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Dysferlin is a membrane-associated protein implicated in membrane resealing; loss of dysferlin leads to muscular dystrophy. We examined the same loss-of-function Dysf mutation in two different mouse strains, 129T2/SvEmsJ (Dysf129) and C57BL/6J (DysfB6). Although there are many genetic differences between these two strains, we focused on polymorphisms in Anxa6 because these variants were previously associated with modifying a pathologically distinct form of muscular dystrophy and increased the production of a truncated annexin A6 protein. Dysferlin deficiency in the C57BL/6J background was associated with increased Evan's Blue dye uptake into muscle and increased serum creatine kinase compared to the 129T2/SvEmsJ background. In the C57BL/6J background, dysferlin loss was associated with enhanced pathologic severity, characterized by decreased mean fiber cross-sectional area, increased internalized nuclei, and increased fibrosis, compared to that in Dysf129 mice. Macrophage infiltrate was also increased in DysfB6 muscle. High-resolution imaging of live myofibers demonstrated that fibers from DysfB6 mice displayed reduced translocation of full-length annexin A6 to the site of laser-induced sarcolemmal disruption compared to Dysf129 myofibers, and impaired translocation of annexin A6 associated with impaired resealing of the sarcolemma. These results provide one mechanism by which the C57BL/6J background intensifies dysferlinopathy, giving rise to a more severe form of muscular dystrophy in the DysfB6 mouse model through increased membrane leak and inflammation.
Muscular dystrophies are a class of genetic myopathies characterized by progressive deterioration of muscle. Loss-of-function mutations in the gene encoding dysferlin (known as DYSF or FER1L1) produce a range of muscle diseases in humans.
Dysferlin belongs to a larger family of proteins, the ferlin family; family members are defined as having multiple C2 domains similar to those found in the classic Ca2+-sensitive phospholipid binding protein synaptotagmin. C2 domains bind negatively charged phospholipids in the presence of Ca2+. Through its carboxy-terminal transmembrane domain, dysferlin localizes to the plasma membrane and to the transverse tubule, a membranous network that coordinates Ca2+ handling and contraction in muscle.
utilized a laser to ablate the sarcolemma and image repair in live myofibers. Dysf-null fibers resealed sarcolemmal disruption more slowly than did normal fibers, as shown by increased extracellular dye influx.
Loss of dysferlin in humans and mice is characterized by significantly elevated serum creatine kinase (CK) levels, a marker of muscle injury and disease, and progressive muscle weakness. Elevated levels of serum CK are evident before the onset of muscle weakness. Additionally, muscle biopsy samples show increased numbers of inflammatory cells, including macrophages, within the muscle.
Additionally, the same genetic lesion, R1905X, has been reported to result in the full range of dysferlin-associated phenotypes, including limb-girdle muscular dystrophy 2B, Miyoshi myopathy, and distal anterior compartment myopathy.
Anxa6 was found to modify muscular dystrophy in mice lacking the dystrophin-associated protein γ-sarcoglycan, and a loss of γ-sarcoglycan produces muscular dystrophy by causing sarcolemmal instability.
Annexin A6 is an atypical annexin with eight annexin repeats, two regions composed of four repeats connected by a middle hinge. Evolutionarily, annexin A6 has been speculated to have arisen from a gene-duplication event combining annexins A5 and A10.
To better understand the role that the genetic background plays in dysferlin-related muscle disease, we evaluated mouse models harboring the same Dysf-null mutation backcrossed onto two different murine background strains, C57BL/6J and 129T2/SvEmsJ, generating DysfB6 and Dysf129 mouse models, respectively. DysfB6 mice have increased serum CK leak and influx of Evans Blue dye (EBD), suggestive of impaired membrane resealing. Consistent with inefficient repair, DysfB6 muscle pathology, characterized by increased central nuclei, decreased myofiber size, elevated immune infiltrate, and increased fibrosis, is more severe than Dysf129 muscle pathology. We conducted laser disruption of the sarcolemma in Dysf myofibers from both genetic backgrounds and found delayed membrane repair marked by dye uptake in DysfB6 compared to Dysf129 muscle, consistent with increased disease severity. Although there are many genetic differences between these strains, the C57BL/6J strain carries the same Anxa6 allele described in the DBA2J background that produces low-level expression of truncated annexin A6.
