Inhibition of plasmin activity with α2-antiplasmin decreases myotube formation and differentiation.

C2C12 myoblasts were grown in DMEM plus 10% FBS and then switched to DMEM supplemented with insulin for 3 days (A, B, C), to induce myotube formation and differentiation, or 18 hours (D), to induce only myogenic differentiation, in the presence or absence of α2-antiplasmin, SB203580, or BSA. (A) The α2-antiplasmin decreases myotube formation. Quantitative effects of plasmin inhibition on myoblast fusion. Near-confluent C2C12 cells were switched to DMEM plus insulin to promote fusion in the absence (○) or presence of α2-antiplasmin (▵), SB203580 (▪), or BSA (■). Fusion is represented as percentage of nuclei in myotubes. Values are mean of 3 independent experiments. (B) Lower-power views of representative fields. Preconfluent cells in DMEM/10% FBS (i), confluent cells in DMEM/insulin (ii), confluent cells in DMEM/insulin plus α2-antiplasmin (iii). Magnifications are × 100. (C) The α2-antiplasmin decreases myogenic differentiation. Northern blot analysis of 5 μg total RNA from C2C12 cells grown as myoblasts in DMEM plus 10% FBS (lane 1) and then switched to DMEM plus insulin for 3 days to induce myoblast differentiation and fusion, in the absence (lane 2) or presence of α2-antiplasmin (lane 3), SB203580 (lane 4), or BSA (lane 5). The probe for each hybridization is indicated. (D) Inhibition of plasmin activity with α2-antiplasmin decreases myogenin expression prior the onset of myotube formation. Northern blot analysis of 10 μg total RNA from C2C12 cells grown as myoblasts in DMEM plus 10% FBS and the switched for 18 hours to DMEM plus 170 nM insulin to induce myogenic differentiation, in the absence (lane 1) or presence of 100 nM α2-antiplasmin (lane 2) or 10 μM SB203580 (lane 3). The probe for each hybridization is indicated.