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Viral-mediated expression of desmin mutants to create mouse models of myofibrillar myopathy

Viral-mediated expression of desmin mutants to create mouse models of myofibrillar myopathy Background: The clinical features of myofibrillar myopathies display a wide phenotypic heterogeneity. To this date, no studies have evaluated this parameter due to the absence of pertinent animal models. By studying two mutants of desmin, which induce subtle phenotypic differences in patients, we address this issue using an animal model based on the use of adeno-associated virus (AAV) vectors carrying mutated desmin cDNA. Methods: After preparation of the vectors, they were injected directly into the tibialis anterior muscles of C57BL/6 mice to allow expression of wild-type (WT) or mutated (R406W or E413K) desmin. Measurements of maximal force were carried out on the muscle in situ and then the injected muscles were analyzed to determine the structural consequences of the desmin mutations on muscle structure (microscopic observations, histology and immunohistochemistry). Results: Injection of AAV carrying WT desmin results in the expression of exogenous desmin in 98% of the muscle fibers without any pathological or functional perturbations. Exogenous WT and endogenous desmin are co-localized and no differences were observed compared to non-injected muscle. Expression of desmin mutants in mouse muscles induce morphological changes of muscle fibers (irregular shape and size) and the appearance of desmin accumulations around the nuclei (for R406W) or in subsarcolemmal regions of fibers (for E413K). These accumulations seem to occur and disrupt the Z-line, and a strong regeneration was observed in muscle expressing the R406W desmin, which is not the case for E413K. Moreover, both mutants of desmin studied here induce a decrease in muscle force generation capacity. Conclusions: In this study we show that AAV-mediated expression of desmin mutants in mouse muscles recapitulate the aggregation features, the decrease in contractile function and the morphological changes observed in patients with myofibrillar myopathy. More importantly, our results suggest that the R406W desmin mutant induces a robust muscle regeneration, which is not the case for the E413K mutant. This difference could help to explain the phenotypic differences observed in patients. Our results highlight the heterogeneous pathogenic mechanisms between different desmin mutants and open the way for new advances in the study of myofibrillar myopathies. Keywords: Myofibrillar myopathy, Intermediate filament, Desmin, AAV vectors, Phenotypic heterogeneity, Desminopathy * Correspondence: onnik.agbulut@univ-paris-diderot.fr Equal contributors Université Paris Diderot, Sorbonne Paris Cité, CNRS EAC4413, Unit of Functional and Adaptive Biology, Laboratory of Stress and Pathologies of the Cytoskeleton, 75013, Paris, France Full list of author information is available at the end of the article © 2013 Joanne 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. Joanne et al. Skeletal Muscle 2013, 3:4 Page 2 of 13 http://www.skeletalmusclejournal.com/content/3/1/4 Background severe with a high risk of heart failure. The E413K muta- Myofibrillar myopathy (MFM) [OMIM:601419] refers to tion was first described by Pruszczyk et al. in 2007, as a a group of genetically heterogeneous chronic neuromus- disease primarily affecting cardiac function with an cular disorders. MFMs are mainly caused by mutations autosomal dominant mode of inheritance [20]. Three in the desmin gene [1], while other forms of MFM are cases from the same family were described. The first caused by mutations in alpha-B-crystallin [2], myotilin symptoms of heart disease developed later than those of [3], ZASP [4], filamin C [5] or BAG3 [6] genes. Rigid the R406W mutation (age of onset at 30 years versus 20 spine syndrome caused by mutation in the SEPN1 gene years). The first signs of muscle weakness appeared is also considered as a MFM [7]. The morphological about 15 years after the first cardiac symptoms and were changes described in MFM result from disturbance of restricted to the lower limbs. Therefore, from these the sarcomeric Z-line and the myofibrils, followed by ab- reports it would appear that the R406W mutation is normal aggregation of proteins predominantly involved more pathogenic than the E413K mutation, since the in the structure of the Z-line. The age of disease onset clinical symptoms develop later and the skeletal muscles can vary from early adolescence to the 60s. The disease are less affected. However, the molecular mechanism is very heterogeneous manifesting in some cases as a underlying these differences in the clinical phenotypes relentlessly progressive skeletal myopathy with no signs cannot be studied with the cellular models that are cur- of cardiac involvement [8,9]. In other cases cardiomyop- rently available, since both desmin mutations R406W athy is a prominent [1] or even exclusive [10] feature. and E413K present the same features. Both are unable to Respiratory insufficiency may also be a major manifest- form filamentous networks on their own, but are able to ation and cause of death [11]. integrate into pre-existing desmin networks and form In 1998, Goldfarb and his co-workers [1] established aggregates, which are concentrated around the nuclei that a mutation in the desmin gene was involved in and throughout the cytoplasm [21]. Several teams, MFM. Since then, more than 50 different mutations re- including ours, have demonstrated that R406W and sponsible for MFM symptoms have been identified in E413K mutations, located in the terminal consensus the desmin gene. Desmin is the major specific inter- motif, alter in a similar manner the dimer interaction, mediate filament protein of skeletal, cardiac and smooth which impairs the filament formation [20-22]. These muscles. It forms a three-dimensional scaffold around observations clearly demonstrate that neither in silico the myofibrillar Z-disc, and connects the contractile ap- nor cellular models are able to explain the molecular paratus and other structural elements of the cell, that is, mechanisms which result in the phenotypic heteroge- the subsarcolemmal cytoskeleton, the nuclei and other neity observed in patients carrying these mutations. organelles [12,13]. It also links the myofibrils laterally by To try to address this issue, several transgenic mouse connecting the Z-discs, and is particularly abundant at the models expressing different mutations of desmin have myotendinous and neuromuscular junctions of skeletal been obtained [23-25]. However, the different promoters muscle and in the intercalated disks of cardiomyocytes and strains used make the comparison quite difficult. [14]. Thus, desmin filaments are essential to maintain cel- Moreover, conventional techniques of transgenesis do lular integrity, for the transmission of force and the not seem compatible to analyze the large number of mechanochemical signaling of muscle cells [15-17]. mutations which have been identified within the desmin In the present study, we examined two missense gene. We therefore chose to use a simpler strategy to desmin mutations, R406W and E413K. A patient with a study, in the same genetic background, different domi- R406W mutation in desmin was first described by Park nant mutations of desmin. In order to develop these et al. in 2000 [18]. In 2004, three new cases were models, we produced an adeno-associated virus (AAV) described and compared [19]. From this study it became vector that allows the expression of a mutant desmin in muscles. Intramuscular injection of this virus allows the apparent that patients with the R406W mutation have many common features. These four patients were of dif- localized transgenic expression of desmin mutants in ferent nationalities and no other member of their fam- C57BL/6 mice. Different analyses were carried out first on the muscle in situ (measurement of muscular force) and ilies was affected by the disease (sporadic de novo mode of inheritance). The first signs of the disease appeared at then the injected muscles were analyzed to determine the around 20 years of age and were characterized by severe structural consequences of the desmin mutations on muscle structure (microscopic observations, histology and cardiac involvement (cardiac arrhythmia and complete atrioventricular block resulting in pacemaker implant- immunohistochemistry). ation). The disease developed with increasing muscle It should be noted that studies on MFM are inherently weakness of the lower limbs. This weakness then limited because of the small number of patients and very extended to the muscles of the upper limbs and the limited access to biopsy. In addition, this strategy of cre- neck. The final outcome of the disease is usually very ating an in vivo model appears to be very interesting Joanne et al. Skeletal Muscle 2013, 3:4 Page 3 of 13 http://www.skeletalmusclejournal.com/content/3/1/4 because of its ease and rapidity of obtaining, and the low determined by real-time PCR (TaqMan, Life Tech- cost associated with its implementation. Using this nologies) using CMV promoter specific primers 0 0 model we aim to reproduce in vivo the phenotypic vari- (forward: 5 -CTACGCCCATTTGCGTCAA-3;re- 0 0 ability observed in patients in order to better understand verse: 5 -GCACCAAAATCAACGGGAC-3;probe: 0 0 the mechanisms behind these variations. 5 -CAAAATGTCGTAACAACTCCGCCCC-3 ). Methods Intramuscular delivery of AAV vectors Plasmids production All procedures have been approved by our institutional The full-length human desmin cDNAs were cloned into Ethics Committee, and conducted according to the pSMD2 plasmid (cytomegalovirus (CMV) promoter and French and European laws, directives and regulations on human β-globin pA) using Xho I restriction site. To dis- animal care (European Commission Directive 86/609/ tinguish the desmin transgene from the endogenous EEC). Our animal facility is fully licensed by the French form, a c-Myc tag was introduced at the 5 end of the competent authorities and has animal welfare insurance. desmin cDNA. It should be noted that the presence of For intramuscular injection, 35 15-week-old female c-Myc at the N-terminal of desmin does not disturb fila- C57BL/6 mice were randomized into four groups: PBS ment assembly or cellular localization [21]. R406W and (n = 6), AAV-DesWT (n = 11), AAV-DesR406W (n = E413K desmin mutations were introduced into the 10) and AAV-DesE413K (n = 8). Animals were pSMD2 plasmid by site-directed mutagenesis using the anesthetized by intraperitoneal injection of pentobarbital Stratagene QuikChange kit (Agilent Technologies, sodium (60 mg/kg). Each tibialis anterior muscle was Massy, France). Escherichia coli Stbl2 strain (Life Tech- injected with 50 μl of either AAV-2/1-Des (10 viral nologies, Saint Aubin, France) was transformed with the genomes/muscle) or sterile PBS solution as a control. different plasmids according to the manufacturer’s instructions for amplification. All plasmids were purified using the endotoxin-free PureYield Plasmid Maxiprep Muscle force measurements System (Promega, Lyon, France), and then verified by Four weeks after injection, mice were anesthetized restriction enzyme digestion and sequencing (Eurofins (pentobarbital sodium, 60 mg/kg) and the limbs were MWF Operon, Ebersberg, Germany). fixed with clamps. The distal tendon of the tibialis anter- ior muscle was attached to a dual-mode lever arm sys- Production, purification and titration of the AAV vectors tem that measures muscle isometric force (300C; Aurora Pseudotyped AAV-2/1 vectors were generated in human Scientific, Ontario, Canada). Great care was taken to en- embryonic kidney 293 cells by the triple transfection sure that the blood and nerve