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Enhanced Muscular Dystrophy from Loss of Dysferlin Is Accompanied by Impaired Annexin A6 Translocation after Sarcolemmal Disruption

Open ArchivePublished:April 09, 2016DOI:https://doi.org/10.1016/j.ajpath.2016.02.005
      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.
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      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.
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      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.

      Materials and Methods

      Animals

      Wild-type 129T2/SvEmsJ (WT129), WT C57BL/6J (WTB6), Dysf129,
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      and DysfB6
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      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.

      Muscle Analysis

      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),
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      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.
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      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.

      Macrophage Visualization

      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.

      Immunoblot Analysis

      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.
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      Prism software (GraphPad) was used for performing a two-way analysis of variance.

      Electroporation, Fiber Preparation, and Laser Damage Assay

      The ANXA6-GFP plasmid was described previously.
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      • Vergara J.
      DNA transfection of mammalian skeletal muscles using in vivo electroporation.
      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.
      • Rayavarapu S.
      • Van der Meulen J.H.
      • Gordish-Dressman H.
      • Hoffman E.P.
      • Nagaraju K.
      • Knoblach S.M.
      Characterization of dysferlin deficient SJL/J mice to assess preclinical drug efficacy: fasudil exacerbates muscle disease phenotype.
      Statistical analysis was performed with Prism software (GraphPad) using an unpaired t-test.

      Results

      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.
      • Ho M.
      • Post C.M.
      • Donahue L.R.
      • Lidov H.G.
      • Bronson R.T.
      • Goolsby H.
      • Watkins S.C.
      • Cox G.A.
      • Brown Jr., R.H.
      Disruption of muscle membrane and phenotype divergence in two novel mouse models of dysferlin deficiency.
      This Dysf allele was previously backcrossed to the C57BL/6 and referred to as Bla/J.
      • Lostal W.
      • Bartoli M.
      • Bourg N.
      • Roudaut C.
      • Bentaib A.
      • Miyake K.
      • Guerchet N.
      • Fougerousse F.
      • McNeil P.
      • Richard I.
      Efficient recovery of dysferlin deficiency by dual adeno-associated vector-mediated gene transfer.
      In separate studies, this same allele was backcrossed in the 129T2/SvEmsJ strain; herein these strains are referred to as DysfB6 and Dysf129, respectively.
      • Demonbreun A.R.
      • Fahrenbach J.P.
      • Deveaux K.
      • Earley J.U.
      • Pytel P.
      • McNally E.M.
      Impaired muscle growth and response to insulin-like growth factor 1 in dysferlin-mediated muscular dystrophy.
      • Lostal W.
      • Bartoli M.
      • Bourg N.
      • Roudaut C.
      • Bentaib A.
      • Miyake K.
      • Guerchet N.
      • Fougerousse F.
      • McNeil P.
      • Richard I.
      Efficient recovery of dysferlin deficiency by dual adeno-associated vector-mediated gene transfer.
      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.
      • Straub V.
      • Rafael J.A.
      • Chamberlain J.S.
      • Campbell K.P.
      Animal models for muscular dystrophy show different patterns of sarcolemmal disruption.
      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.
      • Brancaccio P.
      • Lippi G.
      • Maffulli N.
      Biochemical markers of muscular damage.
      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.
      Figure thumbnail gr1
      Figure 1Myofiber leak is increased in DysfB6 muscle compared to Dysf129 muscle. A: Gross images of Evans Blue dye (EBD) uptake in abdominal muscle show increased EBD uptake (blue streaks) in DysfB6 muscle. B: High-magnification images show that EBD uptake is increased in DysfB6 myofibers (black arrow). C: Myofiber EBD uptake is increased (red; white arrow), a marker of muscle damage, in DysfB6 quadriceps muscles. Nuclei were stained with DAPI. D: At 1 year of age, EBD uptake in quadriceps muscle is increased in DysfB6 mice compared to age-matched Dysf129 mice. E: Serum creatine kinase (CK) is elevated, a marker of muscle damage, in DysfB6 compared to Dysf129. Data are expressed as means ± SEM. n = 6 mice per genotype (D); n ≥ 10 mice per group (E). P < 0.05, ∗∗P < 0.01 versus Dysf129. Scale bars: 10mm (A); 1mm (B); 50 μm (C).

