Abstract
The dystrophin–glycoprotein complex (DGC) is a membrane adhesion complex that provides structural stability at the sarcolemma by linking the myocyte’s internal cytoskeleton and external extracellular matrix. In Duchenne muscular dystrophy (DMD), the absence of dystrophin leads to the loss of the DGC at the sarcolemma, resulting in sarcolemmal instability and progressive muscle damage. Utrophin (UTRN), an autosomal homolog of dystrophin, is upregulated in dystrophic muscle and partially compensates for the loss of dystrophin in muscle from patients with DMD. Here, we examine the interaction between Utr and sarcospan (SSPN), a small transmembrane protein that is a core component of both UTRN–glycoprotein complex (UGC) and DGC. We show that additional loss of SSPN causes an earlier onset of disease in dystrophin-deficient mdx mice by reducing the expression of the UGC at the sarcolemma. In order to further evaluate the role of SSPN in maintaining therapeutic levels of Utr at the sarcolemma, we tested the effect of Utr transgenic overexpression in mdx mice lacking SSPN (mdx:SSPN −/−:Utr-Tg). We found that overexpression of Utr restored SSPN to the sarcolemma in mdx muscle but that the ablation of SSPN in mdx muscle reduced Utr at the membrane. Nevertheless, Utr overexpression reduced central nucleation and improved grip strength in both lines. These findings demonstrate that high levels of Utr transgenic overexpression ameliorate the mdx phenotype independently of SSPN expression but that loss of SSPN may impair Utr-based mechanisms that rely on lower levels of Utr protein.
Introduction
The dystrophin–glycoprotein complex (DGC) is a membrane adhesion complex that provides structural stability at the muscle sarcolemma by linking the myocyte’s internal cytoskeleton and external extracellular matrix (1–3). In Duchenne muscular dystrophy (DMD), the absence or reduction of dystrophin leads to the loss of the DGC at the sarcolemma, resulting in sarcolemmal instability and progressive contraction-induced damage (4,5). This progressive loss of membrane integrity causes muscle fiber degeneration and ultimately leads to muscle cell death. Individuals with DMD experience progressive muscle wasting during childhood and typically lose ambulation in early adolescence (6). Although corticosteroid treatment and supportive care can slow disease progression and increase DMD life expectancy, there is currently no curative treatment available and cardiac and respiratory failures are the major causes of morbidity and mortality for individuals with DMD.
Along with the DGC, two other major adhesion complexes are expressed at the skeletal muscle sarcolemma: the α7β1 integrin complex and the utrophin (UTRN) glycoprotein complex (UGC) (7–9). Both proteins are upregulated in the reduction or absence of dystrophin and can partially protect against damage to the muscle during contraction (9). In healthy muscle, the UGC is primarily localized to the neuromuscular junction, but in DMD, it is upregulated and found throughout the sarcolemma (10). The composition of the DGC and UGC is highly similar and includes the sarcoglycans (α-, β-, γ- and δ-SG), sarcospan (SSPN), dystroglycan (α- and β-DG), the syntrophins and dystrobrevin (11–13). SSPN, a 25-kDa integral membrane protein, forms a tight subcomplex with the SGs that stabilizes α-dystroglycan (α-DG) at the sarcolemma (14–16). Loss of any one of the SGs results in the reduction or loss of the sarcolemmal SG–SSPN subcomplex and causes a spectrum of skeletal and/or cardiac muscle disorders (17). Limb-girdle muscular dystrophy (LGMD) types 2C, 2D, 2E and 2F are caused by recessive mutations in γ-, α-, β- and δ-SG genes, respectively (18–22). LGMD is characterized by progressive proximal skeletal muscle weakness, ranging from mild to severe, and can be accompanied by cardiomyopathy (23,24). Notably, although mutations in SSPN have not been associated with human muscle disease, and SSPN expression is typically reduced or absent in skeletal muscle from LGMD patients (25).
Our group has previously shown that overexpression of SSPN in mdx muscle increases the sarcolemmal abundance of all three skeletal muscle adhesion complexes (26–28). Given this key role of SSPN in mediating sarcolemmal integrity in mdx pathology, we investigate the loss of SSPN expression in mdx skeletal muscle. In order to further evaluate the role of SSPN in maintaining the therapeutic levels of Utr at the sarcolemma, we tested the effect of Utr transgenic overexpression in mdx mice lacking SSPN (mdx:SN). Transgenic Utr overexpression has been well characterized as a near-complete rescue model in the dystrophin mdx mouse, and UTRN-based therapies are currently being tested as a therapeutic approach in DMD. Here, we show that overexpression of Utr restored UGC-associated proteins to the sarcolemma in both mdx and mdx:SN mice. Furthermore, Utr overexpression reduced central nucleation and improved grip strength in both lines. These findings demonstrate that Utr overexpression can rescue the mdx phenotype independently of SSPN expression and suggest that the improvements mediated by SSPN transgenic overexpression may involve pathways in addition to the UGC that are beneficial to muscle.
Results
Loss of SSPN exacerbates dystrophy in mdx mice
To determine the effect of SSPN loss on mdx dystrophic pathology, we crossed mdx mice to Sspn−/− mice (29) to generate mdx:Sspn+/− heterozygous mice. Heterozygous mdx: Sspn+/− mice were then crossed to produce double knockout mdx:Sspn−/− mice (abbreviated as mdx:SN). Double null mice were born in expected ratios (data not shown), gained weight at approximately the same rate as mdx mice (Supplementary Material, Fig. S2) and bred normally. Although survival was not specifically tracked, no excess deaths were noted and some mdx:SN animals survived up to at least 18 months.