supply remained intact method described by Rivière et al., with minor mo- during surgery. Active force measurements were difications [26]. The productions were realized with performed as described previously [27-29]. The sciatic pXX6 adenovirus helper plasmid coding the adenoviral nerve was crushed proximally and stimulated distally by sequences essential for AAV production, the pRep-Cap a bipolar silver electrode using supramaximal square plasmid coding the AAV-1 capsid and the pSMD2- wave pulses of 0.1 ms duration. All isometric mea- DesWT, pSMD2-DesR406W or pSMD2-DesE413K plas- surements were made at L0 (muscle length at which mid coding the different desmin transgenes. After triple maximal force was obtained during the twitch). Force transfection of the different plasmids in 293 cells, cells production in response to tetanic stimulation were suc- were harvested and submitted to three freeze-thaw cessively recorded (pulse frequency from 25, 50 and 100 cycles using a 37°C water bath and dry ice cooled etha- to 143 Hz, 500 ms) and at least 1 minute was allowed nol. The lysate was incubated with Benzonase (50 U/ml; between each contraction. The absolute maximal force Sigma-Aldrich, Saint-Quentin Fallavier, France) for 30 was normalized to the muscle mass to determine specific minutes at 37°C. It was then clarified by centrifugation maximal force. at 6,500 rpm for 20 minutes and passed though a 0.45 μm filter. The lysate was layered over the top of an At the end of the experiments, the animals were iodixanol gradient solution (from 15% to 60%; Sigma- euthanized with an overdose of pentobarbital. After con- Aldrich) and centrifuged for 90 minutes at 59,000 rpm. tractile measurements, the muscles were dissected, The virus was isolated between the 40% and 60% weighed and frozen in isopentane pre-cooled in liquid iodixanol solutions, washed with PBS-MK (PBS, nitrogen or fixed in paraformaldehyde for further ana- MgCl 1 mM, KCl 2.5 mM) solution and concentrated lysis. Force measurements were made in PBS treated on Amicon Ultra-15 centrifugal filter (Millipore, (n = 12) or WT (n = 22), R406W (n = 20) and E413K Molsheim, France). The final viral preparations were (n = 15) desmin expressing tibialis anterior muscles stored in PBS solution at -80°C. The AAV titer was 1 month after AAV vectors injection. Joanne et al. Skeletal Muscle 2013, 3:4 Page 4 of 13 http://www.skeletalmusclejournal.com/content/3/1/4 Histological staining After washing, the specimens were post-fixed for 1 hour Transversal frozen sections of 10 μm thickness were with 1% osmium tetroxide solution, dehydrated and prepared from all of the muscles. Hematoxylin and eosin embedded in epoxy resin. Ultrathin sections (70 nm) staining was used to examine general morphology of the were cut with an ultramicrotome (Leica UC6; Leica muscles. The muscle mitochondrial distribution and Microsystems) and stained for 15 minutes with 4% uranyl localization was examined using succinate dehydrogen- acetate and for 2 minutes with Reynolds’ lead citrate, be- ase (SDH) staining. The extent of fibrosis was assessed by fore observation at 80 kV with a transmission electron Sirius red staining and was expressed as the fibrosis index microscope (TEM) Philips Tecnai 12 BioTWIN (Philips, (ratio of the area of the fibrosis to the area of fibers). All Amsterdam, Netherlands) equipped with an Olympus staining methods were carried out as described by KeenView CCD camera (Shinjuku, Tokyo, Japan). Dubowitz [30]. Images were taken with a microscope (Leica Microsystems, Nanterre, France) equipped with a Western blot digital camera (QImaging, Surrey, Canada). For fibrosis Immunoblotting was carried out on extracts of muscles analysis, nine digital images per muscle (n = 5/group) were snap frozen in liquid nitrogen immediately after dissec- processed with ImageJ software (National Institutes of tion. Frozen muscles were placed into an ice-cold Health, Bethesda, MD, USA) [31]. homogenization buffer containing: 50 mM Tris (pH 7.6), 250 mM NaCl, 3 mM ethylenediaminetetraacetic acid Immunofluorescence (EDTA), 3 mM ethylene glycol tetraacetic acid (EGTA), Transversal (morphometric analysis) and longitudinal 0.5% NP40, 2 mM dithiothreitol, 10 mM sodium (striation pattern analysis) frozen sections of 8 μm thick- orthovanadate, 10 mM NaF, 10 mM glycerophosphate ness were cut using a microtome (Leica Microsystems) and 2% of protease inhibitor cocktail (Sigma-Aldrich). for immunostaining. The sections were incubated with Samples were minced with scissors and then blocking solution (bovine serum albumin, 5%) for 1 hour homogenized using plastic pestles, incubated for 30 and then incubated for 30 minutes with goat anti-mouse minutes on ice, sonicated three times for 5 seconds with immunoglobulin G (IgG) Fab fragment (1:100; Jackson 30 second intervals on ice, and then centrifuged at ImmunoResearch Europe, Newmarket, UK). Following a 12,000 g for 30 minutes at 4°C. Protein concentration PBS wash, the sections were incubated for 90 minutes was measured using the Bradford method with bovine with primary antibodies against perlecan (1:400, rat serum albumin as a standard. Equal amounts of protein monoclonal; Millipore), c-Myc (1:1000, rabbit polyclonal; extracts (25 μg) were separated by SDS-PAGE before Sigma-Aldrich), α-actinin (1:100, mouse monoclonal; electrophoretic transfer onto a nitrocellulose membrane Sigma-Aldrich) or myosin heavy chain (MHC) isoform (Amersham Hybond-ECL; GE Healthcare, Vélizy- neonatal (1:100, rabbit polyclonal) [32]. After washing Villacoublay, France). Western blot analysis was carried in PBS, sections were incubated for 1 hour with second- out using anti-c-Myc antibody (1:1,000, mouse monoclo- ary antibodies (Alexa Fluor; Life Technologies) or nal; Santa Cruz Biotechnology, Heidelberg, Germany) phalloidin-TRITC labeled (Sigma-Aldrich). After washing and anti-pan-actin antibody (1:10,000, mouse monoclo- in PBS, slides were finally mounted in Vectashield with nal; Millipore). Antibody reacting bands were visua- DAPI H-1200 (Vector Laboratories, Peterborough, UK). lized with peroxidase-conjugated secondary antibodies Images were captured using a motorized confocal laser (Thermo-Fisher Scientific, Brebières, France) and a scanning microscope (LSM 700; Carl Zeiss SAS, Le Pecq, chemiluminescent detection system (ECL-Plus; GE France). Morphometric analyses were made using the Healthcare). ImageJ [31] software and a homemade macro. The smallest diameter (minimum Feret diameter) of all the muscle fibers of the whole muscle section was measured. The pattern of striations was analyzed on longitudinal Statistical analysis muscle sections using the Plot Profile function of ImageJ Groups were statistically compared using analysis of [31] software. variance (ANOVA). If necessary, post-hoc analysis was performed using Tukey’s Honestly Significant Difference Electron microscopy (HSD) test. For groups that did not pass tests of normal- Electron microscopy was carried out as described previ- ity (Shapiro-Wilk) and equal variance (Bartlett), non- ously [33]. Briefly, the calf muscles of mice were fixed in parametric tests were used (Kruskal-Wallis) and multiple 2.5% glutaraldehyde buffered in 0.1 M cacodylate at pH comparison were driven using the Dunn procedure and 7.4. After 1 hour, the tibialis anterior muscle was multivariate normal distribution in R software. For stat- dissected and separated in three by a short-axis section istical analysis of fiber size distribution, Kolmogorov- and then fixed overnight at 4°C in the same fixative. Smirnov test was used. Values are means ±SEM. Joanne et al. Skeletal Muscle 2013, 3:4 Page 5 of 13 http://www.skeletalmusclejournal.com/content/3/1/4 Results (Figure 1A, WT) had no effect on muscle structure, Morphological modifications of muscles expressing which was identical to an untreated muscle. On the con- mutated desmin recapitulate MFM trary, expression of the R406W mutant of desmin Since the diagnosis of MFM is most often made by using induced the appearance of darkly stained deposits the results obtained from histological stains of muscle located mainly in the perinuclear regions of the muscle sections, we first conducted a series of histological stains fiber (see arrowhead in Figure 1A, R406W). We also saw of mouse muscles expressing WT or mutated (R406W myofibers with central nuclei indicating that the muscle or E413K) desmin 1 month after injection of the differ- fiber is in the process of regeneration (see asterisks in ent AAV constructions (AAV-DesWT, AAV-DesR406W Figure 1A, R406W) or the nuclei are moving from a per- and AAV-DesE413K) in the tibialis anterior muscle of ipheral to a central location. Expression of E413K mu- C57BL/6 mice (Figure 1A). Normal myofibers with a tant of desmin provoked a similar phenotype but with polygonal shape, peripheral nuclei, intact sarcolemma, more blurred dark deposits irradiating from both the non-fragmented sarcoplasm and homogeneous fiber size subsarcolemmal and perinuclear regions (see arrowhead distribution revealed that overexpression of WT desmin in Figure 1A, E413K) of the muscle fibers. In addition, Figure 1 Morphological perturbations induced by desmin mutants in tibialis anterior muscles. (A) Hematoxylin and eosin staining, (B) succinate dehydrogenase (SDH) staining, (C) immunostaining against c-Myc (green) and perlecan (red), and (D) immunostaining against c-Myc (green); on serial transversal (A, B and C) and longitudinal (D) sections of tibialis anterior muscles expressing WT, R406W or E413K desmin. All analyses were performed 1 month after intramuscular injection of AAV vectors. Note that expression of WT desmin does not seem to modify neither muscle morphology (A to B) nor desmin localization (C to D), whereas expression of R406W and E413K desmin mutants induces accumulation of the desmin in the perinuclear region (see arrowheads). Arrows in (B) illustrate abnormal accumulation of mitochondria observed in both R406W and E413K expressed tibialis anterior muscles. Arrow in (C) illustrates a weaker accumulation of desmin in the case of E413K desmin. Asterisks indicate centronucleated muscle fibers. Nuclei (blue) were stained using DAPI (C to D). Scale bars = 30 μm. AAV, adeno- associated virus; DAPI, 40,6-diamidino-2-phenylindole; SDH, succinate dehydrogenase; WT, wild-type. Joanne et al. Skeletal Muscle 2013, 3:4 Page 6 of 13 http://www.skeletalmusclejournal.com/content/3/1/4 many muscle fibers were irregular in shape in muscles to be pushed towards the subsarcolemmal regions expressing the mutated desmin. (see arrow, Figure 1C, E413K). This observation In a second step, we performed SDH staining on serial corresponded well with the central accumulation of sections to explore the distribution of muscle mitochon- staining seen on SDH staining. We also observed, al- dria (Figure 1B). Muscles treated with the virus though to a lesser extent than for R406W, accumulation expressing WT desmin showed a mosaic of fibers with a in perinuclear regions of the fibers (see arrowhead, relatively dense purple appearance in oxidative fibers Figure 1C, E413K). These features were also observed in and scattered purple speckles in the nonoxidative fibers. the longitudinal sections where the R406W desmin These observations are in perfect accordance to a nor- seemed to strongly accumulate around the nuclei while mal muscle. In contrast, the muscles expressing the E413K desmin formed numerous dots occupying the en- R406W mutant showed a localized absence of staining tire width of the fibers (Figure 1D). Moreover, the align- in perinuclear regions (see arrowhead in Figure 1B, ment of the sarcomeres seemed to be disturbed in areas R406W) that correspond to the accumulation of dark where R406W or E413K desmin were accumulated material revealed by hematoxylin and eosin staining (Figure 