We now show that the DysfB6 strain carries Anxa6 gene polymorphisms and the alternatively spliced Anxa6 transcript seen in the DBA2J background. This alternatively spliced Anxa6 transcript resulted in the expression of a truncated form of annexin A6, A6N32. Upon sarcolemmal disruption, we found impaired translocation of the full-length A6 protein to the site of membrane injury in the C57BL/6J strain compared to those from the 129T2/SvEmsJ background. This cellular defect of impaired annexin A6 translocation provides a molecular explanation for the more severe progression of muscle disease in the DysfB6 mice, through its modulation of membrane leak and immune infiltration.
mice were housed in uniform conditions in a single, barrier facility. Congenic mice, Dysf129;Ltbp4d/d and Dysf129;LtbpP4iIi mice, were generated by crossing DBA2J mice with Dysf129 mice over five generations to generate mice that carried both the Dysf-null and Ltbp4 deletion (Ltbp4d/d) alleles on the 129 background strain. All animals were housed and treated in accordance with the standards set by the Animal Care and Use Committee.
Muscles from WT129, WTB6, Dysf129, and DysfB6 mice were examined at >52 weeks of age. The quadriceps muscles were dissected from tendon to tendon (n ≥ 3 mice). The excised muscle was immediately frozen in liquid nitrogen and stored at −80°C. Sections from the center of the muscle were stained with hematoxylin and eosin. The amount of fibers with internal nuclei was calculated as the number of fibers containing internal nuclei/the total number of fibers counted per image, standardized as a percentage (n ≥ 250 fibers per animal). Mean fiber size and fiber variation were calculated using the cross-sectional area of individual myofibers (n ≥ 1208 fibers per animal; n ≥ 3 animals per genotype). Statistical analysis was performed with Prism software version 6.0h (GraphPad Software, Inc., San Diego, CA) using a two-way analysis of variance test. Dysf129;Ltbp4i/i and Dysf129;Ltbp4d/d muscles were examined using similar methods as described earlier in this paragraph from mice at ≥6 months of age (n ≥ 3).
Fibrosis Quantification Analysis
Quadriceps muscles from >52-week-old WT129, WTB6, Dysf129, and DysfB6 mice were sectioned and stained with Picrosirius red (#24901; Polysciences Inc., Warrington, PA). Representative images of stained muscle sections were taken at ×20 magnification. Using ImageJ plug-in software version 1.50i (NIH, Bethesda, MD),
the amount of Picrosirius red–stained collagen was quantified from at least three fields per animal (n ≥ 3 animals per genotype). Statistical analysis was performed with Prism software (GraphPad) using a two-way analysis of variance test. Muscles from Dysf129;Ltbp4i/i and Dysf129;Ltbp4d/d mice (n ≥ 3) were stained with Masson's trichrome and imaged identically (n ≥ 3).
EBD Imaging and Quantification
EBD uptake into muscle was quantified as described previously.
Briefly, EBD (E-2129; Sigma-Aldrich, St. Louis, MO) was dissolved in phosphate-buffered saline at 10 mg/mL. Each animal received an i.p. injection of EBD at 5 μL/g body weight. Approximately 48 hours after injection, tissues were harvested. For quantification, whole tissue was dissected, finely minced, weighed, and incubated at 55°C in 1 mL of formamide for 2 hours, with shaking. Spectrophotometric absorbance was measured at 620 nm. Statistical analysis was performed with Prism software (GraphPad) using an unpaired t-test.
