Identification and physiological activity of survival factor released from cardiomyocytes during ischaemia and reperfusion

Aims: We carried out a screening of survival factors released from cells exposed to simulated ischaemia and reperfusion (sI/R) using the embryonic rat heart-derived cell line, H9c2 cells, and examined the physiological role of the identified factor. Method and results: The culture medium supernatant of H9c2 cells exposed to sI/R was separated by column chromatography and the fractions examined for survival activity. The protein with survival activity was identified by mass spectrometry, and its physiological role was examined in the models of ischaemia. Cell survival activity was detected in at least three fractions of the cell supernatant collected during sI/R and subjected to a series of column chromatographic steps. Among the proteins measured by mass spectrometry and western blotting, a p36 protein identified as a glycolytic enzyme, lactate dehydrogenase muscle subunit (M-LDH), showed strong survival activity. H₂O₂-induced intracellular calcium overload in H9c2 cells and irregular Ca²⁺ transients in adult rat cardiomyocytes were both found to be inhibited by pretreatment with M-LDH. M-LDH also lowered the frequency and amplitude of early afterdepolarizations induced by H ₂O₂ in adult rat cardiomyocytes and suppressed the ischaemia-reperfusion-induced reduction of cardiac output from mouse working heart preparations. M-LDH was found to increase the phosphorylation of extracellular signal-regulated kinase1/2 (ERK1/2), which plays a role in H9c2 cell survival. Conclusion: M-LDH released from cardiomyocytes after hypoxia and reoxygenation has a role in protecting the heart from oxidative stress-induced injury through an intracellular signal transduction pathway involving ERK1/2.