Latent TGF-β–binding protein 4 modifies muscular dystrophy in mice (original) (raw)
F2 mice display an intermediate phenotype between the two parental strains. We generated F2 mice homozygous for the _Sgcg_-null allele in a mixed genetic background of _129_T2/SvEmsJ and DBA/2J. The phenotypic range was determined using two different measures of the disease progress, membrane permeability and fibrosis. Disruption of the dystrophin glycoprotein complex (DGC) enhances membrane leak and promotes abnormal membrane permeability measured as Evans blue dye content in myofibers in vivo (7). The second assay quantifies the hydroxyproline (HOP) content of muscle as a direct reflection of the degree of fibrosis (7). All analyses were conducted in 8-week-old animals, since this time frame corresponds to an early point in the pathologic process but one at which the muscle phenotype is evident.
The interbred F2 generation (γ-sarcoglycan–null in the F2 generation [_F2-Sgcg_]) displayed a phenotype intermediate between the two parental backgrounds with the γ-sarcoglycan–null allele (Figure 1). 129-Sgcg mice had a milder phenotype, with decreased dye uptake and fibrosis compared with D2-Sgcg mice. The mean dye uptake for the F2-Sgcg mice (n = 186, or n = 372 quadriceps muscles) was 29.9 ± 13.1, close to the midpoint between the 129-Sgcg (15.8 ± 8.9) and the D2-Sgcg (35.5 ± 20.2) strains. We determined fibrosis by measuring HOP content and also observed an intermediate phenotype for the F2-Sgcg animals. The 129-Sgcg quadriceps muscles had 9.6 ± 1.7 nM HOP/mg, while the D2-Sgcg had 24.3 ± 7.7, and the value for the F2-Sgcg was 12.2 ± 3.3.
Mice lacking γ-sarcoglycan (_Sgcg-_null mice) serve as a model for muscular dystrophy. The _Sgcg-_null allele displays a mild phenotype in the 129 background strain (129-Sgcg) and a severe phenotype in the D2 strain (D2-Sgcg). Sgcg mice from the two backgrounds were interbred (F2-Sgcg), and the F2-Sgcg animals displayed an intermediate phenotype. (A) Muscle membrane leak in the quadriceps muscle was measured by Evans blue dye uptake in the 2 parental strains (129-Sgcg and D2-Sgcg) and the intercrossed F2-Sgcg generation. (B) Quadriceps muscle fibrosis was measured by determining HOP content in the two parental strains; the F2-Sgcg mice also revealed an intermediate phenotype. (C) An example of the phenotypic range is shown from diaphragm muscles from 2 F2-Sgcg animals (the two left panels) and 1 wild-type control (right panel). Dye uptake (blue) and fibrotic replacement (white) are shown. (D) Grip strength was measured in 8-week-old animals of the 129 and D2 backgrounds with and without the _Sgcg_-null allele. The D2-Sgcg mice were weaker than 129-Sgcg mice, while the parental strains without the muscular dystrophy gene were not significantly different.
To evaluate whether these pathological features correlated with muscle function, we evaluated grip strength of 8-week _Sgcg_-null mice in both genetic backgrounds. We found that there was no significant difference between the two wild-type parental strains in grip strength performance. _Sgcg_-null mice in the D2 background were weaker than _Sgcg_-null mice in the 129 background (Figure 1D), confirming that these pathological differences also influenced muscle function. For mapping studies, we relied on the pathological quantitative traits, since these traits could be measured more reliably and allowed the assay of multiple muscle groups individually.
Genome-wide mapping identifies a single strong locus, dMOD1, that modifies both membrane leak and fibrosis. We used whole-genome SNP analysis to map genetic regions that segregated with enhanced or suppressed phenotype. SNP genotyping was performed using 328 SNPs that differ between the parental 129 and D2 strains. The whole-genome scan was conducted on F2-Sgcg animals. For genotyping, we selected animals with dye uptake and HOP values at least 1 SD above or below the mean in at least two muscle groups, and we chose animals with concordance in two or more muscle groups to minimize intra-animal variability. In addition, we discarded data from animals with very large intramuscular variability, defined by an SD of greater than 20% of the mean for a given muscle pair. Based on these criteria, DNA samples from 80 of 186 F2-Sgcg animals were analyzed. For skeletal muscle dye uptake measurements, 49 animals were genotyped (25 animals had increased dye uptake and 24 animals had decreased dye uptake). For skeletal muscle HOP, 31 animals were genotyped (18 animals chosen for increased HOP and 13 for decreased HOP).