EBD was injected as described in the previous section. Tissue was harvested and flash-frozen in liquid nitrogen. Muscle was sectioned and stained with F4/80 Alexa Fluor 488 (NB600-404AF488; Novus Biologicals, Littleton, CO) used at a dilution of 1:100. Sections were mounted in Vectashield with DAPI (Vector Laboratories, Burlingame, CA). Images were acquired on an Axio Imager.M2 (Carl Zeiss, Oberkochen, Germany). F4/80+ cells were quantified from at least three fields and four animals per genotype. Statistical analysis was performed with Prism software (GraphPad) using an unpaired t-test.
Proteins transferred to polyvinylidene difluoride membranes were immunoblotted with anti–annexin A6 antibody (ab31026; Abcam, Cambridge, UK) used at a dilution of 1:4000. A secondary antibody, goat anti-rabbit conjugated to horseradish peroxidase (Jackson ImmunoResearch Laboratories, West Grove, PA) was used at 1:5000. Blocking and antibody incubations were performed in StartingBlock T20 Blocking Buffer (Pierce, Rockford, IL). SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific, Rochester, NY) and a BioSpectrum Imaging System (UVP, Upland, CA) were used for imaging. As a loading control, MemCode (Thermo Fisher Scientific) was used for reversibly stained, transferred whole muscle lysate. Images were quantified from at least three mice per genotype using ImageJ software (NIH).
RT-PCR and Quantitative PCR Analysis
RNA was isolated from the abdominal muscles and tibialis anterior muscles of age- and sex-matched WT129, WTB6, Dysf129, and DysfB6 mice (n = 3 per genotype). Tissue was immediately placed in TRIzol (Ambion Diagnostics, Austin, TX) and disrupted using a bead homogenizer, followed by centrifugation for 3 minutes at 12,000 × g at 4°C. One-fifth volume of chloroform was added, and the tubes were shaken by hand and then incubated for 5 minutes at room temperature before centrifugation for 15 minutes at 12,000 × g at 4°C. RNA was extracted from the upper phase. RNA extraction was performed using the Aurum Total RNA Mini Kit (Bio-Rad Laboratories, Segrate, Italy) with column DNase digestion, following the manufacturer's guidelines. cDNA was synthesized using qScript cDNA SuperMix (Quanta Biosciences, Gaithersburg, MD) from 1 μg of RNA per sample, following the manufacturer's guidelines. Quantitative PCR was performed using iTaq universal SYBR Green supermix (Bio-Rad) in a CFX96 Real-Time PCR Detection System (Bio-Rad). Control reactions were performed with RNA processed using the same method but without RT. PCR was performed for Anxa6 exons 9/10 to exon 15 using the following primers: Anxa6_9.10F, 5′-ACAGCACCTACGACTGGTGTTTGA-3′; Anxa6_15R, 5′-CAATTCCCTTCATGGCTTTCCGCA-3′. These reactions detect the full-length and alternate Anxa6 transcripts with expected product sizes of 515 and 168 bp, respectively, for 9.10F and 15R. To specifically detect the alternate A6 transcript, the following primer was used: Anxa6_11.15.16R, 5′-CAGTTCCAATTCCCTTCATGGCTTTCCGCAAAAT-3′, in conjunction with the exon 9/10 forward primers, giving an expected product size for these reactions, 174 bp. For all reactions, 35 cycles were performed with annealing at 63°C for 30 seconds and extension at 72°C for 1 minute.
Statistical analysis was performed using Prism software (GraphPad) using a two-way analysis of variance. Quantitative PCR was performed for alternative splice form Anxa6 transcript using the primers described in RT-PCR and Quantitative PCR Analysis. GAPDH was amplified using the following primers: GAPDHF, 5′-TTGTGATGGGTGTGAACCACGA-3′; GAPDHR, 5′-AGCCCTTCCACAATGCCAAAGT-3′. For all reactions, 45 cycles were performed with annealing and extension at 60°C for 70 seconds total. Melt curves of the reaction products were obtained starting at 65°C and increasing to 95°C with a step size of 0.5°C. Quantitative PCR data were analyzed by determining the relative expression of full-length or alternatively spliced Anxa6 transcripts in comparison to the reference gene (Gapdh), following the mathematical model from Pfaffl et al.