1D). A closer analysis of the Z-lines (α-actinin (Figure 1A). An irregular staining of some fibers and in immunostaining) and thin filaments (phalloidin staining) some cases a strong accumulation of staining in the cen- demonstrated a very clear and localized perturbation of ter of fibers (see arrow in Figure 1B, R406W) were also the alignment at the zones of accumulation of desmin observed. The muscles expressing the E413K desmin (Additional file 1: Figure S1). As seen in Additional mutant also presented many fibers with this abnormal file 1: Figure S1, c-Myc was co-localized with α-actinin internal architecture characterized by a central dark in both WT and mutated desmin expressing muscles; staining, indicating a modified internal repartition of whereas actin, the major component of the I-band, was mitochondria (see arrow in Figure 1B, E413K). In not localized with c-Myc. It should be noted that the addition, we also observed a decrease in the staining of alignment of Z-lines did not seem to be disrupted at the subsarcolemmal and perinuclear regions (see arrowhead sites where desmin was not drastically accumulated, as in Figure 1B, E413K), which also corresponded to the ac- seen in Figure 1D. cumulation revealed by hematoxylin and eosin staining (Figure 1A). Aberrant muscle regeneration and impaired distribution of muscle fiber size Aberrant aggregation of desmin As shown in Figure 1, many muscle fibers expressing Next, we performed a series of immunostaining to detect R406W mutant are centronucleated (see asterisks in the expression and localization of desmin (Figure 1C,D). Figure 1A,C, R406W) suggesting that there is an import- To distinguish the desmin transgene from endogenous ant muscle regeneration. If this phenomenon really occurs, desmin, a c-Myc tag was introduced at the 5 end of the the absence of c-Myc immunostaining in these centro- desmin cDNA. On transversal sections desmin appeared nucleated fibers could be explained by the fact that AAV homogeneously distributed throughout the cytoplasm has never been described to transduce satellite cells. (Figure 1C, WT). On longitudinal sections it was also cor- To confirm that there is an increase of regeneration in rectly and repeatedly aligned like sarcomeres (Figure 1D, muscles expressing mutant desmin, we measured the WT). It should be noted that immunostaining against number of neonatal MHC-positive fibers reported to the desmin on AAV-DesWT treated tibialis anterior showed total number of fibers from tibialis anterior sections no differences compared to untreated control muscles (Figure 2A). For muscles expressing WT desmin, we (data not shown). Furthermore, we observed that there were not able to detect any neonatal MHC-positive were also no observable differences between the fibers. In the case of muscles expressing R406W or immunostaining against c-Myc and that against desmin in E413K desmin there was, however, an increasing number WT expressing tibialis anterior muscle (data not shown), of fibers expressing this marker (Figure 2A). This ratio which confirmed perfect integration of exogenous desmin was larger and significantly different from WT in the into the normal desmin network. case of R406W desmin (20.5%, P <0.01) compared to As seen in Figure 1C,D, compared to immunostaining E413K desmin (5.5%, P >0.05). against c-Myc in WT expressing muscles, we showed To confirm that all of these modifications were not significant differences in muscles expressing the R406W due to a difference in the expression levels of exogenous and E413K desmin mutants. In these muscles the mu- desmin, we performed a Western blot with total protein tant desmin was strongly accumulated around the nuclei extract from AAV-treated muscles. We detected a for the R406W desmin (see arrowhead, Figure 1C, similar amount of exogenous desmin between WT R406W). For desmin E413K we observed a much weaker and E413K expressing muscles, whereas in R406W labeling in the center of the fibers and desmin seems expressing muscles we observed a decrease in the Joanne et al. Skeletal Muscle 2013, 3:4 Page 7 of 13 http://www.skeletalmusclejournal.com/content/3/1/4 A D Figure 2 Regeneration, hyperplasia and decrease in overall fiber size of R406W desmin expressing muscles compared to E413K expressing muscles. (A) Percentage of neonatal myosin heavy chain (MHC) expressing muscle fibers, (B) total number (dots filled bars) and c-Myc expressing (empty bar) muscle fibers, (C) Western blot quantification of c-Myc, (D) size distribution of total and (E) c-Myc expressing muscle fibers; were examined in WT (n = 3), R406W (n = 3) and E413K (n = 3) desmin expressing tibialis anterior muscles 1 month after AAV vectors injection. For all morphometric analysis, all muscle fibers in whole muscle sections were analyzed. Note the strong increase in regeneration of tibialis anterior expressing R406W desmin compared to the slight increase with E413K desmin (A). Note also hyperplasia of R406W desmin expressing muscle demonstrated after counting the exogenous desmin expressing (Myc-positive, empty bars) and the total number of fibers (dots filled bars) (B). To compare size distribution of total (D) and exogenous desmin expressing (Myc-positive) (E) muscle fibers of WT, R406W or E413K desmin muscles, the Gaussian curve was plotted and grouped. Note the overall decrease in the size of both total and exogenous desmin expressing muscle fibers in R406W muscles, and, to a much lesser extent, the decrease in the size of fibers expressing desmin E413K. Asterisks indicate a significant difference compared to the WT (*P ≤0.05, **P ≤0.01, ***P ≤0.001). AAV, adeno-associated virus; MHC, myosin heavy chain; WT, wild-type. amount of exogenous desmin (Figure 2C). We also between the muscles expressing WT (2721±291 fibers) counted and determined the c-Myc expression of each and those expressing the R406W desmin (2163±212 muscle fiber of the entire muscle section. Interestingly, fibers) or E413K (2404±68 fibers). In contrast, compared after having counted the number of c-Myc-positive to the WT (2779±265 fibers), the total number of fibers muscle fibers for each muscle (Figure 2B), we observed increased significantly in the case of muscles expressing that there was no significant (P = 0.252) difference R406W desmin mutant (4546±313 fibers, P = 0.0036), Joanne et al. Skeletal Muscle 2013, 3:4 Page 8 of 13 http://www.skeletalmusclejournal.com/content/3/1/4 which was not the case for muscles expressing E413K yet located at the Z-line (see arrowheads in Figure 3E). (2684±48 fibers, P >0.05). This increase in the total num- These results emphasize that the R406W desmin aggre- ber of muscle fibers in R406W expressing muscles may gates are generated at the perinuclear area and then they also explain the relative decrease in the amount of exogen- spread throughout the whole muscle. ous desmin seen on Western blot because newly generated In E413K expressing muscles, 1 month after AAV- muscle fibers cannot express exogenous desmin. DesE413K injection, ultrastructural examination revealed As expected for a regenerating muscle, the overall dis- granulofilamentous electron-dense material located under tribution of the size of all fibers analyzed from a cross the sarcolemma and between the myofibrils, generally section of tibialis anterior (Figure 2D) treated with AAV- continuous with the Z-lines (see arrowheads in Figure 3F, DesR406W is strongly shifted to the smaller sizes G,H). Some of these aggregates formed ‘ring-like’ (-20.6% of average size compared to WT desmin structures of a shape already seen in patients carrying the expressing fibers). We also observed this phenomenon, E413K desmin mutant (see asterisks in Figure 3G). As but to a lesser extent, with the muscles treated with seen in Figure 3G,H (see arrows), expression of E413K AAV-DesE413K (-3.7%). Focusing this analysis on the desmin also resulted in compressed sarcomeres with fibers expressing only exogenous desmin, we observed narrower Z-lines, presumably reflecting a loss of myofibril very similar results (-14.6% for R406W desmin anchorage. expressing fibers and -2.9% for E413K desmin expressing fibers, respectively). This observation emphasizes that Impaired muscle function muscle regeneration is not the sole mechanism respon- One of the most marked features of patients with MFM sible for the impaired distribution of muscle fiber size is the reduced maximal active force production of the observed in the muscles expressing R406W (Figure 2E). muscles affected by the disease. A good model of MFM must therefore reproduce this feature. We measured iso- Ultrastructural analysis indicates perturbations at Z-lines metric force produced by the tibialis anterior muscles in Using electron microscopy we examined more closely the response to nerve stimulation of mice previously ultrastructure of the tibialis anterior muscles expressing transduced (1 month before) with AAV-DesWT, AAV- WT or mutated desmin. All analyses were performed 1 DesR406W or AAV-DesE413K. These measures revealed month after injection of AAV vectors, with the exception a significant and strong decrease in specific maximal of R406W expressing muscles for which an additional ana- force, that is, maximal force generating capacity, for lysis was also performed 1 week after AAV injection. both R406W (-18.8%, P <0.001) and E413K (-18.1%, P Muscles expressing WT desmin showed no difference <0.001) desmin expressing muscles compared to WT compared to an untreated muscle. Electron microscopy (Figure 4A). It should be noted that we did not observe studies disclosed a normal ultrastructure of WT expres- any significant differences in specific maximal force be- sing muscles in terms of sarcomere organization, Z-line tween the muscle expressing WT desmin and untreated alignment, distribution and localization of mitochondria, muscle (data not shown). The reduced specific maximal and other organelles (Figure 3A). On the contrary, expres- force was not related to a significant increase in fibrosis, sion of the R406W desmin mutant resulted in the appear- that is, reduced contractile materials (Figure 4B). We ance of granulofilamentous electron-dense material (see also analyzed the absolute maximal force of a muscle arrowheads in Figure 3C,D,E) located in the perinuclear that is directly proportional to its specific maximal active regions (Figure 3B,C) and in the intermyofibrillar space force and weight. The reduction of absolute maximal (Figure 3D). Much of this material appeared to be force only observed for muscles expressing desmin associated with the Z-line and perturbation of the sarco- E413K (-24.2%, P <0.001) (Figure 4D) was due to a de- mere organization was observed (Figure 3D). It should be crease in specific maximal force since it appears that the noted that the phenomena was much more pronounced weight of the muscles expressing the E413K mutant near the nucleus (Figure 3C). In the perinuclear regions, were not significantly lower compared to WT (49.9 mg expression of the R406W mutant led to a complete aggre- versus 40.7 mg, P = 0.77) (Figure 4D). We also noticed gation of the Z-line and severe perturbation of sarcomere that the increased weight observed for the R406W group alignment, whereas, far away from the nucleus no ma- (Figure 4C) may be related to the previously observed jor alteration of sarcomere organization was detected hyperplasia, as seen in Figure 2B. (Figure 3B,D). As seen in the Figure 3D, accumulation of electron-dense material at the Z-line also perturbed mito- Discussion chondria localization. Additional ultrastructural analysis The main advantage of the strategy that we have showed that accumulation of electron-dense material presented is that it is easy to quickly obtain a large num- located in the perinuclear area was already detectable 1 ber of viral vectors allowing