We identified one major locus, dMOD1, on chromosome 7 that influenced both membrane permeability and fibrosis (Figure 2). For dMOD1, the peak lod score for dye uptake was 10.17 at SNP marker rs3156053 at 28.01 Mb (build 36.1). This same SNP was also significantly associated with fibrosis in the quadriceps muscles, yielding a lod score of 6.95. Thus, the same SNP was highly linked to both features of muscular dystrophy, membrane permeability and replacement fibrosis. Using a denser chromosome 7 SNP panel, we further refined dMOD1 using the same cohort of F2-Sgcg mice. The peak lod scores increased to 13.59 for membrane leak and 11.32 for fibrosis (Figure 2, A and B, right).
The pathological muscular dystrophy traits of membrane leak and fibrosis independently map to the dMOD1 locus on chromosome 7. (A) lod scores are shown for 328 SNPs for the trait of membrane leak (dye uptake) in the quadriceps muscles. dMOD1 maps to chromosome 7 with a peak lod of 10.17. (B) lod scores across the genome correlated to fibrosis (HOP content) also map to the dMOD1 on chromosome 7 with a peak lod score of 6.95. Chromosomes and SNP positions are indicated on the x axis, and lod scores are on the y axis. The percentages indicate the chance that this event occurred non-randomly. The right panels show lod scores derived from fine mapping of chromosome 7 near dMOD1, with a peak lod score for dye uptake and fibrosis of 13.52 and 11.32, respectively. (C) The degree of membrane leak and fibrosis correlate. Measurements from quadriceps muscle of membrane leak (dye uptake) and fibrosis (HOP content) were plotted (r2 = 0.1029; P < 0.005).
We analyzed the correlation between membrane leak and fibrosis in the same animals. Although the correlation was weak (r2 = 0.1029), a t test showed that the correlation was significant (P < 0.005; Figure 2C). In our previous analysis, we were unable to demonstrate a clear correlation between these two aspects of pathology, membrane leak and fibrosis (7), and assumed that they may be influenced by different modifier loci. This current study was performed with a larger sample size and thus likely accounts for the weak, but statistically evident, correlation. The finding that both traits were influenced by the same strong modifier region on chromosome 7 indicates that the responsible gene can affect both aspects of disease, membrane leak and fibrosis, or that the two aspects of pathology are interdependent on each other.
Given the unexpected result of a single strong locus affecting muscular dystrophy, we analyzed data from individual animals separately for both pathological traits to more fully dissect the genomic interval associated with dMOD1 (Figure 3). We aligned the dye uptake values from mild to severe and analyzed the SNP data, including those genotypes from the high-resolution genotyping. Using only the dye uptake values, the 95% confidence interval for dMOD1 extended from 19.88 Mb to 30.88 Mb. This data set was larger because more animals met criteria of mild or severe disease. This analysis highlights a comparative absence of the homozygous DBA allele, consistent with a dominant effect of the modifier gene on membrane leak. When the HOP values were considered similarly, the mode of genetic inheritance was less clear and was consistent with a potential semidominant mode of inheritance. The 95% confidence interval for dMOD1 HOP content extended from 25.24 Mb to 34.88 Mb. Since these intervals overlap, we assume that a single gene mediates both membrane leak and fibrosis and therefore refine dMOD1 to the interval between 25.24 Mb and 30.88 Mb on chromosome 7 (Figure 3).
Refining the chromosome 7 dMOD1 interval. The upper panel shows SNP data arranged in rank order of membrane leak (dye uptake) in all muscle groups. The lower panel shows SNP data arranged in rank order of fibrosis (HOP uptake) in all muscle groups. Animals on the left were the most severely affected, having high levels of membrane leak and fibrosis, while those on the right were less severely affected. D2 homozygous chromosomes are shown in blue, 129 in red, and the heterozygous chromosomes in yellow. The thick horizontal lines indicate the 95% confidence interval for each trait. Shown in the box are the genetic regions associated with membrane leak and with fibrosis, demonstrating overlap and listing several candidate genes including TGFB1 and Ltbp4. Asterisks indicate markers also used for the whole-genome scan.
