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The superhealing MRL background improves muscular dystrophy

The superhealing MRL background improves muscular dystrophy Background: Mice from the MRL or “superhealing” strain have enhanced repair after acute injury to the skin, cornea, and heart. We now tested an admixture of the MRL genome and found that it altered the course of muscle pathology and cardiac function in a chronic disease model of skeletal and cardiac muscle. Mice lacking γ-sarcoglycan (Sgcg), a dystrophin-associated protein, develop muscular dystrophy and cardiomyopathy similar to their human counterparts with limb girdle muscular dystrophy. With disruption of the dystrophin complex, the muscle plasma membrane becomes leaky and muscles develop increased fibrosis. Methods: MRL/MpJ mice were bred with Sgcg mice, and cardiac function was measured. Muscles were assessed for fibrosis and membrane leak using measurements of hydroxyproline and Evans blue dye. Quantitative trait locus mapping was conducted using single nucleotide polymorphisms distinct between the two parental strains. Results: Introduction of the MRL genome reduced fibrosis but did not alter membrane leak in skeletal muscle of the Sgcg model. The MRL genome was also associated with improved cardiac function with reversal of depressed fractional shortening and the left ventricular ejection fraction. We conducted a genome-wide analysis of genetic modifiers and found that a region on chromosome 2 was associated with cardiac, diaphragm muscle and abdominal muscle fibrosis. Conclusions: These data are consistent with a model where the MRL genome acts in a dominant manner to suppress fibrosis in this chronic disease setting of heart and muscle disease. Keywords: Cardiomyopathy, Fibrosis, MRL, Muscular dystrophy Background injury settings where the MRL background induces more Murphy Roths Large (MRL) mice are an inbred mouse rapid healing, multiple mechanisms have been impli- strain noted to have enhanced healing ability. This MRL cated to explain this phenomenon including decreased strain was initially discovered because of its rapid ability scar formation, altered inflammatory response, reduced to heal ear holes [1,2]. The MRL strain’s capacity to rap- apoptosis, increased proliferation, improved remodeling idly recover from injury has been seen for both digit and, in some settings, enhanced stem cell function wounding and corneal scarring [3,4]. The MRL strain [4,6,12-15]. Genetic data support that many different has been reported to reduce scar formation after acute mechanisms account for enhanced healing since more cardiac injury, including freeze injury and coronary ar- than 40 different genetic loci have been associated with tery ligation [5,6]. However, other studies have suggested aspects of the healing phenotype [14,16]. that larger scale acute cardiac injury cannot be overcome The dystrophin complex is composed of membrane- by the MRL strain’s healing capacity [7-11]. In those associated proteins that mediate membrane stability in heart and skeletal muscle. Mutations that disrupt expres- sion of dystrophin or its associated proteins the sarco- * Correspondence: emcnally@uchicago.edu Department of Medicine, Section of Cardiology, 5841 S. Maryland, MC 6088, glycans proteins cause progressive cardiac and skeletal Chicago, IL 60637, USA 2 muscle degeneration in humans and mouse models. At Department of Human Genetics, The University of Chicago, Chicago, IL the cellular level, the loss of dystrophin or the 60637, USA Full list of author information is available at the end of the article © 2012 Heydemann 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. Heydemann et al. Skeletal Muscle 2012, 2:26 Page 2 of 11 http://www.skeletalmusclejournal.com/content/2/1/26 sarcoglycans leads to a disrupted sarcolemmal mem- Methods brane that is abnormally leaky. Membrane permeability Animals results in increased intracellular calcium that triggers The Sgcg mouse was previously bred for ten generations proteolysis and necrosis. An inflammatory response also into the DBA/2J strain (000671, Jackson Laboratory, Bar contributes to muscle degeneration [17]. Muscle con- Harbor, ME) [18,19]. MRL/MpJ (000486, Jackson La- traction is thought to provoke submicroscopic disrup- boratory, Bar Harbor, ME) mice were bred to the Sgcg +/− tion of the sarcolemma, and in the absence of a normal animals to generate Sgcg F1 mice on a 50% MRL/50% dystrophin complex, this leads to dysfunction and de- DBA/2J background. F1 mice were interbred to generate struction of cardiomyocytes and skeletal myofibers. The an F2 cohort. To increase the number of mutant ani- reduction in contractile cells and the presence of fibrotic mals, Sgcg mice from the F2 generation were bred again scar tissue within the heart led to reduced cardiac con- to produce additional Sgcg mice from an F3 generation. tractile function and congestive heart failure. All mice were housed in uniform conditions in a single Mouse models of dystrophin or sarcoglycan mutations pathogen-free barrier facility. All animals used in this recapitulate the basic pathological defects seen in human study were housed and treated in accordance with the forms of these genetic disorders. Mice lacking γ- standards set by the University of Chicago Animal Care sarcoglycan were engineered by removing the first cod- and Use Committee. The number of mice used for histo- ing exon of the Sgcg gene and model muscular dystrophy pathological analysis is shown in Table 1. [18]. We previously introduced the Sgcg allele into the Mice were sacrificed at either 8 or 32 weeks for ana- DBA/2J genetic background and found that this back- lysis. Muscles were used for either the Evans blue dye ground confers a more severe phenotype [19]. The assay or the HOP assay. Dye uptake assays were per- enhanced severity is seen as increased scar formation, formed on triceps muscles, gastrocnemius/soleus mus- measured as hydroxyproline (HOP) content because this cles and gluteus/hamstrings groups of muscles. Dye modified amino acid is a marker of collagen. In skeletal uptake was performed on half of each quadriceps muscle, increased Evans blue dye uptake is monitored to muscle and half of the abdominal muscles since these reflect membrane leakiness [20]. Using these assays, we muscles are of sufficient size to perform both dye up- showed that the DBA/2J genetic background worsened take and fibrosis assays. Hydroxyproline (HOP) assays the disease process [19]. were performed on the diaphragm muscle, the cardiac Because of its role in wound healing and the basic ventricles isolated as a single unit, half of the abdom- similarities between common forms of cellular injury inal muscles and half of each quadriceps muscle. The and what is seen in muscular dystrophy and cardiomy- amount of muscle assayed varied with the size of the opathy, we introduced the MRL genome into the Sgcg muscle andrangedfrom30mg for thediaphragm model. We hypothesized that the indolent pace of cellu- muscle to 300 mg for the gluteus/hamstring muscle lar damage in this disorder could be abated by the MRL Table 1 Number of mice analyzed background. Sgcg mice were bred to MRL mice to gener- Trait Genotypes ate Sgcg mice on a mixed genetic background with 50% D2 MRL/D2 MRL/D2 contribution of the MRL strain. This breeding strategy Fibrosis Sgcg Sgcg Sgcg , 32wk produced Sgcg mice with 50% genetic background from Quadriceps 25 124 18 the MRL strain and 50% background of the DBA/2J Diaphragm 25 45 10 MRL/D2 strain, and these mice are referred to Sgcg .We Heart 27 125 9 found that a 50% contribution of the MRL genome D2 MRL/D2 MRL/D2 Membrane leak Sgcg Sgcg Sgcg , 32wk reduced fibrosis in the heart and skeletal muscle, con- Quadriceps 37 149 15 sistent with dominant genetic loci in the MRL back- ground. Interestingly, the MRL genome did not Gluteus 11 76 15 consistently reduce membrane leak, suggesting that the Triceps 14 110 15 MRL background does not exert its effect on myocyte Abdominals 14 74 15 membrane stability and instead acts downstream on re- Gastrocnemius/soleus 14 111 15 MRL/D2 modeling. Sgcg mice had improved cardiac func- D2 MRL/D2 Central nuclei Sgcg Sgcg tion so that they were now similar to wild-type mice. Quadriceps 6 7 We conducted a genome-wide scan using informative polymorphisms and identified a region on chromosome Diaphragm 6 4 D2 MRL/D2 2 that was associated with fibrosis in cardiac, diaphragm Fiber size variability Sgcg Sgcg and abdominal muscles. These data demonstrate that Quadriceps 7 9 genes within the MRL background are modifiers for car- Diaphragm 6 4 diopulmonary involvement in muscular dystrophy. Heydemann et al. Skeletal Muscle 2012, 2:26 Page 3 of 11 http://www.skeletalmusclejournal.com/content/2/1/26 group that included the semimembranosus, semitendi- were removed with a topical depilatory agent. Limb leads nosus and biceps femoris. were attached for electrocardiogram gating, and the ani- mals were imaged in the left lateral decubitus position with Evans blue dye uptake assay for membrane leak a Visual Sonics Vevo 770 machine using a 30-MHz high- Evans blue dye (Sigma, E-2129) was performed as frequency transducer. Body temperature was maintained described [19,21]. Evans blue dye (Sigma, E-2129) was using a heated imaging platform and warming lamps. dissolved in phosphate-buffered saline at 10 mg/ml and Anesthesia was variably delivered to maintain heart rates injected intraperitoneally at 5 μl/g body weight. Twenty throughout the procedure at a constant 380–420 beats per to 40 h later, the tissues were harvested, finely minced, minute. Two-dimensional images were recorded in para- weighed and incubated at 55°C in 1 ml formamide for sternal long- and short-axis projections, with guided M- 2 h before spectrophotometric absorbance was measured mode recordings at the midventricular level in both views. at 620 nm [22,23]. Results are reported as absorbance/ LV cavity size and wall thickness were measured in at least mg tissue. three beats from each projection and averaged. LV wall thickness, interventricular septum (IVS) and posterior wall Hydroxyproline assay for fibrosis (PW) thickness, and internal dimensions at diastole and The hydroxyproline (HOP) assay was performed as systole (LVIDd and LVIDs, respectively) were measured. described [19,21,24]. The tissue was minced, weighed LV fractional shortening [(LVIDd – LVIDs)/LVIDd] and and hydrolyzed overnight in 2 ml of 6 M hydrochloric relative wall thickness [(IVS thickness + PW thickness)/ acid at 110°C. Ten μl of this hydrolysate was mixed with LVIDd] were calculated from the M-mode measurements. 