the expression of various week after AAV-DesR406W injection, but they were not desmin mutants and to study the effects of their Joanne et al. Skeletal Muscle 2013, 3:4 Page 9 of 13 http://www.skeletalmusclejournal.com/content/3/1/4 AB CD EF GH Figure 3 Ultrastructural perturbations induced by desmin mutants in tibialis anterior muscles. (A) WT, (B to E) R406W desmin, (F to H) E413K desmin; expressing tibialis anterior muscles, 1 week (E) or 1 month (A to D, F to H) after AAV vectors injection. Note that expression of WT desmin does not seem to modify muscle ultrastructure (A), whereas expression of R406W mutant of desmin induces the appearance of granulofilamentous electron-dense material (arrowheads, B to E) located at the perinuclear regions (B to C), between the intermyofibrillar space and at the Z-line (D); and perturbs mitochondria localization (D). Expression of E413K in the tibialis anterior muscle also induces accumulation of granulofilamentous electron-dense material (arrowheads, F to H) that may form ‘ring-like’ structures (asterisk, G). Arrows indicate compressed sarcomere by the accumulation of granulofilamentous electron-dense material. AAV, adeno-associated virus; M, mitochondria; N, nucleus; WT, wild-type. expression in one genetic background. The idea of creat- in patients with myotilin-related MFM. However, the ing a disease model by directly expressing a mutant in a authors have not performed any functional studies and given organ has already been performed in rodents for consequently cannot evaluate how their mutants affect several proteins [34-37]. Thus, by using electroporation, muscle physiology. Our results on muscle function a similar strategy was recently employed by Keduka reinforce the validity of this strategy since our model et al., to compare in vivo the effects of two mutations of reproduces the pathological features of the disease with myotilin, a protein involved in MFM [38]. They con- the major protein responsible for MFM: desmin. More- clude that this approach recapitulates the pathological over, compared to non-viral transfection techniques such changes and the biochemical characteristics observed as electroporation, this method significantly improves Joanne et al. Skeletal Muscle 2013, 3:4 Page 10 of 13 http://www.skeletalmusclejournal.com/content/3/1/4 AB CD Figure 4 Altered mechanical characteristics induced by desmin mutants in tibialis anterior muscles. (A) Muscle specific maximal active force, (B) muscle fibrosis, (C) muscle weight and (D) absolute maximal active force were examined in WT (white bars), R406W (light grey bars) and E413K (dark grey bars) desmin expressing tibialis anterior muscles 1 month after AAV vectors injection. Note decrease in muscle specific maximal active force of R406W and E413K expressing muscles compared to WT (A). Asterisks indicate a significant difference compared to the WT (*P ≤0.05, **P ≤0.01, ***P ≤0.001). AAV, adeno-associated virus; WT, wild-type. the percentage of transfected fibers [39,40], is easier to areas of the muscle fibers for the R406W mutation of implement in vivo and does not induce the confounding desmin; while for the E413K mutation, small and effect of muscle damage frequently observed with elec- dispersed deposits were located both in the center and troporation. In the present study, we obtained a very at the periphery of muscle fibers. These results are very homogeneous expression of the mutant desmin with similar to those observed on muscle biopsies of patients more than 98% of the muscle fibers being transduced [18-20]. In our model we also observe, for the mutation and expressing the transgene. AAV-mediated expression E413K, the typical aggregates with ‘ring-like’ structures of mutated desmin mimics MFM related disorders and are generally continuous with the Z-lines [20]. This ap- seems to be a good model to study molecular heterogen- proach seems to be adequate and recapitulates extremely eity of MFM pathogenesis. well the morphological modifications of muscles in the Despite the fact that we conducted our study on one context of MFM. muscle (tibialis anterior) at one time point (1 month), Our results also demonstrate that the R406W mutant we have demonstrated that intramuscular injection of induces hyperplasia and an increase in muscle mass that viral vectors to promote the expression of mutant is not accompanied by an increase in the absolute max- desmin is a potentially useful approach, since the results imal active force, as is supposed to be the case for a obtained in this study are very similar to the phenotypes healthy muscle since specific maximal force is reduced. observed in patients. In addition to the loss of muscle Each muscle fiber seems to develop less force. This is specific maximal force, we observe an accumulation of also true in the case of the E413K mutant. Two main granular aggregates in subsarcolemmal and perinuclear mechanisms may explain this decrease in specific Joanne et al. Skeletal Muscle 2013, 3:4 Page 11 of 13 http://www.skeletalmusclejournal.com/content/3/1/4 maximal force: a direct disturbance of the generation of the same molecular pattern, can induce the observed force by the sarcomere or an impaired transmission of differences in regeneration. Taken together, our results the force to the tendons. Our observations demonstrate confirm that AAV-mediated expression of mutants is a that the sarcomeres are locally disturbed where we ob- useful method to explore clinical phenotypic heteroge- serve aggregates of desmin mutants, suggesting that neity of a disease, such as MFM. mutations of desmin, by interfering directly with the Z-line, influence force production of muscle fibers. It is Additional file also possible that a disturbance in the alignment of the sarcomeres compared to the longitudinal axis of the Additional file 1: Figure S1. Z-lines perturbations induced by aggregation of desmin mutants in tibialis anterior muscles. (A) Merge of muscle (pennation angle) or changes in the extracellular (B) immunostaining against c-Myc and (C) immunostaining against matrix may be partly responsible for the decrease in spe- α-actinin (left part) or specific staining of actin using phalloidin (right cific maximal force by affecting the force transmission as part). The plotted graphics reveal the striation pattern of c-Myc (green), α-actinin or phalloidin (red). All analyses were performed 1 month after observed in desmin null mice [41,42]. These points re- intramuscular injection of AAV vectors. Note significant perturbations of main to be clarified to determine how much each mech- Z-line located in the areas where exogenous desmin accumulates. AAV, anism is affected by these mutations of desmin. adeno-associated virus. Finally, in the case of the R406W expressing muscles, we observed focal muscle regeneration, which was Abbreviations confirmed by the presence of immature muscle fibers AAV: Adeno-associated virus; CMV: Cytomegalovirus; DAPI: 4 ,6-diamidino-2- phenylindole; EDTA: Ethylenediaminetetraacetic acid; EGTA: ethylene glycol expressing the neonatal isoform of MHC. This mutation tetraacetic acid; Fab fragment: Antigen-binding fragment; HSD: Honestly of desmin also induces a decrease in the overall size of Significant Difference; IgG: Immunoglobulin G; MFM: Myofibrillar myopathy; fibers. These phenomena, observed 1 month after injec- MHC: Myosin heavy chain; PBS: Phosphate-buffered saline; PCR: Polymerase chain reaction; SDH: Succinate dehydrogenase; SEM: Standard error of the tion of the virus in tibialis anterior muscle, support the mean; TEM: Transmission electron microscope; WT: Wild-type. idea that the R406W mutation induces repeated cycles of de-/regeneration of muscle fibers. During regener- Competing interests ation, lost fibers are replaced by clusters of myotubes, The authors declare that they have no competing interests. formed by satellite cells, which looked like split fibers Authors’ contributions and have, therefore, a reduced size [43]. This may par- PJ carried out the histological, immunostaining and Western blot tially explain the hyperplasia. However, we emphasize experiments and analysis, performed the statistical analysis, participated in that muscle regeneration is not the sole mechanism re- the design of this study and drafted the manuscript. OC carried out the preparation, purification of the plasmids and site-directed mutagenesis. CH sponsible for the hyperplasia and impaired distribution carried out the production, purification and injection of the AAV vectors. AF of muscle fiber size observed in the muscles expressing carried out the muscle force measurements. PV participated in the design of R406W, since we also observed small fibers expressing the study and helped to draft the manuscript. JD and GBB participated in the design of the AAV vectors, titration of viral particles and helped to draft exogenous (c-Myc-positive) desmin. The strong regener- the manuscript. OA designed and coordinated the study, obtained the ation observed in tibialis anterior expressing the R406W funding, carried out the electron microscopy experiment, performed in vivo mutant of desmin may affect the long-term regenerative experiments and analysis and drafted the manuscript. All authors read and approved the final manuscript. capacities of muscles and could explain the disorders observed in MFM. In contrast, the weaker regeneration of Acknowledgements muscles expressing the E413K desmin mutant could ex- This work was supported by the Association Française contre les Myopathies plain the longer asymptomatic period observed in patients. (AFM) contract numbers 14040 and 15454, and the University Paris Diderot, Paris 7, France. OC was supported by fellowships from the Ministère de la Recherche et de la Technologie (MRT) and the AFM. We would like to thank Conclusions Dr Zhenlin Li and Professor Denise Paulin for their helpful advice; Alexis In this study, we show that AAV-mediated expression of Canette from the imaging facility ImagoSeine at the Jacques Monod Institute, Paris, France, for his work in electron microscopy studies; Solenne desmin mutants in mouse muscles recapitulate the ag- Marie for technical assistance during AAV vectors preparation; and gregation features, the contractile function decrease and Dr Philippe Noirez for developing imageJ software macros. the morphological change observed in MFM patients. In Author details somewhat more detail, differences in muscle regener- Université Paris Diderot, Sorbonne Paris Cité, CNRS EAC4413, Unit of ation, distribution of muscle fiber size and muscle force Functional and Adaptive Biology, Laboratory of Stress and Pathologies of the production exist between the R406W and E413K desmin Cytoskeleton, 75013, Paris, France. Department of Aging, Stress and Inflammation, Université Pierre et Marie Curie-Paris 6, Sorbonne Universités, mutants. Above all, our results suggest that the R406W 75005, Paris, France. Université Pierre et Marie Curie-Paris 6, Sorbonne mutant of desmin induced a strong muscle regeneration, Universités, UMR S794, INSERM U974, CNRS UMR7215, Institut de Myologie, which is not the case of the E413K mutant. This diffe- 75013, Paris, France. Université Paris Descartes, Sorbonne Paris Cité, 75006, Paris, France. rence could be at the origin of phenotypic differences observed in patients. It could be interesting, in the Received: 19 October 2012 Accepted: 28 January 2013 future, to understand how these mutations, which affect Published: 20 February 2013 Joanne et al. Skeletal Muscle 2013, 3:4 Page 12 of 13 http://www.skeletalmusclejournal.com/content/3/1/4 References 21. Chourbagi O, Bruston F, Carinci M, Xue Z, Vicart P, Paulin D, Agbulut O: 1. 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Viral-mediated expression of desmin mutants to create mouse models of myofibrillar myopathy