In mdx mice, significant necrosis and inflammation is observed in some limb muscles starting at 3 weeks of age, most prominently in the TA (30–32). This period of damage and myonecrosis is followed by active muscle regeneration, which can be evaluated by the presence of fibers with centralized nuclei. At 3 and 6 weeks of age, hematoxylin and eosin (H&E)-stained transverse cryosections of quadriceps were evaluated for dystrophic pathology (Fig. 1A and B). Few degenerating fibers were seen in 3-week mdx quadriceps, and little-to-no inflammation was evident at this timepoint (Fig. 1A). In contrast, at 3 weeks, degenerating fibers were present in the mdx:SN quadriceps and regions of inflammatory infiltrates were readily apparent (Fig. 1A). In the quadriceps of mdx:SN mice at 3 weeks, there was also a small, but significant increase in central nuclei, indicating an earlier initiation of active regeneration (Fig. 1B). By 6 weeks, both genotypes had a high level of central nucleation in the quadriceps and there was no longer a significant difference in myofiber regeneration (Fig. 1B). However, at 6 weeks, the mdx:SN had more severe dystrophic features, including greater fiber size variability, mild endomysial fibrosis and adipose infiltrates (Fig. 1A and C). Staining against collagen I also revealed large patches of fibrotic deposition (Fig. 2A and Supplementary Material, Fig. S1).
Loss of SSPN results in a more severe dystrophic mdx phenotype. (A) Transverse sections of quadriceps muscle from 3- and 6-week-old mdx and mdx:SN mice were stained with H&E to visualize muscle pathology. Bar, 100 μm. (B) At 3 weeks of age, sections from mdx:SN quadriceps have increased central nucleation relative to mdx muscle (mdx, n = 3; mdx:SN, n = 8). At 6 weeks, there was no significant difference in central nucleation between mdx and mdx:SN. Data represent means ± SEM. Statistics were calculated using Student’s t-test (***P < 0.001). (C) Fiber size (cross-sectional area) variability in tibialis anterior (TA) at 6–8 weeks of age (mdx, n = 3; mdx:SN, n = 3). (D) Transverse sections of diaphragm muscle from 4-week-old, 12-week-old and 14-month old WT, mdx and mdx:SN mice were stained with H&E to visualize muscle pathology. Bar, 100 μm.
Increased fibrosis and loss of membrane stability in mdx:SN muscle. (A) Transverse sections of quadriceps muscle from 6-week-old mdx and mdx:SN mice were stained with antibody against collagen I to assess fibrosis. Bar, 250 μm. (B) Transverse sections of quadriceps muscle from 3- and 6-week-old mdx and mdx:SN mice were stained with eMHC. Representative images from 3-week-old mice are shown here. Bar, 400 μm. (C, D) Fibers positive for eMHC were quantified relative to total number of fibers. (E) In 3-week-old mice, the number of positive EBD fibers was counted in order to quantify sarcolemmal damage. (F) In 6-week-old mice, sections were stained for IgM and positive fibers were counted. (G) Hearts from 12-month-old mice were sectioned and stained with antibody against collagen I. Magnified regions of fibrosis are shown in the figure. Bar, 500 μm.
We next examined the diaphragm, the most severely affected muscle in the mdx mouse. In order to compare diaphragms between animals, sections were taken from the anterolateral right costal muscle in wild-type (WT), mdx and mdx:SN mice at multiple timepoints (Fig. 1D). Notably, the mdx:SN diaphragm exhibited a more severe phenotype at all stages examined. At 4 weeks, there were more cellular infiltrates and fibrosis in mdx:SN relative to mdx. At 12 weeks, the mdx:SN diaphragm was substantially thicker and more fibrotic, with increased regions of necrotic fibers. In aged diaphragms greater than 1 year of age, both mdx and mdx:SN diaphragms were thinned and displayed substantial fibrosis relative to WT, while the mdx:SN diaphragm was thinner and had fewer intact muscle fibers.
In order to further examine the timeline of regeneration in mdx:SN, we stained for expression of embryonic myosin heavy chain, which is expressed in newly regenerated fibers (Fig. 2B). As with the regenerative marker of central nucleation, the number of positive fibers was increased in mdx:SN relative to mdx at 3 weeks, indicating an earlier initiation of active regeneration in the double knockout mice (Fig. 2B–C). There was no longer a significant difference between the groups at 6 weeks (Fig. 2D).
To assess the effect of SSPN loss in the heart, we sectioned hearts from 12-month-old mdx and mdx:SN mice and stained for HE and with antibody against collagen I (Supplementary Material, Fig. 2G). Consistent with skeletal muscle, we found increased regions of cardiac in the mdx:SN heart.
Muscle fiber membrane damage is increased in mdx:SN mice
Along with increased regeneration, loss of membrane stability in mdx muscle leads to overt membrane damage and infiltration into the muscle fiber (4,5,33). In mdx:SN, we measured membrane stability using two different markers of membrane damage, Evans blue dye (EBD) uptake and IgM antibody staining. EBD uptake is an in vivo strategy of injecting dye that binds to albumin forming a large complex (34). In healthy muscle, this complex is carried by blood and does not enter the muscle fiber, while fluorescent dye crosses into the sarcoplasm of damaged fibers (35). Similarly, IgM is found in blood in a complex of approximately 800 kDa and does not cross the membrane into healthy muscle fibers (33). Following dissection and sectioning, muscle can be stained with IgM and evaluated for positive fibers. At 3 weeks, quadriceps exhibited a low average percentage (>3%) of EBD positive fibers and no significant difference between groups, although mdx:SN had higher values (Fig. 2D). However, at 6 weeks, there was a significant increase in IgM-positive fibers, suggesting that loss of SSPN expression leads to more fiber damage at this timepoint (Fig. 2F).
