 2-chloro-adenosine induce apoptosis of myogenic cells via com pletely different metabolic pathways, and are consistent with the hypothesi s that adenosine accumulation in dystrophic muscles may represent a novel p athogenetic pathway in muscle diseases. (C) 2000 Elsevier Science B.V. All rights reserved.