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Dystrophin deficiency exacerbates skeletal muscle pathology in dysferlin-null mice

Dystrophin deficiency exacerbates skeletal muscle pathology in dysferlin-null mice Background: Mutations in the genes coding for either dystrophin or dysferlin cause distinct forms of muscular dystrophy. Dystrophin links the cytoskeleton to the sarcolemma through direct interaction with b-dystroglycan. This link extends to the extracellular matrix by b-dystroglycan’s interaction with a-dystroglycan, which binds extracellular matrix proteins, including laminin a2, agrin and perlecan, that possess laminin globular domains. The absence of dystrophin disrupts this link, leading to compromised muscle sarcolemmal integrity. Dysferlin, on the 2+ other hand, plays an important role in the Ca -dependent membrane repair of damaged sarcolemma in skeletal muscle. Because dysferlin and dystrophin play different roles in maintaining muscle cell integrity, we hypothesized that disrupting sarcolemmal integrity with dystrophin deficiency would exacerbate the pathology in dysferlin-null mice and allow further characterization of the role of dysferlin in skeletal muscle. Methods: To test our hypothesis, we generated dystrophin/dysferlin double-knockout (DKO) mice by breeding mdx mice with dysferlin-null mice and analyzed the effects of a combined deficiency of dysferlin and dystrophin on muscle pathology and sarcolemmal integrity. Results: The DKO mice exhibited more severe muscle pathology than either mdx mice or dysferlin-null mice, and, importantly, the onset of the muscle pathology occurred much earlier than it did in dysferlin-deficient mice. The DKO mice showed muscle pathology of various skeletal muscles, including the mandible muscles, as well as a greater number of regenerating muscle fibers, higher serum creatine kinase levels and elevated Evans blue dye uptake into skeletal muscles. Lengthening contractions caused similar force deficits, regardless of dysferlin expression. However, the rate of force recovery within 45 minutes following lengthening contractions was hampered in DKO muscles compared to mdx muscles or dysferlin-null muscles, suggesting that dysferlin is required for the initial recovery from lengthening contraction-induced muscle injury of the dystrophin-glycoprotein complex-compromised muscles. Conclusions: The results of our study suggest that dysferlin-mediated membrane repair helps to limit the dystrophic changes in dystrophin-deficient skeletal muscle. Dystrophin deficiency unmasks the function of dysferlin in membrane repair during lengthening contractions. Dystrophin/dysferlin-deficient mice provide a very useful model with which to evaluate the effectiveness of therapies designed to treat dysferlin deficiency. Keywords: dysferlin, dystrophin, membrane repair, sarcolemmal integrity Background surface of the plasma membrane [2]. Dystrophin plays an Duchenne muscular dystrophy (DMD) is an X-linked important role in linking the cytoskeleton to the sarco- recessive disease affecting approximately 1 in 3, 500 lemma through the direct interactions of its N-terminus with F-actin and its C-terminus with b-dystroglycan [2]. males and is caused by defects in the dystrophin gene [1]. Dystrophin is an integral component of the dystrophin- This link is extended to the extracellular matrix (ECM) glycoprotein complex (DGC) and is localized to the inner by a-dystroglycan, which binds to laminin a2, agrin and perlecan with high affinity. The dystrophin-mediated * Correspondence: kevin-campbell@uiowa.edu continuous link between the cytoskeleton and the ECM Department of Molecular Physiology and Biophysics, Howard Hughes is reported to play an important role in stabilizing the Medical Institute, Roy J and Lucille A Carver College of Medicine, The sarcolemmal structure, transmitting force laterally and University of Iowa, 285 Newton Road, 4283 CBRB, Iowa City, IA 52242, USA Full list of author information is available at the end of the article © 2011 Han et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Han et al. Skeletal Muscle 2011, 1:35 Page 2 of 11 http://www.skeletalmusclejournal.com/content/1/1/35 preventing the expansion of muscle membrane damage background of DKO mice is a mixture of C57/BL6, 129/ during lengthening contraction (LC) [3-8]. In DMD SVJ and C57BL/10ScSn. The wild-type (WT) littermates patients and mdx mice, which also have a mutation in with the same genetic background were used as controls. the dystrophin gene, loss of dystrophin disrupts the link Mice were maintained at The University of Iowa Animal between the cytoskeleton and the ECM, leading to the Care Unit and treated in accordance with animal use loss of sarcolemmal integrity. This loss of sarcolemmal guidelines. All animal studies were authorized by the Ani- integrity eventually results in muscle degeneration, mal Care, Use, and Review Committee of The University necrosis and fibrosis. As a consequence, DMD patients of Iowa. are confined to a wheelchair in their early teens and die in their early 20s as a result of cardiopulmonary failure Serum creatine kinase assay [9]. Using a Microvette CB 300 (Sarstedt AG & Co, Newton, The plasma membrane provides a physical barrier NC), we collected blood required for quantitative, kinetic between the extracellular space and the intracellular determination of serum CK activity by mouse tail vein environment, and maintenance of this barrier is crucial bleeds from nonanesthetized, restrained mice. Red blood for the survival of any cell. We previously showed that cells were pelleted by centrifugation at 10, 000 rpm for 4 skeletal muscle possesses the ability to repair membrane minutes, and serum was separated, collected and analyzed 2+ wounding in a Ca -dependent manner and that dysferlin immediately without freezing. Serum CK assays were per- plays a critical role in this process [10,11]. Mutations in formed with an enzyme-coupled assay reagent kit (Stanbio dysferlin cause limb-girdle muscular dystrophy type 2B Laboratory, Boerne, TX, USA) according to the manufac- [12,13], Miyoshi myopathy [13] and a distal anterior turer’s instructions. Absorbance at 340 nm was measured compartment myopathy [14]. every 30 seconds for 2 minutes at 37°C so that changes in Dystrophin deficiency renders the muscle susceptible to enzyme activity could be calculated. contraction-induced sarcolemmal injuries [4-8], whereas dysferlin deficiency results in compromised membrane Histological and immunofluorescence analyses repair [10,15-19]. Taking into consideration these two dif- Muscles (masseter, quadriceps, hamstrings, gluteus, gas- ferent roles in maintaining sarcolemmal integrity, we trocnemius, tibialis anterior, iliopsoas and diaphragm) hypothesized that skeletal muscle membrane stability were dissected and frozen in isopentane cooled to -165°C mediated by the DGC and dysferlin-mediated membrane in liquid nitrogen. Seven-micron cryosections were cut repair are both essential for the maintenance of muscle and fixed in 10% neutral buffer formalin for five minutes. membrane integrity and function. On the basis of this After fixation, the slides were washed for five minutes hypothesis, we predicted that a combined deficiency in under running water followed by H & E staining (Surgi- both dysferlin and dystrophin would lead to more severe path Medical Industries, Inc/Leica Microsystems, Rich- muscle pathology due both to an increased susceptibility mond, IL, USA). H & E-stained sections were analyzed by to muscle membrane injuries in the absence of dystrophin light microscopy (Leica Microsystems Inc, Buffalo Grove, and to defective membrane repair in the absence of dysfer- IL, USA; Carl Zeiss Microscopy, LLC, Thornwood, NY, lin. To test this hypothesis, we generated mice that lack USA). Immunofluorescence analyses were also performed both dysferlin and dystrophin. The dystrophin/dysferlin on 7-μm cryosections. Sections were processed for immu- double-knockout (DKO) mice developed more severe nofluorescence microscopy and analyzed with an epifluor- muscle pathology than either mdx mice or dysferlin-null escence microscope (Leica Microsystems Inc, and Carl mice, which is reflected by the higher number of regener- Zeiss Microscopy, LLC). Mouse anti-dysferlin (Hamlet-1, ated muscle fibers, increased serum creatine kinase (CK) Novocastra, Newcastle, UK) mAb and rabbit pAb against levels and more Evans blue dye (EBD) uptake in their b-dystroglycan [22], sarcospan [23], dystrophin (Abcam, muscles. These data show that dysferlin-mediated mem- Cambridge, MA, USA) and laminin a2 chain (AXXORA brane repair limits the severity of dystrophic changes in LLC, San Diego, CA, USA) were used for immunofluores- mdx skeletal muscle. cence analysis. The central nucleated and total muscle fibers were counted on muscle sections costained with Methods anti-laminin a2 antibody and 4’, 6-diamidino-2-phenylin- Mice dole (Sigma-Aldrich, St Louis, MO, USA) using Image-Pro The DKO mice were generated by breeding female mdx/ Plus version 6 software (Media Cybernetics, Inc, Bethesda, C57BL/10ScSn (mdx) mice with male dysferlin-null mice MD, USA). [10] through two generations (Additional file 1, Figure S1). F2 pups were genotyped using recently improved methods Western blot analysis for genotyping the dysferlin allele [20] and the mdx allele Proteins were extracted from 20 to 35 cryosections (30 μm [21] to identify the double-mutant mice. The genetic thick) of quadriceps tissue from each mouse using 250 μl Han et al. Skeletal Muscle 2011, 1:35 Page 3 of 11 