150 μl isopropanol, then 75 μl of 1.4% chloramine-T (Sigma, St Louis, MO) in citrate buffer and oxidized at Genetic and statistical analysis room temperature for 10 min. One ml of a 3:13 solution Single nucleotide polymorphisms (SNPs, n = 1,701) in- of Ehrlich’s reagent (3 g of 4-(dimethylamino) benzalde- formative between the parental DBA/2J and MRL/MpJ MRL/D2 hyde, Sigma, St Louis, MO; 10 ml ethanol; 675 μl sul- strains were genotyped in 80 Sgcg F2-F4 Sgcg ani- furic acid) to isopropanol was added, mixed and mals on the Illumina GoldenGate platform using the incubated for 30 min at 55°C followed by extinction Mutation Mapping and Developmental Analysis Panel measurement at 558 nm. A standard curve (0–5000 nM, (MMDAP) [27] and the Mouse Universal Genotyping trans-4-hydroxy-L-proline, Sigma, St Louis, MO) was Array (GeneSeek, Neogen Corp., Lansing, MI) [28]. R included in each assay. Results are reported as nM package QTLRel was used to perform whole-genome HOP/mg tissue. quantitative trait locus (QTL) mapping for each of the membrane permeability and fibrosis phenotypes and Immunofluorescence microscopy using sex as a covariate [29,30]. Significance thresholds Tissues were flash frozen in liquid nitrogen-cooled iso- were determined by 1,000 permutation tests. The 1.5- pentane and stored at 80°C; 7-μm sections were cut on a LOD drop support interval was calculated using cryostat and fixed to slides in ice-cold 100% methanol. QTLRel. Tests of normality and other statistics were cal- The following antibodies were used: dystrophin NCL- culated in Prism (GraphPad). Build 37.1 was used for DYS2 (Novocastra/Leica), PH3 04–817 (Millipore), CD3 genomic analysis. MON1003-1 (Monsanto), caspase 3 (BD Biosciences), For HOP and dye uptake assays, data were analyzed by MAC1 BD557395 (BD Biosciences) and eMHC F1.652 one-way, unpaired ANOVA with parametric methods (ATCC). The TUNEL kit was from Millipore. Central followed by the Tukey multiple comparison post-test nuclei and fiber size variability was determined blinded (Prism, Graphpad), and p < 0.05 was considered signifi- to genotype by analyzing ten randomly chosen fields of cant. Data from 197 Sgcg animals from an F3 intercross 40× magnification. Fiber size variability was compared between the DBA/2J and 129T2/SvEmsJ backgrounds D2/129 MRL/D2 using each animal’s coefficient of variability (standard (Sgcg ) was compared to the Sgcg cohort. deviation/mean). Results Echocardiography A 50% contribution of the MRL background reduces Twelve-week animals were evaluated by echocardiog- fibrosis in Sgcg mice raphy as described [25,26]. Investigators were blinded to To assess the MRL contribution to muscular dystrophy, genotype. To avoid the stress associated with conscious we used the MRL/MpJ substrain since it contains a wild- restraint, anesthetized animals were studied. Anesthesia type fas allele [1]. We also used mice lacking γ- was induced by 1% isoflurane in a closed chamber sarcoglycan (Sgcg null) since these mice are a model of (Ohmeda Fluotec 3; Matrix Medical, Orchard Park, NY) limb girdle muscular dystrophy 2C [18] and on the in 20% O delivered through a nose cone. Chest hairs DBA/2J background have a more severe phenotype, 2 Heydemann et al. Skeletal Muscle 2012, 2:26 Page 4 of 11 http://www.skeletalmusclejournal.com/content/2/1/26 reminiscent of what is seen in humans [19,21]. MRL/ fibrosis in aged (32 week) animals and found a persistent MRL/D2 MpJ mice were bred to Sgcg null and then interbred to reduction in fibrosis in the Sgcg mice (Figure 2, di- generate Sgcg null mice with a 50% MRL/MpJ and 50% agonal bars). The 129T2/SvEmsJ strain was previously DBA/2J contribution. In Sgcg null mice, like all dys- shown to suppress the severity of muscle pathology in Sgcg trophin complex-associated mutations, fibrosis and col- null mice [33]. In comparison, the MRL background lagen deposition is increased in the heart and muscle, similar to what is seen in human patients with similar mutations [18,31]. A 50% contribution from the MRL background reduced fibrosis in Sgcg heart and muscles (Figure 1). In Sgcg hearts, fibrosis was often seen grossly as large patchy white areas and with the introduction of the MRL background visible fibrosis was diminished so that the hearts were indistinguishable from those of wild-type mice (Additional file 1: Figure S1). Of all the muscle groups analyzed, only diaphragm muscle retained any visible fibrosis. Diaphragm muscle is the most con- sistently damaged muscle in multiple mouse models, in- cluding this model, and the mdx model of Duchenne muscular dystrophy [32]. Therefore, it is possible that the degree of injury in this muscle overwhelms the heal- ing properties of the MRL background. We quantified fibrosis by measuring HOP content as D2 MRL/D2 an indicator of collagen in Sgcg and Sgcg mus- MRL/D2 cles. Sgcg mice have significantly reduced fibrosis D2 compared to Sgcg mice (Figure 2). We also evaluated Figure 2 The MRL genome quantitatively reduces fibrosis in Figure 1 The MRL genome suppresses fibrosis in the heart and Sgcg mice. Hydroxyproline (HOP) is a measure of fibrosis and is D2 muscles of Sgcg mice. Sgcg mice lack the dystrophin-associated represented on the y axis (mM/mg). In the heart, diaphragm and protein, γ-sarcoglycan, and when in the DBA/2J (D2) background quadriceps muscles, fibrosis is significantly (p < 0.001) reduced in MRL/D2 have a more severe phenotype with enhanced membrane leak and intercrossed Sgcg (D2/MRL) animals compared to Sgcg in the D2 fibrosis [21]. A 50% contribution of the MRL genome, referred to as (D2) background at 8 weeks. Fibrosis remains significantly (***p <0.001) MRL/D2 MRL/D2 Sgcg , suppressed fibrosis in both heart and skeletal muscle. reduced in Sgcg animals at the 32-week time point. Heydemann et al. Skeletal Muscle 2012, 2:26 Page 5 of 11 http://www.skeletalmusclejournal.com/content/2/1/26 suppressed fibrosis more than the 129T2/SvEmsJ back- We compared suppression of dye uptake by the MRL ground (Figure 3). The MRL background dramatically background to that induced by the 129T2/SvEmsJ back- reduced fibrosis in the heart and abdominal muscles com- ground in Sgcg mice (Figure 6). The quadriceps and ab- pared to 129T2/SvEmsJ. The MRL background also sup- dominal muscles from Sgcg null mice with a contribution pressed fibrosis more than the 129T2/SvEmsJ background from the 129T2/SvEmsJ background showed increased in the diaphragm and quadriceps muscles, but to a lesser membrane leak compared to Sgcg mice with an MRL con- degree. This finding suggests that the phenotypically bene- tribution. However, the triceps muscle group from the ficial genetic modifiers in theMRL genome maybemore MRL background showed increased dye uptake, and the potent than those in the 129T2/SvEmsJ genome. gluteus/hamstring muscle group showed no significant dif- ference. Thus, there was no consistent suppression of The MRL background does not protect against membrane membrane leakiness by the MRL strain. leak Disruption of the membrane-associated dystrophin com- The MRL genome may promote skeletal muscle plex renders the sarcolemma unusually fragile, leading to regeneration abnormal membrane leakage that is visualized by uptake of The MRL background is thought to exert part of its effect the nonspecific vital tracer Evans blue dye [20]. Upon gross by promoting regeneration [34]. We examined embryonic MRL/D2 inspection, the muscles from the Sgcg mice displayed myosin heavy chain (eMHC) expression as a reflection of MRL/D2 high levels of dye uptake, comparable to what was observed regeneration. Sgcg muscle shows patchy areas with a in the parental Sgcg mice (Additional file 1: Figure S1). On qualitative increase in eMHC-positive fibers compared to a microscopic level, dye-positive cells were readily detect- Sgcg (Figure 7), and wild-type muscle showed no eMHC MRL/D2 able in Sgcg muscle and heart (Figure 4). Evans blue positive fibers (data not shown). In dystrophic skeletal dyelevelsweremeasuredinmultiplemusclegroupsand muscle, ongoing regeneration is thought to offset degener- MRL/D2 were not significantly different between Sgcg and ation. Consistent with this, eMHC-positive regions were D2 Sgcg muscles for quadriceps, triceps, gastrocnemius/ also