      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.
      Figure thumbnail gr2
      Figure 2Histopathology is increased in DysfB6 mice compared to Dysf129 mice. A: DysfB6 quadriceps (Quad) muscles display characteristic features of muscular dystrophy, including internalized nuclei, fibrosis, and immune infiltrate. B and C: The number of largest myofibers is reduced, and the number of smaller-sized fibers is increased, in B6 muscles in both wild-type (WT) and Dysf mutants. D: Cross-sectional area is reduced in Dysf129 and DysfB6 muscle compared with WT controls. Cross-sectional area is reduced in DysfB6 muscle, which expresses annexin A6N32, compared to Dysf129 muscle. E: The percentage of myofibers with internalized nuclei is significantly increased in DysfB6 muscle compared to both WT and Dysf129 muscle. F: Quantification of F4/80+ cells shows an increased macrophage infiltrate per field in DysfB6 muscle compared to Dysf129 muscle. G: Grip strength is reduced in DysfB6 mice. Data are expressed as means ± SEM (E–G). n ≥ 3 mice per genotype (BF); n = 9 mice per genotype (G). P < 0.05. Scale bars = 50 μm. H&E, hematoxylin and eosin.

      Increased Inflammation in the C57BL/6J Background

      Inflammatory infiltration has been described in human muscle biopsy samples with DYSF mutations.
      • Illa I.
      • Serrano-Munuera C.
      • Gallardo E.
      • Lasa A.
      • Rojas-Garcia R.
      • Palmer J.
      • Gallano P.
      • Baiget M.
      • Matsuda C.
      • Brown R.H.
      Distal anterior compartment myopathy: a dysferlin mutation causing a new muscular dystrophy phenotype.
      • Cenacchi G.
      • Fanin M.
      • De Giorgi L.B.
      • Angelini C.
      Ultrastructural changes in dysferlinopathy support defective membrane repair mechanism.
      • Confalonieri P.
      • Oliva L.
      • Andreetta F.
      • Lorenzoni R.
      • Dassi P.
      • Mariani E.
      • Morandi L.
      • Mora M.
      • Cornelio F.
      • Mantegazza R.
      Muscle inflammation and MHC class I up-regulation in muscular dystrophy with lack of dysferlin: an immunopathological study.
      • McNally E.M.
      • Ly C.T.
      • Rosenmann H.
      • Mitrani Rosenbaum S.
      • Jiang W.
      • Anderson L.V.
      • Soffer D.
      • Argov Z.
      Splicing mutation in dysferlin produces limb-girdle muscular dystrophy with inflammation.
      Activated macrophages were increased in the SJL/J mouse model.
      • Nagaraju K.
      • Rawat R.
      • Veszelovszky E.
      • Thapliyal R.
      • Kesari A.
      • Sparks S.
      • Raben N.
      • Plotz P.
      • Hoffman E.P.
      Dysferlin deficiency enhances monocyte phagocytosis: a model for the inflammatory onset of limb-girdle muscular dystrophy 2B.
      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.
      • Mann C.J.
      • Perdiguero E.
      • Kharraz Y.
      • Aguilar S.
      • Pessina P.
      • Serrano A.L.
      • Munoz-Canoves P.
      Aberrant repair and fibrosis development in skeletal muscle.
      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.
      Figure thumbnail gr3
      Figure 3Fibrosis in skeletal muscle is increased in DysfB6 compared to Dysf129. A: At 52 weeks, Picrosirius red staining in quadriceps (Quad) muscle is increased in DysfB6. B: Quantification of collagen shows fibrosis that is greater in mice with the annexin A6N32 (DysfB6 and WTB6) than in mice with full-length annexin A6 (Dysf129 and WT129). Data are expressed as means ± SEM. n = 3 mice per genotype. P < 0.05. Scale bars = 50 μm. WT, wild type.