Briefly, the footpad was injected with 10 μL of hyaluronidase (8 U). Two hours after injection, up to 20 μL of 2 μg/μL of endotoxin-free plasmid DNA was injected into the footpad between the muscle bundle and the epidermis. Voltage was applied. Muscle fibers were isolated and studied 7 days after electroporation to allow for recovery and efficient protein expression within the electroporated muscles. The flexor digitorum brevis muscle bundle was dissected and placed in Dulbecco's Modified Eagle's Medium containing bovine serum albumin plus collagenase solution. Dissociated fibers were plated on confocal microscopy dishes (P35G-1.5-14-C; MatTek, Ashland, MA). FM 4-64 dye (T-13320; Molecular Probes, Eugene, OR) was added at 2.5 μmol/L before imaging. Fibers were irradiated using the region of interest point in the NIS Elements imaging software version 4.30.02 (Nikon Instruments, Melville, NY) on the A1R confocal microscope (Nikon Instruments) using a 405-nm laser set at 100% power for 5 seconds. Images were acquired before damage, on laser damage, every 2 seconds after damage for 20 seconds, and then one image every 10 seconds for 130 seconds. Images were quantified using ImageJ software (NIH). For quantitative analysis of FM dye, fluorescence was measured at the site of injury in individual frames using ImageJ software (NIH) and adjusted to the baseline fluorescence at time 0 calculated at the membrane before damage (F/F0). Suboptimal fibers were excluded from the analysis. Statistical analysis was performed with Prism software (GraphPad) using an unpaired t-test.
Serum CK Measurement
Serum was collected from age-matched Dysf129 and DysfB6 animals from retro-orbital bleeds using heparinized capillary tubes (Fisher Scientific, Pittsburgh, PA) into serum separator tubes (Becton, Dickinson, and Company, Franklin Lakes, NJ) and centrifuged for 10 minutes at 8000 × g. The plasma fractions were frozen and stored at −80°C and then assayed later using the EnzyChrom Creatine Kinase Assay Kit (ECPK-100; BioAssay Systems, Hayward, CA). Activity was measured in the Synergy HTX Multi-Mode Microplate Reader (BioTek Instruments, Winooski, VT). Statistical analysis was performed with Prism software (GraphPad) using an unpaired t-test.
Grip Strength Measurement
Grip strength was assessed using a grip strength meter (Columbus Instruments, Columbus, OH) consisting of a horizontal bar attached to a force meter. Forelimbs grip the bar in 10 consecutive measurements within 2 minutes. Forces were recorded and normalized to body weight as described previously.
Statistical analysis was performed with Prism software (GraphPad) using an unpaired t-test.
Membrane Leak Is Increased in DysfB6 Myofibers
The AJ mouse strain housed at Jackson Laboratories carries a spontaneous retro-transposon insertion in intron 4 of the Dysf gene, causing a null allele of the Dysf gene and progressive muscular dystrophy.
Although some properties of these mice have been reported individually, myopathic progression has not been directly compared between these two mouse strains. EBD uptake in muscle reflects myofibers that have increased sarcolemmal leak as well as degenerating fibers. Normal muscle is impermeable to this vital tracer, whereas muscular dystrophies with sarcolemmal defects display enhanced EBD uptake.
EBD uptake was visually apparent in DysfB6 muscle, seen as blue striations in the abdominal muscles (Figure 1A). A higher-magnification image is shown in Figure 1B illustrating the presence of EBD following the myofiber pattern. By immunofluorescence microscopy, the quadriceps muscle showed increased numbers of EBD-positive myofibers compared to Dysf129 muscle (Figure 1C). Whole quadriceps muscle was minced and EBD uptake was quantified; DysfB6 muscle contained more EBD than did Dysf129 muscle (P < 0.01) (Figure 1D). Elevated serum CK is a marker of muscle injury and disease in both mice and humans, including the dysferlinopathies.
