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First published online 20 February 2007
doi: 10.1242/jcs.03360

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Disrupted mechanical stability of the dystrophin-glycoprotein complex causes severe muscular dystrophy in sarcospan transgenic mice

Angela K. Peter^1,*, Gaynor Miller^1,* and Rachelle H. Crosbie^1,2,

¹ Department of Physiological Science, University of California, Los Angeles, CA 90095, USA
² Molecular Biology Institute, University of California, Los Angeles, CA 90095, USA

View larger version (23K): [in this window] [in a new window]   Fig. 1. SSPN-Tg mice display kyphosis and reduced body mass. (A) Schematic diagram of construct used to generate SSPN-Tg mice. A human skeletal muscle -actin promoter (HSA) was used to control muscle-specific expression of the human SSPN (hSSPN) transgene. A SV40 VP1 intron serves as a splice acceptor and is located downstream of the HSA promoter. Polyadenylation sites (pA) were inserted at the 3' end of the hSSPN cDNA. (B) Representative photographs of phenotypic Tg and non-Tg littermates reveal dramatic differences in body size and length. Severe kyphosis is evident in the phenotypic Tg mouse, as illustrated in the side view. Phenotypic SSPN-Tg mice appear atrophic and have limited mobility. Non-phenotypic SSPN-Tg mice were indistinguishable from their non-Tg controls (data not shown). (C) Phenotypic SSPN-Tg mice are nearly 50% lighter than non-Tg controls. Body mass (g) of 4-week-old non-Tg (n=14), non-phenotypic SSPN-Tg (n=3), and phenotypic SSPN-Tg (n=5) is plotted. Values are means and s.e.m.; *P=0.0015.

View larger version (91K): [in this window] [in a new window]   Fig. 2. SSPN-Tg mice display muscle pathology. (A) Transverse cryosections of quadriceps (Quad), tibialis anterior (TA), soleus (Sol), and diaphragm (Diaph) muscles from non-Tg and phenotypic SSPN-Tg (line 37.5) mice were stained with Hematoxylin and Eosin. SSPN-Tg muscle exhibits a greater variation in fiber diameter in comparison to non-Tgs. Pathological features were not observed in non-phenotypic SSPN-Tg muscle (data not shown). Bar, 20 µm. (B) Central nucleation (%) was quantified for quadriceps and tibialis anterior muscles isolated from 4-week-old phenotypic SSPN-Tg mice and non-Tg littermate controls. SSPN-Tg mice display between 17- and 20-fold more fibers with centrally placed nuclei compared to non-Tgs. Values are means and s.e.m. of total fibers counted. *P0.05. (C) DNA fragmentation was analyzed by TdT-dUTP labeling. Apoptotic myonuceli (red, arrows) were present only in phenotypic SSPN-Tg mice and were never observed in non-Tg or non-phenotypic SSPN-Tg. Both apoptotic nuclei shown lie within the same myofiber outlined with -DG (green). Bar, 50 µm. (D) Quadriceps muscle from non-Tg and phenotypic SSPN-Tg mice was stained with antibodies to collagen III and collagen IV. Small areas of fibrosis in the SSPN-Tg muscle are denoted with an asterisk. Bar, 20 µm.

View larger version (49K): [in this window] [in a new window]   Fig. 3. SSPN-Tg mice do not exhibit sarcolemma instability. (A) Evans Blue tracer assay for infiltration of blood serum proteins into the muscle fiber. Intraperitoneal injection of Evans Blue dye can be detected in muscle fibers with damaged sarcolemma. Transverse cross sections of quadriceps muscle were imaged using a fluorescence microscope equipped with green activation filters. An image from mdx muscle, which has severe membrane damage, is shown as a positive control for Evans Blue staining. Evans Blue dye was not detected in phenotypic or non-phenotypic (data not shown) SSPN-Tg mice. Bar, 50 µm. (B) Analysis of muscle-specific creatine kinase in circulating blood serum. Creatine kinase activities were evaluated in serum samples from non-Tg, mdx, and phenotypic SSPN-Tg mice. SSPN-Tg mice do not exhibit leakage of muscle-specific creatine kinase into the blood serum. Values are means and s.e.m. of total mice analyzed. *P=0.002 compared to control.