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Life Sciences; Cell Biology; Developmental Biology; Biochemistry, general; Systems Biology; Biotechnology
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Abstract

Background: The clinical features of myofibrillar myopathies display a wide phenotypic heterogeneity. To this date, no studies have evaluated this parameter due to the absence of pertinent animal models. By studying two mutants of desmin, which induce subtle phenotypic differences in patients, we address this issue using an animal model based on the use of adeno-associated virus (AAV) vectors carrying mutated desmin cDNA. Methods: After preparation of the vectors, they were injected directly into the tibialis anterior muscles of C57BL/6 mice to allow expression of wild-type (WT) or mutated (R406W or E413K) desmin. Measurements of maximal force were carried out on the muscle in situ and then the injected muscles were analyzed to determine the structural consequences of the desmin mutations on muscle structure (microscopic observations, histology and immunohistochemistry). Results: Injection of AAV carrying WT desmin results in the expression of exogenous desmin in 98% of the muscle fibers without any pathological or functional perturbations. Exogenous WT and endogenous desmin are co-localized and no differences were observed compared to non-injected muscle. Expression of desmin mutants in mouse muscles induce morphological changes of muscle fibers (irregular shape and size) and the appearance of desmin accumulations around the nuclei (for R406W) or in subsarcolemmal regions of fibers (for E413K). These accumulations seem to occur and disrupt the Z-line, and a strong regeneration was observed in muscle expressing the R406W desmin, which is not the case for E413K. Moreover, both mutants of desmin studied here induce a decrease in muscle force generation capacity. Conclusions: In this study we show that AAV-mediated expression of desmin mutants in mouse muscles recapitulate the aggregation features, the decrease in contractile function and the morphological changes observed in patients with myofibrillar myopathy. More importantly, our results suggest that the R406W desmin mutant induces a robust muscle regeneration, which is not the case for the E413K mutant. This difference could help to explain the phenotypic differences observed in patients. Our results highlight the heterogeneous pathogenic mechanisms between different desmin mutants and open the way for new advances in the study of myofibrillar myopathies. Keywords: Myofibrillar myopathy, Intermediate filament, Desmin, AAV vectors, Phenotypic heterogeneity, Desminopathy * Correspondence: onnik.agbulut@univ-paris-diderot.fr Equal contributors Université Paris Diderot, Sorbonne Paris Cité, CNRS EAC4413, Unit of Functional and Adaptive Biology, Laboratory of Stress and Pathologies of the Cytoskeleton, 75013, Paris, France Full list of author information is available at the end of the article © 2013 Joanne 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. Joanne et al. Skeletal Muscle 2013, 3:4 Page 2 of 13 http://www.skeletalmusclejournal.com/content/3/1/4 Background severe with a high risk of heart failure. The E413K muta- Myofibrillar myopathy (MFM) [OMIM:601419] refers to tion was first described by Pruszczyk et al. in 2007, as a a group of genetically heterogeneous chronic neuromus- disease primarily affecting cardiac function with an cular disorders. MFMs are mainly caused by mutations autosomal dominant mode of inheritance [20]. Three in the desmin gene [1], while other forms of MFM are cases from the same family were described. The first caused by mutations in alpha-B-crystallin [2], myotilin symptoms of heart disease developed later than those of [3], ZASP [4], filamin C [5] or BAG3 [6] genes. Rigid the R406W mutation (age of onset at 30 years versus 20 spine syndrome caused by mutation in the SEPN1 gene years). The first signs of muscle weakness appeared is also considered as a MFM [7]. The morphological about 15 years after the first cardiac symptoms and were changes described in MFM result from disturbance of restricted to the lower limbs. Therefore, from these the sarcomeric Z-line and the myofibrils, followed by ab- reports it would appear that the R406W mutation is normal aggregation of proteins predominantly involved more pathogenic than the E413K mutation, since the in the structure of the Z-line. The age of disease onset clinical symptoms develop later and the skeletal muscles can vary from early adolescence to the 60s. The disease are less affected. However, the molecular mechanism is very heterogeneous manifesting in some cases as a underlying these differences in the clinical phenotypes relentlessly progressive skeletal myopathy with no signs cannot be studied with the cellular models that are cur- of cardiac involvement [8,9]. In other cases cardiomyop- rently available, since both desmin mutations R406W athy is a prominent [1] or even exclusive [10] feature. and E413K present the same features. Both are unable to Respiratory insufficiency may also be a major manifest- form filamentous networks on their own, but are able to ation and cause of death [11]. integrate into pre-existing desmin networks and form In 1998, Goldfarb and his co-workers [1] established aggregates, which are concentrated around the nuclei that a mutation in the desmin gene was involved in and throughout the cytoplasm [21]. Several teams, MFM. Since then, more than 50 different mutations re- including ours, have demonstrated that R406W and sponsible for MFM symptoms have been identified in E413K mutations, located in the terminal consensus the desmin gene. Desmin is the major specific inter- motif, alter in a similar manner the dimer interaction, mediate filament protein of skeletal, cardiac and smooth which impairs the filament formation [20-22]. These muscles. It forms a three-dimensional scaffold around observations clearly demonstrate that neither in silico the myofibrillar Z-disc, and connects the contractile ap- nor cellular models are able to explain the molecular paratus and other structural elements of the cell, that is, mechanisms which result in the phenotypic heteroge- the subsarcolemmal cytoskeleton, the nuclei and other neity observed in patients carrying these mutations. organelles [12,13]. It also links the myofibrils laterally by To try to address this issue, several transgenic mouse connecting the Z-discs, and is particularly abundant at the models expressing different mutations of desmin have myotendinous and neuromuscular junctions of skeletal been obtained [23-25]. However, the different promoters muscle and in the intercalated disks of cardiomyocytes and strains used make the comparison quite difficult. [14]. Thus, desmin filaments are essential to maintain cel- Moreover, conventional techniques of transgenesis do lular integrity, for the transmission of force and the not seem compatible to analyze the large number of mechanochemical signaling of muscle cells [15-17]. mutations which have been identified within the desmin In the present study, we examined two missense gene. We therefore chose to use a simpler strategy to desmin mutations, R406W and E413K. A patient with a study, in the same genetic background, different domi- R406W mutation in desmin was first described by Park nant mutations of desmin. In order to develop these et al. in 2000 [18]. In 2004, three new cases were models, we produced an adeno-associated virus (AAV) described and compared [19]. From this study it became vector that allows the expression of a mutant desmin in muscles. Intramuscular injection of this virus allows the apparent that patients with the R406W mutation have many common features. These four patients were of dif- localized transgenic expression of desmin mutants in ferent nationalities and no other member of their fam- C57BL/6 mice. Different analyses were carried out first on the muscle in situ (measurement of muscular force) and ilies was affected by the disease (sporadic de novo mode of inheritance). The first signs of the disease appeared at then the injected muscles were analyzed to determine the around 20 years of age and were characterized by severe structural consequences of the desmin mutations on muscle structure (microscopic observations, histology and cardiac involvement (cardiac arrhythmia and complete atrioventricular block resulting in pacemaker implant- immunohistochemistry). ation). The disease developed with increasing muscle It should be noted that studies on MFM are inherently weakness of the lower limbs. This weakness then limited because of the small number of patients and very extended to the muscles of the upper limbs and the limited access to biopsy. In addition, this strategy of cre- neck. The final outcome of the disease is usually very ating an in vivo model appears to be very interesting Joanne et al. Skeletal Muscle 2013, 3:4 Page 3 of 13 http://www.skeletalmusclejournal.com/content/3/1/4 because of its ease and rapidity of obtaining, and the low determined by real-time PCR (TaqMan, Life Tech- cost associated with its implementation. Using this nologies) using CMV promoter specific primers 0 0 model we aim to reproduce in vivo the phenotypic vari- (forward: 5 -CTACGCCCATTTGCGTCAA-3;re- 0 0 ability observed in patients in order to better understand verse: 5 -GCACCAAAATCAACGGGAC-3;probe: 0 0 the mechanisms behind these variations. 5 -CAAAATGTCGTAACAACTCCGCCCC-3 ). Methods Intramuscular delivery of AAV vectors Plasmids production All procedures have been approved by our institutional The full-length human desmin cDNAs were cloned into Ethics Committee, and conducted according to the pSMD2 plasmid (cytomegalovirus (CMV) promoter and French and European laws, directives and regulations on human β-globin pA) using Xho I restriction site. To dis- animal care (European Commission Directive 86/609/ tinguish the desmin transgene from the endogenous EEC). Our animal facility is fully licensed by the French form, a c-Myc tag was introduced at the 5 end of the competent authorities and has animal welfare insurance. desmin cDNA. It should be noted that the presence of For intramuscular injection, 35 15-week-old female c-Myc at the N-terminal of desmin does not disturb fila- C57BL/6 mice were randomized into four groups: PBS ment assembly or cellular localization [21]. R406W and (n = 6), AAV-DesWT (n = 11), AAV-DesR406W (n = E413K desmin mutations were introduced into the 10) and AAV-DesE413K (n = 8). Animals were pSMD2 plasmid by site-directed mutagenesis using the anesthetized by intraperitoneal injection of pentobarbital Stratagene QuikChange kit (Agilent Technologies, sodium (60 mg/kg). Each tibialis anterior muscle was Massy, France). Escherichia coli Stbl2 strain (Life Tech- injected with 50 μl of either AAV-2/1-Des (10 viral nologies, Saint Aubin, France) was transformed with the genomes/muscle) or sterile PBS solution as a control. different plasmids according to the manufacturer’s instructions for amplification. All plasmids were purified using the endotoxin-free PureYield Plasmid Maxiprep Muscle force measurements System (Promega, Lyon, France), and then verified by Four weeks after injection, mice were anesthetized restriction enzyme digestion and sequencing (Eurofins (pentobarbital sodium, 60 mg/kg) and the limbs were MWF Operon, Ebersberg, Germany). fixed with clamps. The distal tendon of the tibialis anter- ior muscle was attached to a dual-mode lever arm sys- Production, purification and titration of the AAV vectors tem that measures muscle isometric force (300C; Aurora Pseudotyped AAV-2/1 vectors were generated in human Scientific, Ontario, Canada). Great care was taken to en- embryonic kidney 293 cells by the triple transfection sure that the blood and nerve supply remained