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First published online 15 March 2005
doi: 10.1242/jcs.01717

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-Sarcoglycan deficiency increases cell contractility, apoptosis and MAPK pathway activation but does not affect adhesion

¹ Pennsylvania Muscle Institute, University of Pennsylvania Medical Center, D-700 Richards Building, 3700 Hamilton Walk, Philadelphia, PA 19104-6083, USA
² Department of Chemical and Biomolecular Engineering, Towne Building, 220 South 33rd Street, University of Pennsylvania, Philadelphia, PA 19104-6393, USA
³ Department of Physiology, University of Pennsylvania Medical Center, 3700 Hamilton Walk, Philadelphia, PA 19104-6085, USA
⁴ Graduate Group in Cell and Molecular Biology, University of Pennsylvania Medical Center, 3620 Hamilton Walk, Philadelphia, PA 19104-6058, USA

View larger version (14K): [in a new window]   Fig. 1. Apoptosis of primary cells. SG-deficient myotubes on rigid substrate exhibited greater apoptosis than wild-type C57 myotubes at day 7. Both C57 and SG–/– 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 SG–/– at day 7 was significantly greater (17-fold) (P<0.001) than the relative intensity at day 2, indicating that SG–/– cells cultured on rigid substrate apoptosed to a far greater extent than normal cells under normal conditions.

View larger version (36K): [in a new window]   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 SG–/– 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 SG–/– cells were striated as C57 cells. For both cell types, only the upper cell was striated. Bar, 20 µm.

View larger version (21K): [in a new window]   Fig. 3. Relaxation dynamics of normal and SG-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 SG–/– (10 cells) were binned by time, averaged, and fit by L/Linit = 1 – A (1 – e–t/) with the indicated R2 values. Based on the fit of the binned data, AC57=0.16 and ASG=0.31. Error bars represent the s.e. of the binned means. Although SG–/– cells relaxed faster and to a greater extent than C57 cells, SG–/– cells showed more heterogeneity.

View larger version (57K): [in a new window]   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 SG–/– cells had a high width ratio as C57 cells. Bar, 20 µm.

View larger version (20K): [in a new window]   Fig. 5. Dynamic adhesive strength of primary myotubes. C57 and SG–/– myotubes have similar dynamic adhesive strength. (A) Forced peeling was done with a large-bore micropipette. The shear stress, cell, 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 SG–/– cells revealed no significant difference in peeling dynamics. The R2 for the C57 and SG–/– fits are 0.71 and 0.84, respectively.

View larger version (31K): [in a new window]   Fig. 6. Phosphoprotein comparisons of normal and SG-deficient TA muscle. (A) Immunoblots of tissue lysates show significant activation of ERK-1 and downregulation of adducin- phosphorylation with no changes in cofilin in SG–/– tissue compared with control tissue. (B) Blots of stretched and unstretched SG–/– tissue show hyperactivation of p38 MAPK, ERK1/2, MAPKAP2 and Smad1 in stretched tissue.

View larger version (39K): [in a new window]   Fig. 7. Sarcoglycans and signaling. (A) Schematics of sarcolemma scaffolding in normal and SG–/– muscle cells. Filamin normally associates with sarcoglycans, but in SG–/– 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 SG–/– 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.