Grip strength and voluntary ambulation are decreased in mdx:SN mice
To determine the effect of SSPN loss on mdx muscle strength, forelimb grip strength was tested at 6 weeks for mdx and mdx:SN mice. Each mouse was tested in five consecutive trials, and all animals performed progressively worse over the five trials. At 6 weeks, there was no significant difference between mdx and mdx:SN, and both genotypes were weaker at all timepoints compared with WT (Fig. 3A). We further tested the mice at a more advanced timepoints and found that there was a significant decrease in mdx:SN grip strength relative to age-matched mdx at both 6 months and 1 year of age (Fig. 3A).
Loss of SSPN exacerbates weakness in aged mdx:SN mice. (A) Forelimb grip strength normalized to body weight at 6 weeks of age, 6 months and 1 year (fifth consecutive trial reported). (B) Post-exercise 6 min walking distances at 6 months of age were recorded using an open field activity chamber (n = 3 for both conditions). Data represent means ± SEM. Statistics calculated using one-way ANOVA followed by Tukey’s multiple comparison’s test (*P < 0.05 and **P < 0.01).
In addition to grip strength, voluntary ambulation was used as a further metric of muscle function. Following mild exercise, mdx mice show reduced activity (36). Our laboratory has developed an ambulation assay that can be readily coupled with the mild exertion of grip strength testing (28,37). Immediately following grip strength testing, mice are placed in an open field chamber and are allowed to freely explore for 6 min. The movement of the mouse is recorded, and the total distance traveled by each mouse is calculated. We have previously shown that after grip strength testing trials, mdx mice typically remain motionless for a few minutes and then begin to slowly explore the field chamber at a much slower rate than WT mice (28,37). Here, we found that at the 6-month timepoint, mdx:SN mice moved far less than age-matched mdx mice, and the total distance traveled was reduced by 69% relative to mdx controls (Fig. 3B), revealing that recovery from a grip strength exercise protocol is much slower in mdx mice lacking SSPN. Taken together, these data reveal that SSPN contributes to the compensatory response in mdx muscle (Table 1).
SSPN is necessary for compensatory responses in muscular dystrophy
| SSPN-null mice crossed with: | ||||
|---|---|---|---|---|
| Genotypes: | Itga mice (α7 integrin null mice) | mdx mice (Dmd-deficient mice) | mdx:Itga mice (α7 integrin and dystrophin double null mice) | myd mice (LARGE-deficient mice) |
| Question: | Does elevation of α7 integrin compensate for reduction of other known laminin-binding complexes (UGC and DGC) in SSPN-nulls? SSPN-null and Itga-null mice exhibit very mild muscle phenotypes at baseline with normal lifespans. α7 integrin expression is increased in SSPN-null muscle relative to WT controls, while the UGC and DGC expression levels are decreased in the SSPN-nulls | Is the increased UGC and α7β1 expression at the mdx sarcolemma affected by loss of SSPN? The UGC and α7β1 integrin are increased at the sarcolemma in mdx muscle as part of a compensatory mechanism in response to the loss of dystrophin in mdx muscle | Does additional loss of SSPN in DKO muscle affect DGC expression? α7β1 integrin is increased at the sarcolemma in mdx muscle as part of a compensatory mechanism in response to the loss of dystrophin in mdx muscle. Loss of α7 integrin in mdx muscle exacerbates disease pathology, even though levels of UGC are increased in the DKO muscle | Does loss of SSPN affect UGC and DGC expression in myd mice? Spontaneous mutation in the LARGE gene causes muscular dystrophy in mice and humans, resulting in hypoglycosylation of α-DG that diminishes laminin-binding (47). LARGE-deficient (myd) mice exhibit increased UGC expression (27) |
| Outcome: | Yes. DKO mice die prematurely and DKO muscle exhibits histopathology. Loss of SSPN in α7 integrin nulls further decreases DGC and UGC expression in addition to abolishing Akt signaling (45) | Yes. Loss of SSPN affects expression of the UGC and α7β1 integrin at the mdx sarcolemma (27; this study). DKO mice exhibit earlier onset of pathology and increased membrane fragility relative to mdx mice (this study) | Yes. Loss of SSPN diminishes Utr expression and abolishes association of SGs and Utr with DG. SSPN is necessary for protein–protein interactions within the UGC (45) | Yes. Loss of SSPN from myd muscle decreases expression of all adhesion complexes (27) |
| SSPN-null mice crossed with: | ||||
|---|---|---|---|---|
| Genotypes: | Itga mice (α7 integrin null mice) | mdx mice (Dmd-deficient mice) | mdx:Itga mice (α7 integrin and dystrophin double null mice) | myd mice (LARGE-deficient mice) |
| Question: | Does elevation of α7 integrin compensate for reduction of other known laminin-binding complexes (UGC and DGC) in SSPN-nulls? SSPN-null and Itga-null mice exhibit very mild muscle phenotypes at baseline with normal lifespans. α7 integrin expression is increased in SSPN-null muscle relative to WT controls, while the UGC and DGC expression levels are decreased in the SSPN-nulls | Is the increased UGC and α7β1 expression at the mdx sarcolemma affected by loss of SSPN? The UGC and α7β1 integrin are increased at the sarcolemma in mdx muscle as part of a compensatory mechanism in response to the loss of dystrophin in mdx muscle | Does additional loss of SSPN in DKO muscle affect DGC expression? α7β1 integrin is increased at the sarcolemma in mdx muscle as part of a compensatory mechanism in response to the loss of dystrophin in mdx muscle. Loss of α7 integrin in mdx muscle exacerbates disease pathology, even though levels of UGC are increased in the DKO muscle | Does loss of SSPN affect UGC and DGC expression in myd mice? Spontaneous mutation in the LARGE gene causes muscular dystrophy in mice and humans, resulting in hypoglycosylation of α-DG that diminishes laminin-binding (47). LARGE-deficient (myd) mice exhibit increased UGC expression (27) |
