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- Fig. 1.
Apoptosis of primary cells. γSG-deficient myotubes on rigid substrate exhibited greater apoptosis than wild-type C57 myotubes at day 7. Both C57 and γSG–/– cells were split, plated in parallel on soft collagen-substrates or rigid IPN-patterned coverslips, and later tested for apoptosis by TUNEL assay at day 2 and day 7. The mean intensity of nuclei for the cells on coverslips was divided by the mean intensity for the cells grown on the collagen-substrates for each day. Error bars represent the s.e.m. of the intensities of at least 50 cells. The relative intensity for γSG–/– at day 7 was significantly greater (17-fold) (P<0.001) than the relative intensity at day 2, indicating that γSG–/– cells cultured on rigid substrate apoptosed to a far greater extent than normal cells under normal conditions.
- Table 1.
Protein expression of cells on IPN-patterned glass
C57 γSG-/- Lower layer Upper layer Lower layer Upper layer γSG +++ +++ - - αSG + + - + Myosin + +++ - ++ Utrophin +++ +++++ + + - Fig. 2.
Myosin expression and striation in primary cells. A fraction of primary cells crawled on top of other cells while in culture. (A) A sketch of the two-cell system indicating that only the upper cell was striated. (B,C) Two different fields with γSG–/– cells growing on top of other cells. The upper layer of cells is in focus in (B,C) and the lower layer of cells is in focus in (B′,C′). There is ∼6 μm height difference between the two focal planes. The arrowheads point to nuclei that are in focus in the lower layer. Myosin appeared highly expressed and often exhibited some structure (B) or complete striation (C) only in cells growing on top of other cells. The arrow in B indicates a fiber of structured myosin in the myotube and the arrow in C indicates a fully striated myofiber. (D) Almost twice as many γSG–/– cells were striated as C57 cells. For both cell types, only the upper cell was striated. Bar, 20 μm.
- Fig. 3.
Relaxation dynamics of normal and γSG-deficient myotubes.(A) Length relaxation, or self-peeling, occurred when one end of the cell was mechanically detached with a micropipette at t=0. (B) Data for 6-day-old C57 (15 cells) and γSG–/– (10 cells) were binned by time, averaged, and fit by L/Linit = 1 – A (1 – e–t/τ) with the indicated R2 values. Based on the fit of the binned data, AC57=0.16 and AγSG=0.31. Error bars represent the s.e. of the binned means. Although γSG–/– cells relaxed faster and to a greater extent than C57 cells, γSG–/– cells showed more heterogeneity.
- Fig. 4.
Cell width ratios. (A) The mid-width of cells Wmid was compared with the width near the end Wend. (B) Twice as many γSG–/– cells had a high width ratio as C57 cells. Bar, 20 μm.
- Fig. 5.
Dynamic adhesive strength of primary myotubes. C57 and γSG–/– myotubes have similar dynamic adhesive strength. (A) Forced peeling was done with a large-bore micropipette. The shear stress, τcell, of the aspirating fluid imposed a tension, Tpeel, which peeled the cell from the substrate at a velocity Vpeel. The flow rate, Q, was held constant by a syringe pump, but Tpeel increased linearly with the length of cell inside the micropipette, Lasp. The fluid velocity uz within the pipette is parabolic in profile as sketched. (B) A typical peeling run exhibits dips in the velocity as a function of tension. These dips correspond to physical focal adhesions (PFA, empty squares) along the length of the cell. The velocity envelope that defines `peeling' (filled squares) can be compared between cell types. (C) A comparison of peeling data for normal (C57) and γSG–/– cells revealed no significant difference in peeling dynamics. The R2 for the C57 and γSG–/– fits are 0.71 and 0.84, respectively.
- Fig. 6.
Phosphoprotein comparisons of normal and γSG-deficient TA muscle. (A) Immunoblots of tissue lysates show significant activation of ERK-1 and downregulation of adducin-γ phosphorylation with no changes in cofilin in γSG–/– tissue compared with control tissue. (B) Blots of stretched and unstretched γSG–/– tissue show hyperactivation of p38 MAPK, ERK1/2, MAPKAP2 and Smad1 in stretched tissue.
- Table 2.