DysfB6 mice had higher levels of serum CK compared to Dysf129 mice at 7 months, a time of early disease onset (P < 0.05) (Figure 1E). These data show that DysfB6 muscle has increased myofiber leak, consistent with poor myofiber resealing, compared to Dysf129.
DysfB6 Muscle Pathology in the C57BL/6J Background
To determine whether the genetic background modifies muscle disease in the absence of dysferlin, muscle from both Dysf129 and DysfB6 mice were analyzed. Muscle from 52-week-old Dysf mice displayed characteristic myopathic features, including internalized nuclei, fibrosis, and immune infiltrate (Figure 2A). DysfB6 muscles had an increased number of smaller-sized fibers and a reduction in largest myofibers compared to Dysf129 muscle and WT control (Figure 2, B and C). These data correlated with a reduction in the mean cross-sectional area. Both Dysf129 and DysfB6 myofiber size were reduced compared to strain-matched controls, and DysfB6 muscle also showed reduced cross-sectional area compared to Dysf129 muscle (P < 0.05) (Figure 2D). Consistent with a more severe myopathic phenotype, DysfB6 muscle contained an increased percentage of myofibers with internalized nuclei compared to both WT controls and Dysf129 muscle (P < 0.05) (Figure 2E). These data indicate that the phenotype of dysferlin-mediated muscular dystrophy is enhanced in the C57BL/6J background compared to the 129T2/SvEmsJ background.
Increased Inflammation in the C57BL/6J Background
Inflammatory infiltration has been described in human muscle biopsy samples with DYSF mutations.
F4/80 conjugated to Alexa Fluor 488 is a marker of activated macrophages, and F4/80 immunofluorescence imaging showed increased macrophage infiltration, represented as number of F4/80+ cells per field, in DysfB6 muscle compared to Dysf129 muscle (P < 0.05) (Figure 2F). The percentage of F4/80+ cells per total nuclei was also significantly increased in DysfB6 muscle (data not shown). Furthermore, DysfB6 muscle represented as F4/80+ cells per fiber was also increased compared to Dysf129 muscle (data not shown). Consistent with the increased pathology and membrane leak, an increased number of macrophages are present in muscle from the C57BL/6J background.
Decreased Strength in DysfB6 Mice Compared to Dysf129 Mice
To examine the effect of genetic background on muscle strength in combination with the loss of dysferlin, we examined grip strength. DysfB6 mice had reduced grip strength when normalized to body weight compared to age-matched and sex-matched Dysf129 mice (2.59 versus 2.92 kgf/kg, respectively; P < 0.05) (Figure 2G).
DysfB6 Muscle Has Increased Levels of Fibrosis Compared to Dysf129 Muscle
Fibrosis, or the increase in interstitial collagen deposition, is a pathologic feature that is increased in many forms of muscular dystrophies.
Muscle samples were stained with Picrosirius red and imaged to determine the fibrosis composition. At 52 weeks, DysfB6 muscles had grossly increased Picrosirius red staining compared to Dysf129 muscle and WT controls (Figure 3A). The amount of Picrosirius red was quantified using ImageJ plug-in software (NIH). DysfB6 muscle showed statistically elevated levels of fibrosis compared to Dysf129 muscle and WT controls (P < 0.05) (Figure 3B). These data show that the genetic background modifies fibrosis with increased muscle fibrosis in DysfB6 mice compared to Dysf129 mice.
Decreased Membrane Repair in DysfB6 Myofibers
Dysferlin is a protein known to be required for efficient sarcolemmal repair, and a lack of dysferlin delays muscle membrane resealing.
We evaluated whether the genetic background alters membrane by conducting laser wounding on isolated myofibers from Dysf129 and DysfB6 mice in the presence of FM 1-43, a lipophilic dye that under normal conditions exhibits minimal fluorescence but increases fluorescence after damage as it binds the phospholipid membrane (Figure 4A). We hypothesized that if a difference is seen between these two strains, it is not due to the identical dysferlin lesion but due to another genetic variant that is distinct between these two background strains. After laser-induced membrane damage, FM 1-43 dye uptake was elevated over time in DysfB6 myofibers compared to that in age-matched Dysf129 myofibers (Figure 4B). Myofibers lacking dysferlin on the C57BL/6J background had significantly elevated FM-dye influx at 100 seconds after injury (P < 0.05) (Figure 4C). These data show that genetic background plays a distinct role in influencing the process of membrane repair in dysferlinopathies.