      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.
      • Bansal D.
      • Miyake K.
      • Vogel S.S.
      • Groh S.
      • Chen C.C.
      • Williamson R.
      • McNeil P.L.
      • Campbell K.P.
      Defective membrane repair in dysferlin-deficient muscular dystrophy.
      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.
      Figure thumbnail gr4
      Figure 4B6 background contributes to plasma membrane repair in the absence of dysferlin. A: Myofibers from dysferlin-null mice (Dysf129 and DysfB6) were isolated and subjected to laser-induced injury in the presence of FM 1-43. Representative images after injury demonstrate FM 1-43 uptake that is increased in DysfB6 fibers (arrow). B: Over time, FM 1-43 uptake is increased in DysfB6 fibers compared to Dysf129 myofibers. C: At 100 seconds after damage, FM 1-43 dye uptake is significantly greater in DysfB6 animals lacking Dysf and containing the A6N32 splice variant than in Dysf129 myofibers, which do not express the A6N32 splice variant at an appreciable level. Data are expressed as means ± SEM. n = 3 mice per genotype (B and C); n ≥ 7 fibers isolated (B and C). P < 0.05. Scale bars = 4 μm.

      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.
      • Swaggart K.A.
      • Demonbreun A.R.
      • Vo A.H.
      • Swanson K.E.
      • Kim E.Y.
      • Fahrenbach J.P.
      • Holley-Cuthrell J.
      • Eskin A.
      • Chen Z.
      • Squire K.
      • Heydemann A.
      • Palmer A.A.
      • Nelson S.F.
      • McNally E.M.
      Annexin A6 modifies muscular dystrophy by mediating sarcolemmal repair.
      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.
      • Swaggart K.A.
      • Demonbreun A.R.
      • Vo A.H.
      • Swanson K.E.
      • Kim E.Y.
      • Fahrenbach J.P.
      • Holley-Cuthrell J.
      • Eskin A.
      • Chen Z.
      • Squire K.
      • Heydemann A.
      • Palmer A.A.
      • Nelson S.F.
      • McNally E.M.
      Annexin A6 modifies muscular dystrophy by mediating sarcolemmal repair.
      Figure thumbnail gr5
      Figure 5Dysferlin and annexin A6 (A6) are involved in skeletal muscle membrane repair. A: Annexin A6 is a 68-kDa, Ca2+-dependent membrane binding protein that contains eight annexin repeats. Annexin A6N32 (A6N32) is a 32-kDa, truncated splice form of the annexin A6 protein. Dysferlin is a 237-kDa, calcium-dependent phospholipid-binding protein that contains seven C2 domains and a carboxyl-terminal transmembrane domain. B: Sanger sequencing confirms the single-nucleotide polymorphism (G) in Anxa6 in wild-type (WT)B6 and DysfB6 mouse strains. This sequence is associated with the alternative Anxa6 transcript that encodes A6N32.
      • Swaggart K.A.
      • Demonbreun A.R.
      • Vo A.H.
      • Swanson K.E.
      • Kim E.Y.
      • Fahrenbach J.P.
      • Holley-Cuthrell J.
      • Eskin A.
      • Chen Z.
      • Squire K.
      • Heydemann A.
      • Palmer A.A.
      • Nelson S.F.
      • McNally E.M.
      Annexin A6 modifies muscular dystrophy by mediating sarcolemmal repair.
      C: Immunoblot analysis with the anti–annexin A6 antibody recognizes full-length annexin A6 and A6N32. Low-exposure imaging demonstrates that full-length annexin A6 is more highly expressed in Dysf-null mice compared to strain-matched WT controls. D: On longer exposure, annexin A6N32 is visible (arrows) in DysfB6 muscle lysates. The 43-kDa protein band was used as a loading control using MemCode reversible stain (Thermo Fisher Scientific, Rochester, NY).
      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.
      • Swaggart K.A.
      • Demonbreun A.R.
      • Vo A.H.
      • Swanson K.E.
      • Kim E.Y.
      • Fahrenbach J.P.
      • Holley-Cuthrell J.
      • Eskin A.
      • Chen Z.
      • Squire K.
      • Heydemann A.
      • Palmer A.A.
      • Nelson S.F.
      • McNally E.M.
      Annexin A6 modifies muscular dystrophy by mediating sarcolemmal repair.
      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).
      Figure thumbnail gr6
      Figure 6Annexin A6 (A6) transcripts are expressed differentially between the B6 and 129 mouse strains. Annexin A6 is highly expressed in muscle. A single-nucleotide polymorphism in the B6 genome results in an alternative splice variant of annexin A6, A6N32. A: RT-PCR shows amplification of full-length annexin A6, running at 515 bp, in both backgrounds. Primers specific for the A6N32 splice form show increased amplification in the B6 background, running at 174 bp. B: Quantitative PCR from abdominal muscle shows expression of full-length annexin A6 that is significantly increased on the B6 strains compared to the 129 strains in both wild-type (WT) and Dysf mice. C: By quantitative PCR from abdominal muscle, A6N32 transcripts are increased in WTB6 and DysfB6 mice compared to mice on the 129 background. D: Similar to findings in abdominal muscle, quantitative PCR from tibialis anterior (TA) muscle shows expression of full-length annexin A6 that is significantly increased on the B6 strains compared to the 129 strains in both WT and Dysf mice. E: In TA muscle, A6N32 transcripts are increased in WTB6 and DysfB6 mice compared to WT129 and Dysf129 mice. Data are expressed as means ± SEM. n = 3 mice per genotype. P < 0.05. Abs, abdominal muscle.