| Outcome: | Yes. DKO mice die prematurely and DKO muscle exhibits histopathology. Loss of SSPN in α7 integrin nulls further decreases DGC and UGC expression in addition to abolishing Akt signaling (45) | Yes. Loss of SSPN affects expression of the UGC and α7β1 integrin at the mdx sarcolemma (27; this study). DKO mice exhibit earlier onset of pathology and increased membrane fragility relative to mdx mice (this study) | Yes. Loss of SSPN diminishes Utr expression and abolishes association of SGs and Utr with DG. SSPN is necessary for protein–protein interactions within the UGC (45) | Yes. Loss of SSPN from myd muscle decreases expression of all adhesion complexes (27) |
SSPN is necessary for compensatory responses in muscular dystrophy
| SSPN-null mice crossed with: | ||||
|---|---|---|---|---|
| Genotypes: | Itga mice (α7 integrin null mice) | mdx mice (Dmd-deficient mice) | mdx:Itga mice (α7 integrin and dystrophin double null mice) | myd mice (LARGE-deficient mice) |
| Question: | Does elevation of α7 integrin compensate for reduction of other known laminin-binding complexes (UGC and DGC) in SSPN-nulls? SSPN-null and Itga-null mice exhibit very mild muscle phenotypes at baseline with normal lifespans. α7 integrin expression is increased in SSPN-null muscle relative to WT controls, while the UGC and DGC expression levels are decreased in the SSPN-nulls | Is the increased UGC and α7β1 expression at the mdx sarcolemma affected by loss of SSPN? The UGC and α7β1 integrin are increased at the sarcolemma in mdx muscle as part of a compensatory mechanism in response to the loss of dystrophin in mdx muscle | Does additional loss of SSPN in DKO muscle affect DGC expression? α7β1 integrin is increased at the sarcolemma in mdx muscle as part of a compensatory mechanism in response to the loss of dystrophin in mdx muscle. Loss of α7 integrin in mdx muscle exacerbates disease pathology, even though levels of UGC are increased in the DKO muscle | Does loss of SSPN affect UGC and DGC expression in myd mice? Spontaneous mutation in the LARGE gene causes muscular dystrophy in mice and humans, resulting in hypoglycosylation of α-DG that diminishes laminin-binding (47). LARGE-deficient (myd) mice exhibit increased UGC expression (27) |
| Outcome: | Yes. DKO mice die prematurely and DKO muscle exhibits histopathology. Loss of SSPN in α7 integrin nulls further decreases DGC and UGC expression in addition to abolishing Akt signaling (45) | Yes. Loss of SSPN affects expression of the UGC and α7β1 integrin at the mdx sarcolemma (27; this study). DKO mice exhibit earlier onset of pathology and increased membrane fragility relative to mdx mice (this study) | Yes. Loss of SSPN diminishes Utr expression and abolishes association of SGs and Utr with DG. SSPN is necessary for protein–protein interactions within the UGC (45) | Yes. Loss of SSPN from myd muscle decreases expression of all adhesion complexes (27) |
| SSPN-null mice crossed with: | ||||
|---|---|---|---|---|
| Genotypes: | Itga mice (α7 integrin null mice) | mdx mice (Dmd-deficient mice) | mdx:Itga mice (α7 integrin and dystrophin double null mice) | myd mice (LARGE-deficient mice) |
| Question: | Does elevation of α7 integrin compensate for reduction of other known laminin-binding complexes (UGC and DGC) in SSPN-nulls? SSPN-null and Itga-null mice exhibit very mild muscle phenotypes at baseline with normal lifespans. α7 integrin expression is increased in SSPN-null muscle relative to WT controls, while the UGC and DGC expression levels are decreased in the SSPN-nulls | Is the increased UGC and α7β1 expression at the mdx sarcolemma affected by loss of SSPN? The UGC and α7β1 integrin are increased at the sarcolemma in mdx muscle as part of a compensatory mechanism in response to the loss of dystrophin in mdx muscle | Does additional loss of SSPN in DKO muscle affect DGC expression? α7β1 integrin is increased at the sarcolemma in mdx muscle as part of a compensatory mechanism in response to the loss of dystrophin in mdx muscle. Loss of α7 integrin in mdx muscle exacerbates disease pathology, even though levels of UGC are increased in the DKO muscle | Does loss of SSPN affect UGC and DGC expression in myd mice? Spontaneous mutation in the LARGE gene causes muscular dystrophy in mice and humans, resulting in hypoglycosylation of α-DG that diminishes laminin-binding (47). LARGE-deficient (myd) mice exhibit increased UGC expression (27) |
| Outcome: | Yes. DKO mice die prematurely and DKO muscle exhibits histopathology. Loss of SSPN in α7 integrin nulls further decreases DGC and UGC expression in addition to abolishing Akt signaling (45) | Yes. Loss of SSPN affects expression of the UGC and α7β1 integrin at the mdx sarcolemma (27; this study). DKO mice exhibit earlier onset of pathology and increased membrane fragility relative to mdx mice (this study) | Yes. Loss of SSPN diminishes Utr expression and abolishes association of SGs and Utr with DG. SSPN is necessary for protein–protein interactions within the UGC (45) | Yes. Loss of SSPN from myd muscle decreases expression of all adhesion complexes (27) |
Localization of major muscle adhesion complexes in mdx:SN muscle. Transverse sections of whole quadriceps muscle from 6-week-old WT, mdx and mdx:SN were probed with antibodies against DMD, UTRN, β1D integrin (Itgb), β-DG, sarcoglycans (α- and β-SG) and SSPN and were visualized using indirect fluorescence microscopy. The fluorescence signal was adjusted to visualize the reduced levels of sarcolemmal proteins in mdx muscle for comparison to mdx:SN samples. Bar, 100 μm.