intact method described by Rivière et al., with minor mo- during surgery. Active force measurements were difications [26]. The productions were realized with performed as described previously [27-29]. The sciatic pXX6 adenovirus helper plasmid coding the adenoviral nerve was crushed proximally and stimulated distally by sequences essential for AAV production, the pRep-Cap a bipolar silver electrode using supramaximal square plasmid coding the AAV-1 capsid and the pSMD2- wave pulses of 0.1 ms duration. All isometric mea- DesWT, pSMD2-DesR406W or pSMD2-DesE413K plas- surements were made at L0 (muscle length at which mid coding the different desmin transgenes. After triple maximal force was obtained during the twitch). Force transfection of the different plasmids in 293 cells, cells production in response to tetanic stimulation were suc- were harvested and submitted to three freeze-thaw cessively recorded (pulse frequency from 25, 50 and 100 cycles using a 37°C water bath and dry ice cooled etha- to 143 Hz, 500 ms) and at least 1 minute was allowed nol. The lysate was incubated with Benzonase (50 U/ml; between each contraction. The absolute maximal force Sigma-Aldrich, Saint-Quentin Fallavier, France) for 30 was normalized to the muscle mass to determine specific minutes at 37°C. It was then clarified by centrifugation maximal force. at 6,500 rpm for 20 minutes and passed though a 0.45 μm filter. The lysate was layered over the top of an At the end of the experiments, the animals were iodixanol gradient solution (from 15% to 60%; Sigma- euthanized with an overdose of pentobarbital. After con- Aldrich) and centrifuged for 90 minutes at 59,000 rpm. tractile measurements, the muscles were dissected, The virus was isolated between the 40% and 60% weighed and frozen in isopentane pre-cooled in liquid iodixanol solutions, washed with PBS-MK (PBS, nitrogen or fixed in paraformaldehyde for further ana- MgCl 1 mM, KCl 2.5 mM) solution and concentrated lysis. Force measurements were made in PBS treated on Amicon Ultra-15 centrifugal filter (Millipore, (n = 12) or WT (n = 22), R406W (n = 20) and E413K Molsheim, France). The final viral preparations were (n = 15) desmin expressing tibialis anterior muscles stored in PBS solution at -80°C. The AAV titer was 1 month after AAV vectors injection. Joanne et al. Skeletal Muscle 2013, 3:4 Page 4 of 13 http://www.skeletalmusclejournal.com/content/3/1/4 Histological staining After washing, the specimens were post-fixed for 1 hour Transversal frozen sections of 10 μm thickness were with 1% osmium tetroxide solution, dehydrated and prepared from all of the muscles. Hematoxylin and eosin embedded in epoxy resin. Ultrathin sections (70 nm) staining was used to examine general morphology of the were cut with an ultramicrotome (Leica UC6; Leica muscles. The muscle mitochondrial distribution and Microsystems) and stained for 15 minutes with 4% uranyl localization was examined using succinate dehydrogen- acetate and for 2 minutes with Reynolds’ lead citrate, be- ase (SDH) staining. The extent of fibrosis was assessed by fore observation at 80 kV with a transmission electron Sirius red staining and was expressed as the fibrosis index microscope (TEM) Philips Tecnai 12 BioTWIN (Philips, (ratio of the area of the fibrosis to the area of fibers). All Amsterdam, Netherlands) equipped with an Olympus staining methods were carried out as described by KeenView CCD camera (Shinjuku, Tokyo, Japan). Dubowitz [30]. Images were taken with a microscope (Leica Microsystems, Nanterre, France) equipped with a Western blot digital camera (QImaging, Surrey, Canada). For fibrosis Immunoblotting was carried out on extracts of muscles analysis, nine digital images per muscle (n = 5/group) were snap frozen in liquid nitrogen immediately after dissec- processed with ImageJ software (National Institutes of tion. Frozen muscles were placed into an ice-cold Health, Bethesda, MD, USA) [31]. homogenization buffer containing: 50 mM Tris (pH 7.6), 250 mM NaCl, 3 mM ethylenediaminetetraacetic acid Immunofluorescence (EDTA), 3 mM ethylene glycol tetraacetic acid (EGTA), Transversal (morphometric analysis) and longitudinal 0.5% NP40, 2 mM dithiothreitol, 10 mM sodium (striation pattern analysis) frozen sections of 8 μm thick- orthovanadate, 10 mM NaF, 10 mM glycerophosphate ness were cut using a microtome (Leica Microsystems) and 2% of protease inhibitor cocktail (Sigma-Aldrich). for immunostaining. The sections were incubated with Samples were minced with scissors and then blocking solution (bovine serum albumin, 5%) for 1 hour homogenized using plastic pestles, incubated for 30 and then incubated for 30 minutes with goat anti-mouse minutes on ice, sonicated three times for 5 seconds with immunoglobulin G (IgG) Fab fragment (1:100; Jackson 30 second intervals on ice, and then centrifuged at ImmunoResearch Europe, Newmarket, UK). Following a 12,000 g for 30 minutes at 4°C. Protein concentration PBS wash, the sections were incubated for 90 minutes was measured using the Bradford method with bovine with primary antibodies against perlecan (1:400, rat serum albumin as a standard. Equal amounts of protein monoclonal; Millipore), c-Myc (1:1000, rabbit polyclonal; extracts (25 μg) were separated by SDS-PAGE before Sigma-Aldrich), α-actinin (1:100, mouse monoclonal; electrophoretic transfer onto a nitrocellulose membrane Sigma-Aldrich) or myosin heavy chain (MHC) isoform (Amersham Hybond-ECL; GE Healthcare, Vélizy- neonatal (1:100, rabbit polyclonal) [32]. After washing Villacoublay, France). Western blot analysis was carried in PBS, sections were incubated for 1 hour with second- out using anti-c-Myc antibody (1:1,000, mouse monoclo- ary antibodies (Alexa Fluor; Life Technologies) or nal; Santa Cruz Biotechnology, Heidelberg, Germany) phalloidin-TRITC labeled (Sigma-Aldrich). After washing and anti-pan-actin antibody (1:10,000, mouse monoclo- in PBS, slides were finally mounted in Vectashield with nal; Millipore). Antibody reacting bands were visua- DAPI H-1200 (Vector Laboratories, Peterborough, UK). lized with peroxidase-conjugated secondary antibodies Images were captured using a motorized confocal laser (Thermo-Fisher Scientific, Brebières, France) and a scanning microscope (LSM 700; Carl Zeiss SAS, Le Pecq, chemiluminescent detection system (ECL-Plus; GE France). Morphometric analyses were made using the Healthcare). ImageJ [31] software and a homemade macro. The smallest diameter (minimum Feret diameter) of all the muscle fibers of the whole muscle section was measured. The pattern of striations was analyzed on longitudinal Statistical analysis muscle sections using the Plot Profile function of ImageJ Groups were statistically compared using analysis of [31] software. variance (ANOVA). If necessary, post-hoc analysis was performed using Tukey’s Honestly Significant Difference Electron microscopy (HSD) test. For groups that did not pass tests of normal- Electron microscopy was carried out as described previ- ity (Shapiro-Wilk) and equal variance (Bartlett), non- ously [33]. Briefly, the calf muscles of mice were fixed in parametric tests were used (Kruskal-Wallis) and multiple 2.5% glutaraldehyde buffered in 0.1 M cacodylate at pH comparison were driven using the Dunn procedure and 7.4. After 1 hour, the tibialis anterior muscle was multivariate normal distribution in R software. For stat- dissected and separated in three by a short-axis section istical analysis of fiber size distribution, Kolmogorov- and then fixed overnight at 4°C in the same fixative. Smirnov test was used. Values are means ±SEM. Joanne et al. Skeletal Muscle 2013, 3:4 Page 5 of 13 http://www.skeletalmusclejournal.com/content/3/1/4 Results (Figure 1A, WT) had no effect on muscle structure, Morphological modifications of muscles expressing which was identical to an untreated muscle. On the con- mutated desmin recapitulate MFM trary, expression of the R406W mutant of desmin Since the diagnosis of MFM is most often made by using induced the appearance of darkly stained deposits the results obtained from histological stains of muscle located mainly in the perinuclear regions of the muscle sections, we first conducted a series of histological stains fiber (see arrowhead in Figure 1A, R406W). We also saw of mouse muscles expressing WT or mutated (R406W myofibers with central nuclei indicating that the muscle or E413K) desmin 1 month after injection of the differ- fiber is in the process of regeneration (see asterisks in ent AAV constructions (AAV-DesWT, AAV-DesR406W Figure 1A, R406W) or the nuclei are moving from a per- and AAV-DesE413K) in the tibialis anterior muscle of ipheral to a central location. Expression of E413K mu- C57BL/6 mice (Figure 1A). Normal myofibers with a tant of desmin provoked a similar phenotype but with polygonal shape, peripheral nuclei, intact sarcolemma, more blurred dark deposits irradiating from both the non-fragmented sarcoplasm and homogeneous fiber size subsarcolemmal and perinuclear regions (see arrowhead distribution revealed that overexpression of WT desmin in Figure 1A, E413K) of the muscle fibers. In addition, Figure 1 Morphological perturbations induced by desmin mutants in tibialis anterior muscles. (A) Hematoxylin and eosin staining, (B) succinate dehydrogenase (SDH) staining, (C) immunostaining against c-Myc (green) and perlecan (red), and (D) immunostaining against c-Myc (green); on serial transversal (A, B and C) and longitudinal (D) sections of tibialis anterior muscles expressing WT, R406W or E413K desmin. All analyses were performed 1 month after intramuscular injection of AAV vectors. Note that expression of WT desmin does not seem to modify neither muscle morphology (A to B) nor desmin localization (C to D), whereas expression of R406W and E413K desmin mutants induces accumulation of the desmin in the perinuclear region (see arrowheads). Arrows in (B) illustrate abnormal accumulation of mitochondria observed in both R406W and E413K expressed tibialis anterior muscles. Arrow in (C) illustrates a weaker accumulation of desmin in the case of E413K desmin. Asterisks indicate centronucleated muscle fibers. Nuclei (blue) were stained using DAPI (C to D). Scale bars = 30 μm. AAV, adeno- associated virus; DAPI, 40,6-diamidino-2-phenylindole; SDH, succinate dehydrogenase; WT, wild-type. Joanne et al. Skeletal Muscle 2013, 3:4 Page 6 of 13 http://www.skeletalmusclejournal.com/content/3/1/4 many muscle fibers were irregular in shape in muscles to be pushed towards the subsarcolemmal regions expressing the mutated desmin. (see arrow, Figure 1C, E413K). This observation In a second step, we performed SDH staining on serial corresponded well with the central accumulation of sections to explore the distribution of muscle mitochon- staining seen on SDH staining. We also observed, al- dria (Figure 1B). Muscles treated with the virus though to a lesser extent than for R406W, accumulation expressing WT desmin showed a mosaic of fibers with a in perinuclear regions of the fibers (see arrowhead, relatively dense purple appearance in oxidative fibers Figure 1C, E413K). These features were also observed in and scattered purple speckles in the nonoxidative fibers. the longitudinal sections where the R406W desmin These observations are in perfect accordance to a nor- seemed to strongly accumulate around the nuclei while mal muscle. In contrast, the muscles expressing the E413K desmin formed numerous dots occupying the en- R406W mutant showed a localized absence of staining tire width of the fibers (Figure 1D). Moreover, the align- in perinuclear regions (see arrowhead in Figure 1B, ment of the sarcomeres seemed to be disturbed in areas R406W) that correspond to the accumulation of dark where R406W or E413K desmin were accumulated material revealed by hematoxylin and eosin staining (Figure 1D). A closer