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MUSCLE CELL BIOLOGY AND CELL MOTILITY

Induction of group VIA phospholipase A₂ activity during in vitro ischemia in C2C12 myotubes is associated with changes in the level of its splice variants

¹Department of Molecular Biology, University of Copenhagen, and ²Eye Pathology Institute, Copenhagen, Denmark

Submitted 10 January 2007 ; accepted in final form 2 September 2007

The involvement of group VI Ca²⁺-independent PLA₂s (iPLA₂-VI) in in vitro ischemia [oxygen and glucose deprivation (OGD)] in mouse C2C12 myotubes was investigated. OGD induced a time-dependent (0–6 h) increase in bromoenol lactone (BEL)-sensitive iPLA₂ activity, which was suppressed by specific short interfering (si)RNA knockdown of iPLA₂-VIA. OGD was associated with an increase in iPLA₂-VIA protein levels, whereas mRNA levels were unchanged. The levels of iPLA₂-VIB mRNA and protein were not increased by OGD. RT-PCR and Western blot analysis identified a mouse iPLA₂-VIA homolog to catalytically inactive 50-kDa iPLA₂-VIA-ankyrin variants previously identified in humans. Both the mRNA and protein levels of this 50-kDa variant were reduced significantly within 1 h following OGD. In C2C12 myoblasts, iPLA₂-VIA seemed to predominantly reside at the endoplasmatic reticulum, where it accumulated further during OGD. A time-dependent reduction in cell viability during the early OGD period (3 h) was partially prevented by iPLA₂-VIA knockdown or pharmacological inhibition (10 µM BEL), whereas iPLA₂-VIA overexpression had no effect on cell viability. Taken together, these data demonstrate that OGD in C2C12 myotubes is associated with an increase in iPLA₂-VIA activity that decreases cell viability. iPLA₂-VIA activation may be modulated by changes in the levels of active and inactive iPLA₂-VIA isoforms.

hypoxia; Ca²⁺-independent phospholipase A₂-β; Ca²⁺-independent phospholipase A₂-