Consistent with the mdx:SN genotype, SSPN and dystrophin (Fig. 4) were not detected in skeletal muscle by immunofluorescent staining. Since UTRN and integrin are upregulated at the sarcolemma in mdx muscle overexpressing SSPN (26,28), we examined the expression of UGC components and β1D integrin to determine if additional ablation of SSPN in the mdx context would affect the expression of the compensatory adhesion complexes that are upregulated in dystrophin deficiency. SSPN forms a tight subcomplex with the SGs and loss of any one of the SGs at the sarcolemma results in the absence of the entire SG–SSPN subcomplex within the DGC, although cases of partial loss of the subcomplex in human LGMD cases have been reported. Ablation of SSPN in mdx muscle caused reduction of α-sarcoglycan (α-SG) in mdx:SN samples, although no major change in β-SG was noted (Fig. 4). Furthermore, we found that β-dystroglycan (β-DG) and integrin β1 levels were decreased in in mdx:SN muscles relative to controls (Fig. 4). Taken together, these results support that SSPN contributes to the compensatory mechanisms in mdx muscle (Table 1).
Rescue of mdx pathology by UTRN overexpression is not dependent on SSPN
Given the increased severity of pathology in mdx mice lacking SSPN, we were interested in whether overexpression of Utr could rescue the mdx:SN phenotype. The mdx:utr-Tg rescue model (‘Fiona’ line) is a well-established transgenic model expressing high levels of Utr (38). These mice exhibit near-normal muscle histology and improved physiological measures (38–40). No differences in body weight were observed at 6 weeks between mdx:utr-Tg and mdx:SN:utr-Tg mice, and mice from both lines appeared similar to WT control mice (Supplementary Material, Fig. S3).
At 6 weeks, H&E staining of quadriceps in both mdx:utr-Tg and mdx:SN:utr-Tg histologically resembled WT muscle, with no obvious dystrophic features such as fibrosis, regenerating fibers or fiber size variation (Fig. 5A). Notably, the more severe pathology seen in mdx:SN muscle at 6 weeks, including fat deposition and inflammatory infiltrates, was not observed with expression of the Utr transgene. Central nucleation was counted across whole quadriceps in order to evaluate degeneration/regeneration. All conditions had little-to-no central nucleation, demonstrating that at 6 weeks of age, the Utr transgene was able to fully rescue this feature of mdx pathology with or without SSPN expression (Fig. 5B).
Transgenic expression of Utr improves dystrophic phenotype in mdx and mdx:SN mice. (A) Transverse sections of whole quadriceps muscle from 6-week-old WT, mdx:utr-Tg and mdx:SN:Utr-Tg mice were stained with H&E to visualize muscle pathology. Bar, 100 μm. (B) Fibers with central nucleation were quantitated in quadriceps muscle and expressed as relative to total fibers for each genotype. (C) Forelimb grip strength was measured across five trials and normalized to body weight at 6 weeks of age (fifth consecutive trial reported). Statistics calculated using one-way ANOVA followed by Tukey’s multiple comparison’s test.
When forelimb grip strength was tested, both mdx:utr-Tg and mdx:SN:utr-Tg mice performed significantly better that mdx alone (Fig. 5C). There was no significant difference between mdx:utr-Tg and mdx:SN:utr-Tg, suggesting that the functional improvements resulting from the Utr transgene are independent of SSPN expression. It is worth noting that, in our assay, the performance of mdx:utr-Tg was significantly decreased relative to WT mice. While some studies have reported that utr-Tg improves mdx physiological performance to WT levels in whole body tension escape assays (38,41), Duan and colleagues (42) demonstrated that mdx:utr-Tg are susceptible to injury following treadmill exercise. Ervasti and colleagues (40) later showed that mdx:utr-Tg mice have reduced activity after mild exercise and that mdx:utr-Tg muscle is susceptible to eccentric contraction-induced force loss in vivo. Of note, there was no difference between mdx:utr-Tg and mdx:SN:utr-Tg performance in any of the five trial repetitions.
Indirect immunofluorescence analysis of transverse quadriceps cryosections revealed that dystrophin and SSPN were absent from the sarcolemma mdx:SN:utr-Tg muscle, as expected (Fig. 6). The Utr transgenic expression was robustly evident at the mdx sarcolemma, as expected, and also increased SSPN at the sarcolemma, but not completely back to WT levels (Fig. 6). Interestingly, ablation of SSPN in mdx:utr-Tg muscle decreased Utr localization to the sarcolemma relative to mdx:utr-Tg controls, but we conclude that the levels of Utr remaining at the sarcolemma are sufficient to ameliorate disease pathology. The observed reduction in Utr expression in cases of SSPN deficiency is consistent with other double knockout studies with SSPN (Table 1). Both full length and truncated Utr transgenes are able to ameliorate disease in mdx and in mdx:utr-null mice, and loss of SSPN does not hamper Utr function in preventing muscle degeneration or improving grip strength (Table 2). Thus, we conclude that SSPN is not required for Utr transgenic rescue of dystrophin deficiency at these high levels of Utr overexpression. It is feasible that loss of SSPN may impair Utr-based mechanisms that rely on lower levels of Utr protein.
