Sarcospan reduces dystrophic pathology: stabilization of the utrophin-glycoprotein complex

Abstract

Mutations in the dystrophin gene cause Duchenne muscular dystrophy and result in the loss of dystrophin and the entire dystrophin-glycoprotein complex (DGC) from the sarcolemma. We show that sarcospan (SSPN), a unique tetraspanin-like component of the DGC, ameliorates muscular dystrophy in dystrophin-deficient mdx mice. SSPN stabilizes the sarcolemma by increasing levels of the utrophin-glycoprotein complex (UGC) at the extrasynaptic membrane to compensate for the loss of dystrophin. Utrophin is normally restricted to the neuromuscular junction, where it replaces dystrophin to form a functionally analogous complex. SSPN directly interacts with the UGC and functions to stabilize utrophin protein without increasing utrophin transcription. These findings reveal the importance of protein stability in the prevention of muscular dystrophy and may impact the future design of therapeutics for muscular dystrophies.

Figure 1.

SSPN stabilizes the UGC at the sarcolemma. Transverse cryosections of quadriceps muscle from non-Tg (wild type), SSPN-Tg (wild type), mdx, and SSPN-Tg:mdx mice were stained with the antibodies to dystrophin (Dys), utrophin (Utrn), DGs (α- and β-DG), mouse SSPN (mSSPN), transgenic hSSPN, and SGs (α- and β-SG). Protein staining was visualized by indirect immunofluorescence. Bar, 100 μm.

Figure 2.

SSPN regulates UGC levels by interacting directly with this complex. (A) Skeletal muscle from mdx and SSPN-Tg:mdx tissue was solubilized in modified radioimmunoprecipitation assay buffer, and 60-μg protein samples were resolved by SDS-PAGE and immunoblotted using antibodies against dystrophin (Dys), utrophin (Utrn), DGs (α- and β-DG), exogenous SSPN (hSSPN), and SGs (α-, β-, and γ-SG). As expected, exogenous hSSPN was only detected in SSPN-Tg:mdx muscle. Expression levels of DGs and SGs were dramatically elevated in SSPN-Tg:mdx muscle samples. Utrophin, a homologue of dystrophin, is up-regulated in SSPN-Tg:mdx tissue. Equal loading of mdx and SSPN-Tg:mdx lysates was confirmed by Coomassie blue (CB) staining. Molecular masses of individual proteins are indicated in parentheses. (B) Quantitative RT-PCR analysis was used to determine the level of utrophin mRNA in both SSPN-Tg:mdx and mdx tissue. Data are normalized to GAPDH controls and are represented relative to control values (100%). Utrophin mRNA levels were not statistically different (standard t test; P = 0.258) between SSPN-Tg:mdx and mdx controls, suggesting that SSPN-Tg expression did not affect utrophin transcription. Values represent mean ± SEM (error bars). (C) SSPN is a core component of the UGC in SSPN-Tg:mdx mice. Purified UGC proteins were separated by ultracentrifugation through 5–30% sucrose gradients. Fraction 1 represents the lightest region of the gradient. Fractions were analyzed by immunoblotting with antibodies to utrophin (Utrn), DGs (α- and β-DG), SGs (α-, and γ-SG), and exogenous SSPN (hSSPN) as indicated. SSPN and utrophin are enriched in fraction 4, demonstrating that SSPN is a core component of the UGC. The molecular mass of each component is identical to that indicated in A.

Figure 3.

SSPN-Tg:mdx mice display near normal muscle pathology. (A) Transverse cryosections of whole quadriceps muscle from age-matched mdx and SSPN-Tg:mdx mice were stained with H&E to visualize muscle pathology. Many necrotic patches are visible in mdx quadriceps. Necrotic fibers were never observed in SSPN-Tg:mdx quadriceps. Boxed regions indicated on H&E staining of quadriceps muscle from mdx and SSPN-Tg:mdx mice are shown at higher magnifications in B. Note the presence of numerous myofibers with central nucleation as well as the variation in fiber size evident in mdx tissue. Muscle from SSPN-Tg:mdx muscle displays reduced central nucleation and improvements in fiber size variation. Bar, 100 μm.

Figure 4.

SSPN improves muscle degeneration and sarcolemmal stability. (A) Central nucleation (percent of total fibers) was quantified for quadriceps isolated from mdx and SSPN-Tg:mdx (n = 4). SSPN-Tg:mdx mice display a threefold reduction in central nuclei compared with mdx age-matched controls. Each value represents mean ± SEM (error bars) of the total quadriceps analyzed (*, P = 6.0 × 10⁻⁴). (B) To examine infiltration of blood serum proteins into damaged muscle fibers, mdx and SSPN-Tg:mdx mice were intraperitoneally injected with Evans blue dye, a marker for membrane instability. mdx quadriceps displays many Evans blue dye–positive fibers (visualized by red fluorescence), which is a marker for membrane damage. Evans blue dye was not detected in muscle from SSPN-Tg:mdx mice, demonstrating that SSPN expression restored membrane stability in dystrophin-deficient muscle. Bar, 20 μm.