analysis of the Z-lines (α-actinin (Figure 1A). An irregular staining of some fibers and in immunostaining) and thin filaments (phalloidin staining) some cases a strong accumulation of staining in the cen- demonstrated a very clear and localized perturbation of ter of fibers (see arrow in Figure 1B, R406W) were also the alignment at the zones of accumulation of desmin observed. The muscles expressing the E413K desmin (Additional file 1: Figure S1). As seen in Additional mutant also presented many fibers with this abnormal file 1: Figure S1, c-Myc was co-localized with α-actinin internal architecture characterized by a central dark in both WT and mutated desmin expressing muscles; staining, indicating a modified internal repartition of whereas actin, the major component of the I-band, was mitochondria (see arrow in Figure 1B, E413K). In not localized with c-Myc. It should be noted that the addition, we also observed a decrease in the staining of alignment of Z-lines did not seem to be disrupted at the subsarcolemmal and perinuclear regions (see arrowhead sites where desmin was not drastically accumulated, as in Figure 1B, E413K), which also corresponded to the ac- seen in Figure 1D. cumulation revealed by hematoxylin and eosin staining (Figure 1A). Aberrant muscle regeneration and impaired distribution of muscle fiber size Aberrant aggregation of desmin As shown in Figure 1, many muscle fibers expressing Next, we performed a series of immunostaining to detect R406W mutant are centronucleated (see asterisks in the expression and localization of desmin (Figure 1C,D). Figure 1A,C, R406W) suggesting that there is an import- To distinguish the desmin transgene from endogenous ant muscle regeneration. If this phenomenon really occurs, desmin, a c-Myc tag was introduced at the 5 end of the the absence of c-Myc immunostaining in these centro- desmin cDNA. On transversal sections desmin appeared nucleated fibers could be explained by the fact that AAV homogeneously distributed throughout the cytoplasm has never been described to transduce satellite cells. (Figure 1C, WT). On longitudinal sections it was also cor- To confirm that there is an increase of regeneration in rectly and repeatedly aligned like sarcomeres (Figure 1D, muscles expressing mutant desmin, we measured the WT). It should be noted that immunostaining against number of neonatal MHC-positive fibers reported to the desmin on AAV-DesWT treated tibialis anterior showed total number of fibers from tibialis anterior sections no differences compared to untreated control muscles (Figure 2A). For muscles expressing WT desmin, we (data not shown). Furthermore, we observed that there were not able to detect any neonatal MHC-positive were also no observable differences between the fibers. In the case of muscles expressing R406W or immunostaining against c-Myc and that against desmin in E413K desmin there was, however, an increasing number WT expressing tibialis anterior muscle (data not shown), of fibers expressing this marker (Figure 2A). This ratio which confirmed perfect integration of exogenous desmin was larger and significantly different from WT in the into the normal desmin network. case of R406W desmin (20.5%, P <0.01) compared to As seen in Figure 1C,D, compared to immunostaining E413K desmin (5.5%, P >0.05). against c-Myc in WT expressing muscles, we showed To confirm that all of these modifications were not significant differences in muscles expressing the R406W due to a difference in the expression levels of exogenous and E413K desmin mutants. In these muscles the mu- desmin, we performed a Western blot with total protein tant desmin was strongly accumulated around the nuclei extract from AAV-treated muscles. We detected a for the R406W desmin (see arrowhead, Figure 1C, similar amount of exogenous desmin between WT R406W). For desmin E413K we observed a much weaker and E413K expressing muscles, whereas in R406W labeling in the center of the fibers and desmin seems expressing muscles we observed a decrease in the Joanne et al. Skeletal Muscle 2013, 3:4 Page 7 of 13 http://www.skeletalmusclejournal.com/content/3/1/4 A D Figure 2 Regeneration, hyperplasia and decrease in overall fiber size of R406W desmin expressing muscles compared to E413K expressing muscles. (A) Percentage of neonatal myosin heavy chain (MHC) expressing muscle fibers, (B) total number (dots filled bars) and c-Myc expressing (empty bar) muscle fibers, (C) Western blot quantification of c-Myc, (D) size distribution of total and (E) c-Myc expressing muscle fibers; were examined in WT (n = 3), R406W (n = 3) and E413K (n = 3) desmin expressing tibialis anterior muscles 1 month after AAV vectors injection. For all morphometric analysis, all muscle fibers in whole muscle sections were analyzed. Note the strong increase in regeneration of tibialis anterior expressing R406W desmin compared to the slight increase with E413K desmin (A). Note also hyperplasia of R406W desmin expressing muscle demonstrated after counting the exogenous desmin expressing (Myc-positive, empty bars) and the total number of fibers (dots filled bars) (B). To compare size distribution of total (D) and exogenous desmin expressing (Myc-positive) (E) muscle fibers of WT, R406W or E413K desmin muscles, the Gaussian curve was plotted and grouped. Note the overall decrease in the size of both total and exogenous desmin expressing muscle fibers in R406W muscles, and, to a much lesser extent, the decrease in the size of fibers expressing desmin E413K. Asterisks indicate a significant difference compared to the WT (*P ≤0.05, **P ≤0.01, ***P ≤0.001). AAV, adeno-associated virus; MHC, myosin heavy chain; WT, wild-type. amount of exogenous desmin (Figure 2C). We also between the muscles expressing WT (2721±291 fibers) counted and determined the c-Myc expression of each and those expressing the R406W desmin (2163±212 muscle fiber of the entire muscle section. Interestingly, fibers) or E413K (2404±68 fibers). In contrast, compared after having counted the number of c-Myc-positive to the WT (2779±265 fibers), the total number of fibers muscle fibers for each muscle (Figure 2B), we observed increased significantly in the case of muscles expressing that there was no significant (P = 0.252) difference R406W desmin mutant (4546±313 fibers, P = 0.0036), Joanne et al. Skeletal Muscle 2013, 3:4 Page 8 of 13 http://www.skeletalmusclejournal.com/content/3/1/4 which was not the case for muscles expressing E413K yet located at the Z-line (see arrowheads in Figure 3E). (2684±48 fibers, P >0.05). This increase in the total num- These results emphasize that the R406W desmin aggre- ber of muscle fibers in R406W expressing muscles may gates are generated at the perinuclear area and then they also explain the relative decrease in the amount of exogen- spread throughout the whole muscle. ous desmin seen on Western blot because newly generated In E413K expressing muscles, 1 month after AAV- muscle fibers cannot express exogenous desmin. DesE413K injection, ultrastructural examination revealed As expected for a regenerating muscle, the overall dis- granulofilamentous electron-dense material located under tribution of the size of all fibers analyzed from a cross the sarcolemma and between the myofibrils, generally section of tibialis anterior (Figure 2D) treated with AAV- continuous with the Z-lines (see arrowheads in Figure 3F, DesR406W is strongly shifted to the smaller sizes G,H). Some of these aggregates formed ‘ring-like’ (-20.6% of average size compared to WT desmin structures of a shape already seen in patients carrying the expressing fibers). We also observed this phenomenon, E413K desmin mutant (see asterisks in Figure 3G). As but to a lesser extent, with the muscles treated with seen in Figure 3G,H (see arrows), expression of E413K AAV-DesE413K (-3.7%). Focusing this analysis on the desmin also resulted in compressed sarcomeres with fibers expressing only exogenous desmin, we observed narrower Z-lines, presumably reflecting a loss of myofibril very similar results (-14.6% for R406W desmin anchorage. expressing fibers and -2.9% for E413K desmin expressing fibers, respectively). This observation emphasizes that Impaired muscle function muscle regeneration is not the sole mechanism respon- One of the most marked features of patients with MFM sible for the impaired distribution of muscle fiber size is the reduced maximal active force production of the observed in the muscles expressing R406W (Figure 2E). muscles affected by the disease. A good model of MFM must therefore reproduce this feature. We measured iso- Ultrastructural analysis indicates perturbations at Z-lines metric force produced by the tibialis anterior muscles in Using electron microscopy we examined more closely the response to nerve stimulation of mice previously ultrastructure of the tibialis anterior muscles expressing transduced (1 month before) with AAV-DesWT, AAV- WT or mutated desmin. All analyses were performed 1 DesR406W or AAV-DesE413K. These measures revealed month after injection of AAV vectors, with the exception a significant and strong decrease in specific maximal of R406W expressing muscles for which an additional ana- force, that is, maximal force generating capacity, for lysis was also performed 1 week after AAV injection. both R406W (-18.8%, P <0.001) and E413K (-18.1%, P Muscles expressing WT desmin showed no difference <0.001) desmin expressing muscles compared to WT compared to an untreated muscle. Electron microscopy (Figure 4A). It should be noted that we did not observe studies disclosed a normal ultrastructure of WT expres- any significant differences in specific maximal force be- sing muscles in terms of sarcomere organization, Z-line tween the muscle expressing WT desmin and untreated alignment, distribution and localization of mitochondria, muscle (data not shown). The reduced specific maximal and other organelles (Figure 3A). On the contrary, expres- force was not related to a significant increase in fibrosis, sion of the R406W desmin mutant resulted in the appear- that is, reduced contractile materials (Figure 4B). We ance of granulofilamentous electron-dense material (see also analyzed the absolute maximal force of a muscle arrowheads in Figure 3C,D,E) located in the perinuclear that is directly proportional to its specific maximal active regions (Figure 3B,C) and in the intermyofibrillar space force and weight. The reduction of absolute maximal (Figure 3D). Much of this material appeared to be force only observed for muscles expressing desmin associated with the Z-line and perturbation of the sarco- E413K (-24.2%, P <0.001) (Figure 4D) was due to a de- mere organization was observed (Figure 3D). It should be crease in specific maximal force since it appears that the noted that the phenomena was much more pronounced weight of the muscles expressing the E413K mutant near the nucleus (Figure 3C). In the perinuclear regions, were not significantly lower compared to WT (49.9 mg expression of the R406W mutant led to a complete aggre- versus 40.7 mg, P = 0.77) (Figure 4D). We also noticed gation of the Z-line and severe perturbation of sarcomere that the increased weight observed for the R406W group alignment, whereas, far away from the nucleus no ma- (Figure 4C) may be related to the previously observed jor alteration of sarcomere organization was detected hyperplasia, as seen in Figure 2B. (Figure 3B,D). As seen in the Figure 3D, accumulation of electron-dense material at the Z-line also perturbed mito- Discussion chondria localization. Additional ultrastructural analysis The main advantage of the strategy that we have showed that accumulation of electron-dense material presented is that it is easy to quickly obtain a large num- located in the perinuclear area was already detectable 1 ber of viral vectors allowing the expression