Utr localization to the sarcolemma is diminished in the absence of SSPN. Transverse sections of whole quadriceps muscle from 6-week-old WT, mdx:utr-Tg and mdx:SSPN−/−:utr-Tg were stained with antibodies against Dys, Utr, integrin β1D (Itgb), β-DG and SSPN. Loss of dystrophin and SSPN in the mdx:SSPN−/−:utr-Tg confirms expected expression across genotypes. Bar, 50 μm.
Discussion
In this study, we examined the effect of SSPN loss on muscle pathology and function in mdx mice (Table 1). Our analysis revealed that while mdx:SN mice are viable and fertile, they have a more severe dystrophic phenotype than mdx mice, with an earlier onset of both histological and functional changes in skeletal muscle. H&E histology revealed an increase in several hallmarks of dystrophic pathology including inflammation, muscle fiber degeneration, lipid deposits and fibrosis. By 6 weeks, mdx:SN mice show a decrease in membrane stability, while regeneration (measured both by percent of fibers with central nuclei and eMHC) is no longer significantly increased relative to mdx. This may indicate that the acute phase of damage has stabilized by the 6 week timepoint in mdx:SN muscle; alternatively, this could be due to a reduced regenerative capacity as seen in more severe dystrophic models such as D2-mdx mouse (43,44). Interestingly, a previous study from our laboratory demonstrated that loss of SSPN in WT muscle impairs regeneration following acute muscle injury (27). This suggests that, in addition to the increased membrane fragility observed in mdx:SN fibers, reduced regeneration may play a role in the progression of mdx:SN dystrophy.
The initial characterization of SSPN knockout mice reported no effect on muscle physiology in mice up to 4 weeks of age (29). However, further studies have revealed skeletal muscle and cardiac phenotypes that are exacerbated by age or injury (27,45,46). While there are no pathological changes, or loss of force production seen in limb muscles tested at 4 weeks or 4.5 months, diaphragm muscle had a decrease in specific force and was more susceptible to eccentric contraction damage relative to WT at 4.5 months (29,45). In the present study, we found that although grip strength was not affected at the 6 weeks timepoint, mdx:SN mice are significantly weaker than mdx mice at both 6 months and 1 year of age, and they have reduced open field activity following mild exercise. Previously, our laboratory showed that UGC complexes from SSPN-null muscle are more readily dissociated by fractionation than UGC complexes from WT muscle (27), and some components of the DGC/UGC complexes are less abundant at the sarcolemma in skeletal and cardiac muscle (45,46). Given that SSPN is a core component of the UGC as well as the DGC (16,26), a likely explanation for this finding is that the loss of SSPN reduces the stability of compensatory UGC complexes at the sarcolemma. The reduced expression of the UGC is a common theme that is observed upon SSPN deficiency in many double knockout mouse studies (Table 1). Examples include reduced sarcolemma expression of the UGC in muscle from mice lacking both integrin α7 and SSPN (45) as well as double knockout mice lacking both SSPN and LARGE (27), the putative GlcNAC glycosyltransferase that causes muscular dystrophy in the myd mouse (47). Interestingly, loss of SSPN does not appear have as much of an effect on the localization of β-SG to the sarcolemma in mdx muscle. However, the loss of α-SG and reduction of β-DG are expected to completely disrupt protein–protein interactions within the UGC as we have demonstrated in both skeletal and cardiac muscles (26,27,45,46).
In order to further examine the association of SSPN and UTRN in protecting the mdx sarcolemma, we tested whether Utr overexpression could rescue mdx pathology in the absence of SSPN. Interestingly, we detected no difference in dystrophic pathology between mdx:utr-Tg muscle with or without SSPN expression (Table 2). This suggests that Utr-mediated rescue is not dependent on SSPN expression. This finding is of particular interest, given that UTRN overexpression and SSPN overexpression have both been shown to ameliorate mdx pathology and are both ongoing targets for therapeutic development in DMD (48,49). Our findings raise the possibility that Utr and SSPN may alleviate muscle damage through different mechanisms and that simultaneously targeting Utr and SSPN could have additive therapeutic benefits for patients with DMD. For example, while Utr overexpression ameliorates both mdx and utr−/−:mdx mice (Table 2), SSPN overexpression is only able to rescue mdx pathology. One major benefit of both SSPN and UTRN as single or combined therapeutic targets in DMD is their potential to treat patients regardless of specific dystrophin mutation.
Drug-mediated upregulation of UTRN expression has been extensively investigated in pre-clinical studies and represents an appealing therapeutic strategy that could be utilized regardless of DMD mutation. Although the small molecule Utr modulator ezutromid failed to meet endpoints in a recent Phase 2 trial (50,51), there is new focus on developing second-generation Utr modulators with more favorable pharmacokinetic profiles (52,53) and repurposing existing drugs for Utr upregulation (49,54). Our laboratory has similarly developed a high-through screening platform for identifying novel enhancers of SSPN expression (48). Given our finding that SSPN expression is not required for UTRN rescue in mdx, it is possible that a combinatorial therapy approach for these small molecule modulators may hold promise for the long-term protection of muscle in DMD. It is currently unknown whether Utr upregulation would benefit other muscular dystrophies, including the sarcoglycanopathies or the dystroglycanopathies (Table 2).
An important caveat to this part of our study is that the mdx:utr-Tg mouse expresses transgenic Utr at a high level, reported to be between 3- and 10-fold over WT levels of Utr (40). It may be that, at more modest levels of Utr overexpression, loss of SSPN would reveal deficits that are not seen at this high, non-physiologic level of Utr transgene. Our findings demonstrate that Utr transgenic overexpression can rescue the mdx phenotype independently of SSPN expression, providing further insight into the mechanism of Utr-mediated rescue of dystrophic pathology.