http://www.skeletalmusclejournal.com/content/1/1/35 of PBS plus 1% Triton X-100, 0.5% SDS and protease inhi- muscles were stimulated for 300 milliseconds. The stimu- bitors. The protein samples in the supernatant were mixed lation frequency was increased until the force reached a with 80 μl of 5× Laemmli sample buffer, and 70-μlfinal plateau at maximum isometric tetanic force (P ). The sus- samples were resolved by SDS-PAGE on 3% to 15% linear ceptibility to LC-induced injury was assessed by subjecting gradient gels and transferred onto polyvinylidene fluoride each muscle to eight LCs at a rate of one LC every three Immobilon-FL membrane (Millipore, Billerica, MA, USA). minutes [3,25,26]. Each LC consisted of maximally activat- The membranes were blocked with fish gelatin in ing the muscle at a fixed length for 100 milliseconds, then Tris-buffered saline (TBS) and incubated with primary stretching the muscle at a strain of 30% of L at a strain a antibodies (mouse anti-dysferlin mAb Hamlet, mouse velocity of 1 L /second. Muscle activation ceased upon anti-b-dystroglycan 8D5 mAb, mouse anti-dystrophin achieving the 30% strain and was returned to L at the mAb and rabbit anti-dihydropyridine receptor (anti- same velocity. To assess the force deficit generated by this DHPR) a2 pAb [24]). Blots were washed with TBS + 0.1% assay, a measurement of P was taken three minutes after Tween 20 and incubated with infrared dye-conjugated sec- the last LC and repeated at 15, 30 and 45 minutes. The ondary antibodies (Pierce Biotechnology/Thermo Fisher total fiber cross-sectional area and specific P (kN/m ) Scientific,Inc,Rockford,IL,USA).Afterwashing,blots were calculated based on measurements of muscle mass, were captured using the Odyssey Imaging System (LI- L and P . A generalized linear model with repeated-mea- f 0 COR, Lincoln, NE, USA). sures analysis of variance (ANOVA) using SPSS software (SPSS, Inc, Chicago, IL, USA) was used to determine Evans blue dye uptake whether time after LC, dysferlin and dystrophin were sig- Evans blue dye (Sigma-Aldrich) was dissolved in PBS (10 nificant factors. mg/ml) and sterilized through a 0.2-μm pore size filter. Themicewereanesthetizedbyketamineinjection (0.1 Statistics ml/10 g body weight), and 0.05 ml/10 g body weight dye Data were calculated according to analysis of variance solution was injected intraperitoneally. The animals were (ANOVA)and areexpressedasmeans±SEM. Where killed 24 hours after injection, and their skeletal muscles appropriate, the significance of differences between multi- were isolated and frozen in isopentane cooled to -165°C ple mouse models was assessed using one-way ANOVA in liquid nitrogen. Microscopic evaluation of EBD uptake with Student-Newman-Keuls posttests, and the signifi- was performed on 7-μm muscle cryosections. Muscle cance of differences between two experimental groups cryosections were fixed in cold acetone at -20°C for were assessed using an unpaired two-tailed Student’s 10 minutes, washed with PBS, coverslipped in VECTA- t-test. P < 0.05 was accepted as significant. SHIELD Mounting Medium (Vector Laboratories, Burlingame, CA, USA) and evaluated by fluorescence Results microscopy. Disrupted dystrophin and dysferlin expression in skeletal muscles of DKO mice Force measurement Both immunofluorescence and Western blot analyses con- Contractile properties were measured in vitro on extensor firmed the complete loss of both dystrophin and dysferlin digitorum longus (EDL) muscles from WT, dysferlin-null, in the skeletal muscle membranes of the DKO mice (Fig- mdx and DKO mice (12 to 22 weeks of age) as described ures 1A and 1B) [15]. Mutations in dystrophin disrupt the previously [3,25]. The mice were anesthetized with an stability of the entire DGC at the sarcolemma, which in intraperitoneal injection of 2% avertin (0.0015 ml/g body turn renders the muscle susceptible to contraction- weight), and thoracotomy was performed. EDL muscles induced injuries [2]. To assess the expression of DGC were immersed in an oxygenated bath (95% O ,5%CO ) components in DKO muscle, we performed immunofluor- 2 2 that contained Ringer’s solution (pH 7.4) at 25°C. For each escence and immunoblot experiments using the specific muscle, one end tendon was tied securely with a 6-0 antibodies against b-dystroglycan and sarcospan, two suture to a dual-mode servomotor (Aurora Scientific, Inc, DGC components. The WT and dysferlin-null muscles Aurora, ON, Canada) and the other tendon was clamped showed normal expression of b-dystroglycan and sarco- to a fixed post. Using twitches with a pulse duration of 0.2 span (Figures 1A and 1C), suggesting a stable DGC at the milliseconds, the voltage or current of stimulation was sarcolemma. The mdx and DKO muscles showed a reduc- increased to achieve a maximum twitch and then tion in the expression of both proteins (Figures 1A and increased slightly. Twitches were then used to adjust the 1C), suggesting a disrupted DGC in the muscles of these muscle length to the optimum length for force develop- mice. The expression levels of non-DGC proteins DHPR ment (L ). Fiber length (L ) was determined by multiplying a2 and laminin a2 were also examined, and we found that o f L by the ratio of fiber length to muscle length (0.45) they were not reduced in the DKO muscles (Figures 1A [3,25,26]. The muscle length was set at L ,and EDL and 1C). o Han et al. Skeletal Muscle 2011, 1:35 Page 4 of 11 http://www.skeletalmusclejournal.com/content/1/1/35 Figure 1 Disrupted expression of dysferlin and dystrophin in skeletal muscle of dystrophin/dysferlin double-knockout mice. (A) Western blot showing the expression pattern of dysferlin (DysF), dystrophin (Dyst), b-dystroglycan (b-DG) and dihydropyridine receptor a2 (DHPRa2) in skeletal muscle tissue lysates from wild-type (WT), dysferlin-null, mdx and dystrophin/dysferlin double-knockout (DKO) mice. (B) Expression of dystrophin and dysferlin in skeletal muscles from WT, dysferlin-null, mdx and DKO mice were examined by immunofluorescence staining. (C) b-DG and sarcospan (SSPN) were greatly diminished at the sarcolemma of mdx and DKO muscles, but laminin a2 (Lam2) staining was not reduced. Scale bars: 100 μm. Han et al. Skeletal Muscle 2011, 1:35 Page 5 of 11 http://www.skeletalmusclejournal.com/content/1/1/35 Increased muscle histopathology in the DKO mice The quadriceps muscle sections were also analyzed at Defects in either dysferlin or dystrophin lead to deficits six months of age in DKO mice and compared with the in skeletal muscle [1,12,13]. Therefore, we carefully muscles isolated from age-matched controls, dysferlin- examined the function and histopathology of skeletal null and mdx mice. The DKO quadriceps muscle tissues muscles in the DKO mice. We observed that many of exhibited more severe muscular dystrophic pathology the DKO mice developed malocclusion. Histological compared with dysferlin-null and mdx mice (Figure 3). examination of the DKO mandible muscles by H & E Centrally nucleated muscle fibers, which indicate regen- staining revealed greater dystrophic features than either erating fibers, were quantified in quadriceps muscles the mdx or dysferlin-null masseter muscles (Figure 2). from six-month-old WT, dysferlin-null, mdx and DKO Because rodent incisors grow continuously from birth mice. Of the total muscle fibers, 28% ± 3% in dysferlin- and are kept worn down and sharp by continuously null mice, 55% ± 2% in mdx mice and 73% ± 2% in DKO gnawing[27], themoreseveremusclepathology ofthe mice were centrally nucleated compared to 1.3% ± 0.2% mandibular muscles in DKO mice suggests increased in WT mice (six mice per group) (Figure 3B). Muscle his- muscle weakness of these muscles as a reason for the topathology of aged DKO mice was also performed, long incisor growth. The front teeth of mice with mal- including the triceps, gastrocnemius, diaphragm, tibialis occlusion were regularly clipped, allowing them access anterior, iliopsoas, hamstring and gluteus muscles (Addi- to and ingestion of food. tional file 2, Figure S2). All muscles from DKO mice Figure 2 H & E staining of masseter muscle sections from WT, dysferlin-null, mdx and DKO mice at six months of age. Scale bar: 100 μm. Han et al. Skeletal Muscle 2011, 1:35 Page 6 of 11 http://www.skeletalmusclejournal.com/content/1/1/35 Figure 3 Histopathological analyses of quadriceps muscle sections from DKO mice. (A) H & E-stained quadriceps muscle sections from dysferlin-null, mdx and DKO mice at six months of age. Scale bar: 100 μm. (B) Quantitative analysis of centrally nucleated muscle fibers (CNF) in quadriceps muscles from WT, dysferlin-null, mdx and DKO mice (n = 6 per group) at six months of age. Each group was significantly different from all the other groups. For clarity, significance is shown only for the comparisons with the DKO mice. ***P < 0.001. exhibited severe pathology, including widespread muscle presence of EBD-positive fibers indicates the presence of necrosis, fibrosis and fatty replacement. These results are membrane disruptions in muscle fibers. Individual EBD- consistent with our hypothesis that dystrophin and dys- positive muscle fibers were scattered throughout the ferlin play nonredundant roles in maintaining muscle muscle sections of dysferlin-null mice, and the muscle function. sections of mdx mice showed clusters of EBD-positive fibers [28] (Figure 4A). Interestingly, the DKO mice Severely compromised muscle sarcolemmal integrity in showed both patterns of dye uptake: individual and clus- the DKO mice ters of EBD-positive muscle fibers (Figure 4A). To examine the