View larger version (42K): [in this window] [in a new window]   Fig. 4. DGC proteins are increased upon SSPN overexpression. Immunoblot analysis of total proteins extracted from skeletal muscle of non-Tg (lane 1), non-phenotypic SSPN-Tg line 29.1 (lane 2), and phenotypic SSPN-Tg line 37.5 (lane 3) mice. Skeletal muscle was solubilized using 3% SDS buffer and protein samples (60 µg) were separated by SDS-PAGE and transferred to nitrocellulose membranes. Equal loading of protein samples was confirmed by Coomassie Blue (CB) stain. Immunoblots were probed with antibodies to laminin (Lam), dystrophin (Dys), - and -DG, -, -, and -SG, exogenous SSPN (hSSPN), endogenous SSPN (mSSPN) and caveolin-3 (cav-3), as indicated.

View larger version (116K): [in this window] [in a new window]   Fig. 5. SSPN expression does not perturb sarcolemma localization of the DGC. Immunohistochemical analysis of DGC and its associated proteins on transverse cryosections of quadriceps muscle from phenotypic SSPN-Tg and non-Tg mice. Sections were stained with antibodies to dystrophin (Dys), - and -DG, -, -, and -SG, caveolin-3 (Cav-3), and laminin (Lam). Staining of endogenous mouse SSPN (mSSPN) and exogenous human SSPN (hSSPN) is also shown. Bar, 20 µm.

View larger version (47K): [in this window] [in a new window]   Fig. 6. SSPN induces aggregation of the SGs. (A) Schematic diagram to illustrate the experimental procedure. Following two 1% digitonin solubilizations (Supe #1 and Supe #2), insoluble protein aggregates were extracted using 3% SDS homogenization buffer. (B) Non-phenotypic and phenotypic SSPN-Tg (SSPN-Tg) muscle solubilized fractions contain more DGC proteins than non-Tg (Non-Tg) controls. Digitonin solubilized proteins (60 µg) were analyzed by 12% SDS-PAGE, transferred to nitrocellulose, and analyzed for components of the DGC by immunoblot analysis. Coomassie Blue (CB) stain was used to confirm equal loading of protein samples. (C) SDS solubilized proteins (60 µg) from non-Tg (Non-Tg) and SSPN-Tg (SSPN-Tg) were separated on 12% SDS-PAGE and transferred to nitrocellulose membranes. Representative data are shown from non-phenotypic (line 31.6) and phenotypic (line 31.7) SSPN-Tg mice. Nitrocellulose membranes were separately stained for -DG and -DG, -SG, -SG and -SG, as well as the SSPN transgene (hSSPN). Equal protein loading was confirmed by Coomassie Blue (CB) stain. (D) Quantification of transgene (hSSPN) solubilization. Percentage of solubilized protein was determined by relative densitometry. Relative levels of hSSPN in the soluble and insoluble fractions were analyzed for the phenotypic and non-phenotypic samples. Data is presented as percentages relative to the total level of transgene expression in the soluble and insoluble fractions combined. (E) SSPN homo-oligomerization in non-phenotypic and phenotypic insoluble fractions. SDS solubilized proteins (60 µg) from non-phenotypic and phenotypic SSPN-Tg muscle were analyzed by SDS-PAGE under reducing (R) or non-reducing (NR) conditions and transferred to nitrocellulose. Membranes were probed with antibodies recognizing transgene (hSSPN) expression. Higher-order SSPN oligomers were present only in extracts from phenotypic SSPN-Tg muscle.