Materials and Methods
Animal models
WT and mdx mice were purchased from Jackson Laboratories (Bar Harbor, ME), and SSPN-null mice (Sspntm1Kcam, MGI:2387936) were a generous gift from Kevin P. Campbell, PhD (University of Iowa Carver College of Medicine, Howard Hughes Medical Institute, Iowa City, IA) (29). mdx:Utr-Tg (mdx:utr-Tg) mice were a gift from James M. Ervasti, PhD (University of Minnesota, Minneapolis, MN), with permission from Kay E. Davies, PhD (University of Oxford, Oxford, UK), and were maintained on the mdx background. To generate mdx:SN and mdx:SN:utr-Tg mice, mdx:utr-Tg females (C57Bl/10ScSn background) were crossed with SSPN-null males (C57BL/6 J background), generating heterozygous mdx:SSPN-het or mdx:SSPN-het:utr-Tg males and females. Littermates were crossed to generate homozygous mdx:SN or mdx:SN:utr-Tg and were maintained by sibling crosses (B6/B10ScSn background). Double knockouts (DKOs) were viable and fertile, and the line was maintained with homozygous crosses (mdx:SN to mdx:SN:utr-Tg). Mice were maintained in the Terasaki Life Sciences Vivarium, and all procedures were carried out in accordance with guidelines set by the University of California, Los Angeles Institutional Animal Care and Use Committee.
UTRN ameliorates dystrophin-deficient muscular dystrophy in an SSPN-independent manner
| Utr-Tg mice crossed with: | |||||
|---|---|---|---|---|---|
| Genotypes: | mdx mice (dystrophin-deficient mice) | utr−/−:mdx mice (Utr and dystrophin double null mice) | mdx:SSPN-null mice (SSPN and dystrophin double null mice) | Sarcoglycan-null mice (α-, β-, γ- or δ-SG null mice are models for LGMD) | myd mice (LARGE-deficient mice represent one of many models for dystroglycanopathy) |
| Question: | Does Utr rescue skeletal and cardiac muscle disease in the DMD murine model? | Can Utr replace both for Utr and Dys to rescue disease? | Can Utr ‘rescue’ mdx disease in the absence of SSPN? | Can Utr rescue muscular dystrophy caused by absence of the SG–SSPN subcomplex? | Can Utr address hypoglycosylation deficiency of α-DG? |
| Outcome: | Yes. Expression of full length and mini-Utr reduces membrane instability, prevents degeneration/regeneration cycles and reduces fibrosis (38,55,56) | Yes. Overexpression of a truncated Utr transgene in utr−/−:mdx skeletal muscle prevented premature death, decreased central nucleation and supported expression of the UGC at the sarcolemma (58) | Yes. mdx:SSPN-null:utr-Tg mice were identical to Utr-Tg:mdx mice in amelioration of pathology. Ablation of SSPN reduced localization of Utr at the sarcolemma (this study) | Unknown | Unknown |
| Utr-Tg mice crossed with: | |||||
|---|---|---|---|---|---|
| Genotypes: | mdx mice (dystrophin-deficient mice) | utr−/−:mdx mice (Utr and dystrophin double null mice) | mdx:SSPN-null mice (SSPN and dystrophin double null mice) | Sarcoglycan-null mice (α-, β-, γ- or δ-SG null mice are models for LGMD) | myd mice (LARGE-deficient mice represent one of many models for dystroglycanopathy) |
| Question: | Does Utr rescue skeletal and cardiac muscle disease in the DMD murine model? | Can Utr replace both for Utr and Dys to rescue disease? | Can Utr ‘rescue’ mdx disease in the absence of SSPN? | Can Utr rescue muscular dystrophy caused by absence of the SG–SSPN subcomplex? | Can Utr address hypoglycosylation deficiency of α-DG? |
| Outcome: | Yes. Expression of full length and mini-Utr reduces membrane instability, prevents degeneration/regeneration cycles and reduces fibrosis (38,55,56) | Yes. Overexpression of a truncated Utr transgene in utr−/−:mdx skeletal muscle prevented premature death, decreased central nucleation and supported expression of the UGC at the sarcolemma (58) | Yes. mdx:SSPN-null:utr-Tg mice were identical to Utr-Tg:mdx mice in amelioration of pathology. Ablation of SSPN reduced localization of Utr at the sarcolemma (this study) | Unknown | Unknown |
UTRN ameliorates dystrophin-deficient muscular dystrophy in an SSPN-independent manner
| Utr-Tg mice crossed with: | |||||
|---|---|---|---|---|---|
| Genotypes: | mdx mice (dystrophin-deficient mice) | utr−/−:mdx mice (Utr and dystrophin double null mice) | mdx:SSPN-null mice (SSPN and dystrophin double null mice) | Sarcoglycan-null mice (α-, β-, γ- or δ-SG null mice are models for LGMD) | myd mice (LARGE-deficient mice represent one of many models for dystroglycanopathy) |
| Question: | Does Utr rescue skeletal and cardiac muscle disease in the DMD murine model? | Can Utr replace both for Utr and Dys to rescue disease? | Can Utr ‘rescue’ mdx disease in the absence of SSPN? | Can Utr rescue muscular dystrophy caused by absence of the SG–SSPN subcomplex? | Can Utr address hypoglycosylation deficiency of α-DG? |
| Outcome: | Yes. Expression of full length and mini-Utr reduces membrane instability, prevents degeneration/regeneration cycles and reduces fibrosis (38,55,56) | Yes. Overexpression of a truncated Utr transgene in utr−/−:mdx skeletal muscle prevented premature death, decreased central nucleation and supported expression of the UGC at the sarcolemma (58) | Yes. mdx:SSPN-null:utr-Tg mice were identical to Utr-Tg:mdx mice in amelioration of pathology. Ablation of SSPN reduced localization of Utr at the sarcolemma (this study) | Unknown | Unknown |