sarcolemmal integrity of the skeletal Membrane disruptions and muscle damage not only muscle fibers in the DKO mice, we injected EBD and allow EBD uptake but also lead to leakage of cytosolic analyzed its uptake into the quadriceps skeletal muscles. contents such as CK, which can then be detected in the EBD is a membrane-impermeable dye that binds serum serum. Measurements of serum CK levels provide an albumin. A cell can uptake this dye only if the plasma index of the active skeletal muscle necrosis and the pre- membrane of the cell is compromised. Therefore, the sence of membrane disruptions. Our analysis revealed Han et al. Skeletal Muscle 2011, 1:35 Page 7 of 11 http://www.skeletalmusclejournal.com/content/1/1/35 Figure 4 Evans blue dye uptake analyses of quadriceps muscles and serum CK measurements. (A) Evans blue dye (EBD) fluorescence photomicrographs of quadriceps muscle sections from dysferlin-null, mdx and DKO mice at six months of age. Scale bar: 100 μm. (B) Serum creatine kinase (CK) levels were significantly different (P < 0.001) between DKO mice (n = 6) and the other groups (n = 5, 4 and 6 for WT, dysferlin-null and mdx mice, respectively). Values for mdx mice were significantly different from those for WT mice (P = 0.018). For clarity, significance is shown only for the comparisons with the DKO mice. ***P < 0.001. that the DKO mice had serum CK levels twofold higher m for dysferlin-null muscle, suggesting that the absence than those of mdx mice and tenfold higher than those of dysferlin alone does not lead to a deficit of specific of dysferlin-null mice (Figure 4B). Taken together, these force. The specific forces for mdx mice (166 ± 8 kN/m ) data suggest that the dystrophin and dysferlin double- were significantly lower than those for WT mice. The deficiency results in decreased sarcolemmal integrity. absence of dysferlin had no effect in the mdx back- ground, as indicated by a lack of significant difference in Severe functional deficits in the DKO muscles specific forces of DKO muscles (153 ± 11 kN/m ) com- To examine whether a double-deficiency of dysferlin pared to those of mdx muscles (Figure 5A). However, and dystrophin also leads to a more severe functional dysferlin had a significant role in the force recovery fol- disturbance, we measured the force production of EDL lowing LCs, particularly in the context of an mdx back- muscles and their responses to LCs. The specific forces ground (Figure 5B). Both the WT and dysferlin-null were 219 ± 7 kN/m for WT muscle and 212 ± 8 kN/ muscles recovered marginally within 45 minutes post- Han et al. Skeletal Muscle 2011, 1:35 Page 8 of 11 http://www.skeletalmusclejournal.com/content/1/1/35 Figure 5 Effects of dysferlin and dystrophin deficiencies on skeletal muscle contractile properties. We investigated the extensor digitorum longus (EDL) muscles taken from WT (n = 4), dysferlin-null (n = 6), mdx (n = 8) and DKO mice (n = 3). (A) Tetanic force measurements prior to LC. *P < 0.05 and **P < 0.01. (B) Tetanic force recovery following LC. Dysferlin deficiency had a significant effect on force recovery in the absence of dystrophin, as indicated by a significant interaction (P = 0.04) between dysferlin, dystrophin and time after LC on the basis of analysis of variance. (C) Summary of estimated maximal recovery levels following LC. *P < 0.05. (D) Summary of force recovery rate following LC. *P < 0.05. LC (WT: 69% ± 4% at 3 minutes vs 79% ± 2% at 45 DKO muscles (0.032 ± 0.012/minute) was significantly minutes; dysferlin-null: 65% ± 4% at 3 minutes vs 71% ± lower than in mdx muscles (0.080 ± 0.006/minute), 4% at 45 minutes). Although the force in mdx muscles although there was no difference in the maximum was greatly diminished at 3 minutes post-LC, it gradu- recovery levels (Figures 5C and 5D). These data suggest ally recovered from 22% ± 4% at 3 minutes to 61% ± 5% that dysferlin deficiency impairs the force recovery fol- at 45 minutes (Figure 5B). Interestingly, the DKO mus- lowing LCs in mdx mice, indicating that compromised cles also recovered significantly from 16% ± 2% at 3 membrane repair [10] has consequences for post-LC minutes to 52% ± 1% at 45 minutes (Figure 5B). How- force generation. ever, dysferlin deficiency significantly slowed the recov- ery of force in the absence of dystrophin (Figure 5B). To Discussion estimate the maximal recovery level and the recovery Skeletal muscle is associated with significant stress and rate, we fitted the data to a one-phase association equa- strain during muscle contraction. Previous studies have tion. Particularly, we found that the recovery rate in shown at least two mechanisms that skeletal muscle Han et al. Skeletal Muscle 2011, 1:35 Page 9 of 11 http://www.skeletalmusclejournal.com/content/1/1/35 cells utilize to maintain sarcolemmal integrity: a tightly resembling that seen in DMD patients, in contrast to a associated basal lamina mediated by the dystroglycan mild, slowly progressing cardiac manifestation in mdx complex limits the extent of plasma membrane damage mice [15]. Thus the DKO mouse model is well-suited [3] and membrane repair machinery involving multiple to the study of the molecular mechanisms of and ther- proteins that actively restore membrane integrity follow- apy for cardiomyopathy in DMD. Our present study ing limited levels of membrane disruption, such as dys- reveals for the first time that dystrophin deficiency ferlin [10,15,18]; calpain [29]; annexins A1, A2 and A5 unmasks a role for dysferlin in repairing membrane [18,30,31]; and MG53 [32,33]. A defect in either damage during the initial injury elicited by LCs. Com- pared to WT skeletal muscle, mdx muscle had a much mechanism is detrimental to the muscle, as manifested by the fact that genetic mutations in either the DGC greater force deficit measured three minutes after LC components (for review, see [34]) or dysferlin [12-14] (78% in mdx vs 31% in WT). However, by 45 minutes, cause various muscular dystrophies. In the present work the force in mdx muscles recovered from 22% to over using mouse models, we have further demonstrated that 60% of the preinjury level, whereas the WT and dysfer- a double-deficiency in both dystrophin and dysferlin lin-null muscles gained only 10% (from 69% to 79%) results in more severe muscular dystrophy. The results and 6% (from 65% to 71%), respectively. These data of our work are in agreement with those of Shaw et al. suggest that, unlike WT and dysferlin-null muscle, in [35], who showed that mice overexpressing the Cox- which LCs induce little membrane damage, mdx muscle sackie virus and adenovirus receptor transgene demon- undergoes more severe membrane damage, which strate decreased expression levels of dystrophin and requires an active membrane repair process to restore dysferlin, which may lead to an associated myopathy. membrane integrity. DKO muscles are also repair-com- Our work provides genetic evidence that dysferlin and petent, as the force also recovered from 16% at 3 min- the DGC are independent pathways for the maintenance utes to 52% at 45 minutes. However, by comparing the of sarcolemmal integrity in muscle and further high- recovery rate constants of mdx muscles and DKO mus- lights the importance of these two pathways in muscle cles, we found that the recovery rate of the DKO mus- health. cles was less than half the rate of mdx muscles. Contraction-induced injury is characterized by two dis- Therefore, our data derived from using a LC assay tinct phases: an initial injury and a delayed secondary reveal that dystrophin deficiency sufficiently unmasks injury from the inflammatory response. The initial injury the membrane repair role of dysferlin. The DKO mouse consists of mechanical disruption of sarcomeres followed would be a very useful model with which to determine 2+ by impaired excitation-contraction coupling and Ca sig- the efficacy of a therapeutic treatment designed for 2+ dysferlinopathy. naling and finally by activation of Ca -sensitive degrada- tion pathways [36]. Interestingly, for muscle with an intact DGC, whether membrane damage occurs during the initial Conclusion injury phase is unclear; but if it exists, it is dysferlin-inde- Taken together, our results show that both DGC- pendent [25,37,38]. Several hours to days following the mediated membrane stability and dysferlin-mediated initial injury, infiltration of inflammatory factors damage membrane repair contribute to the function and mainte- muscle fibers and release reactive oxygen species (ROS), nance of skeletal muscle. The studies presented here which aid in clearing disrupted myofibrils but also damage suggestthatthe DKOmouse modelmay be avaluable previously undamaged myofibrils. At this stage, ROS tool in the development of therapies designed to treat potentially cause damage to the sarcolemma, possibly dysferlinopathies. through lipid peroxidation [39,40]. Investigators in pre- vious studies have demonstrated that dysferlin is impor- Additional material tant in limiting the extent of secondary injury. For example, three days following a protocol of small strain Additional file 1: Figure S1 Breeding strategy to generate dystrophin/dysferlin double-knockout mice. Male dysferlin-null mice LCs, muscles of dysferlin-deficient mice displayed were mated with mdx female mice, then F1 heterozygous males and increased macrophage infiltration, decreased ability to seal females were bred to generate F2 males and females. Of the F2 males off a membrane-impermeable dye and increased force def- and females, 12.5% are predicted to be DKO mice. icits [38,41]. These data