of various week after AAV-DesR406W injection, but they were not desmin mutants and to study the effects of their Joanne et al. Skeletal Muscle 2013, 3:4 Page 9 of 13 http://www.skeletalmusclejournal.com/content/3/1/4 AB CD EF GH Figure 3 Ultrastructural perturbations induced by desmin mutants in tibialis anterior muscles. (A) WT, (B to E) R406W desmin, (F to H) E413K desmin; expressing tibialis anterior muscles, 1 week (E) or 1 month (A to D, F to H) after AAV vectors injection. Note that expression of WT desmin does not seem to modify muscle ultrastructure (A), whereas expression of R406W mutant of desmin induces the appearance of granulofilamentous electron-dense material (arrowheads, B to E) located at the perinuclear regions (B to C), between the intermyofibrillar space and at the Z-line (D); and perturbs mitochondria localization (D). Expression of E413K in the tibialis anterior muscle also induces accumulation of granulofilamentous electron-dense material (arrowheads, F to H) that may form ‘ring-like’ structures (asterisk, G). Arrows indicate compressed sarcomere by the accumulation of granulofilamentous electron-dense material. AAV, adeno-associated virus; M, mitochondria; N, nucleus; WT, wild-type. expression in one genetic background. The idea of creat- in patients with myotilin-related MFM. However, the ing a disease model by directly expressing a mutant in a authors have not performed any functional studies and given organ has already been performed in rodents for consequently cannot evaluate how their mutants affect several proteins [34-37]. Thus, by using electroporation, muscle physiology. Our results on muscle function a similar strategy was recently employed by Keduka reinforce the validity of this strategy since our model et al., to compare in vivo the effects of two mutations of reproduces the pathological features of the disease with myotilin, a protein involved in MFM [38]. They con- the major protein responsible for MFM: desmin. More- clude that this approach recapitulates the pathological over, compared to non-viral transfection techniques such changes and the biochemical characteristics observed as electroporation, this method significantly improves Joanne et al. Skeletal Muscle 2013, 3:4 Page 10 of 13 http://www.skeletalmusclejournal.com/content/3/1/4 AB CD Figure 4 Altered mechanical characteristics induced by desmin mutants in tibialis anterior muscles. (A) Muscle specific maximal active force, (B) muscle fibrosis, (C) muscle weight and (D) absolute maximal active force were examined in WT (white bars), R406W (light grey bars) and E413K (dark grey bars) desmin expressing tibialis anterior muscles 1 month after AAV vectors injection. Note decrease in muscle specific maximal active force of R406W and E413K expressing muscles compared to WT (A). Asterisks indicate a significant difference compared to the WT (*P ≤0.05, **P ≤0.01, ***P ≤0.001). AAV, adeno-associated virus; WT, wild-type. the percentage of transfected fibers [39,40], is easier to areas of the muscle fibers for the R406W mutation of implement in vivo and does not induce the confounding desmin; while for the E413K mutation, small and effect of muscle damage frequently observed with elec- dispersed deposits were located both in the center and troporation. In the present study, we obtained a very at the periphery of muscle fibers. These results are very homogeneous expression of the mutant desmin with similar to those observed on muscle biopsies of patients more than 98% of the muscle fibers being transduced [18-20]. In our model we also observe, for the mutation and expressing the transgene. AAV-mediated expression E413K, the typical aggregates with ‘ring-like’ structures of mutated desmin mimics MFM related disorders and are generally continuous with the Z-lines [20]. This ap- seems to be a good model to study molecular heterogen- proach seems to be adequate and recapitulates extremely eity of MFM pathogenesis. well the morphological modifications of muscles in the Despite the fact that we conducted our study on one context of MFM. muscle (tibialis anterior) at one time point (1 month), Our results also demonstrate that the R406W mutant we have demonstrated that intramuscular injection of induces hyperplasia and an increase in muscle mass that viral vectors to promote the expression of mutant is not accompanied by an increase in the absolute max- desmin is a potentially useful approach, since the results imal active force, as is supposed to be the case for a obtained in this study are very similar to the phenotypes healthy muscle since specific maximal force is reduced. observed in patients. In addition to the loss of muscle Each muscle fiber seems to develop less force. This is specific maximal force, we observe an accumulation of also true in the case of the E413K mutant. Two main granular aggregates in subsarcolemmal and perinuclear mechanisms may explain this decrease in specific Joanne et al. Skeletal Muscle 2013, 3:4 Page 11 of 13 http://www.skeletalmusclejournal.com/content/3/1/4 maximal force: a direct disturbance of the generation of the same molecular pattern, can induce the observed force by the sarcomere or an impaired transmission of differences in regeneration. Taken together, our results the force to the tendons. Our observations demonstrate confirm that AAV-mediated expression of mutants is a that the sarcomeres are locally disturbed where we ob- useful method to explore clinical phenotypic heteroge- serve aggregates of desmin mutants, suggesting that neity of a disease, such as MFM. mutations of desmin, by interfering directly with the Z-line, influence force production of muscle fibers. It is Additional file also possible that a disturbance in the alignment of the sarcomeres compared to the longitudinal axis of the Additional file 1: Figure S1. Z-lines perturbations induced by aggregation of desmin mutants in tibialis anterior muscles. (A) Merge of muscle (pennation angle) or changes in the extracellular (B) immunostaining against c-Myc and (C) immunostaining against matrix may be partly responsible for the decrease in spe- α-actinin (left part) or specific staining of actin using phalloidin (right cific maximal force by affecting the force transmission as part). The plotted graphics reveal the striation pattern of c-Myc (green), α-actinin or phalloidin (red). All analyses were performed 1 month after observed in desmin null mice [41,42]. These points re- intramuscular injection of AAV vectors. Note significant perturbations of main to be clarified to determine how much each mech- Z-line located in the areas where exogenous desmin accumulates. AAV, anism is affected by these mutations of desmin. adeno-associated virus. Finally, in the case of the R406W expressing muscles, we observed focal muscle regeneration, which was Abbreviations confirmed by the presence of immature muscle fibers AAV: Adeno-associated virus; CMV: Cytomegalovirus; DAPI: 4 ,6-diamidino-2- phenylindole; EDTA: Ethylenediaminetetraacetic acid; EGTA: ethylene glycol expressing the neonatal isoform of MHC. This mutation tetraacetic acid; Fab fragment: Antigen-binding fragment; HSD: Honestly of desmin also induces a decrease in the overall size of Significant Difference; IgG: Immunoglobulin G; MFM: Myofibrillar myopathy; fibers. These phenomena, observed 1 month after injec- MHC: Myosin heavy chain; PBS: Phosphate-buffered saline; PCR: Polymerase chain reaction; SDH: Succinate dehydrogenase; SEM: Standard error of the tion of the virus in tibialis anterior muscle, support the mean; TEM: Transmission electron microscope; WT: Wild-type. idea that the R406W mutation induces repeated cycles of de-/regeneration of muscle fibers. During regener- Competing interests ation, lost fibers are replaced by clusters of myotubes, The authors declare that they have no competing interests. formed by satellite cells, which looked like split fibers Authors’ contributions and have, therefore, a reduced size [43]. This may par- PJ carried out the histological, immunostaining and Western blot tially explain the hyperplasia. However, we emphasize experiments and analysis, performed the statistical analysis, participated in that muscle regeneration is not the sole mechanism re- the design of this study and drafted the manuscript. OC carried out the preparation, purification of the plasmids and site-directed mutagenesis. CH sponsible for the hyperplasia and impaired distribution carried out the production, purification and injection of the AAV vectors. AF of muscle fiber size observed in the muscles expressing carried out the muscle force measurements. PV participated in the design of R406W, since we also observed small fibers expressing the study and helped to draft the manuscript. JD and GBB participated in the design of the AAV vectors, titration of viral particles and helped to draft exogenous (c-Myc-positive) desmin. The strong regener- the manuscript. OA designed and coordinated the study, obtained the ation observed in tibialis anterior expressing the R406W funding, carried out the electron microscopy experiment, performed in vivo mutant of desmin may affect the long-term regenerative experiments and analysis and drafted the manuscript. All authors read and approved the final manuscript. capacities of muscles and could explain the disorders observed in MFM. In contrast, the weaker regeneration of Acknowledgements muscles expressing the E413K desmin mutant could ex- This work was supported by the Association Française contre les Myopathies plain the longer asymptomatic period observed in patients. (AFM) contract numbers 14040 and 15454, and the University Paris Diderot, Paris 7, France. OC was supported by fellowships from the Ministère de la Recherche et de la Technologie (MRT) and the AFM. We would like to thank Conclusions Dr Zhenlin Li and Professor Denise Paulin for their helpful advice; Alexis In this study, we show that AAV-mediated expression of Canette from the imaging facility ImagoSeine at the Jacques Monod Institute, Paris, France, for his work in electron microscopy studies; Solenne desmin mutants in mouse muscles recapitulate the ag- Marie for technical assistance during AAV vectors preparation; and gregation features, the contractile function decrease and Dr Philippe Noirez for developing imageJ software macros. the morphological change observed in MFM patients. In Author details somewhat more detail, differences in muscle regener- Université Paris Diderot, Sorbonne Paris Cité, CNRS EAC4413, Unit of ation, distribution of muscle fiber size and muscle force Functional and Adaptive Biology, Laboratory of Stress and Pathologies of the production exist between the R406W and E413K desmin Cytoskeleton, 75013, Paris, France. Department of Aging, Stress and Inflammation, Université Pierre et Marie Curie-Paris 6, Sorbonne Universités, mutants. Above all, our results suggest that the R406W 75005, Paris, France. Université Pierre et Marie Curie-Paris 6, Sorbonne mutant of desmin induced a strong muscle regeneration, Universités, UMR S794, INSERM U974, CNRS UMR7215, Institut de Myologie, which is not the case of the E413K mutant. This diffe- 75013, Paris, France. Université Paris Descartes, Sorbonne Paris Cité, 75006, Paris, France. rence could be at the origin of phenotypic differences observed in patients. It could be interesting, in the Received: 19 October 2012 Accepted: 28 January 2013 future, to understand how these mutations, which affect Published: 20 February 2013 Joanne et al. Skeletal Muscle 2013, 3:4 Page 12 of 13 http://www.skeletalmusclejournal.com/content/3/1/4 References 21. Chourbagi O, Bruston F, Carinci M, Xue Z, Vicart P, Paulin D, Agbulut O: 1. 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Skeletal MuscleSpringer Journals

Published: Feb 20, 2013

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