Phosphoscreen of activation in γSG-/- muscle relative to normal muscle
Phosphoprotein (site) γSG-/- (% Normal) Stress/stretch, mechano-transduction Apoptosis signal Viability signal Hyperactivation (P)-ERK1(T185/Y187) +126 Hornberger et al., 2004 Subramaniam et al., 2004; Cheung et al., 2004 (P)-MAPKAPK2 (T222) +114 Krook et al., 2000 (P)-FAK (Y577) +114 Lunn et al., 2004 Grace et al., 2003 Kurenova et al., 2004; Lin et al., 2004 (P)-MYPT1/2 (T696) +53 (P)-FAK (Y397) +38 Lunn et al., 2004 Grace et al., 2003; Lesay et al., 2001 Frisch et al., 1996 No significant change in activation (P)-PKC-e (S729) +16 Ni et al., 2003; Mansour et al., 2004 Jung et al., 2004 (P)-PAX (Y31) +10 (P)-NR1 (S896) +3 (P)-Histone H3 (S28) -2 (P)-CREB (S133) -3 (P)-PKCa (S657) -4 (P)-PAX (Y118) -8 (P)-Cofilin 2 (S3) -16 Decreased activation (P)-AcCoA Carb. (S79) -26 (P)-Adducin-α (S662) -40 (P)-SMAD-1 (S463) -40 (P)-p38 MAPK (T180/Y182) -54 (P)-MAPKAP-2 (T334) -78 (P)-Adducin-γ (S662) -74 - Table 3.
Stretch activation of normal muscle and γSG-/- *
Phosphoprotein (site) Normal (%) stretched/unstretched γSG-/- (%) stretched/unstretched Stress/stretch mechano-transduction Apoptosis signal Activation (P)-p38 MAPK (T180/Y182) +556 +900 Martineau et al., 2001; Azuma et al., 2001; Fanning et al., 2003 Deschesnes et al., 2001 (P)-ERK1/2 (T202/Y204) +204 +846 Jansen et al., 2004 Subramaniam et al., 2004; Cheung et al., 2004 (P)-ERK1/2 (T185/Y187) +738 +797 Jansen et al., 2004; Hornberger et al., 2004 Subramaniam et al., 2004; Cheung et al., 2004 (P)-MAPKAPK2 (T334) +162 +586 Krook et al., 2000; Mizutani et al., 2004 (P)-Smad1 (S463) -13 +39 Suzawa et al., 2002 -
↵* After 20 minutes at ∼10% stretch.
-
- Fig. 7.
Sarcoglycans and signaling. (A) Schematics of sarcolemma scaffolding in normal and γSG–/– muscle cells. Filamin normally associates with sarcoglycans, but in γSG–/– cells, sarcolemma filamin expression is considerably increased (Thompson et al., 2000). A relationship between cytoskeletal stresses and signaling via phosphorylation is posited. DG, dystroglycan; ECM, extracellular matrix; SG, sarcoglycan. The integrin adhesion complex proteins talin, paxillin, vinculin, etc., may be perturbed by the filamin over-expression, affecting signalling. (B) Proposed signaling circuit that incorporates changes in phosphorylation in γSG–/– tissue (+ or – in red) as well as our in vitro results for contractility and viability. Filamin-B overexpression has been shown to accelerate myotube differentiation (van der Flier et al., 2002) and is also known to influence MAP-kinase signaling (Awata et al., 2001), extending perhaps to ERK-1/2. Filamin also interacts with cvHSP, a homolog of hsp27 (Krief et al., 1999) and (P)-hsp27 is a substrate of (P)-MAPKAPK2 (Ibitayo et al., 1999), which is in turn a substrate of (P)-p38 MAPK (Ryder et al., 2000). Both (P)-hsp27 and (P)-MYPT1/2 are associated with sustained muscle contraction (Ibitayo et al., 1999; Sward et al., 2000). (P)-ERK-1/2 phosphorylates FAK (Carragher et al., 2003). Both (P)-FAK (Laprise et al., 2002) and (P)-ERK-1/2 have been linked to myocyte apoptosis (Cheung and Slack, 2004; Subramaniam et al., 2004). Dashed lines indicate proposed connections, thick lines indicate stretch-induced signals.
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