indicate that defects in membrane Additional file 2: Figure S2 Histopathology analyses of various muscles from dystrophin/dysferlin double-knockout mice.H&E- repair or alteration in repair result in chemoattraction of stained muscle sections of triceps (TC), gastrocnemius (GA), diaphragm immune cells [38,41]. (DIA), tibialis anterior (TA), iliopsoas (IP), hamstring (HS) and gluteus (GT) Previously, we demonstrated that a combined defi- muscles from dystrophin/dysferlin double-knockout (DKO) mice at one and one-half years of age. Scale bar: 100 μm. ciency in dysferlin and dystrophin results in the devel- opment of a pronounced early-onset cardiomyopathy Han et al. Skeletal Muscle 2011, 1:35 Page 10 of 11 http://www.skeletalmusclejournal.com/content/1/1/35 7. Weller B, Karpati G, Carpenter S: Dystrophin-deficient mdx muscle fibers Abbreviations are preferentially vulnerable to necrosis induced by experimental CK: creatine kinase; DGC: dystrophin-glycoprotein complex; DHPR: lengthening contractions. J Neurol Sci 1990, 100:9-13. dihydropyridine receptor; DKO: dystrophin/dysferlin double-knockout; DMD: 8. Dellorusso C, Crawford RW, Chamberlain JS, Brooks SV: Tibialis anterior Duchenne muscular dystrophy; EBD: Evans blue dye; ECM: extracellular muscles in mdx mice are highly susceptible to contraction-induced matrix; EDL: extensor digitorum longus; H & E: hematoxylin and eosin; LC: injury. J Muscle Res Cell Motil 2001, 22:467-475. lengthening contraction; mAb: monoclonal antibody; pAb: polyclonal 9. Moser H: Duchenne muscular dystrophy: pathogenetic aspects and antibody; PBS: phosphate-buffered saline; ROS: reactive oxygen species. genetic prevention. Hum Genet 1984, 66:17-40. 10. Bansal D, Miyake K, Vogel SS, Groh S, Chen CC, Williamson R, McNeil PL, Acknowledgements Campbell KP: Defective membrane repair in dysferlin-deficient muscular We thank Keith Garringer, Samantha K Watkins, Sally Prouty and David P dystrophy. Nature 2003, 423:168-172. Venzke for technical support and Drs Yvonne Kobayashi, Li Xu and Piming Zhao for critical reading and discussion. This work was supported in part by 11. Han R, Campbell KP: Dysferlin and muscle membrane repair. Curr Opin American Heart Association Scientist Development grant 10SDG4140138 (to Cell Biol 2007, 19:409-416. RH), Muscular Dystrophy Association Research Grant MDA171667 (to RH), 12. Bashir R, Britton S, Strachan T, Keers S, Vafiadaki E, Lako M, Richard I, Paul D Wellstone Muscular Dystrophy Cooperative Research Center grant Marchand S, Bourg N, Argov Z, Sadeh M, Mahjneh I, Marconi G, Passos- 1U54NS053672 (to KPC), Muscular Dystrophy Association grant MDA3936 (to Bueno MR, Moreira Ede S, Zatz M, Beckmann JS, Bushby K: A gene related KPC), Muscular Dystrophy Association Development grants MDA200826 (to to Caenorhabditis elegans spermatogenesis factor fer-1 is mutated in JRL) and MDA67814 (to EPR) and US Department of Defense grant limb-girdle muscular dystrophy type 2B. Nat Genet 1998, 20:37-42. W81XWH-05-1-0079. The authors declare no conflicts of interest. KPC is an 13. Liu J, Aoki M, Illa I, Wu C, Fardeau M, Angelini C, Serrano C, Urtizberea JA, investigator at the Howard Hughes Medical Institute. Hentati F, Hamida MB, Bohlega S, Culper EJ, Amato AA, Bossie K, Oeltjen J, Bejaoui K, McKenna-Yasek D, Hosler BA, Schurr E, Arahata K, de Jong PJ, Author details Brown RH Jr: Dysferlin, a novel skeletal muscle gene, is mutated in Department of Cell and Molecular Physiology, Stritch School of Medicine, Miyoshi myopathy and limb girdle muscular dystrophy. Nat Genet 1998, Loyola University Medical Center, 2160 S 1st Avenue, Maywood, IL 60558, 20:31-36. USA. Department of Molecular Physiology and Biophysics, Howard Hughes 14. Illa I, Serrano-Munuera C, Gallardo E, Lasa A, Rojas-García R, Palmer J, Medical Institute, Roy J and Lucille A Carver College of Medicine, The Gallano P, Baiget M, Matsuda C, Brown RH: Distal anterior compartment University of Iowa, 285 Newton Road, 4283 CBRB, Iowa City, IA 52242, USA. myopathy: a dysferlin mutation causing a new muscular dystrophy Department of Neurology, Howard Hughes Medical Institute, Roy J and phenotype. Ann Neurol 2001, 49:130-134. Lucille A Carver College of Medicine, The University of Iowa, 285 Newton 15. Han R, Bansal D, Miyake K, Muniz VP, Weiss RM, McNeil PL, Campbell KP: Dysferlin-mediated membrane repair protects the heart from stress- Road, 4283 CBRB, Iowa City, IA 52242, USA. Department of Internal induced left ventricular injury. J Clin Invest 2007, 117:1805-1813. Medicine, Howard Hughes Medical Institute, Roy J and Lucille A Carver 16. Cenacchi G, Fanin M, De Giorgi LB, Angelini C: Ultrastructural changes in College of Medicine, The University of Iowa, 285 Newton Road, 4283 CBRB, dysferlinopathy support defective membrane repair mechanism. J Clin Iowa City, IA 52242, USA. Pathol 2005, 58:190-195. 17. Cai C, Weisleder N, Ko JK, Komazaki S, Sunada Y, Nishi M, Takeshima H, Authors’ contributions Ma J: Membrane repair defects in muscular dystrophy are linked to RH conceived the study, carried out the histopathological studies, altered interaction between MG53, caveolin-3, and dysferlin. J Biol Chem participated in the sequence alignment and drafted the manuscript. ER 2009, 284:15894-15902. carried out the force measurement experiments and performed the 18. Lennon NJ, Kho A, Bacskai BJ, Perlmutter SL, Hyman BT, Brown RH Jr: statistical analysis. JL carried out the immunoassays. DB initiated the mouse Dysferlin interacts with annexins A1 and A2 and mediates sarcolemmal breeding and participated in sequence alignment. KC conceived the study, wound-healing. J Biol Chem 2003, 278:50466-50473. participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript. 19. Hino M, Hamada N, Tajika Y, Funayama T, Morimura Y, Sakashita T, Yokota Y, Fukamoto K, Kobayashi Y, Yorifuji H: Insufficient membrane Competing interests fusion in dysferlin-deficient muscle fibers after heavy-ion irradiation. Cell The authors declare that they have no competing interests. Struct Funct 2009, 34:11-15. 20. Han R, Kobuke K, Anderson M, Beltrán-Valero de Bernabé D, Kobayashi Y, Received: 25 October 2011 Accepted: 1 December 2011 Yang B, Campbell K: Improved genotyping of the dysferlin null mouse. Published: 1 December 2011 Protoc Exch 2011. 21. Shin JH, Hakim CH, Zhang K, Duan D: Genotyping mdx, mdx3cv, and mdx4cv mice by primer competition polymerase chain reaction. Muscle References Nerve 2011, 43:283-286. 1. Koenig M, Hoffman EP, Bertelson CJ, Monaco AP, Feener C, Kunkel LM: 22. Lim LE, Duclos F, Broux O, Bourg N, Sunada Y, Allamand V, Meyer J, Complete cloning of the Duchenne muscular dystrophy (DMD) cDNA Richard I, Moomaw C, Slaughter C, Tomé FMS, Fardeau M, Jackson CE, and preliminary genomic organization of the DMD gene in normal and Beckmann JS, Campbell KP: β-sarcoglycan: characterization and role in affected individuals. Cell 1987, 50:509-517. limb-girdle muscular dystrophy linked to 4q12. Nat Genet 1995, 2. Cohn RD, Campbell KP: Molecular basis of muscular dystrophies. Muscle 11:257-265. Nerve 2000, 23:1456-1471. 23. Lebakken CS, Venzke DP, Hrstka RF, Consolino CM, Faulkner JA, 3. Han R, Kanagawa M, Yoshida-Moriguchi T, Rader EP, Ng RA, Michele DE, Williamson RA, Campbell KP: Sarcospan-deficient mice maintain normal Muirhead DE, Kunz S, Moore SA, Iannaccone ST, Miyake K, McNeil PL, muscle function. Mol Cell Biol 2000, 20:1669-1677. Mayer U, Oldstone MB, Faulkner JA, Campbell KP: Basal lamina strengthens 24. Gurnett CA, Kahl SD, Anderson RD, Campbell KP: Absence of the skeletal cell membrane integrity via the laminin G domain-binding motif of α- muscle sarcolemma chloride channel ClC-1 in myotonic mice. J Biol dystroglycan. Proc Natl Acad Sci USA 2009, 106:12573-12579. Chem 1995, 270:9035-9038. 4. Petrof BJ, Shrager JB, Stedman HH, Kelly AM, Sweeney HL: Dystrophin 25. Han R, Frett EM, Levy JR, Rader EP, Lueck JD, Bansal D, Moore SA, Ng R, protects the sarcolemma from stresses developed during muscle Beltrán-Valero de Bernabé D, Faulkner JA, Campbell KP: Genetic ablation of contraction. Proc Natl Acad Sci USA 1993, 90:3710-3714. complement C3 attenuates muscle pathology in dysferlin-deficient mice. 5. Clarke MS, Khakee R, McNeil PL: Loss of cytoplasmic basic fibroblast J Clin Invest 2010, 120:4366-4374. growth factor from physiologically wounded myofibers of normal and 26. Kobayashi YM, Rader EP, Crawford RW, Iyengar NK, Thedens DR, dystrophic muscle. J Cell Sci 1993, 106:121-133. Faulkner JA, Parikh SV, Weiss RM, Chamberlain JS, Moore SA, Campbell KP: 6. Moens P, Baatsen PH, Maréchal G: Increased susceptibility of EDL muscles Sarcolemma-localized nNOS is required to maintain activity after mild from mdx mice to damage induced by contractions with stretch. J exercise. Nature 2008, 456:511-515. Muscle Res Cell Motil 1993, 14:446-451. Han et al. Skeletal Muscle 2011, 1:35 Page 11 of 11 http://www.skeletalmusclejournal.com/content/1/1/35 27. Caton J, Tucker AS: Current knowledge of tooth development: patterning and mineralization of the murine dentition. J Anat 2009, 214:502-515. 28. Straub V, Rafael JA, Chamberlain JS, Campbell KP: Animal models for muscular dystrophy show different patterns of sarcolemmal disruption. J Cell Biol 1997, 139:375-385. 29. Mellgren RL, Zhang W, Miyake K, McNeil PL: Calpain is required for the rapid, calcium-dependent repair of wounded plasma membrane. J Biol Chem 2007, 282:2567-2575. 30. McNeil AK, Rescher U, Gerke V, McNeil PL: Requirement for annexin A1 in plasma membrane repair. J Biol Chem 2006, 281:35202-35207. 