positive for Evans blue dye uptake, indicative of soleus, gluteus and abdominals muscle groups (Figure 5). muscle damage. We also evaluated phosphorylated histone We also measured dye uptake in older animals at 32 weeks 3 (PH3) as a reflection of mitotic index. No clear differ- MRL/D2 to assess disease progression. At 32 weeks, the skeletal ences for PH3 staining were seen between Sgcg and muscles continued to show comparable levels of membrane Sgcg muscle. Regenerating skeletal muscle, whether from leak as what was seen at 8 weeks (Figure 5). The gluteus/ trauma or muscular dystrophy, is identified by the presence hamstring group of muscles showed an increased of dye of myofibers with centrally positioned nuclei. In muscular uptake compared to the 8-week animals. This increase was dystrophy, ongoing regeneration is also marked by MRL/D2 not seen for other muscle groups. increased fiber size variability. Sgcg mice have an Figure 3 The reduction of fibrosis in intercrossed Sgcg animals is specific to the MRL genome. Hydroxyproline (HOP) is a measure of fibrosis and is represented on the y axis (mM/mg). Fibrosis is significantly reduced in the heart (***p < 0.001), diaphragm (p < 0.001), quadriceps (*p = 0.041) and abdominal muscles (p < 0.001) of Sgcg animals intercrossed in the D2/MRL background compared to those intercrossed in the D2/129 background. Heydemann et al. Skeletal Muscle 2012, 2:26 Page 6 of 11 http://www.skeletalmusclejournal.com/content/2/1/26 Improved cardiac function from a 50% contribution of the MRL background We performed 2D and M mode echocardiography on MRL/D2 D2 Sgcg and Sgcg mice at 12 weeks of age. Both frac- tional shortening and the left ventricular ejection fraction D2 were significantly reduced in Sgcg mice, indicating that the increased fibrosis impairs cardiac function (Table 2). Fractional shortening and the left ventricular ejection frac- MRL/D2 MRL/D2 tion were similar between Sgcg and WT mice, consistent with improved function mediated by 50% of the MRL genome. Wall thickness was also increased in Sgcg mice compared to the wild type of the same background. In contrast, wall thickness was not different between MRL/D2 MRL/D2 Sgcg and WT mice. Chromosome 2 associates with reduced fibrosis Sgcg heart and diaphragm muscle We conducted a genome-wide scan using markers that were informative in the two parental strains DBA/2J and MRL/MpJ (Figure 8). QTLRel was used to identify regions of association; this analysis takes into account the relatedness of individual animals in the cohort [29,30]. A region on chromosome 2 was identified that associated with fibrosis in the heart. The 1.5-LOD drop interval of this region spans from 64.8008−73.1758 cM in mouse genome build 37.1. This same region was also associated with fibrosis in the diaphragm muscle and also for fibrosis in the abdominal muscles. It should be noted that the significance of these associations is sug- Figure 4 Membrane leak is not corrected by the presence of gestive (p < 0.63) when using the stringent QTLRel ana- the MRL genome in Sgcg heart and muscle. Evans blue dye is found in cardiomyocytes and skeletal myofibers reflecting lysis. However, the overlapping intervals found in heart, membrane leakiness. Dye uptake occurs in patchy pattern diaphragm and abdominal muscles provide additional throughout the heart and muscle and is seen as opacified cells that support that this interval modifies fibrosis. fluoresce red. Nuclei are shown in blue and dystrophin in green. Ltbp4 polymorphism does not account for the MRL increased number of central nuclei in quadriceps and dia- healing properties D2 phragm muscle compared to Sgcg mice (Figure 7). These The DBA/2J strain contains an insertion/deletion poly- same muscles also showed increased fiber size variability morphism within the Ltbp4 gene [21] that modifies both when there is a contribution from the MRL background. membrane permeability and fibrosis, as two independent These data are consistent with a model in which favorable traits, in Sgcg muscular dystrophy. Ltbp4 encodes latent matrix remodeling may support enhanced regeneration. TGFβ-binding protein 4, and TGFβ proteins have been We also assessed apoptosis using a TUNEL assay and extensively linked to fibrosis in many disease states in- caspase 3 staining and again found no differences be- cluding muscular dystrophy [35,36]. Most murine MRL/D2 tween Sgcg and Sgcg muscle, suggesting that strains, including the MRL/MpJ strain used here, contain gross differences in programmed cell death are unlikely the protective Ltbp4 allele with an additional 12 amino MRL/D2 to account for the improved healing of the MRL strain acids inserted in exon 12. Because the Sgcg cohort (Additional file 1: Figure S2). We also characterized used here contained both the protective (Ltbp allele) whether T cell infiltration or macrophage infiltration and the disease-enhancing allele (Ltbp4 ), we tested MRL/D2 differed qualitatively between Sgcg and Sgcg whether Ltbp4 genotype correlated with fibrosis in the MRL/D2 muscle, but we found no clear differences between Sgcg cohort. Table 3 shows that neither membrane MRL/D2 Sgcg and Sgcg muscle, suggesting other features permeability nor fibrosis is correlated with the Ltbp4 MRL/DBA2J contribute to the MRL background’seffectonmuscular genotype in the Sgcg cohort. Therefore, Ltbp4 dystrophy (Additional file 1: Figure S2). does not account for the suppressive effect of the MRL Heydemann et al. Skeletal Muscle 2012, 2:26 Page 7 of 11 http://www.skeletalmusclejournal.com/content/2/1/26 Figure 5 The MRL genome does not significantly reduce membrane damage in Sgcg muscle. Evans blue dye uptake is a measure of membrane damage and is represented on the y axis (absorbance/mg). In the quadriceps, triceps, abdominal and gastrocnemius/soleus muscles, D2 MRL/D2 membrane damage is not significantly different between Sgcg animals and intercrossed Sgcg animals at both the 8- and 32-week time points. In the gluteus/hamstring muscles, membrane damage is significantly (**p < 0.01) increased at the 32-week time point in intercrossed Sgcg MRL/D2 animals compared to the 8-week time point. Figure 6 The MRL genome has a variable ability to reduce membrane leak in muscular dystrophy compared to the 129T2/SvEmsJ strain. Evans blue dye uptake was measured in multiple muscle groups represented on the y axis (absorbance/mg). Membrane damage is D2/MRL reduced in the quadriceps (**p = 0.0053) and abdominal (**p = 0.0012) muscles of intercrossed Sgcg animals compared to intercrossed D2/129 MRL/D2 Sgcg mice. Membrane damage is increased in the triceps muscle (*p = 0.0298) of intercrossed Sgcg animals. There is no difference in membrane damage in the gluteus/hamstring muscle. Heydemann et al. Skeletal Muscle 2012, 2:26 Page 8 of 11 http://www.skeletalmusclejournal.com/content/2/1/26 Figure 7 Regeneration may be enhanced in the MRL background. Embryonic myosin heavy chain (eMHC) staining was increased in MRL/D2 Sgcg compared to Sgcg muscle, and eMHC fibers were seen in areas where dye uptake was seen. However, phosphorylated histone 3 (PH3), MRL/D2 a marker of mitotic index, was not increased in Sgcg compared to Sgcg muscle. Skeletal muscle central nucleation and fiber size variability MRL/D2 D2 are enhanced by the MRL background. Sgcg muscle demonstrated an increase in centrally nucleated fibers compared to Sgcg mice. Fiber size variability was reflected in the coefficient of variability of fiber diameter measurements. The solid line indicates significance (p < 0.05). These data support that the MRL exerts its effect in skeletal muscle, at least in part, by promoting regeneration. background, and other genetic modifiers account for this The MRL strain typically shows the improved healing difference. capability in its first 2–6 months of life. After 6 months of age, wild-type MRL mice develop autoimmune disorders; Discussion it is for this reason that we conducted studies using a 50% The MRL genome protects against fibrosis but not contribution of the MRL strain. Quantitative trait mapping membrane leak has been used to define many different genetic regions Mutations in the dystrophin or sarcoglycan genes share a associated with the healing properties [14]. The auto- common pathological mechanism characterized by disrup- immune properties have been genetically separated from tion of the plasma membrane of cardiomyocytes and skel- the healing properties since the four to six different “auto- etal myofibers. Membrane leak is an early step in the immune” genetic loci do not overlap with those associated pathological process while fibrosis is thought to be a sec- with improved healing. In our studies, only 50% of the ondary response. Our study suggests that there are genetic MRL genome was capable of suppressing fibrosis and im- pathways that target fibrosis without altering the sarco- proving function. In the course of these studies, we never MRL/D2 lemmal leak properties. In the heart, reduced fibrosis was identified a single Sgcg animal that had markedly ele- correlated with functional benefit, although cardiac func- vated fibrosis. This stands in contrast to what is normally tion may be improved from both cardiac intrinsic as well observed in the Sgcg animals where outliers with markedly as cardiac extrinsic features. For example, improved skel- elevated fibrosis are often seen. The maximum values for MRL/D2 etal muscle function may contribute to improved