Induction of group VIA phospholipase A2activity during in vitro ischemia in C2C12 myotubes is associated with changes in the level of its splice variants

Abstract

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 iPLA2activity, 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.