31. Bouter A, Gounou C, Bérat R, Tan S, Gallois B, Granier T, d’Estaintot BL, Pöschl E, Brachvogel B, Brisson AR: Annexin-A5 assembled into two- dimensional arrays promotes cell membrane repair. Nat Commun 2011, 2:270. 32. Cai C, Masumiya H, Weisleder N, Matsuda N, Nishi M, Hwang M, Ko JK, Lin P, Thornton A, Zhao X, Pan Z, Komazaki S, Brotto M, Takeshima H, Ma J: MG53 nucleates assembly of cell membrane repair machinery. Nat Cell Biol 2009, 11:56-64. 33. Wang X, Xie W, Zhang Y, Lin P, Han L, Han P, Wang Y, Chen Z, Ji G, Zheng M, Weisleder N, Xiao RP, Takeshima H, Ma J, Cheng H: Cardioprotection of ischemia/reperfusion injury by cholesterol- dependent MG53-mediated membrane repair. Circ Res 2010, 107:76-83. 34. Barresi R, Campbell KP: Dystroglycan: from biosynthesis to pathogenesis of human disease. J Cell Sci 2006, 119:199-207. 35. Shaw CA, Larochelle N, Dudley RWR, Lochmüller H, Danialou G, Petrof BJ, Karpati G, Holland PC, Nalbantoglu J: Simultaneous dystrophin and dysferlin deficiencies associated with high-level expression of the coxsackie and adenovirus receptor in transgenic mice. Am J Pathol 2006, 169:2148-2160. 36. Peake J, Nosaka K, Suzuki K: Characterization of inflammatory responses to eccentric exercise in humans. Exerc Immunol Rev 2005, 11:64-85. 37. Chiu YH, Hornsey MA, Klinge L, Jørgensen LH, Laval SH, Charlton R, Barresi R, Straub V, Lochmüller H, Bushby K: Attenuated muscle regeneration is a key factor in dysferlin-deficient muscular dystrophy. Hum Mol Genet 2009, 18:1976-1989. 38. Roche JA, Lovering RM, Bloch RJ: Impaired recovery of dysferlin-null skeletal muscle after contraction-induced injury in vivo. Neuroreport 2008, 19:1579-1584. 39. Aoi W, Naito Y, Takanami Y, Kawai Y, Sakuma K, Ichikawa H, Yoshida N, Yoshikawa T: Oxidative stress and delayed-onset muscle damage after exercise. Free Radic Biol Med 2004, 37:480-487. 40. Liao P, Zhou J, Ji LL, Zhang Y: Eccentric contraction induces inflammatory responses in rat skeletal muscle: role of tumor necrosis factor-α. Am J Physiol Regul Integr Comp Physiol 2010, 298:R599-R607. 41. Roche JA, Lovering RM, Roche R, Ru LW, Reed PW, Bloch RJ: Extensive mononuclear infiltration and myogenesis characterize recovery of dysferlin-null skeletal muscle from contraction-induced injuries. Am J Physiol Cell Physiol 2010, 298:C298-C312. doi:10.1186/2044-5040-1-35 Cite this article as: Han et al.: Dystrophin deficiency exacerbates skeletal muscle pathology in dysferlin-null mice. Skeletal Muscle 2011 1:35. Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color figure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Skeletal Muscle Springer Journals

Dystrophin deficiency exacerbates skeletal muscle pathology in dysferlin-null mice

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Copyright © 2011 by Han et al; licensee BioMed Central Ltd.
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Life Sciences; Cell Biology; Developmental Biology; Biochemistry, general; Systems Biology; Biotechnology
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Abstract

Background: Mutations in the genes coding for either dystrophin or dysferlin cause distinct forms of muscular dystrophy. Dystrophin links the cytoskeleton to the sarcolemma through direct interaction with b-dystroglycan. This link extends to the extracellular matrix by b-dystroglycan’s interaction with a-dystroglycan, which binds extracellular matrix proteins, including laminin a2, agrin and perlecan, that possess laminin globular domains. The absence of dystrophin disrupts this link, leading to compromised muscle sarcolemmal integrity. Dysferlin, on the 2+ other hand, plays an important role in the Ca -dependent membrane repair of damaged sarcolemma in skeletal muscle. Because dysferlin and dystrophin play different roles in maintaining muscle cell integrity, we hypothesized that disrupting sarcolemmal integrity with dystrophin deficiency would exacerbate the pathology in dysferlin-null mice and allow further characterization of the role of dysferlin in skeletal muscle. Methods: To test our hypothesis, we generated dystrophin/dysferlin double-knockout (DKO) mice by breeding mdx mice with dysferlin-null mice and analyzed the effects of a combined deficiency of dysferlin and dystrophin on muscle pathology and sarcolemmal integrity. Results: The DKO mice exhibited more severe muscle pathology than either mdx mice or dysferlin-null mice, and, importantly, the onset of the muscle pathology occurred much earlier than it did in dysferlin-deficient mice. The DKO mice showed muscle pathology of various skeletal muscles, including the mandible muscles, as well as a greater number of regenerating muscle fibers, higher serum creatine kinase levels and elevated Evans blue dye uptake into skeletal muscles. Lengthening contractions caused similar force deficits, regardless of dysferlin expression. However, the rate of force recovery within 45 minutes following lengthening contractions was hampered in DKO muscles compared to mdx muscles or dysferlin-null muscles, suggesting that dysferlin is required for the initial recovery from lengthening contraction-induced muscle injury of the dystrophin-glycoprotein complex-compromised muscles. Conclusions: The results of our study suggest that dysferlin-mediated membrane repair helps to limit the dystrophic changes in dystrophin-deficient skeletal muscle. Dystrophin deficiency unmasks the function of dysferlin in membrane repair during lengthening contractions. Dystrophin/dysferlin-deficient mice provide a very useful model with which to evaluate the effectiveness of therapies designed to treat dysferlin deficiency. Keywords: dysferlin, dystrophin, membrane repair, sarcolemmal integrity Background surface of the plasma membrane [2]. Dystrophin plays an Duchenne muscular dystrophy (DMD) is an X-linked important role in linking the cytoskeleton to the sarco- recessive disease affecting approximately 1 in 3, 500 lemma through the direct interactions of its N-terminus with F-actin and its C-terminus with b-dystroglycan [2]. males and is caused by defects in the dystrophin gene [1]. Dystrophin is an integral component of the dystrophin- This link is extended to the extracellular matrix (ECM) glycoprotein complex (DGC) and is localized to the inner by a-dystroglycan, which binds to laminin a2, agrin and perlecan with high affinity. The dystrophin-mediated * Correspondence: kevin-campbell@uiowa.edu continuous link between the cytoskeleton and the ECM Department of Molecular Physiology and Biophysics, Howard Hughes is reported to play an important role in stabilizing the Medical Institute, Roy J and Lucille A Carver College of Medicine, The sarcolemmal structure, transmitting force laterally and University of Iowa, 285 Newton Road, 4283 CBRB, Iowa City, IA 52242, USA Full list of author information is available at the end of the article © 2011 Han et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Han et al. Skeletal Muscle 2011, 1:35 Page 2 of 11 http://www.skeletalmusclejournal.com/content/1/1/35 preventing the expansion of muscle membrane damage background of DKO mice is a mixture of C57/BL6, 129/ during lengthening contraction (LC) [3-8]. In DMD SVJ and C57BL/10ScSn. The wild-type (WT) littermates patients and mdx mice, which also have a mutation in with the same genetic background were used as controls. the dystrophin gene, loss of dystrophin disrupts the link Mice were maintained at The University of Iowa Animal between the cytoskeleton and the ECM, leading to the Care Unit and treated in accordance with animal use loss of sarcolemmal integrity. This loss of sarcolemmal guidelines. All animal studies were authorized by the Ani- integrity eventually results in muscle degeneration, mal Care, Use, and Review Committee of The University necrosis and fibrosis. As a consequence, DMD patients of Iowa. are confined to a wheelchair in their early teens and die in their early 20s as a result of cardiopulmonary failure Serum creatine kinase assay [9]. Using a Microvette CB 300 (Sarstedt AG & Co, Newton, The plasma membrane provides a physical barrier NC), we collected blood required for quantitative, kinetic between the extracellular space and the intracellular determination of serum CK activity by mouse tail vein environment, and maintenance of this barrier is crucial bleeds from nonanesthetized, restrained mice. Red blood for the survival of any cell. We previously showed that cells were pelleted by centrifugation at 10, 000 rpm for 4 skeletal muscle possesses the ability to repair membrane minutes, and serum was separated, collected and analyzed 2+ wounding in a Ca -dependent manner and that dysferlin immediately without freezing. Serum CK assays were per- plays a critical role in this process [10,11]. Mutations in formed with an enzyme-coupled assay reagent kit (Stanbio dysferlin cause limb-girdle muscular dystrophy type 2B Laboratory, Boerne, TX, USA) according to the manufac- [12,13], Miyoshi myopathy [13] and a distal anterior turer’s instructions. Absorbance at 340 nm was measured compartment myopathy [14]. every 30 seconds for 2 minutes at 37°C so that changes in Dystrophin deficiency renders the muscle susceptible to enzyme activity could be calculated. contraction-induced sarcolemmal injuries [4-8], whereas dysferlin deficiency results in compromised membrane Histological and immunofluorescence analyses repair [10,15-19]. Taking into consideration these two dif- Muscles (masseter, quadriceps, hamstrings, gluteus, gas- ferent roles in maintaining sarcolemmal integrity, we trocnemius, tibialis anterior, iliopsoas and diaphragm) hypothesized that skeletal muscle