Critical limb ischemia causes severe damage to the skeletal muscle. This study develops a reproducible model of myotube
ischemia by simulating, in vitro, the critical parameters that occur in skeletal muscle
ischemia. Monolayers of C2C12 myoblasts were differentiated into mature myotubes and exposed to nutrition depletion,
hypoxia and
hypercapnia for variable time periods. A range of
culture media and gas mixture combinations were used to obtain an optimum ischemic environment. Nuclear staining, cleaved
caspase-3 and
lactate dehydrogenase (LDH) release assay were used to assess apoptosis and myotube survival. HIF-1α concentration of cell lysates, pH of
conditioned media as well as partial pressures of
oxygen (PO₂) and
carbon dioxide (PCO₂) in the media were used to confirm ischemic simulation. Culturing myotubes in depleted media, in a gas mixture containing 20% CO+80% N₂ for 6-12 h increased the PCO₂ and decreased the pH and PO₂ of
culture media. This attempts to mimic the in vivo ischemic state of skeletal muscle. These conditions were used to study the potential tissue-protective effects of
erythropoietin (EPO) in C2C12 myotubes exposed to
ischemia.
EPO (60 ng/ml) suppressed LDH release, decreased cleaved
caspase-3 and reduced the number of apoptotic nuclei, suggesting significantly decreased
ischemia-induced apoptosis in myotubes (P<0.01) and a potential role in tissue protection. Additional therapeutic agents designed for tissue protection can also be evaluated using this model.