      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.
      • Swaggart K.A.
      • Demonbreun A.R.
      • Vo A.H.
      • Swanson K.E.
      • Kim E.Y.
      • Fahrenbach J.P.
      • Holley-Cuthrell J.
      • Eskin A.
      • Chen Z.
      • Squire K.
      • Heydemann A.
      • Palmer A.A.
      • Nelson S.F.
      • McNally E.M.
      Annexin A6 modifies muscular dystrophy by mediating sarcolemmal repair.
      Overexpression of exogenous A6N32 reduced the formation of annexin A6 aggregates at the site of injury, and is associated with increased FM dye influx.
      • Swaggart K.A.
      • Demonbreun A.R.
      • Vo A.H.
      • Swanson K.E.
      • Kim E.Y.
      • Fahrenbach J.P.
      • Holley-Cuthrell J.
      • Eskin A.
      • Chen Z.
      • Squire K.
      • Heydemann A.
      • Palmer A.A.
      • Nelson S.F.
      • McNally E.M.
      Annexin A6 modifies muscular dystrophy by mediating sarcolemmal repair.
      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.
      • Swaggart K.A.
      • Demonbreun A.R.
      • Vo A.H.
      • Swanson K.E.
      • Kim E.Y.
      • Fahrenbach J.P.
      • Holley-Cuthrell J.
      • Eskin A.
      • Chen Z.
      • Squire K.
      • Heydemann A.
      • Palmer A.A.
      • Nelson S.F.
      • McNally E.M.
      Annexin A6 modifies muscular dystrophy by mediating sarcolemmal repair.
      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.
      Figure thumbnail gr7
      Figure 7Reduced full-length annexin A6 (A6) translocation during membrane repair in DysfB6-null myofibers. Dysf129 and DysfB6 myofibers were electroporated with annexin A6-GFP plasmid, followed by harvest and live cell imaging after laser ablation. A: In Dysf129 myofibers, annexin A6 (green) translocated rapidly to the site of laser disruption, forming a tight aggregate over the site of disruption (long arrow, A and B), as seen in the merged differential interference contrast (DIC) image. In contrast, in DysfB6 myofibers that endogenously express A6N32, annexin A6 (green) translocated less well, forming a smaller-sized aggregate (short arrow) at the site of membrane disruption. These data indicate that annexin A6 translocation occurred in the absence of dysferlin. B: On Z-stack projections of injured myofibers, the size of the A6 aggregate is reduced in DysfB6 compared to Dysf129 myofibers. C: The size of the A6 aggregate is reduced in DysfB6 myofibers compared to Dysf129 myofibers. Data are expressed as means ± SEM. n = 3 mice per genotype (C); n ≥ 8 fibers isolated (C). ∗∗P < 0.01. Scale bars: 4 μm (A); 8 μm (B).