membrane stability were dissected and frozen in isopentane cooled to -165°C mediated by the DGC and dysferlin-mediated membrane in liquid nitrogen. Seven-micron cryosections were cut repair are both essential for the maintenance of muscle and fixed in 10% neutral buffer formalin for five minutes. membrane integrity and function. On the basis of this After fixation, the slides were washed for five minutes hypothesis, we predicted that a combined deficiency in under running water followed by H & E staining (Surgi- both dysferlin and dystrophin would lead to more severe path Medical Industries, Inc/Leica Microsystems, Rich- muscle pathology due both to an increased susceptibility mond, IL, USA). H & E-stained sections were analyzed by to muscle membrane injuries in the absence of dystrophin light microscopy (Leica Microsystems Inc, Buffalo Grove, and to defective membrane repair in the absence of dysfer- IL, USA; Carl Zeiss Microscopy, LLC, Thornwood, NY, lin. To test this hypothesis, we generated mice that lack USA). Immunofluorescence analyses were also performed both dysferlin and dystrophin. The dystrophin/dysferlin on 7-μm cryosections. Sections were processed for immu- double-knockout (DKO) mice developed more severe nofluorescence microscopy and analyzed with an epifluor- muscle pathology than either mdx mice or dysferlin-null escence microscope (Leica Microsystems Inc, and Carl mice, which is reflected by the higher number of regener- Zeiss Microscopy, LLC). Mouse anti-dysferlin (Hamlet-1, ated muscle fibers, increased serum creatine kinase (CK) Novocastra, Newcastle, UK) mAb and rabbit pAb against levels and more Evans blue dye (EBD) uptake in their b-dystroglycan [22], sarcospan [23], dystrophin (Abcam, muscles. These data show that dysferlin-mediated mem- Cambridge, MA, USA) and laminin a2 chain (AXXORA brane repair limits the severity of dystrophic changes in LLC, San Diego, CA, USA) were used for immunofluores- mdx skeletal muscle. cence analysis. The central nucleated and total muscle fibers were counted on muscle sections costained with Methods anti-laminin a2 antibody and 4’, 6-diamidino-2-phenylin- Mice dole (Sigma-Aldrich, St Louis, MO, USA) using Image-Pro The DKO mice were generated by breeding female mdx/ Plus version 6 software (Media Cybernetics, Inc, Bethesda, C57BL/10ScSn (mdx) mice with male dysferlin-null mice MD, USA). [10] through two generations (Additional file 1, Figure S1). F2 pups were genotyped using recently improved methods Western blot analysis for genotyping the dysferlin allele [20] and the mdx allele Proteins were extracted from 20 to 35 cryosections (30 μm [21] to identify the double-mutant mice. The genetic thick) of quadriceps tissue from each mouse using 250 μl Han et al. Skeletal Muscle 2011, 1:35 Page 3 of 11 http://www.skeletalmusclejournal.com/content/1/1/35 of PBS plus 1% Triton X-100, 0.5% SDS and protease inhi- muscles were stimulated for 300 milliseconds. The stimu- bitors. The protein samples in the supernatant were mixed lation frequency was increased until the force reached a with 80 μl of 5× Laemmli sample buffer, and 70-μlfinal plateau at maximum isometric tetanic force (P ). The sus- samples were resolved by SDS-PAGE on 3% to 15% linear ceptibility to LC-induced injury was assessed by subjecting gradient gels and transferred onto polyvinylidene fluoride each muscle to eight LCs at a rate of one LC every three Immobilon-FL membrane (Millipore, Billerica, MA, USA). minutes [3,25,26]. Each LC consisted of maximally activat- The membranes were blocked with fish gelatin in ing the muscle at a fixed length for 100 milliseconds, then Tris-buffered saline (TBS) and incubated with primary stretching the muscle at a strain of 30% of L at a strain a antibodies (mouse anti-dysferlin mAb Hamlet, mouse velocity of 1 L /second. Muscle activation ceased upon anti-b-dystroglycan 8D5 mAb, mouse anti-dystrophin achieving the 30% strain and was returned to L at the mAb and rabbit anti-dihydropyridine receptor (anti- same velocity. To assess the force deficit generated by this DHPR) a2 pAb [24]). Blots were washed with TBS + 0.1% assay, a measurement of P was taken three minutes after Tween 20 and incubated with infrared dye-conjugated sec- the last LC and repeated at 15, 30 and 45 minutes. The ondary antibodies (Pierce Biotechnology/Thermo Fisher total fiber cross-sectional area and specific P (kN/m ) Scientific,Inc,Rockford,IL,USA).Afterwashing,blots were calculated based on measurements of muscle mass, were captured using the Odyssey Imaging System (LI- L and P . A generalized linear model with repeated-mea- f 0 COR, Lincoln, NE, USA). sures analysis of variance (ANOVA) using SPSS software (SPSS, Inc, Chicago, IL, USA) was used to determine Evans blue dye uptake whether time after LC, dysferlin and dystrophin were sig- Evans blue dye (Sigma-Aldrich) was dissolved in PBS (10 nificant factors. mg/ml) and sterilized through a 0.2-μm pore size filter. Themicewereanesthetizedbyketamineinjection (0.1 Statistics ml/10 g body weight), and 0.05 ml/10 g body weight dye Data were calculated according to analysis of variance solution was injected intraperitoneally. The animals were (ANOVA)and areexpressedasmeans±SEM. Where killed 24 hours after injection, and their skeletal muscles appropriate, the significance of differences between multi- were isolated and frozen in isopentane cooled to -165°C ple mouse models was assessed using one-way ANOVA in liquid nitrogen. Microscopic evaluation of EBD uptake with Student-Newman-Keuls posttests, and the signifi- was performed on 7-μm muscle cryosections. Muscle cance of differences between two experimental groups cryosections were fixed in cold acetone at -20°C for were assessed using an unpaired two-tailed Student’s 10 minutes, washed with PBS, coverslipped in VECTA- t-test. P < 0.05 was accepted as significant. SHIELD Mounting Medium (Vector Laboratories, Burlingame, CA, USA) and evaluated by fluorescence Results microscopy. Disrupted dystrophin and dysferlin expression in skeletal muscles of DKO mice Force measurement Both immunofluorescence and Western blot analyses con- Contractile properties were measured in vitro on extensor firmed the complete loss of both dystrophin and dysferlin digitorum longus (EDL) muscles from WT, dysferlin-null, in the skeletal muscle membranes of the DKO mice (Fig- mdx and DKO mice (12 to 22 weeks of age) as described ures 1A and 1B) [15]. Mutations in dystrophin disrupt the previously [3,25]. The mice were anesthetized with an stability of the entire DGC at the sarcolemma, which in intraperitoneal injection of 2% avertin (0.0015 ml/g body turn renders the muscle susceptible to contraction- weight), and thoracotomy was performed. EDL muscles induced injuries [2]. To assess the expression of DGC were immersed in an oxygenated bath (95% O ,5%CO ) components in DKO muscle, we performed immunofluor- 2 2 that contained Ringer’s solution (pH 7.4) at 25°C. For each escence and immunoblot experiments using the specific muscle, one end tendon was tied securely with a 6-0 antibodies against b-dystroglycan and sarcospan, two suture to a dual-mode servomotor (Aurora Scientific, Inc, DGC components. The WT and dysferlin-null muscles Aurora, ON, Canada) and the other tendon was clamped showed normal expression of b-dystroglycan and sarco- to a fixed post. Using twitches with a pulse duration of 0.2 span (Figures 1A and 1C), suggesting a stable DGC at the milliseconds, the voltage or current of stimulation was sarcolemma. The mdx and DKO muscles showed a reduc- increased to achieve a maximum twitch and then tion in the expression of both proteins (Figures 1A and increased slightly. Twitches were then used to adjust the 1C), suggesting a disrupted DGC in the muscles of these muscle length to the optimum length for force develop- mice. The expression levels of non-DGC proteins DHPR ment (L ). Fiber length (L ) was determined by multiplying a2 and laminin a2 were also examined, and we found that o f L by the ratio of fiber length to muscle length (0.45) they were not reduced in the DKO muscles (Figures 1A [3,25,26]. The muscle length was set at L ,and EDL and 1C). o Han et al. Skeletal Muscle 2011, 1:35 Page 4 of 11 http://www.skeletalmusclejournal.com/content/1/1/35 Figure 1 Disrupted expression of dysferlin and dystrophin in skeletal muscle of dystrophin/dysferlin double-knockout mice. (A) Western blot showing the expression pattern of dysferlin (DysF), dystrophin (Dyst), b-dystroglycan (b-DG) and dihydropyridine receptor a2 (DHPRa2) in skeletal muscle tissue lysates from wild-type (WT), dysferlin-null, mdx and dystrophin/dysferlin double-knockout (DKO) mice. (B) Expression of dystrophin and dysferlin in skeletal muscles from WT, dysferlin-null, mdx and DKO mice were examined by immunofluorescence staining. (C) b-DG and sarcospan (SSPN) were greatly diminished at the sarcolemma of mdx and DKO muscles, but laminin a2 (Lam2) staining was not reduced. Scale bars: 100 μm. Han et al. Skeletal Muscle 2011, 1:35 Page 5 of 11 http://www.skeletalmusclejournal.com/content/1/1/35 Increased muscle histopathology in the DKO mice The quadriceps muscle sections were also analyzed at Defects in either dysferlin or dystrophin lead to deficits six months of age in DKO mice and compared with the in skeletal muscle [1,12,13]. Therefore, we carefully muscles isolated from age-matched controls, dysferlin- examined the function and histopathology of skeletal null and mdx mice. The DKO quadriceps muscle tissues muscles in the DKO mice. We observed that many of exhibited more severe muscular