cardiac fibrosis in Sgcg mice were all well below what was D2 function, particularly when considering the contribution measured in Sgcg mice. This is consistent with multiple, of the respiratory musculature. dominant genetic loci imparting the improved healing cap- abilities, similar to what has been observed for the ear hole repair properties in the MRL strain [37]. The finding that Table 2 Cardiac function in Sgcg mice MRL/D2 improvement persists in the Sgcg mice, with almost Fractional shortening complete suppression of fibrosis at 32 weeks, suggests that D2 a Sgcg (n = 4) 33.87 ± 4.80 a persistent healing effect can arise from the MRL strain. D2 a WT (n = 5) 44.58 ± 7.65 MRL/D2 Sgcg (n = 5) 46.00 ± 14.4 Chromosome 2 candidate genes MRL/D2 WT (n = 5) 48.37 ± 11.21 When considering the overlapping region on chromo- p = 0.046. some 2 from the heart, diaphragm and abdominal Heydemann et al. Skeletal Muscle 2012, 2:26 Page 9 of 11 http://www.skeletalmusclejournal.com/content/2/1/26 fibrosis data, the interval contains 49 known genes. There are additional predicted and unnamed genes and genes encoding olfactory receptors that are not consid- ered likely candidates. Within the interval is Jag1, encod- ing jagged the ligand for the Notch receptor. Loss of function of Notch 3 leads to muscle hyperplasia, espe- cially when subjected to repetitive injury [38]. Ex vivo activation of Notch signaling helps maintain donor cell engraftment during myoblast transfer [39]. Another gene linked to growth and healing in the interval is Bmp2-en- coding bone morphogenetic protein 2. The BMPs medi- ate musculoskeletal regeneration [40], and given the MRL background effect on multiple cells and tissues, BMP2 is well positioned to contribute to the MRL superhealing response. That said, this chromosome 2 interval has not previously been linked to other MRL healing properties. The strict criteria imposed by the QTLRel algorithm makes it unlikely that these results derive from relatedness of animals within the cohort. Cardiac injury and repair in the MRL strain The ability of the heart to recover after injury has been studied in the MRL strain using cryoinjury, left anterior descending ligation and ischemia reperfusion methods [5,6,8-10,14,15]. However, scar reduction has been noted after some forms of injury while not after others. In common to all these studies is an acute injury model where a normal heart was substantially damaged in a single setting. The MRL’s ability to heal the heart may be limited such that larger amounts of injury may be insur- mountable, as proposed by Naseem [6]. Our data sup- port that lower, although persistent, levels of injury can be managed by the MRL strain where there is significant reduction in fibrosis and a corresponding functional im- provement in cardiac function. Mechanisms for MRL healing Figure 8 Fibrosis in the heart, diaphragm and abdominal A number of mechanisms likely act in concert to achieve muscles is modified by a locus on chromosome 2. Genome-wide MRL/D2 suppression of fibrosis and improvement of function. In association was examined for fibrosis in Sgcg progeny in the (A) cardiac muscle (n = 65), (B) diaphragm (n = 78) and (C) skeletal muscle, where muscle stem cells robustly regen- abdominal muscle (n = 78). Chromosomes are plotted on the x axis, erate muscle after injury, there is indirect evidence of and the informative SNPs tested (n = 1,707) are displayed as open enhanced regeneration. Specifically, there is an increase circles that alternate color by chromosome; LOD scores are in centrally nucleated myofibers, which is thought to re- represented on the y axis. Overlapping regions on chromosome 2 showed suggestive (p < 0.63) association with fibrosis in heart, flect enhanced myoblast fusion. However, our data do diaphragm and abdominal muscle. not distinguish whether the MRL background exerts its effect on muscle regenerative cells, or by creating a more supportive extracellular matrix or both. MRL animals heal with embryonic characteristics with enhanced blas- MRL/D2 Table 3 Ltbp4 genotype in Sgcg mice tema formation and metabolic and gene expression fea- tures consistent with an earlier developmental state [41]. Dye uptake Fibrosis D/D Altered protease expression has also been noted in the Ltbp4 (n = 14) 3.90 ± 0.83 6.00 ± 1.81 MRL mouse in response to damage [5]. Recent work D/I Ltbp4 (n = 34) 4.18 ± 0.84 8.05 ± 3.02 characterizing the immune infiltrate in muscular dys- I/I Ltbp (n = 47) 4.00 ± 0.85 6.64 ± 2.15 trophy found that reducing osteopontin was effective at Heydemann et al. Skeletal Muscle 2012, 2:26 Page 10 of 11 http://www.skeletalmusclejournal.com/content/2/1/26 suppressing fibrosis [17]. Together these data may favor 2. McBrearty BA, Clark LD, Zhang XM, Blankenhorn EP, Heber-Katz E: Genetic analysis of a mammalian wound-healing trait. Proc Natl Acad Sci U S A matrix-associated modifications that can modulate 1998, 95:11792–11797. muscle fibrosis and function. 3. Gourevitch DL, Clark L, Bedelbaeva K, Leferovich J, Heber-Katz E: Dynamic changes after murine digit amputation: the MRL mouse digit shows waves of tissue remodeling, growth, and apoptosis. Wound Repair Regen Conclusions 2009, 17:447–455. Herein the MRL background was shown to reduce fibrosis 4. Ueno M, Lyons BL, Burzenski LM, Gott B, Shaffer DJ, Roopenian DC, Shultz LD: Accelerated wound healing of alkali-burned corneas in MRL mice is in a chronic model of muscular dystrophy and cardiomy- associated with a reduced inflammatory signature. Invest Ophthalmol Vis opathy, the Sgcg mouse. Although membrane leak was still Sci 2005, 46:4097–4106. evident in Sgcg mice sharing a portion of the MRL gen- 5. Leferovich JM, Bedelbaeva K, Samulewicz S, Zhang XM, Zwas D, Lankford EB, Heber-Katz E: Heart regeneration in adult MRL mice. Proc Natl Acad Sci ome, the reduction in fibrosis was associated with USA 2001, 98:9830–9835. improved cardiac function. The identification of gene(s) 6. Naseem RH, Meeson AP, Michael Dimaio J, White MD, Kallhoff J, Humphries from the MRL genome will help identify pathways import- C, Goetsch SC, De Windt LJ, Williams MA, Garry MG, Garry DJ: Reparative myocardial mechanisms in adult C57BL/6 and MRL mice following ant for chronic repair of myopathic processes. injury. Physiol Genomics 2007, 30:44–52. 7. Abdullah I, Lepore JJ, Epstein JA, Parmacek MS, Gruber PJ: MRL mice fail to heal the heart in response to ischemia-reperfusion injury. Wound Repair Additional file Regen 2005, 13:205–208. 8. Cimini M, Fazel S, Fujii H, Zhou S, Tang G, Weisel RD, Li RK: The MRL mouse Additional file 1: Figure S1. Shown are gross images from Sgcg vs. heart does not recover ventricular function after a myocardial infarction. MRL/D2 Sgcg mice. Evans blue dye uptake could be readily seen in the Cardiovasc Pathol 2008, 17:32–39. quadriceps and diaphragm muscles and did not appear grossly altered 9. Grisel P, Meinhardt A, Lehr HA, Kappenberger L, Barrandon Y, Vassalli G: The by the presence of the MRL background. In contrast, fibrosis was visually MRL mouse repairs both cryogenic and ischemic myocardial infarcts MRL/D2 reduced in the quadriceps and heart of Sgcg compared to Sgcg with scar. Cardiovasc Pathol 2008, 17:14–22. MRL/D2 mice. The diaphragm muscle retained evidence of fibrosis in Sgcg , 10. Robey TE, Murry CE: Absence of regeneration in the MRL/MpJ mouse but the white stripes of fibrosis were smaller, and intact diaphragm heart following infarction or cryoinjury. Cardiovasc Pathol 2008, 17:6–13. muscle was still evident compared to the near total replacement of 11. Oh YS, Thomson LE, Fishbein MC, Berman DS, Sharifi B, Chen PS: Scar diaphragm muscle in Sgcg mice. Figure S2. Shown is staining for formation after ischemic myocardial injury in MRL mice. Cardiovasc Pathol apoptosis with TUNEL and caspase indicating no gross differences 2004, 13:203–206. MRL/D2 between Sgcg and Sgcg muscle. CD3 and MAC1 staining to 12. Harty M, Neff AW, King MW, Mescher AL: Regeneration or scarring: an examine T cell and macrophage infiltrate also did not appear grossly immunologic perspective. Dev Dyn 2003, 226:268–279. altered by the presence of the MRL background. 13. Gourevitch D, Clark L, Chen P, Seitz A, Samulewicz SJ, Heber-Katz E: Matrix metalloproteinase activity correlates with blastema formation in the regenerating MRL mouse ear hole model. Dev Dyn 2003, 226:377–387. Abbreviations 14. Heber-Katz E, Leferovich J, Bedelbaeva K, Gourevitch D, Clark L: The scarless BMP: Bone morphogenetic protein; D2: (DBA/2J); DMD: Duchenne muscular heart and the MRL mouse. Philos Trans R Soc Lond B Biol Sci 2004, dystrophy; HOP: Hydroxyproline; LTBP4: Latent TGFβ-binding protein; 359:785–793. MRL: Murphy Roth Large; QTL: Quantitative trait loci; SNP: Single nucleotide 15. Bedelbaeva K, Gourevitch D, Clark L, Chen P, Leferovich JM, Heber-Katz E: polymorphism; TGFβ: Transforming growth factor β. The MRL mouse heart healing response shows donor dominance in allogeneic fetal liver chimeric mice. Cloning Stem Cells 2004, 6:352–363. Competing interest 16. Heydemann A: The super super-healing MRL mouse strain. Front Biol The authors have no competing interests related to this work. 2012, in press. 