View larger version (39K): [in this window] [in a new window]   Fig. 7. -DG is destabilized in phenotypic SSPN-Tg muscle. (A) Schematic diagram showing the steps in the SDS titration experiment. Skeletal muscle from non-Tg and SSPN-Tg mice was subjected to sequential extractions in lysis buffer with increasing SDS concentrations. Skeletal muscle was homogenized in 0.25% SDS and centrifuged to separate soluble (supe) from insoluble (pellet) proteins. The pellet was resuspended in a 0.5% SDS buffer for a second round of protein extraction. This process was repeated with 1%, 2% and 3% SDS. (B) Protein supernatants from each SDS titration were separated using SDS-polyacrylamide gels and transferred to nitrocellulose membranes. Immunoblots were stained with antibodies to laminin (Lam), - and -DG, -, -, and -SG, hSSPN, and mSSPN as indicated. 1% SDS was required to extract -DG in non-Tg muscle, which represents a strong and stable attachment of -DG to the DGC. In non-phenotypic SSPN-Tg muscle, -DG is removed under lower stringency conditions (0.5% SDS), suggesting that -DG is weakened by presence of SSPN overexpression. Finally, -DG is readily extracted from phenotypic SSPN-Tg muscle with 0.25% SDS, supporting the hypothesis that SSPN destabilizes -DG. (C) Densitometry of -DG staining from immunoblots. Relative levels of -DG found in the supernatant fraction after treatment with 0.25%, 0.50% and 1.0% was quantified for all muscle samples analyzed. (D) Schematic models illustrating -DG attachment to the DGC in non-Tg, non-phenotypic SSPN-Tg, and phenotypic SSPN-Tg.

View larger version (79K): [in this window] [in a new window]   Fig. 8. Disruption of basement membrane organization in SSPN-Tg muscle. Immunostaining of transverse quadriceps sections from non-Tg, non-phenotypic SSPN-Tg (line 31.6), and phenotypic SSPN-Tg (line 37.5) mice. Sections were co-stained with (A) monoclonal antibodies against perlecan (green) and collagen VI (red) or (B) antibodies against perlecan (green) and laminin (red). Merged images of green and red fluorescence are shown in the far right panel. In normal muscle (non-Tg and non-phenotypic SSPN-Tg), perlecan completely co-localizes with collagen VI (A) or laminin (B) at the basement membrane. In muscle from the phenotypic SSPN-Tg mice, collagen VI is present in the basement membrane but appears patchy and reduced in intensity relative to the perlecan and laminin. Collagen VI staining is increased in the interstitial and perivascular space (arrows). Collagen VI and laminin are not co-localized with perlecan in phenotypic SSPN-Tg mice, as demonstrated in merged images. Bar, 50 µm.

View larger version (141K): [in this window] [in a new window]   Fig. 9. Electron micrographs reveal structural defects in basement membrane. (A) Electron micrographs of longitudinal sections of EDL muscles isolated from phenotypic SSPN-Tg and non-Tg mice. Two neighboring muscle fibers are shown in each field. The sarcolemma is denoted by arrows. Bar, 1 µm. (B) Electron micrographs were taken at higher magnifications to visualize the basement membrane. The distance between adjacent sarcolemmas is greater in the SSPN-Tg muscle than in the non-Tg muscle (white arrows). The extracellular matrix is visualized as a tight linear structure between two neighboring sarcolemmas in the non-Tg tissue. However, the extracellular matrix is randomly placed in dense, disorganized clumps in the phenotypic SSPN-Tg muscle (black arrows). Bar, 200 nm.

View larger version (157K): [in this window] [in a new window]   Fig. 10. Schematic diagram showing a pathogenetic mechanism for SSPN-mediated disruption of the DGC. The data in the current report support a model of SSPN-induced dysfunction of the DGC. We propose that SSPN (green) overexpression at the sarcolemma (1) causes clustering of the SGs (yellow) into insoluble aggregates (2). Perturbation of the SG-SSPN subcomplex within the DGC impairs its ability to properly anchor -DG (red) at the sarcolemma (3). The destabilization of -DG attachment to the sarcolemma leads to perturbation of basement membrane (blue) assembly (4). It is feasible that disruption of the basement membrane leads to aberrant cellular signaling (5), which may be responsible for the increased levels of apoptosis in phenotypic SSPN-Tg mice. By this pathogenetic mechanism, SSPN disrupts protein interactions within and across the membrane bilayer leading to a severe phenotype that is reminiscent of congenital MD.