dystrophic pathology the DKO mice developed malocclusion. Histological compared with dysferlin-null and mdx mice (Figure 3). examination of the DKO mandible muscles by H & E Centrally nucleated muscle fibers, which indicate regen- staining revealed greater dystrophic features than either erating fibers, were quantified in quadriceps muscles the mdx or dysferlin-null masseter muscles (Figure 2). from six-month-old WT, dysferlin-null, mdx and DKO Because rodent incisors grow continuously from birth mice. Of the total muscle fibers, 28% ± 3% in dysferlin- and are kept worn down and sharp by continuously null mice, 55% ± 2% in mdx mice and 73% ± 2% in DKO gnawing[27], themoreseveremusclepathology ofthe mice were centrally nucleated compared to 1.3% ± 0.2% mandibular muscles in DKO mice suggests increased in WT mice (six mice per group) (Figure 3B). Muscle his- muscle weakness of these muscles as a reason for the topathology of aged DKO mice was also performed, long incisor growth. The front teeth of mice with mal- including the triceps, gastrocnemius, diaphragm, tibialis occlusion were regularly clipped, allowing them access anterior, iliopsoas, hamstring and gluteus muscles (Addi- to and ingestion of food. tional file 2, Figure S2). All muscles from DKO mice Figure 2 H & E staining of masseter muscle sections from WT, dysferlin-null, mdx and DKO mice at six months of age. Scale bar: 100 μm. Han et al. Skeletal Muscle 2011, 1:35 Page 6 of 11 http://www.skeletalmusclejournal.com/content/1/1/35 Figure 3 Histopathological analyses of quadriceps muscle sections from DKO mice. (A) H & E-stained quadriceps muscle sections from dysferlin-null, mdx and DKO mice at six months of age. Scale bar: 100 μm. (B) Quantitative analysis of centrally nucleated muscle fibers (CNF) in quadriceps muscles from WT, dysferlin-null, mdx and DKO mice (n = 6 per group) at six months of age. Each group was significantly different from all the other groups. For clarity, significance is shown only for the comparisons with the DKO mice. ***P < 0.001. exhibited severe pathology, including widespread muscle presence of EBD-positive fibers indicates the presence of necrosis, fibrosis and fatty replacement. These results are membrane disruptions in muscle fibers. Individual EBD- consistent with our hypothesis that dystrophin and dys- positive muscle fibers were scattered throughout the ferlin play nonredundant roles in maintaining muscle muscle sections of dysferlin-null mice, and the muscle function. sections of mdx mice showed clusters of EBD-positive fibers [28] (Figure 4A). Interestingly, the DKO mice Severely compromised muscle sarcolemmal integrity in showed both patterns of dye uptake: individual and clus- the DKO mice ters of EBD-positive muscle fibers (Figure 4A). To examine the sarcolemmal integrity of the skeletal Membrane disruptions and muscle damage not only muscle fibers in the DKO mice, we injected EBD and allow EBD uptake but also lead to leakage of cytosolic analyzed its uptake into the quadriceps skeletal muscles. contents such as CK, which can then be detected in the EBD is a membrane-impermeable dye that binds serum serum. Measurements of serum CK levels provide an albumin. A cell can uptake this dye only if the plasma index of the active skeletal muscle necrosis and the pre- membrane of the cell is compromised. Therefore, the sence of membrane disruptions. Our analysis revealed Han et al. Skeletal Muscle 2011, 1:35 Page 7 of 11 http://www.skeletalmusclejournal.com/content/1/1/35 Figure 4 Evans blue dye uptake analyses of quadriceps muscles and serum CK measurements. (A) Evans blue dye (EBD) fluorescence photomicrographs of quadriceps muscle sections from dysferlin-null, mdx and DKO mice at six months of age. Scale bar: 100 μm. (B) Serum creatine kinase (CK) levels were significantly different (P < 0.001) between DKO mice (n = 6) and the other groups (n = 5, 4 and 6 for WT, dysferlin-null and mdx mice, respectively). Values for mdx mice were significantly different from those for WT mice (P = 0.018). For clarity, significance is shown only for the comparisons with the DKO mice. ***P < 0.001. that the DKO mice had serum CK levels twofold higher m for dysferlin-null muscle, suggesting that the absence than those of mdx mice and tenfold higher than those of dysferlin alone does not lead to a deficit of specific of dysferlin-null mice (Figure 4B). Taken together, these force. The specific forces for mdx mice (166 ± 8 kN/m ) data suggest that the dystrophin and dysferlin double- were significantly lower than those for WT mice. The deficiency results in decreased sarcolemmal integrity. absence of dysferlin had no effect in the mdx back- ground, as indicated by a lack of significant difference in Severe functional deficits in the DKO muscles specific forces of DKO muscles (153 ± 11 kN/m ) com- To examine whether a double-deficiency of dysferlin pared to those of mdx muscles (Figure 5A). However, and dystrophin also leads to a more severe functional dysferlin had a significant role in the force recovery fol- disturbance, we measured the force production of EDL lowing LCs, particularly in the context of an mdx back- muscles and their responses to LCs. The specific forces ground (Figure 5B). Both the WT and dysferlin-null were 219 ± 7 kN/m for WT muscle and 212 ± 8 kN/ muscles recovered marginally within 45 minutes post- Han et al. Skeletal Muscle 2011, 1:35 Page 8 of 11 http://www.skeletalmusclejournal.com/content/1/1/35 Figure 5 Effects of dysferlin and dystrophin deficiencies on skeletal muscle contractile properties. We investigated the extensor digitorum longus (EDL) muscles taken from WT (n = 4), dysferlin-null (n = 6), mdx (n = 8) and DKO mice (n = 3). (A) Tetanic force measurements prior to LC. *P < 0.05 and **P < 0.01. (B) Tetanic force recovery following LC. Dysferlin deficiency had a significant effect on force recovery in the absence of dystrophin, as indicated by a significant interaction (P = 0.04) between dysferlin, dystrophin and time after LC on the basis of analysis of variance. (C) Summary of estimated maximal recovery levels following LC. *P < 0.05. (D) Summary of force recovery rate following LC. *P < 0.05. LC (WT: 69% ± 4% at 3 minutes vs 79% ± 2% at 45 DKO muscles (0.032 ± 0.012/minute) was significantly minutes; dysferlin-null: 65% ± 4% at 3 minutes vs 71% ± lower than in mdx muscles (0.080 ± 0.006/minute), 4% at 45 minutes). Although the force in mdx muscles although there was no difference in the maximum was greatly diminished at 3 minutes post-LC, it gradu- recovery levels (Figures 5C and 5D). These data suggest ally recovered from 22% ± 4% at 3 minutes to 61% ± 5% that dysferlin deficiency impairs the force recovery fol- at 45 minutes (Figure 5B). Interestingly, the DKO mus- lowing LCs in mdx mice, indicating that compromised cles also recovered significantly from 16% ± 2% at 3 membrane repair [10] has consequences for post-LC minutes to 52% ± 1% at 45 minutes (Figure 5B). How- force generation. ever, dysferlin deficiency significantly slowed the recov- ery of force in the absence of dystrophin (Figure 5B). To Discussion estimate the maximal recovery level and the recovery Skeletal muscle is associated with significant stress and rate, we fitted the data to a one-phase association equa- strain during muscle contraction. Previous studies have tion. Particularly, we found that the recovery rate in shown at least two mechanisms that skeletal muscle Han et al. Skeletal Muscle 2011, 1:35 Page 9 of 11 http://www.skeletalmusclejournal.com/content/1/1/35 cells utilize to maintain sarcolemmal integrity: a tightly resembling that seen in DMD patients, in contrast to a associated basal lamina mediated by the dystroglycan mild, slowly progressing cardiac manifestation in mdx complex limits the extent of plasma membrane damage mice [15]. Thus the DKO mouse model is well-suited [3] and membrane repair machinery involving multiple to the study of the molecular mechanisms of and ther- proteins that actively restore membrane integrity follow- apy for cardiomyopathy in DMD. Our present study ing limited levels of membrane disruption, such as dys- reveals for the first time that dystrophin deficiency ferlin [10,15,18]; calpain [29]; annexins A1, A2 and A5 unmasks a role for dysferlin in repairing membrane [18,30,31]; and MG53 [32,33]. A defect in either damage during the initial injury elicited by LCs. Com- pared to WT skeletal muscle, mdx muscle had a much mechanism is detrimental to the muscle, as manifested by the fact that genetic mutations in either the DGC greater force deficit measured three minutes after LC components (for review, see [34]) or dysferlin [12-14] (78% in mdx vs 31% in WT). However, by 45 minutes, cause various muscular dystrophies. In the present work the force in mdx muscles recovered from 22% to over using mouse models, we have further demonstrated that 60% of the preinjury level, whereas the WT and dysfer- a double-deficiency in both dystrophin and dysferlin lin-null muscles gained only 10% (from 69% to 79%) results in more severe muscular dystrophy. The results and 6% (from 65% to 71%), respectively. These data of our work are in agreement with those of Shaw et al. suggest that, unlike WT and dysferlin-null muscle, in [35], who showed that mice overexpressing the Cox- which LCs induce little membrane damage, mdx muscle sackie virus and adenovirus receptor transgene demon- undergoes more severe membrane damage, which strate decreased expression levels of dystrophin and requires an active membrane repair process to restore dysferlin, which