17. Spencer MJ, Montecino-Rodriguez E, Dorshkind K, Tidball JG: Helper (CD4 (+)) and cytotoxic (CD8(+)) T cells promote the pathology of dystrophin- Authors’ contributions deficient muscle. Clin Immunol 2001, 98:235–243. AH conducted the phenotypic analysis of muscle. KAS conducted the 18. Hack AA, Ly CT, Jiang F, Clendenin CJ, Sigrist KS, Wollmann RL, McNally genome-wide SNP analysis and phenotype analysis. GK performed the EM: Gamma-sarcoglycan deficiency leads tomusclemembranedefects echocardiographic analysis. MH oversaw the breeding. JHC assisted with and apoptosis independent of dystrophin. JCellBiol 1998, phenotypic analysis. EMM conceived the experiments, analyzed the data and 142:1279–1287. wrote the manuscript. All authors read and approved the final manuscript. 19. Heydemann A, Huber JM, Demonbreun A, Hadhazy M, McNally EM: Genetic background influences muscular dystrophy. Neuromuscul Disord 2005, Acknowledgements 15:601–609. Supported by NIH R01HL61322 (EMM), R01HL102322 (AH) and NIH 20. Straub V, Rafael JA, Chamberlain JS, Campbell KP: Animal models for K08HL098565 (GK). muscular dystrophy show different patterns of sarcolemmal disruption. J Cell Biol 1997, 139:375–385. Author details 21. Heydemann A, Ceco E, Lim JE, Hadhazy M, Ryder P, Moran JL, Beier DR, Department of Medicine, Section of Cardiology, 5841 S. Maryland, MC 6088, Palmer AA, McNally EM: Latent TGF-beta-binding protein 4 modifies Chicago, IL 60637, USA. Department of Human Genetics, The University of muscular dystrophy in mice. J Clin Invest 2009, 119:3703–3712. Chicago, Chicago, IL 60637, USA. Current address: Department of Physiology 22. Matsuda R, Nishikawa A, Tanaka H: Visualization of dystrophic muscle and Biophysics, University of Illinois at Chicago, COMRB 2035, MC 901, 835 fibers in mdx mouse by vital staining with Evans blue: evidence of South Wolcott Ave, Chicago, IL 60612-7352, USA. apoptosis in dystrophin-deficient muscle. J Biochem (Tokyo) 1995, 118:959–964. Received: 24 May 2012 Accepted: 8 October 2012 23. 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Wheeler MT, Korcarz CE, Collins KA, Lapidos KA, Hack AA, Lyons MR, Zarnegar S, Earley JU, Lang RM, McNally EM: Secondary coronary artery vasospasm promotes cardiomyopathy progression. Am J Pathol 2004, 164:1063–1071. 27. Moran JL, Bolton AD, Tran PV, Brown A, Dwyer ND, Manning DK, Bjork BC, Li C, Montgomery K, Siepka SM, Vitaterna MH, Takahashi JS, Wiltshire T, Kwiatkowski DJ, Kucherlapati R, Beier DR: Utilization of a whole genome SNP panel for efficient genetic mapping in the mouse. Genome Res 2006, 16:436–440. 28. Collaborative Cross Consortium: The genome architecture of the Collaborative Cross mouse genetic reference population. Genetics 2012, 190:389–401. 29. Cheng R, Abney M, Palmer AA, Skol AD: QTLRel: an R package for genome-wide association studies in which relatedness is a concern. BMC Genet 2011, 12:66. 30. 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Naviaux RK, Le TP, Bedelbaeva K, Leferovich J, Gourevitch D, Sachadyn P, Zhang XM, Clark L, Heber-Katz E: Retained features of embryonic metabolism in the adult MRL mouse. Mol Genet Metab 2009, 96:133–144. • Convenient online submission • Thorough peer review doi:10.1186/2044-5040-2-26 • No space constraints or color figure charges Cite this article as: Heydemann et al.: The superhealing MRL background improves muscular dystrophy. Skeletal Muscle 2012 2:26. • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Skeletal Muscle Springer Journals

The superhealing MRL background improves muscular dystrophy

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Copyright © 2012 by Heydemann et al.; licensee BioMed Central Ltd.
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Life Sciences; Cell Biology; Developmental Biology; Biochemistry, general; Systems Biology; Biotechnology
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23216833
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Abstract

Background: Mice from the MRL or “superhealing” strain have enhanced repair after acute injury to the skin, cornea, and heart. We now tested an admixture of the MRL genome and found that it altered the course of muscle pathology and cardiac function in a chronic disease model of skeletal and cardiac muscle. Mice lacking γ-sarcoglycan (Sgcg), a dystrophin-associated protein, develop muscular dystrophy and cardiomyopathy similar to their human counterparts with limb girdle muscular dystrophy. With disruption of the dystrophin complex, the muscle plasma membrane becomes leaky and muscles develop increased fibrosis. Methods: MRL/MpJ mice were bred with Sgcg mice, and cardiac function was measured. Muscles were assessed for fibrosis and membrane leak using measurements of hydroxyproline and Evans blue dye. Quantitative trait locus mapping was conducted using single nucleotide polymorphisms distinct between the two parental strains. Results: Introduction of the MRL genome reduced fibrosis but did not alter membrane leak in skeletal muscle of the Sgcg model. The MRL genome was also associated with improved cardiac function with reversal of depressed fractional shortening and the left ventricular ejection fraction. We conducted a genome-wide analysis of genetic modifiers and found that a region on chromosome 2 was associated with cardiac, diaphragm muscle and abdominal muscle fibrosis. Conclusions: These data are consistent with a model where the MRL genome acts in a dominant manner to suppress fibrosis in this chronic disease setting of heart and muscle disease. Keywords: Cardiomyopathy, Fibrosis, MRL, Muscular dystrophy Background injury settings where the MRL background induces more Murphy Roths Large (MRL) mice are an inbred mouse rapid healing, multiple mechanisms have been impli- strain noted to have enhanced healing ability. This MRL cated to explain this phenomenon including decreased strain was initially discovered because of its rapid ability scar formation, altered inflammatory response, reduced to heal ear holes [1,2]. The MRL strain’s capacity to rap- apoptosis, increased proliferation, improved remodeling idly recover from injury has been seen for both digit and, in some settings, enhanced stem cell function wounding and corneal scarring [3,4]. The MRL strain [4,6,12-15]. Genetic data support that many different has been reported to reduce scar formation after acute mechanisms account for enhanced healing since more cardiac injury, including freeze injury and coronary ar- than 40 different genetic loci have been associated with tery ligation [5,6]. However, other studies have suggested aspects of the healing phenotype [14,16]. that larger scale acute cardiac injury cannot be overcome The dystrophin complex is composed of membrane- by the MRL strain’s healing capacity [7-11]. In those associated proteins that mediate membrane stability in heart and skeletal muscle. Mutations that disrupt expres- sion of dystrophin or its associated proteins the sarco- * Correspondence: emcnally@uchicago.edu Department of Medicine, Section of Cardiology, 5841 S. Maryland, MC 6088, glycans proteins cause progressive cardiac and skeletal Chicago, IL 60637, USA 2 muscle degeneration in humans and mouse models. At Department of Human Genetics, The University of Chicago, Chicago, IL the cellular level, the loss of dystrophin or the 60637, USA Full list of author information is available at the end of the article © 2012 Heydemann 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. Heydemann et al. Skeletal Muscle 2012, 2:26 Page 2 of 11 http://www.skeletalmusclejournal.com/content/2/1/26 sarcoglycans leads to a disrupted sarcolemmal mem- Methods brane that is abnormally leaky. Membrane permeability Animals results in increased intracellular calcium that triggers The Sgcg mouse was previously bred for ten generations proteolysis and necrosis. An inflammatory response also into the DBA/2J strain (000671, Jackson Laboratory, Bar contributes to muscle degeneration [17]. Muscle con- Harbor, ME) [18,19]. MRL/MpJ (000486, Jackson La- traction is thought to provoke submicroscopic disrup- boratory, Bar Harbor, ME) mice were bred to the Sgcg +/− tion of the sarcolemma, and in the absence of a normal animals to generate Sgcg F1 mice on a 50% MRL/50% dystrophin complex, this leads to dysfunction and de- DBA/2J background. F1 mice were interbred to generate struction of cardiomyocytes and skeletal myofibers. The an F2 cohort. To increase the number of mutant ani- reduction in contractile cells and the presence of fibrotic mals, Sgcg mice from the F2 generation were bred again scar tissue within the heart led to reduced cardiac con- to produce additional Sgcg mice from an F3 generation. tractile function and congestive heart failure. All mice were housed in uniform conditions in a single Mouse models of dystrophin or sarcoglycan mutations pathogen-free barrier facility. All animals used in this recapitulate the basic pathological defects seen in human study were housed and treated in accordance with the forms of these genetic disorders. Mice lacking γ- standards set by the University of Chicago Animal Care sarcoglycan were engineered by removing the first cod- and Use Committee. The number of mice used for histo- ing exon of the Sgcg gene and model muscular dystrophy pathological analysis is shown in Table 1. [18]. We previously introduced the Sgcg allele into the Mice were sacrificed at either 8 or 32 weeks for ana- DBA/2J genetic background and found that this back- lysis. Muscles were used for either the Evans blue dye ground confers a more severe phenotype [19]. The assay or the HOP assay. Dye uptake assays were per- enhanced severity is seen as increased scar formation, formed on triceps