      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.
      • Heydemann A.
      • Ceco E.
      • Lim J.E.
      • Hadhazy M.
      • Ryder P.
      • Moran J.L.
      • Beier D.R.
      • Palmer A.A.
      • McNally E.M.
      Latent TGF-beta-binding protein 4 modifies muscular dystrophy in mice.
      Notably, LTBP4 also modifies years of ambulation in human Duchenne muscular dystrophy.
      • Flanigan K.M.
      • Ceco E.
      • Lamar K.M.
      • Kaminoh Y.
      • Dunn D.M.
      • Mendell J.R.
      • King W.M.
      • Pestronk A.
      • Florence J.M.
      • Mathews K.D.
      • Finkel R.S.
      • Swoboda K.J.
      • Gappmaier E.
      • Howard M.T.
      • Day J.W.
      • McDonald C.
      • McNally E.M.
      • Weiss R.B.
      United Dystrophinopathy Project
      LTBP4 genotype predicts age of ambulatory loss in Duchenne muscular dystrophy.
      In inbred mouse strains, the insertion allele is more common.
      • Heydemann A.
      • Ceco E.
      • Lim J.E.
      • Hadhazy M.
      • Ryder P.
      • Moran J.L.
      • Beier D.R.
      • Palmer A.A.
      • McNally E.M.
      Latent TGF-beta-binding protein 4 modifies muscular dystrophy in mice.
      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.
      Figure thumbnail gr8
      Figure 8Ltbp4 does not alter Dysf histopathology or permeability. We have shown previously that Ltbp4 modifies muscular dystrophy in both mice and humans.
      • Heydemann A.
      • Ceco E.
      • Lim J.E.
      • Hadhazy M.
      • Ryder P.
      • Moran J.L.
      • Beier D.R.
      • Palmer A.A.
      • McNally E.M.
      Latent TGF-beta-binding protein 4 modifies muscular dystrophy in mice.
      • Flanigan K.M.
      • Ceco E.
      • Lamar K.M.
      • Kaminoh Y.
      • Dunn D.M.
      • Mendell J.R.
      • King W.M.
      • Pestronk A.
      • Florence J.M.
      • Mathews K.D.
      • Finkel R.S.
      • Swoboda K.J.
      • Gappmaier E.
      • Howard M.T.
      • Day J.W.
      • McDonald C.
      • McNally E.M.
      • Weiss R.B.
      United Dystrophinopathy Project
      LTBP4 genotype predicts age of ambulatory loss in Duchenne muscular dystrophy.
      Congenic mice were generated that carry the previously identified Ltbp4 modifier allele (Ltbp4d/d) on the Dysf129 background. A: Dysf quadriceps muscles with (Dysf129;Ltbp4i/i) and without (Dysf129;Ltbp4d/d) the Ltbp4 deletion allele display characteristic features of muscular dystrophy, including internalized nuclei, fibrosis, and immune infiltrate, at ≥6 months. B: The percentage of myofibers with internalized nuclei is similar between Dysf129;Ltbp4d/d muscle and Dysf129;Ltbp4i/i muscle. C: Masson's trichrome (MT) staining, marking collagen content blue, is similar in quadriceps muscle at ≥6 months. D: In abdominal muscle, Evans Blue dye uptake is similar between Dysf129;Ltbp4d/d and age-matched Dysf129;Ltbp4i/i mice. Data are expressed as means ± SEM. n ≥ 4 mice per genotype (A–C); n ≥ 8 mice per genotype (D). Scale bars = 50 μm. H&E, hematoxylin and eosin.