Candidate gene analysis of dMOD1. Muscular dystrophy is characterized by ongoing degeneration and active regeneration within the same muscle group. Given the complexity of this process, finding a single genomic modifier locus of very high significance was unexpected. The D2 strain is known to be associated with dystrophic calcification. Notably, dMOD1 falls near the Dyscalc locus, a region previously linked to vascular and myocardial calcinosis (8). Dyscalc1 is a major contributor to dystrophic cardiovascular calcification in susceptible strains, affecting both the vessels and myocardium (8). Two genes have been independently implicated in Dyscalc, Abcc6 and Emp3 (9, 10). Both genes were excluded as accounting for dMOD1 gene using known disease-associated polymorphisms and finding that both parental strains had the susceptible alleles of each gene (data not shown).
TGF-β signaling has been extensively implicated in fibrosis in many different cell and tissue types, including muscle, and enhanced fibrosis was a feature we used to map dMOD1. Increased TGF-β signaling has been observed in muscular dystrophy (11). Moreover, inhibition of TGF-β signaling improves muscle pathology and function in the mdx mouse model of muscular dystrophy (12). Two genes involved in the TGF-β pathway are within the dMOD1 interval, TGFB1 and Ltbp4. We fully sequenced the coding regions, promoter, and flanking intronic regions of TGFB1. However, there were no polymorphisms in the TGFB1 gene between the parental strains D2 and 129.
An insertion/deletion polymorphism in Ltbp4 segregates with severity of muscular dystrophy pathology. TGF-β1 is a secreted growth factor that is held latent in the extracellular matrix (13). LTBP4 is a member of the latent TGF-β–binding protein multigene family that contains 4 LTBPs and 3 fibrillins (14, 15). Ltbp4 is preferentially expressed in cardiac, skeletal, and smooth muscle (14, 15). We evaluated gene expression profiles for Ltbp4 and found that Ltbp4 expression is induced upon myoblast differentiation and with regeneration after injury. In the mouse, a hypomorphic, loss-of-function allele of Ltbp4 develops pulmonary fibrosis, cardiomyopathy, and colon cancer (16). Like other LTBPs, LTBP4 is part of the large latent complex that sequesters TGF-β in the extracellular matrix, limiting its availability to TGF-β receptors (17, 18).
Because of the reported involvement of TGF-β1 in muscular dystrophy and its general role in fibrosis, we evaluated Ltbp4 in both parental strains. We found a 36-bp polymorphism within exon 12 of Ltbp4 (Figure 4). The protected 129 strain had 36 additional bp (Ltbp4+36), and the severely affected D2 strain was deleted for these 36 bp (Ltbp4Δ36). Closer examination revealed a repeating DNA sequence that is duplicated in the 129 strain, and this repeating structure may have predisposed to the apparent insertion into 129. This repeated sequence and the deletion/insertion are unrelated to the intron-exon boundary, since the deletion occurs wholly within an exon and not at the intron-exon border.
An insertion/deletion polymorphism in Ltbp4 predicts the phenotype in muscular dystrophy. Ltbp4 encodes a TGF-β–binding protein expressed in skeletal and cardiac muscle. (A) The gene structure is shown for Ltbp4. Exons 11, 12, 13 (red bars) of Ltbp4 encode a proline-rich region. (B) 129-Sgcg mice, with a milder phenotype, have a 36-bp insertion that encodes an extended proline-rich region, while severely affected D2-Sgcg mice have a deletion of 36 bp. The insertion/deletion occurs wholly within exon 12. (C) Ltbp4+36 correlates with reduced membrane permeability and reduced fibrosis in F2-Sgcg mice. Congenic _Sgcg_-null mice in the C57BL/6J background or in the CD1 background have a mild phenotype comparable to that of 129-Sgcg mice (7), and these mice also have the protective insertion Ltbp4+36 allele. mdx mice, the model for DMD, in the C57BL/10 background, also have the protective Ltbp4+36 allele.