Truncated Annexin A6 Is Expressed in DysfB6 Tissue
Annexin A6 (A6) is a 68-kDa, atypical annexin that contains eight annexin repeats split by a hinge region. Anxa6, encoding annexin A6, was recently found to modify muscular dystrophy caused by mutant γ-sarcoglycan, a protein that mediates membrane stability.
Specifically, the DBA2J strain was shown to harbor a cryptic A-to-G splice site in Anxa6. This allele results in a truncated annexin A6 protein product of approximately 32 kDa, predicted to contain the first four annexin repeat domains (Figure 5A). The region of chromosome 11, which harbors the Anxa6 gene, is shared between the DBA2J and C57BL/6J strains, but is not shared by the 129T2/SvEmsJ strain. Sanger sequencing confirmed that both the WTB6 and DysfB6 background strains contained the G variant in Anxa6, resulting in the altered cryptic splice donor, whereas the WT129 and Dysf129 contained the A nucleotide (Figure 5B). Immunoblot analysis was performed with an antibody against the amino-terminus of annexin A6 that recognizes both the full-length annexin A6 protein and the truncated annexin A6 protein (A6N32) (Figure 5C). The expression of full-length annexin A6 protein was twofold greater in Dysf-null muscle compared to WT in both genetic backgrounds, whereas the expression of A6N32 was 20-fold greater in DysfB6 muscle compared to Dysf129 muscle (Figure 5, C and D). Total protein bands running near 43 kDa are shown as a loading control. This low-level expression of A6N32 protein was comparable to that previously observed in the DBA2J background in Sgcg-null muscle.
To examine how the presence of the single-nucleotide polymorphism in Anxa6 alters annexin A6 transcript expression, RT-PCR was performed on RNA isolated from abdominal muscle samples from WT129, WTB6, Dysf129, and DysfB6 mice. Previously these single-nucleotide polymorphisms were associated with the presence of an alternative transcript that joins the middle of exon 11 to the middle of exon 15, resulting in a premature stop codon.
Primers designed to detect both full length and the alternative transcript and primers specific to only the alterative splice product were individually tested. Products of expected size were observed, and qualitatively, C57BL/6J muscle expressed an increased amount of alternative transcript compared to 129T2/SvEmsJ muscle (Figure 6A). To quantify the difference in transcript expression, abdominal muscle from WT and mutant mice in the two backgrounds was compared by quantitative PCR. WTB6 and DysfB6 muscle contained significantly increased amounts of full-length and alternative transcripts compared to mice on the 129 background (P < 0.05) (Figure 6, B and C). RNA isolated from tibialis anterior muscle generated annexin A6 and A6N32 transcript profile patterns similar to those generated from abdominal muscle (P < 0.05) (Figure 6, D and E).
DysfB6 Myofibers Have Delayed Annexin A6 Translocation to Sites of Sarcolemmal Disruption Compared to Dysf129 Myofibers
Given the more severe pathology of the DysfB6 mouse model compared to the Dysf129 model, we assessed the effect of genetic background on full-length annexin A6 translocation in the absence of dysferlin. Delayed annexin A6 translocation was previously observed when truncated annexin A6, A6N32, was introduced into WT129 myofibers.
To examine whether the genetic background altered annexin A6 translocation to the site of injury, annexin A6-GFP plasmid was electroporated in DysfB6 or Dysf129 muscles followed by harvest and live cell imaging after laser-induced sarcolemmal wounding. In Dysf129 myofibers, full-length annexin A6 translocated rapidly to the membrane, forming a tight aggregation at the site of laser wounding, similar to previous findings observed in WT fibers.