muscles, gastrocnemius/soleus mus- measured as hydroxyproline (HOP) content because this cles and gluteus/hamstrings groups of muscles. Dye modified amino acid is a marker of collagen. In skeletal uptake was performed on half of each quadriceps muscle, increased Evans blue dye uptake is monitored to muscle and half of the abdominal muscles since these reflect membrane leakiness [20]. Using these assays, we muscles are of sufficient size to perform both dye up- showed that the DBA/2J genetic background worsened take and fibrosis assays. Hydroxyproline (HOP) assays the disease process [19]. were performed on the diaphragm muscle, the cardiac Because of its role in wound healing and the basic ventricles isolated as a single unit, half of the abdom- similarities between common forms of cellular injury inal muscles and half of each quadriceps muscle. The and what is seen in muscular dystrophy and cardiomy- amount of muscle assayed varied with the size of the opathy, we introduced the MRL genome into the Sgcg muscle andrangedfrom30mg for thediaphragm model. We hypothesized that the indolent pace of cellu- muscle to 300 mg for the gluteus/hamstring muscle lar damage in this disorder could be abated by the MRL Table 1 Number of mice analyzed background. Sgcg mice were bred to MRL mice to gener- Trait Genotypes ate Sgcg mice on a mixed genetic background with 50% D2 MRL/D2 MRL/D2 contribution of the MRL strain. This breeding strategy Fibrosis Sgcg Sgcg Sgcg , 32wk produced Sgcg mice with 50% genetic background from Quadriceps 25 124 18 the MRL strain and 50% background of the DBA/2J Diaphragm 25 45 10 MRL/D2 strain, and these mice are referred to Sgcg .We Heart 27 125 9 found that a 50% contribution of the MRL genome D2 MRL/D2 MRL/D2 Membrane leak Sgcg Sgcg Sgcg , 32wk reduced fibrosis in the heart and skeletal muscle, con- Quadriceps 37 149 15 sistent with dominant genetic loci in the MRL back- ground. Interestingly, the MRL genome did not Gluteus 11 76 15 consistently reduce membrane leak, suggesting that the Triceps 14 110 15 MRL background does not exert its effect on myocyte Abdominals 14 74 15 membrane stability and instead acts downstream on re- Gastrocnemius/soleus 14 111 15 MRL/D2 modeling. Sgcg mice had improved cardiac func- D2 MRL/D2 Central nuclei Sgcg Sgcg tion so that they were now similar to wild-type mice. Quadriceps 6 7 We conducted a genome-wide scan using informative polymorphisms and identified a region on chromosome Diaphragm 6 4 D2 MRL/D2 2 that was associated with fibrosis in cardiac, diaphragm Fiber size variability Sgcg Sgcg and abdominal muscles. These data demonstrate that Quadriceps 7 9 genes within the MRL background are modifiers for car- Diaphragm 6 4 diopulmonary involvement in muscular dystrophy. Heydemann et al. Skeletal Muscle 2012, 2:26 Page 3 of 11 http://www.skeletalmusclejournal.com/content/2/1/26 group that included the semimembranosus, semitendi- were removed with a topical depilatory agent. Limb leads nosus and biceps femoris. were attached for electrocardiogram gating, and the ani- mals were imaged in the left lateral decubitus position with Evans blue dye uptake assay for membrane leak a Visual Sonics Vevo 770 machine using a 30-MHz high- Evans blue dye (Sigma, E-2129) was performed as frequency transducer. Body temperature was maintained described [19,21]. Evans blue dye (Sigma, E-2129) was using a heated imaging platform and warming lamps. dissolved in phosphate-buffered saline at 10 mg/ml and Anesthesia was variably delivered to maintain heart rates injected intraperitoneally at 5 μl/g body weight. Twenty throughout the procedure at a constant 380–420 beats per to 40 h later, the tissues were harvested, finely minced, minute. Two-dimensional images were recorded in para- weighed and incubated at 55°C in 1 ml formamide for sternal long- and short-axis projections, with guided M- 2 h before spectrophotometric absorbance was measured mode recordings at the midventricular level in both views. at 620 nm [22,23]. Results are reported as absorbance/ LV cavity size and wall thickness were measured in at least mg tissue. three beats from each projection and averaged. LV wall thickness, interventricular septum (IVS) and posterior wall Hydroxyproline assay for fibrosis (PW) thickness, and internal dimensions at diastole and The hydroxyproline (HOP) assay was performed as systole (LVIDd and LVIDs, respectively) were measured. described [19,21,24]. The tissue was minced, weighed LV fractional shortening [(LVIDd – LVIDs)/LVIDd] and and hydrolyzed overnight in 2 ml of 6 M hydrochloric relative wall thickness [(IVS thickness + PW thickness)/ acid at 110°C. Ten μl of this hydrolysate was mixed with LVIDd] were calculated from the M-mode measurements. 150 μl isopropanol, then 75 μl of 1.4% chloramine-T (Sigma, St Louis, MO) in citrate buffer and oxidized at Genetic and statistical analysis room temperature for 10 min. One ml of a 3:13 solution Single nucleotide polymorphisms (SNPs, n = 1,701) in- of Ehrlich’s reagent (3 g of 4-(dimethylamino) benzalde- formative between the parental DBA/2J and MRL/MpJ MRL/D2 hyde, Sigma, St Louis, MO; 10 ml ethanol; 675 μl sul- strains were genotyped in 80 Sgcg F2-F4 Sgcg ani- furic acid) to isopropanol was added, mixed and mals on the Illumina GoldenGate platform using the incubated for 30 min at 55°C followed by extinction Mutation Mapping and Developmental Analysis Panel measurement at 558 nm. A standard curve (0–5000 nM, (MMDAP) [27] and the Mouse Universal Genotyping trans-4-hydroxy-L-proline, Sigma, St Louis, MO) was Array (GeneSeek, Neogen Corp., Lansing, MI) [28]. R included in each assay. Results are reported as nM package QTLRel was used to perform whole-genome HOP/mg tissue. quantitative trait locus (QTL) mapping for each of the membrane permeability and fibrosis phenotypes and Immunofluorescence microscopy using sex as a covariate [29,30]. Significance thresholds Tissues were flash frozen in liquid nitrogen-cooled iso- were determined by 1,000 permutation tests. The 1.5- pentane and stored at 80°C; 7-μm sections were cut on a LOD drop support interval was calculated using cryostat and fixed to slides in ice-cold 100% methanol. QTLRel. Tests of normality and other statistics were cal- The following antibodies were used: dystrophin NCL- culated in Prism (GraphPad). Build 37.1 was used for DYS2 (Novocastra/Leica), PH3 04–817 (Millipore), CD3 genomic analysis. MON1003-1 (Monsanto), caspase 3 (BD Biosciences), For HOP and dye uptake assays, data were analyzed by MAC1 BD557395 (BD Biosciences) and eMHC F1.652 one-way, unpaired ANOVA with parametric methods (ATCC). The TUNEL kit was from Millipore. Central followed by the Tukey multiple comparison post-test nuclei and fiber size variability was determined blinded (Prism, Graphpad), and p < 0.05 was considered signifi- to genotype by analyzing ten randomly chosen fields of cant. Data from 197 Sgcg animals from an F3 intercross 40× magnification. Fiber size variability was compared between the DBA/2J and 129T2/SvEmsJ backgrounds D2/129 MRL/D2 using each animal’s coefficient of variability (standard (Sgcg ) was compared to the Sgcg cohort. deviation/mean). Results Echocardiography A 50% contribution of the MRL background reduces Twelve-week animals were evaluated by echocardiog- fibrosis in Sgcg mice raphy as described [25,26]. Investigators were blinded to To assess the MRL contribution to muscular dystrophy, genotype. To avoid the stress associated with conscious we used the MRL/MpJ substrain since it contains a wild- restraint, anesthetized animals were studied. Anesthesia type fas allele [1]. We also used mice lacking γ- was induced by 1% isoflurane in a closed chamber sarcoglycan (Sgcg null) since these mice are a model of (Ohmeda Fluotec 3; Matrix Medical, Orchard Park, NY) limb girdle muscular dystrophy 2C [18] and on the in 20% O delivered through a nose cone. Chest hairs DBA/2J background have a more severe phenotype, 2 Heydemann et al. Skeletal Muscle 2012, 2:26 Page 4 of 11 http://www.skeletalmusclejournal.com/content/2/1/26 reminiscent of what is seen in humans [19,21]. MRL/ fibrosis in aged (32 week) animals and found a persistent MRL/D2 MpJ mice were bred to Sgcg null and then interbred to reduction in fibrosis in the Sgcg mice (Figure 2, di- generate Sgcg null mice with a 50% MRL/MpJ and 50% agonal bars). The 129T2/SvEmsJ strain was previously DBA/2J contribution. In Sgcg null mice, like all dys- shown to suppress the severity of muscle pathology in Sgcg trophin complex-associated mutations, fibrosis and col- null mice [33]. In comparison, the MRL background lagen deposition is increased in the heart and muscle, similar to what is seen in human patients with similar mutations [18,31]. A 50% contribution from the MRL background reduced fibrosis in Sgcg heart and muscles (Figure 1). In Sgcg hearts, fibrosis was often seen grossly as large patchy white areas and with the introduction of the MRL background visible fibrosis was diminished so that the hearts were indistinguishable from those of wild-type mice (Additional file 1: Figure S1). Of all the muscle groups analyzed, only diaphragm muscle retained any visible fibrosis. Diaphragm muscle is the most con- sistently damaged muscle in multiple mouse models, in- cluding this model, and the mdx model of Duchenne muscular dystrophy [32]. Therefore, it is possible that the degree of injury in this muscle overwhelms the heal- ing properties of the MRL background. We