We confirmed that the Ltbp4 insertion/deletion segregates with phenotypic variability in muscular dystrophy by examining mixed-background γ-sarcoglycan–null animals with a range of values for membrane permeability defects and fibrosis. Those animals with the lowest values for dye uptake and fibrosis carried the Ltbp4+36 allele (Figure 4C). We generated congenic strains for the γ-sarcoglycan–null allele in the C57BL/6J and CD1 VAF+ backgrounds. Membrane permeability and fibrosis in mice of the congenic strains B6-Sgcg and CD1-Sgcg were comparable to what was seen in 129-Sgcg mice (7), and these strains were also found to harbor the protective allele (Ltbp4+36) (Figure 4C). The mdx model was also found to carry the protective Ltbp4+36 allele (Figure 4C). To ensure that the segregation of Ltbp4 with disease phenotype was not unique to the restricted cohort of animals used for mapping, we replicated these results in an independently bred cohort of additional F2-Sgcg mice from 129-Sgcg and D2-Sgcg parental strains. We genotyped the Ltbp4 allele in this new cohort and found a highly significant correlation between the Ltbp4_Δ_36 allele and the degree of membrane permeability and fibrosis (Table 1).
Ltbp4 genotypes in an independent cohort of _Sgcg_-null mice
Insertion of 12 amino acids in the proline-rich region protects against proteolysis. LTBP4 interacts with the matrix via its aminoterminal segment (19). At the carboxyl terminus, the third cysteine-rich region binds TGF-β1 (17, 18). In between these two domains is a proline-rich region thought to be a target for proteases (14, 15), although the range of proteases responsible for this cleavage is not well defined. Cleavage releases TGF-β from the matrix and renders it accessible to TGF-β receptors, where the ligand can engage the receptor and mediate TGF-β effects (20, 21). The severe phenotype in the D2 background was associated with a deletion of 12 amino acids in the proline-rich region of LTBP4.
To evaluate differential proteolytic sensitivity of LTBP4 from the D2 and 129 strains, we cultured muscle fibroblasts from the parental strains as well as those carrying the _Sgcg_-null allele. Exposure to either pancreatin or plasmin resulted in more digestion of full-length LTBP4 from the D2 background compared with the 129 background, irrespective of the presence of the _Sgcg_-null allele (Figure 5, A and B). To attribute this differential proteolytic susceptibility to the insertion or deletion of 12 amino acids in the proline-rich region, we expressed portions of LTBP4 in vitro. The regions expressed are depicted in Figure 5C; the in vitro products were radiolabeled with [35S]cysteine found only at the carboxyl terminus of the fragments. The 129 and D2 proline-rich regions of LTBP4 were expressed and treated with plasmin, an enzyme implicated in the cleavage of LTBP4 and release of TGF-β (15). Prior to proteolysis, the expressed regions displayed distinct electrophoretic patterns, with the smaller protein migrating more slowly, consistent with altered conformation. The LTBP4 region from the 129 strain (+12 amino acids) was minimally susceptible to proteolysis even at high doses of plasmin (Figure 5E). In contrast, the region of LTBP4 from the D2 strain (deleted for 12 amino acids) displayed a greater susceptibility to plasmin degradation (Figure 5A). Enhanced susceptibility to proteolysis should be associated with increased TGF-β release from the matrix and increased availability to engage cell-surface TGF-β receptors and activate canonical TGF-β signaling.
The 12-amino-acid deletion increases LTBP4 protease susceptibility. (A and B) Total protein was extracted from D2 and 129 fibroblasts with and without the _Sgcg_-null allele, digested, and immunoblotted with an antibody directed toward the amino terminus of LTBP4. (A) Digests with 12 μg of pancreatin. Each pair represents Sgcg mutant, then wild-type. (B) Digestion with 25 μg of plasmin. Each set of 4 represents 3 mutants, then a wild-type. LTBP4 from the D2 strain was digested more easily than LTBP4 from the 129 strain. The percent digested refers to the proteolyzed lower product. On average, plasmin digested 38% of _129_-derived LTBP4 versus 57% of _D2_-derived LTBP4. (C and D) In vitro expression constructs. _D2_- and _129_-derived LTBP4 proline-rich sequences were expressed in vitro. The LTBP4 sequence is underlined; the insertion is shown in gray; and the deletion is represented by dashed lines. The position of the cysteine residues in the expressed sequences is indicated by the asterisks. (E) The constructs were expressed in vitro with [35S]cysteine and then exposed to plasmin, a protease implicated in cleavage of LTBP4 (15). The _D2_-derived sequences are digested at low levels of plasmin, while only the highest level of plasmin can begin to produce the fully digested product from the _129_-derived sequence. The slower migration of the 12-amino-acid-deleted proline-rich region, prior to digestion, is consistent with an altered conformational state that is more susceptible to proteolysis. The arrow indicates the cleavage product.