| Utr-Tg mice crossed with: | |||||
|---|---|---|---|---|---|
| Genotypes: | mdx mice (dystrophin-deficient mice) | utr−/−:mdx mice (Utr and dystrophin double null mice) | mdx:SSPN-null mice (SSPN and dystrophin double null mice) | Sarcoglycan-null mice (α-, β-, γ- or δ-SG null mice are models for LGMD) | myd mice (LARGE-deficient mice represent one of many models for dystroglycanopathy) |
| Question: | Does Utr rescue skeletal and cardiac muscle disease in the DMD murine model? | Can Utr replace both for Utr and Dys to rescue disease? | Can Utr ‘rescue’ mdx disease in the absence of SSPN? | Can Utr rescue muscular dystrophy caused by absence of the SG–SSPN subcomplex? | Can Utr address hypoglycosylation deficiency of α-DG? |
| Outcome: | Yes. Expression of full length and mini-Utr reduces membrane instability, prevents degeneration/regeneration cycles and reduces fibrosis (38,55,56) | Yes. Overexpression of a truncated Utr transgene in utr−/−:mdx skeletal muscle prevented premature death, decreased central nucleation and supported expression of the UGC at the sarcolemma (58) | Yes. mdx:SSPN-null:utr-Tg mice were identical to Utr-Tg:mdx mice in amelioration of pathology. Ablation of SSPN reduced localization of Utr at the sarcolemma (this study) | Unknown | Unknown |
Grip strength test
Forelimb grip strength was measured using a digital force gage (Columbus Instruments, Columbus, OH) using methods described previously (28,37). Five trials were performed with 1 min of rest between each trial. For each trial, the mouse grasped the pull bar connected to the grip strength meter and was gently pulled back until the pull bar was released. This procedure was repeated five times per trial and the peak tension (N) was recorded for each trial.
Open field activity
Ambulation in 6-month-old mice was recorded immediately following grip strength testing (mild exercise protocol, described in (28,37)). Mice were placed in individual recording chambers and were allowed to freely move for 6 min. Distance traveled by each animal was determined using frame-by-frame object tracking in Kinovea open source video analysis software (Version 0.8.15, www.kinovea.org).
Histology and immunofluorescence assays
Muscles were mounted in OCT (Tissue-Tek) and were flash frozen in liquid nitrogen-cooled isopentane and were stored at −80°C until further processing. Transverse 10 μm transverse cryosections were stained with H&E as described previously (28). In H&E-stained sections, centrally nucleated and total fibers were counted in order to determine the level of regeneration (number of fibers with central nuclei divided by total number of fibers). For antibody staining, sections were blocked with 3% BSA in PBS for 30 min at room temperature. Avidin/biotin blocking kit (Vector Laboratories) was used according to manufacturer’s instructions. For antibodies raised in mouse, Mouse on Mouse blocking reagent (Vector Laboratories) was used according to manufacturer’s instructions. Sections were incubated in primary antibody in PBS at 4°C overnight with the following antibodies: dystrophin (MANDYS1; 1:5; Development Studies Hybridoma Bank), Utr (NCL-DRP2, 1:100; Leica), β-DG (MANDAG2; 1:50; Development Studies Hybridoma Bank), α-SG (VP-A105; 1:30; Vector Laboratories), β-SG (VP-B206; 1:30; Vector Laboratories), α7 integrin (C-20; 1:100; Santa Cruz), laminin (L9393; 1:500; Sigma-Aldrich), β1D integrin (MAB1900; 1:20; Millipore) and SSPN (E2; 1:100). Primary antibodies were detected by biotinylated anti-rabbit (BA-1000; 1:500; Vector Laboratories) and biotinylated anti-mouse (BA-9200; 1:500; Vector Laboratories). Fluorescein-conjugated avidin D (A-2001; 1:500; Vector Laboratories) was used to detect biotinylated secondary antibodies. All sections were mounted in Vectashield (Vector Laboratories) and were visualized using an Axioplan 2 fluorescence microscope with Axiovision 3.0 software (Zeiss Inc.). In quadriceps sections from mice treated with EBD, both EBD-positive fibers and total and total fibers outlined with anti-laminin were counted and percent of positive fibers was calculated (positive fibers divided by the total number of fibers in the section).
Statistical analysis
Statistical analysis was performed using GraphPad Prism software (GraphPad Software Inc., Version 8.3.0). All data are presented as mean ± SEM. Significance was determined by two-tailed Student’s t-test or one-way ANOVA followed by Tukey’s multiple comparison’s test. P < 0.05 was considered statistically significant.
Acknowledgements
This work was supported by the National Institutes of Health [R01 AR048179 to R.H.C., T32 GM07104, IRACDA Award K12 GM106996 to J.L.M., T32 AR059033 to J.L.M. and E.M.G., F32 AR069469 to E.M.G.]; the Edith Hyde Fellowship to J.L.M; the Eureka Predoctoral Training Fellowship to J.L.M.; the Muscular Dystrophy Association USA [274143 to R.H.C.]. SSPN-null mice (Sspntm1Kcam, MGI:2387936) were a generous gift from Kevin P. Campbell, PhD, and the mdx:Utr-Tg (mdx:utr-Tg; Fiona line) mice were a generous gift from James M. Ervasti, PhD, with permission from Kay E. Davies, PhD.
Conflict of Interest statement. None declared.
References
Author notes
Co-second authors