quantified fibrosis by measuring HOP content as D2 MRL/D2 an indicator of collagen in Sgcg and Sgcg mus- MRL/D2 cles. Sgcg mice have significantly reduced fibrosis D2 compared to Sgcg mice (Figure 2). We also evaluated Figure 2 The MRL genome quantitatively reduces fibrosis in Figure 1 The MRL genome suppresses fibrosis in the heart and Sgcg mice. Hydroxyproline (HOP) is a measure of fibrosis and is D2 muscles of Sgcg mice. Sgcg mice lack the dystrophin-associated represented on the y axis (mM/mg). In the heart, diaphragm and protein, γ-sarcoglycan, and when in the DBA/2J (D2) background quadriceps muscles, fibrosis is significantly (p < 0.001) reduced in MRL/D2 have a more severe phenotype with enhanced membrane leak and intercrossed Sgcg (D2/MRL) animals compared to Sgcg in the D2 fibrosis [21]. A 50% contribution of the MRL genome, referred to as (D2) background at 8 weeks. Fibrosis remains significantly (***p <0.001) MRL/D2 MRL/D2 Sgcg , suppressed fibrosis in both heart and skeletal muscle. reduced in Sgcg animals at the 32-week time point. Heydemann et al. Skeletal Muscle 2012, 2:26 Page 5 of 11 http://www.skeletalmusclejournal.com/content/2/1/26 suppressed fibrosis more than the 129T2/SvEmsJ back- We compared suppression of dye uptake by the MRL ground (Figure 3). The MRL background dramatically background to that induced by the 129T2/SvEmsJ back- reduced fibrosis in the heart and abdominal muscles com- ground in Sgcg mice (Figure 6). The quadriceps and ab- pared to 129T2/SvEmsJ. The MRL background also sup- dominal muscles from Sgcg null mice with a contribution pressed fibrosis more than the 129T2/SvEmsJ background from the 129T2/SvEmsJ background showed increased in the diaphragm and quadriceps muscles, but to a lesser membrane leak compared to Sgcg mice with an MRL con- degree. This finding suggests that the phenotypically bene- tribution. However, the triceps muscle group from the ficial genetic modifiers in theMRL genome maybemore MRL background showed increased dye uptake, and the potent than those in the 129T2/SvEmsJ genome. gluteus/hamstring muscle group showed no significant dif- ference. Thus, there was no consistent suppression of The MRL background does not protect against membrane membrane leakiness by the MRL strain. leak Disruption of the membrane-associated dystrophin com- The MRL genome may promote skeletal muscle plex renders the sarcolemma unusually fragile, leading to regeneration abnormal membrane leakage that is visualized by uptake of The MRL background is thought to exert part of its effect the nonspecific vital tracer Evans blue dye [20]. Upon gross by promoting regeneration [34]. We examined embryonic MRL/D2 inspection, the muscles from the Sgcg mice displayed myosin heavy chain (eMHC) expression as a reflection of MRL/D2 high levels of dye uptake, comparable to what was observed regeneration. Sgcg muscle shows patchy areas with a in the parental Sgcg mice (Additional file 1: Figure S1). On qualitative increase in eMHC-positive fibers compared to a microscopic level, dye-positive cells were readily detect- Sgcg (Figure 7), and wild-type muscle showed no eMHC MRL/D2 able in Sgcg muscle and heart (Figure 4). Evans blue positive fibers (data not shown). In dystrophic skeletal dyelevelsweremeasuredinmultiplemusclegroupsand muscle, ongoing regeneration is thought to offset degener- MRL/D2 were not significantly different between Sgcg and ation. Consistent with this, eMHC-positive regions were D2 Sgcg muscles for quadriceps, triceps, gastrocnemius/ also positive for Evans blue dye uptake, indicative of soleus, gluteus and abdominals muscle groups (Figure 5). muscle damage. We also evaluated phosphorylated histone We also measured dye uptake in older animals at 32 weeks 3 (PH3) as a reflection of mitotic index. No clear differ- MRL/D2 to assess disease progression. At 32 weeks, the skeletal ences for PH3 staining were seen between Sgcg and muscles continued to show comparable levels of membrane Sgcg muscle. Regenerating skeletal muscle, whether from leak as what was seen at 8 weeks (Figure 5). The gluteus/ trauma or muscular dystrophy, is identified by the presence hamstring group of muscles showed an increased of dye of myofibers with centrally positioned nuclei. In muscular uptake compared to the 8-week animals. This increase was dystrophy, ongoing regeneration is also marked by MRL/D2 not seen for other muscle groups. increased fiber size variability. Sgcg mice have an Figure 3 The reduction of fibrosis in intercrossed Sgcg animals is specific to the MRL genome. Hydroxyproline (HOP) is a measure of fibrosis and is represented on the y axis (mM/mg). Fibrosis is significantly reduced in the heart (***p < 0.001), diaphragm (p < 0.001), quadriceps (*p = 0.041) and abdominal muscles (p < 0.001) of Sgcg animals intercrossed in the D2/MRL background compared to those intercrossed in the D2/129 background. Heydemann et al. Skeletal Muscle 2012, 2:26 Page 6 of 11 http://www.skeletalmusclejournal.com/content/2/1/26 Improved cardiac function from a 50% contribution of the MRL background We performed 2D and M mode echocardiography on MRL/D2 D2 Sgcg and Sgcg mice at 12 weeks of age. Both frac- tional shortening and the left ventricular ejection fraction D2 were significantly reduced in Sgcg mice, indicating that the increased fibrosis impairs cardiac function (Table 2). Fractional shortening and the left ventricular ejection frac- MRL/D2 MRL/D2 tion were similar between Sgcg and WT mice, consistent with improved function mediated by 50% of the MRL genome. Wall thickness was also increased in Sgcg mice compared to the wild type of the same background. In contrast, wall thickness was not different between MRL/D2 MRL/D2 Sgcg and WT mice. Chromosome 2 associates with reduced fibrosis Sgcg heart and diaphragm muscle We conducted a genome-wide scan using markers that were informative in the two parental strains DBA/2J and MRL/MpJ (Figure 8). QTLRel was used to identify regions of association; this analysis takes into account the relatedness of individual animals in the cohort [29,30]. A region on chromosome 2 was identified that associated with fibrosis in the heart. The 1.5-LOD drop interval of this region spans from 64.8008−73.1758 cM in mouse genome build 37.1. This same region was also associated with fibrosis in the diaphragm muscle and also for fibrosis in the abdominal muscles. It should be noted that the significance of these associations is sug- Figure 4 Membrane leak is not corrected by the presence of gestive (p < 0.63) when using the stringent QTLRel ana- the MRL genome in Sgcg heart and muscle. Evans blue dye is found in cardiomyocytes and skeletal myofibers reflecting lysis. However, the overlapping intervals found in heart, membrane leakiness. Dye uptake occurs in patchy pattern diaphragm and abdominal muscles provide additional throughout the heart and muscle and is seen as opacified cells that support that this interval modifies fibrosis. fluoresce red. Nuclei are shown in blue and dystrophin in green. Ltbp4 polymorphism does not account for the MRL increased number of central nuclei in quadriceps and dia- healing properties D2 phragm muscle compared to Sgcg mice (Figure 7). These The DBA/2J strain contains an insertion/deletion poly- same muscles also showed increased fiber size variability morphism within the Ltbp4 gene [21] that modifies both when there is a contribution from the MRL background. membrane permeability and fibrosis, as two independent These data are consistent with a model in which favorable traits, in Sgcg muscular dystrophy. Ltbp4 encodes latent matrix remodeling may support enhanced regeneration. TGFβ-binding protein 4, and TGFβ proteins have been We also assessed apoptosis using a TUNEL assay and extensively linked to fibrosis in many disease states in- caspase 3 staining and again found no differences be- cluding muscular dystrophy [35,36]. Most murine MRL/D2 tween Sgcg and Sgcg muscle, suggesting that strains, including the MRL/MpJ strain used here, contain gross differences in programmed cell death are unlikely the protective Ltbp4 allele with an additional 12 amino MRL/D2 to account for the improved healing of the MRL strain acids inserted in exon 12. Because the Sgcg cohort (Additional file 1: Figure S2). We also characterized used here contained both the protective (Ltbp allele) whether T cell infiltration or macrophage infiltration and the disease-enhancing allele (Ltbp4 ), we tested MRL/D2 differed qualitatively between Sgcg and Sgcg whether Ltbp4 genotype correlated with fibrosis in the MRL/D2 muscle, but we found no clear differences between Sgcg cohort. Table 3 shows that neither membrane MRL/D2 Sgcg and Sgcg muscle, suggesting other features permeability nor fibrosis is correlated with the Ltbp4 MRL/DBA2J contribute to the MRL background’seffectonmuscular genotype in the Sgcg cohort. Therefore, Ltbp4 dystrophy (Additional file 1: Figure S2). does not account for the suppressive effect of the MRL Heydemann et al. Skeletal Muscle 2012, 2:26 Page 7 of 11 http://www.skeletalmusclejournal.com/content/2/1/26 Figure 5 The MRL genome does not significantly reduce membrane damage in Sgcg muscle. Evans blue dye uptake is a measure of membrane damage and is represented on the y axis (absorbance/mg). In the quadriceps, triceps, abdominal and gastrocnemius/soleus muscles, D2 MRL/D2 membrane damage is not significantly different between Sgcg animals and intercrossed Sgcg animals