With differential interference contrast imaging, accumulation of annexin A6 was observed at the site of injury associated with the myofiber (Figure 7A). In contrast, translocation of annexin A6-GFP was reduced in DysfB6 myofibers (Figure 7A). The reduced size of the A6 aggregate in DysfB6 myofibers is visible in the confocal Z-stack projections (Figure 7B). At 150 seconds after wounding, the size of the DysfB6 annexin A6 aggregate at the site of laser wounding was significantly reduced compared to that observed in Dysf129 myofibers (P < 0.01) (Figure 7C). The presence of a genetic background that expressed A6N32 inhibited the translocation of full-length annexin A6 in Dysf-null muscle.
Ltbp4 Does Not Modify Dysferlin-Mediated Muscular Dystrophy
Like Anxa6, Ltbp4, encoding the latent transforming growth factor β–binding protein 4, was similarly identified in a genome-wide scan for modifiers of muscular dystrophy in which an insertion/deletion polymorphism alters the proline-rich hinge region of the Ltbp4 protein.
The DBA2J strain harbors the deleterious Ltbp4 deletion allele (Ltbp4d/d). The deleterious Ltbp4d/d allele was backcrossed into the Dysf129 mouse strain, generating congenic Dysf129 mice with, Dysf129;Ltbp4i/i, and without, Dysf129;Ltbp4d/d. The pathology in these two lines was compared in mice ≥6 months of age. Both Dysf129;Ltbp4d/d and Dysf129;Ltbp4i/i muscle displayed characteristic myopathic features of dysferlinopathy, including internalized nuclei, variable fiber size, and immune infiltrate (Figure 8A). However, the presence of Ltbp4d/d did not change the percentage of myofibers with internalized nuclei (Figure 8B). Fibrosis was assessed through Masson's trichrome staining. No evidence of increased fibrosis was attributable to the Ltbp4 polymorphism (Figure 8C). Membrane leak was assayed through EBD uptake. Dysf129;Ltbp4d/d muscle contained an amount of EBD uptake similar to that of Dysf129;Ltbp4i/i muscle (Figure 8D). These data suggest that the presence of the Ltbp4d/d did not intensify Dysf-mediated muscular dystrophy in mice, unlike the annexin–dysferlin interaction, which mediates muscle disease progression.
Genetic Modifiers of Dysferlinopathy
Muscular dystrophy is defined as a progressive loss of muscle fibers that outpaces the ability for muscle regeneration. Muscle tissue is generally replaced with fibrosis and fatty infiltration, resulting in progressive muscle weakness. To date, over 40 genes have been established as primary gene mutations resulting in dystrophy. Despite the identification of a large number of disease-causing genes, phenotypic outcome associated with a genetic lesion is variable, with a range of age of onset, timing of loss of ambulation, and muscle group involvement. Genetic modifiers act in combination with the primary genetic mutation to influence the outcome or severity of disease. Herein, we utilized two mouse strains harboring the same dysferlin loss-of-function mutation on two different backgrounds, DysfB6 and Dysf129. There are many genetic differences between these strains that can contribute to disease phenotype. In a strict sense, genetic modifiers should have little effect on basal phenotypes, but should instead manifest their effect in the context of disease or other primary phenotype.
We previously mapped two genetic modifiers of muscular dystrophy, Ltbp4 and Anxa6, in mice lacking the dystrophin-associated protein γ-sarcoglycan.
Here, we evaluated the contribution of the Ltbp4 modifier using a congenic approach, but found no discernable effect in mice lacking dysferlin. This observation does not exclude an interaction between Ltbp4 and dysferlinopathy, but certainly the findings suggest that any effect from this locus is modest at best. The Ltbp4 polymorphism acts by regulating latent transforming growth factor β release and its ability to interact with cell surface receptors triggering an increase in intracellular transforming growth factor β signaling. Elevated transforming growth factor β levels are associated with dystrophin-linked dystrophy, and this finding may suggest that transforming growth factor β–mediated responses have less effect in dysferlin deficiency. The observed decreases in SMAD3 and SMAD4 in dysferlinopathy are consistent with this observation.