may lead to an associated myopathy. membrane integrity. DKO muscles are also repair-com- Our work provides genetic evidence that dysferlin and petent, as the force also recovered from 16% at 3 min- the DGC are independent pathways for the maintenance utes to 52% at 45 minutes. However, by comparing the of sarcolemmal integrity in muscle and further high- recovery rate constants of mdx muscles and DKO mus- lights the importance of these two pathways in muscle cles, we found that the recovery rate of the DKO mus- health. cles was less than half the rate of mdx muscles. Contraction-induced injury is characterized by two dis- Therefore, our data derived from using a LC assay tinct phases: an initial injury and a delayed secondary reveal that dystrophin deficiency sufficiently unmasks injury from the inflammatory response. The initial injury the membrane repair role of dysferlin. The DKO mouse consists of mechanical disruption of sarcomeres followed would be a very useful model with which to determine 2+ by impaired excitation-contraction coupling and Ca sig- the efficacy of a therapeutic treatment designed for 2+ dysferlinopathy. naling and finally by activation of Ca -sensitive degrada- tion pathways [36]. Interestingly, for muscle with an intact DGC, whether membrane damage occurs during the initial Conclusion injury phase is unclear; but if it exists, it is dysferlin-inde- Taken together, our results show that both DGC- pendent [25,37,38]. Several hours to days following the mediated membrane stability and dysferlin-mediated initial injury, infiltration of inflammatory factors damage membrane repair contribute to the function and mainte- muscle fibers and release reactive oxygen species (ROS), nance of skeletal muscle. The studies presented here which aid in clearing disrupted myofibrils but also damage suggestthatthe DKOmouse modelmay be avaluable previously undamaged myofibrils. At this stage, ROS tool in the development of therapies designed to treat potentially cause damage to the sarcolemma, possibly dysferlinopathies. through lipid peroxidation [39,40]. Investigators in pre- vious studies have demonstrated that dysferlin is impor- Additional material tant in limiting the extent of secondary injury. For example, three days following a protocol of small strain Additional file 1: Figure S1 Breeding strategy to generate dystrophin/dysferlin double-knockout mice. Male dysferlin-null mice LCs, muscles of dysferlin-deficient mice displayed were mated with mdx female mice, then F1 heterozygous males and increased macrophage infiltration, decreased ability to seal females were bred to generate F2 males and females. Of the F2 males off a membrane-impermeable dye and increased force def- and females, 12.5% are predicted to be DKO mice. icits [38,41]. These data indicate that defects in membrane Additional file 2: Figure S2 Histopathology analyses of various muscles from dystrophin/dysferlin double-knockout mice.H&E- repair or alteration in repair result in chemoattraction of stained muscle sections of triceps (TC), gastrocnemius (GA), diaphragm immune cells [38,41]. (DIA), tibialis anterior (TA), iliopsoas (IP), hamstring (HS) and gluteus (GT) Previously, we demonstrated that a combined defi- muscles from dystrophin/dysferlin double-knockout (DKO) mice at one and one-half years of age. Scale bar: 100 μm. ciency in dysferlin and dystrophin results in the devel- opment of a pronounced early-onset cardiomyopathy Han et al. Skeletal Muscle 2011, 1:35 Page 10 of 11 http://www.skeletalmusclejournal.com/content/1/1/35 7. Weller B, Karpati G, Carpenter S: Dystrophin-deficient mdx muscle fibers Abbreviations are preferentially vulnerable to necrosis induced by experimental CK: creatine kinase; DGC: dystrophin-glycoprotein complex; DHPR: lengthening contractions. J Neurol Sci 1990, 100:9-13. dihydropyridine receptor; DKO: dystrophin/dysferlin double-knockout; DMD: 8. Dellorusso C, Crawford RW, Chamberlain JS, Brooks SV: Tibialis anterior Duchenne muscular dystrophy; EBD: Evans blue dye; ECM: extracellular muscles in mdx mice are highly susceptible to contraction-induced matrix; EDL: extensor digitorum longus; H & E: hematoxylin and eosin; LC: injury. J Muscle Res Cell Motil 2001, 22:467-475. lengthening contraction; mAb: monoclonal antibody; pAb: polyclonal 9. Moser H: Duchenne muscular dystrophy: pathogenetic aspects and antibody; PBS: phosphate-buffered saline; ROS: reactive oxygen species. genetic prevention. Hum Genet 1984, 66:17-40. 10. Bansal D, Miyake K, Vogel SS, Groh S, Chen CC, Williamson R, McNeil PL, Acknowledgements Campbell KP: Defective membrane repair in dysferlin-deficient muscular We thank Keith Garringer, Samantha K Watkins, Sally Prouty and David P dystrophy. Nature 2003, 423:168-172. Venzke for technical support and Drs Yvonne Kobayashi, Li Xu and Piming Zhao for critical reading and discussion. This work was supported in part by 11. Han R, Campbell KP: Dysferlin and muscle membrane repair. Curr Opin American Heart Association Scientist Development grant 10SDG4140138 (to Cell Biol 2007, 19:409-416. RH), Muscular Dystrophy Association Research Grant MDA171667 (to RH), 12. Bashir R, Britton S, Strachan T, Keers S, Vafiadaki E, Lako M, Richard I, Paul D Wellstone Muscular Dystrophy Cooperative Research Center grant Marchand S, Bourg N, Argov Z, Sadeh M, Mahjneh I, Marconi G, Passos- 1U54NS053672 (to KPC), Muscular Dystrophy Association grant MDA3936 (to Bueno MR, Moreira Ede S, Zatz M, Beckmann JS, Bushby K: A gene related KPC), Muscular Dystrophy Association Development grants MDA200826 (to to Caenorhabditis elegans spermatogenesis factor fer-1 is mutated in JRL) and MDA67814 (to EPR) and US Department of Defense grant limb-girdle muscular dystrophy type 2B. Nat Genet 1998, 20:37-42. W81XWH-05-1-0079. The authors declare no conflicts of interest. KPC is an 13. Liu J, Aoki M, Illa I, Wu C, Fardeau M, Angelini C, Serrano C, Urtizberea JA, investigator at the Howard Hughes Medical Institute. Hentati F, Hamida MB, Bohlega S, Culper EJ, Amato AA, Bossie K, Oeltjen J, Bejaoui K, McKenna-Yasek D, Hosler BA, Schurr E, Arahata K, de Jong PJ, Author details Brown RH Jr: Dysferlin, a novel skeletal muscle gene, is mutated in Department of Cell and Molecular Physiology, Stritch School of Medicine, Miyoshi myopathy and limb girdle muscular dystrophy. Nat Genet 1998, Loyola University Medical Center, 2160 S 1st Avenue, Maywood, IL 60558, 20:31-36. USA. Department of Molecular Physiology and Biophysics, Howard Hughes 14. Illa I, Serrano-Munuera C, Gallardo E, Lasa A, Rojas-García R, Palmer J, Medical Institute, Roy J and Lucille A Carver College of Medicine, The Gallano P, Baiget M, Matsuda C, Brown RH: Distal anterior compartment University of Iowa, 285 Newton Road, 4283 CBRB, Iowa City, IA 52242, USA. myopathy: a dysferlin mutation causing a new muscular dystrophy Department of Neurology, Howard Hughes Medical Institute, Roy J and phenotype. Ann Neurol 2001, 49:130-134. Lucille A Carver College of Medicine, The University of Iowa, 285 Newton 15. Han R, Bansal D, Miyake K, Muniz VP, Weiss RM, McNeil PL, Campbell KP: Dysferlin-mediated membrane repair protects the heart from stress- Road, 4283 CBRB, Iowa City, IA 52242, USA. Department of Internal induced left ventricular injury. J Clin Invest 2007, 117:1805-1813. Medicine, Howard Hughes Medical Institute, Roy J and Lucille A Carver 16. Cenacchi G, Fanin M, De Giorgi LB, Angelini C: Ultrastructural changes in College of Medicine, The University of Iowa, 285 Newton Road, 4283 CBRB, dysferlinopathy support defective membrane repair mechanism. J Clin Iowa City, IA 52242, USA. Pathol 2005, 58:190-195. 17. Cai C, Weisleder N, Ko JK, Komazaki S, Sunada Y, Nishi M, Takeshima H, Authors’ contributions Ma J: Membrane repair defects in muscular dystrophy are linked to RH conceived the study, carried out the histopathological studies, altered interaction between MG53, caveolin-3, and dysferlin. J Biol Chem participated in the sequence alignment and drafted the manuscript. ER 2009, 284:15894-15902. carried out the force measurement experiments and performed the 18. Lennon NJ, Kho A, Bacskai BJ, Perlmutter SL, Hyman BT, Brown RH Jr: statistical analysis. JL carried out the immunoassays. DB initiated the mouse Dysferlin interacts with annexins A1 and A2 and mediates sarcolemmal breeding and participated in sequence alignment. KC conceived the study, wound-healing. J Biol Chem 2003, 278:50466-50473. participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript. 19. Hino M, Hamada N, Tajika Y, Funayama T, Morimura Y, Sakashita T, Yokota Y, Fukamoto K, Kobayashi Y, Yorifuji H: Insufficient membrane Competing interests fusion in dysferlin-deficient muscle fibers after heavy-ion irradiation. Cell The authors declare that they have no competing interests. Struct Funct 2009, 34:11-15. 20. 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Roche JA, Lovering RM, Roche R, Ru LW, Reed PW, Bloch RJ: Extensive mononuclear infiltration and myogenesis characterize recovery of dysferlin-null skeletal muscle from contraction-induced injuries. Am J Physiol Cell Physiol 2010, 298:C298-C312. doi:10.1186/2044-5040-1-35 Cite this article as: Han et al.: Dystrophin deficiency exacerbates skeletal muscle pathology in dysferlin-null mice. Skeletal Muscle 2011 1:35. Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color figure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit

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Skeletal MuscleSpringer Journals

Published: Dec 1, 2011

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