To elucidate the mechanisms of
ischemia-mediated
myopathy using in vitro model, changes of
purine nucleotides,
membrane lipid peroxidation(
TBARS), intracellular
calcium ([Ca2+]i)levels, generation of
free radicals, and
deoxyribonucleic acid (DNA) fragmentation were examined in mouse-derived C2C12 myotubes under the condition with an inhibition of glycolytic and oxidative metabolism as the ischemic condition. In
purine nucleotides, intracellular
adenosine triphosphate (
ATP) and
guanosine triphosphate (
GTP) concentrations rapidly and significantly decreased after the treatment with
ischemia. No remarkable differences were observed in other
purine nucleotides, with the exception of
inosine monophosphate (
IMP) and extracellular
hypoxanthine levels, both of which increased significantly during the
ischemia. The
lactate dehydrogenase activity in culture supernatant of C2C12 myotubes increased significantly from 2 to 4 hr after the
ischemia. On the generation of
free radicals, no spectrum was detected in supernatants throughout the observation period, whereas supernatant
TBARS concentration increased rapidly and significantly after the
ischemia. The relative intensity of [Ca2+]i significantly increased after the
ischemia. On the fragmented
deoxyribonucleic acid(
DNA), no TUNEL positive cells was detected in C2C12 myotubes after 1 hr of the
ischemia, however the positive cell percentage subsequently increased. From these results, it was suggested that the ischemic condition induced changes of membrane permeability and increase of [Ca2+]i, both of which lead to cell membrane damage, although a
free radical generation was not detected. The ischemic condition also induced the release of substrate
hypoxanthine for
free radical generation and might initiate the apoptotic pathway in C2C12 myotubes.