At the same time, we took advantage of the availability of the dysferlin-null allele in two genetic backgrounds, C57BL/6J and 129T2/SvEmsJ. By multiple measures, the C57BL/6J background enhances or intensifies the loss of dysferlinopathy, confirming that this is a better model for evaluating the relatively slowly progressive form of muscular dystrophy in mice. Mapping the modifier loci in dysferlinopathy requires substantial intercrossing of these genetic backgrounds, and this mapping is hampered by a late-onset phenotype in dysferlinopathic mice.
Annexin A6 Translocation to Sites of Sarcolemmal Disruption Is Delayed in the C57BL/6J Background
There are substantial data to support an interaction between dysferlin and the annexin complex. Annexins A1 and A2 were previously observed to interact with dysferlin.
Muscular dystrophy has a distinct presentation in zebrafish compared to mammalian muscle, owing to the shorter lifespan and less fibrous replacement than that normally observed in mammalian muscle disease. We previously introduced the truncated annexin A6, A6N32, into 129T2/SVEmsJ muscle, which lacks appreciable expression of this truncated protein.
We found that low-level expression of A6N32 produced a phenotype of delayed annexin A6 trafficking akin to what we now observe in DysfB6 muscle, with a similar delay in translocation and the same reduced size of annexin A6 aggregates at the site of injury. We found that DysfB6 mice, which harbor the deleterious Anxa6 allele, express the truncated protein product, A6N32. In this genetic background, there was enhancement of the muscular dystrophy phenotype and reduced translocation of annexin A6 to sites of sarcolemmal disruption.
Gene mutations that disrupt either sarcolemmal stability or sarcolemmal repair result in increased cellular permeability. A leaky plasma membrane allows for the influx of Ca2+ as well as efflux of cytoplasmic contents, eventually leading to cellular dysfunction and death if not repaired by the resealing machinery. CK leak from muscle into serum is a general phenomenon; recent studies suggest that a number of muscle proteins leak into serum, including titin, malate dehydrogenase 2, carbonic anhydrase III, and myosin heavy chain 1.
The absence of dystrophin renders the sarcolemma fragile and susceptible to injury, whereas the loss of dysferlin impairs membrane trafficking, including that associated with resealing of sarcolemmal disruptions. The observation of increased FM dye leak in DysfB6 compared to Dysf129 fibers is consistent with the elevated serum CK and also is consistent with the molecular signature of delayed resealing observed in the wounding assay. The degree to which delayed sarcolemmal repair contributes to dysferlin-mediated pathology has been questioned, because repair does still occur in the absence of dysferlin.
Annexins are known to homo- and hetero-oligomerize, and we hypothesize that low levels of A6N32 inhibit the formation of higher-order annexin structures that are needed to orchestrate efficient resealing.
a direct interaction between annexin A6 and dysferlin is not required for this inhibition if the larger annexin structure is abnormal. We previously showed co-localization between dysferlin and annexin A6 in muscular dystrophy,
showed that the Dysf-null mice on the AJ background have increased macrophage infiltration and muscle damage after large strain injury compared to A/WySnJ controls. Annexins, especially annexin A1, are linked to inflammation.
showed that A6Δ192, lacking six of the eight annexin domains, was also sufficient for blocking the function of full-length A6 in fibroblast endocytosis and exocytosis, further confirming that truncated annexin A6 can exert a dominant-negative role.
Moreira Ede S.
A gene related to Caenorhabditis elegans spermatogenesis factor fer-1 is mutated in limb-girdle muscular dystrophy type 2B.
Supported by NIH grants NS047726 , NS072027 , and AR052646 (all to E.M.M.). Multiphoton microscopy was performed at the Northwestern University Center for Advanced Microscopy on a Nikon A1R multiphoton microscope acquired through the support of NIH grant 1S10OD010398-01 .