      Discussion

      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.
      • Swaggart K.A.
      • Demonbreun A.R.
      • Vo A.H.
      • Swanson K.E.
      • Kim E.Y.
      • Fahrenbach J.P.
      • Holley-Cuthrell J.
      • Eskin A.
      • Chen Z.
      • Squire K.
      • Heydemann A.
      • Palmer A.A.
      • Nelson S.F.
      • McNally E.M.
      Annexin A6 modifies muscular dystrophy by mediating sarcolemmal repair.
      • Heydemann A.
      • Ceco E.
      • Lim J.E.
      • Hadhazy M.
      • Ryder P.
      • Moran J.L.
      • Beier D.R.
      • Palmer A.A.
      • McNally E.M.
      Latent TGF-beta-binding protein 4 modifies muscular dystrophy in mice.
      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.
      • Wenzel K.
      • Zabojszcza J.
      • Carl M.
      • Taubert S.
      • Lass A.
      • Harris C.L.
      • Ho M.
      • Schulz H.
      • Hummel O.
      • Hubner N.
      • Osterziel K.J.
      • Spuler S.
      Increased susceptibility to complement attack due to down-regulation of decay-accelerating factor/CD55 in dysferlin-deficient muscular dystrophy.
      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.
      • Lennon N.J.
      • Kho A.
      • Bacskai B.J.
      • Perlmutter S.L.
      • Hyman B.T.
      • Brown Jr., R.H.
      Dysferlin interacts with annexins A1 and A2 and mediates sarcolemmal wound-healing.
      Furthermore, a genetic interaction was previously observed in zebrafish engineered with both dysferlin and Anxa6 alleles.
      • Roostalu U.
      • Strahle U.
      In vivo imaging of molecular interactions at damaged sarcolemma.
      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.
      • Swaggart K.A.
      • Demonbreun A.R.
      • Vo A.H.
      • Swanson K.E.
      • Kim E.Y.
      • Fahrenbach J.P.
      • Holley-Cuthrell J.
      • Eskin A.
      • Chen Z.
      • Squire K.
      • Heydemann A.
      • Palmer A.A.
      • Nelson S.F.
      • McNally E.M.
      Annexin A6 modifies muscular dystrophy by mediating sarcolemmal repair.
      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.
      • Ayoglu B.
      • Chaouch A.
      • Lochmuller H.
      • Politano L.
      • Bertini E.
      • Spitali P.
      • Hiller M.
      • Niks E.H.
      • Gualandi F.
      • Ponten F.
      • Bushby K.
      • Aartsma-Rus A.
      • Schwartz E.
      • Le Priol Y.
      • Straub V.
      • Uhlen M.
      • Cirak S.
      • t Hoen P.A.
      • Muntoni F.
      • Ferlini A.
      • Schwenk J.M.
      • Nilsson P.
      • Al-Khalili Szigyarto C.
      Affinity proteomics within rare diseases: a BIO-NMD study for blood biomarkers of muscular dystrophies.
      • Rouillon J.
      • Zocevic A.
      • Leger T.
      • Garcia C.
      • Camadro J.M.
      • Udd B.
      • Wong B.
      • Servais L.
      • Voit T.
      • Svinartchouk F.
      Proteomics profiling of urine reveals specific titin fragments as biomarkers of Duchenne muscular dystrophy.
      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.
      • Cooper S.T.
      • Head S.I.
      Membrane Injury and Repair in the Muscular Dystrophies.
      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.
      • Zaks W.J.
      • Creutz C.E.
      Ca(2+)-dependent annexin self-association on membrane surfaces.
      Although annexins A1 and A2 have been shown to interact with dysferlin,
      • Lennon N.J.
      • Kho A.
      • Bacskai B.J.
      • Perlmutter S.L.
      • Hyman B.T.
      • Brown Jr., R.H.
      Dysferlin interacts with annexins A1 and A2 and mediates sarcolemmal wound-healing.
      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,
      • Swaggart K.A.
      • Demonbreun A.R.
      • Vo A.H.
      • Swanson K.E.
      • Kim E.Y.
      • Fahrenbach J.P.
      • Holley-Cuthrell J.
      • Eskin A.
      • Chen Z.
      • Squire K.
      • Heydemann A.
      • Palmer A.A.
      • Nelson S.F.
      • McNally E.M.
      Annexin A6 modifies muscular dystrophy by mediating sarcolemmal repair.
      but we expect that this interface includes a complex and dynamic set of interactions regulated by Ca2+ and lipid content (Figure 9).
      Figure thumbnail gr9
      Figure 9Schematic model for the annexin–dysferlin interaction. A and B: After membrane damage (A), annexin A6 (A6) aggregates at the site of disruption (B). Dysferlin (DYSF) has previously been shown to be recruited to sites of membrane damage.
      • Bansal D.
      • Miyake K.
      • Vogel S.S.
      • Groh S.
      • Chen C.C.
      • Williamson R.
      • McNeil P.L.
      • Campbell K.P.
      Defective membrane repair in dysferlin-deficient muscular dystrophy.
      C: In the absence of dysferlin, annexin A6 is still recruited to the sarcolemma injury site. D: However, in the presence of truncated A6N32, annexin A6 forms smaller aggregates, and there is more membrane leak, resulting in increased inflammation and creatine kinase (CK) leak. EBD, Evans Blue dye.