TGF-β signaling is increased by deletion of 12 amino acids in LTBP4. Upon ligand interaction, TGF-β receptors activate SMAD signaling, whereby SMAD2/3 is phosphorylated and translocated to the nucleus to drive gene expression (13). The Ltbp4 alleles from 129 and D2 backgrounds should reflect a variable capacity to sequester inactive TGF-β and therefore should elicit differential TGF-β–induced signaling because more TGF-β is available for the ligand-receptor interaction. To evaluate this possibility, we isolated muscle fibroblasts from 129-Sgcg and D2-Sgcg mice and then exposed these cells to identical doses of exogenously added TGF-β. We chose fibroblasts for this assay because γ-sarcoglycan is not expressed in fibroblasts, and therefore TGF-β signaling in this assay is isolated from the ongoing disease process itself which can itself activate TGF-β signaling. We maintained the fibroblast cultures at confluence for 5 days, since this condition is shown to be associated with increased LTBP4 expression and deposition into the matrix (22). TGF-β signaling in this assay is dependent on the presence of intact LTBP4 (23). After exposure to TGF-β, fibroblasts from D2-Sgcg mice displayed significantly more SMAD2/3 phosphorylation than those from 129-Sgcg mice (Figure 6A). This increased signaling was not dependent on the presence of the _Sgcg_-null allele, since wild-type DBA also had increased p-SMAD2/3 for the same dose of TGF-β. Increased TGF-β signaling did not arise from altered LTBP4 protein levels, since LTBP4 protein levels were similar in the 2 strains (Figure 6B). Therefore, these data are consistent with a model wherein increased TGF-β–induced signaling in the D2 strain derives from enhanced proteolytic susceptibility of LTBP4 and release of TGF-β, leading to SMAD activation.
Increased proteolytic cleavage is associated with enhanced TGF-β availability and SMAD signaling, accounting for the more severe phenotype. (A) Fibroblasts were cultured from D2-Sgcg and 129-Sgcg muscle. Fibroblasts were exposed to TGF-β, and the amount of phosphorylated SMAD was determined. Fibroblasts from the severely affected D2-Sgcg muscle respond to TGF-β with enhanced p-SMAD signaling. Coomassie-stained actin is the loading control. (B) The amount of LTBP4 protein is similar in D2 and 129 fibroblasts. Muscle fibroblasts were isolated and subjected to immunoblotting with an anti-LTBP4 antibody. The graph represents the densitometer readings of p-SMAD normalized to actin, and the highest value of the 8 animals was set to 100%.
TGF-β signaling is enhanced in the presence of the Ltbp4Δ36 allele. We examined SMAD signaling within the heart and muscle of mutant animals in the enhanced and suppressed genetic backgrounds. We found that TGF-β signaling was enhanced within muscle nuclei, consistent with a TGF-β effect on both fibroblasts and the neighboring myofibers. Within skeletal muscle, the myofiber nuclei of the severe D2-Sgcg strain had increased p-SMAD staining (Figure 7, green nuclei). The centrally placed nuclei within dystrophic skeletal muscle indicate newly regenerated fibers, and these fibers consistently displayed the most intense signaling compared with other nuclei within the same sections. Within cardiac muscle, the D2-Sgcg hearts also showed p-SMAD–positive nuclei (Figure 7). Within the hearts, p-SMAD–positive nuclei were clustered in regions of damage and were more often seen in the right ventricle. Neither wild-type strain showed activation of SMAD signaling (data not shown). These data support a model whereby LTBP4 is preferentially cleaved in the D2-Sgcg background, leading to enhanced TGF-β availability and increased SMAD signaling.
Increased TGF-β signaling within skeletal myofibers and cardiomyocytes in the D2 background. p-SMAD2 (green) is increased in the myonuclei of D2-Sgcg compared with 129-Sgcg skeletal and cardiac muscle. In skeletal muscle, the centrally placed nuclei, indicative of recent regeneration, show the most intense staining (green). Scale bars: 10 μm.