      Annexins and Inflammation

      DYSF gene mutations have been described with an increased macrophage infiltrate in muscle, suggesting that impaired trafficking and perhaps myofiber leak account for increased inflammation.
      • Illa I.
      • Serrano-Munuera C.
      • Gallardo E.
      • Lasa A.
      • Rojas-Garcia R.
      • Palmer J.
      • Gallano P.
      • Baiget M.
      • Matsuda C.
      • Brown R.H.
      Distal anterior compartment myopathy: a dysferlin mutation causing a new muscular dystrophy phenotype.
      • Gallardo E.
      • Rojas-Garcia R.
      • de Luna N.
      • Pou A.
      • Brown Jr., R.H.
      • Illa I.
      Inflammation in dysferlin myopathy: immunohistochemical characterization of 13 patients.
      Dysf-null myofibers have increased leak, and this leak is an important signal for recruiting inflammatory cells.
      • Mariano A.
      • Henning A.
      • Han R.
      Dysferlin-deficient muscular dystrophy and innate immune activation.
      Roche et al
      • Roche J.A.
      • Tulapurkar M.E.
      • Mueller A.L.
      • van Rooijen N.
      • Hasday J.D.
      • Lovering R.M.
      • Bloch R.J.
      Myofiber damage precedes macrophage infiltration after in vivo injury in dysferlin-deficient a/j mouse skeletal muscle.
      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.
      • Damazo A.S.
      • Yona S.
      • D'Acquisto F.
      • Flower R.J.
      • Oliani S.M.
      • Perretti M.
      Critical protective role for annexin 1 gene expression in the endotoxemic murine microcirculation.
      Annexins are known to be cleaved and secreted; however, the exact mechanism by which annexins are externalized remains unknown because annexins lack an externalization sequence.
      • Wallner B.P.
      • Mattaliano R.J.
      • Hession C.
      • Cate R.L.
      • Tizard R.
      • Sinclair L.K.
      • Foeller C.
      • Chow E.P.
      • Browing J.L.
      • Ramachandran K.L.
      • Pepinsky R.B.
      Cloning and expression of human lipocortin, a phospholipase A2 inhibitor with potential anti-inflammatory activity.
      • Christmas P.
      • Callaway J.
      • Fallon J.
      • Jones J.
      • Haigler H.T.
      Selective secretion of annexin 1, a protein without a signal sequence, by the human prostate gland.
      • Deora A.B.
      • Kreitzer G.
      • Jacovina A.T.
      • Hajjar K.A.
      An annexin 2 phosphorylation switch mediates p11-dependent translocation of annexin 2 to the cell surface.
      Kamal et al
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      Annexin VI-mediated loss of spectrin during coated pit budding is coupled to delivery of LDL to lysosomes.
      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.

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