at both the 8- and 32-week time points. In the gluteus/hamstring muscles, membrane damage is significantly (**p < 0.01) increased at the 32-week time point in intercrossed Sgcg MRL/D2 animals compared to the 8-week time point. Figure 6 The MRL genome has a variable ability to reduce membrane leak in muscular dystrophy compared to the 129T2/SvEmsJ strain. Evans blue dye uptake was measured in multiple muscle groups represented on the y axis (absorbance/mg). Membrane damage is D2/MRL reduced in the quadriceps (**p = 0.0053) and abdominal (**p = 0.0012) muscles of intercrossed Sgcg animals compared to intercrossed D2/129 MRL/D2 Sgcg mice. Membrane damage is increased in the triceps muscle (*p = 0.0298) of intercrossed Sgcg animals. There is no difference in membrane damage in the gluteus/hamstring muscle. Heydemann et al. Skeletal Muscle 2012, 2:26 Page 8 of 11 http://www.skeletalmusclejournal.com/content/2/1/26 Figure 7 Regeneration may be enhanced in the MRL background. Embryonic myosin heavy chain (eMHC) staining was increased in MRL/D2 Sgcg compared to Sgcg muscle, and eMHC fibers were seen in areas where dye uptake was seen. However, phosphorylated histone 3 (PH3), MRL/D2 a marker of mitotic index, was not increased in Sgcg compared to Sgcg muscle. Skeletal muscle central nucleation and fiber size variability MRL/D2 D2 are enhanced by the MRL background. Sgcg muscle demonstrated an increase in centrally nucleated fibers compared to Sgcg mice. Fiber size variability was reflected in the coefficient of variability of fiber diameter measurements. The solid line indicates significance (p < 0.05). These data support that the MRL exerts its effect in skeletal muscle, at least in part, by promoting regeneration. background, and other genetic modifiers account for this The MRL strain typically shows the improved healing difference. capability in its first 2–6 months of life. After 6 months of age, wild-type MRL mice develop autoimmune disorders; Discussion it is for this reason that we conducted studies using a 50% The MRL genome protects against fibrosis but not contribution of the MRL strain. Quantitative trait mapping membrane leak has been used to define many different genetic regions Mutations in the dystrophin or sarcoglycan genes share a associated with the healing properties [14]. The auto- common pathological mechanism characterized by disrup- immune properties have been genetically separated from tion of the plasma membrane of cardiomyocytes and skel- the healing properties since the four to six different “auto- etal myofibers. Membrane leak is an early step in the immune” genetic loci do not overlap with those associated pathological process while fibrosis is thought to be a sec- with improved healing. In our studies, only 50% of the ondary response. Our study suggests that there are genetic MRL genome was capable of suppressing fibrosis and im- pathways that target fibrosis without altering the sarco- proving function. In the course of these studies, we never MRL/D2 lemmal leak properties. In the heart, reduced fibrosis was identified a single Sgcg animal that had markedly ele- correlated with functional benefit, although cardiac func- vated fibrosis. This stands in contrast to what is normally tion may be improved from both cardiac intrinsic as well observed in the Sgcg animals where outliers with markedly as cardiac extrinsic features. For example, improved skel- elevated fibrosis are often seen. The maximum values for MRL/D2 etal muscle function may contribute to improved cardiac fibrosis in Sgcg mice were all well below what was D2 function, particularly when considering the contribution measured in Sgcg mice. This is consistent with multiple, of the respiratory musculature. dominant genetic loci imparting the improved healing cap- abilities, similar to what has been observed for the ear hole repair properties in the MRL strain [37]. The finding that Table 2 Cardiac function in Sgcg mice MRL/D2 improvement persists in the Sgcg mice, with almost Fractional shortening complete suppression of fibrosis at 32 weeks, suggests that D2 a Sgcg (n = 4) 33.87 ± 4.80 a persistent healing effect can arise from the MRL strain. D2 a WT (n = 5) 44.58 ± 7.65 MRL/D2 Sgcg (n = 5) 46.00 ± 14.4 Chromosome 2 candidate genes MRL/D2 WT (n = 5) 48.37 ± 11.21 When considering the overlapping region on chromo- p = 0.046. some 2 from the heart, diaphragm and abdominal Heydemann et al. Skeletal Muscle 2012, 2:26 Page 9 of 11 http://www.skeletalmusclejournal.com/content/2/1/26 fibrosis data, the interval contains 49 known genes. There are additional predicted and unnamed genes and genes encoding olfactory receptors that are not consid- ered likely candidates. Within the interval is Jag1, encod- ing jagged the ligand for the Notch receptor. Loss of function of Notch 3 leads to muscle hyperplasia, espe- cially when subjected to repetitive injury [38]. Ex vivo activation of Notch signaling helps maintain donor cell engraftment during myoblast transfer [39]. Another gene linked to growth and healing in the interval is Bmp2-en- coding bone morphogenetic protein 2. The BMPs medi- ate musculoskeletal regeneration [40], and given the MRL background effect on multiple cells and tissues, BMP2 is well positioned to contribute to the MRL superhealing response. That said, this chromosome 2 interval has not previously been linked to other MRL healing properties. The strict criteria imposed by the QTLRel algorithm makes it unlikely that these results derive from relatedness of animals within the cohort. Cardiac injury and repair in the MRL strain The ability of the heart to recover after injury has been studied in the MRL strain using cryoinjury, left anterior descending ligation and ischemia reperfusion methods [5,6,8-10,14,15]. However, scar reduction has been noted after some forms of injury while not after others. In common to all these studies is an acute injury model where a normal heart was substantially damaged in a single setting. The MRL’s ability to heal the heart may be limited such that larger amounts of injury may be insur- mountable, as proposed by Naseem [6]. Our data sup- port that lower, although persistent, levels of injury can be managed by the MRL strain where there is significant reduction in fibrosis and a corresponding functional im- provement in cardiac function. Mechanisms for MRL healing Figure 8 Fibrosis in the heart, diaphragm and abdominal A number of mechanisms likely act in concert to achieve muscles is modified by a locus on chromosome 2. Genome-wide MRL/D2 suppression of fibrosis and improvement of function. In association was examined for fibrosis in Sgcg progeny in the (A) cardiac muscle (n = 65), (B) diaphragm (n = 78) and (C) skeletal muscle, where muscle stem cells robustly regen- abdominal muscle (n = 78). Chromosomes are plotted on the x axis, erate muscle after injury, there is indirect evidence of and the informative SNPs tested (n = 1,707) are displayed as open enhanced regeneration. Specifically, there is an increase circles that alternate color by chromosome; LOD scores are in centrally nucleated myofibers, which is thought to re- represented on the y axis. Overlapping regions on chromosome 2 showed suggestive (p < 0.63) association with fibrosis in heart, flect enhanced myoblast fusion. However, our data do diaphragm and abdominal muscle. not distinguish whether the MRL background exerts its effect on muscle regenerative cells, or by creating a more supportive extracellular matrix or both. MRL animals heal with embryonic characteristics with enhanced blas- MRL/D2 Table 3 Ltbp4 genotype in Sgcg mice tema formation and metabolic and gene expression fea- tures consistent with an earlier developmental state [41]. Dye uptake Fibrosis D/D Altered protease expression has also been noted in the Ltbp4 (n = 14) 3.90 ± 0.83 6.00 ± 1.81 MRL mouse in response to damage [5]. Recent work D/I Ltbp4 (n = 34) 4.18 ± 0.84 8.05 ± 3.02 characterizing the immune infiltrate in muscular dys- I/I Ltbp (n = 47) 4.00 ± 0.85 6.64 ± 2.15 trophy found that reducing osteopontin was effective at Heydemann et al. Skeletal Muscle 2012, 2:26 Page 10 of 11 http://www.skeletalmusclejournal.com/content/2/1/26 suppressing fibrosis [17]. Together these data may favor 2. McBrearty BA, Clark LD, Zhang XM, Blankenhorn EP, Heber-Katz E: Genetic analysis of a mammalian wound-healing trait. 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Proc Natl Acad Sci ome, the reduction in fibrosis was associated with USA 2001, 98:9830–9835. improved cardiac function. The identification of gene(s) 6. Naseem RH, Meeson AP, Michael Dimaio J, White MD, Kallhoff J, Humphries from the MRL genome will help identify pathways import- C, Goetsch SC, De Windt LJ, Williams MA, Garry MG, Garry DJ: Reparative myocardial mechanisms in adult C57BL/6 and MRL mice following ant for chronic repair of myopathic processes. injury. Physiol Genomics 2007, 30:44–52. 7. Abdullah I, Lepore JJ, Epstein JA, Parmacek MS, Gruber PJ: MRL mice fail to heal the heart in response to ischemia-reperfusion injury. Wound Repair Additional file Regen 2005, 13:205–208. 8. Cimini M, Fazel S, Fujii H, Zhou S, Tang G, Weisel RD, Li RK: The MRL mouse Additional file 1: Figure S1. Shown are gross images from Sgcg vs. heart does not recover ventricular function after a myocardial infarction. MRL/D2 Sgcg mice. Evans blue dye uptake could be readily seen in the Cardiovasc Pathol 2008, 17:32–39. quadriceps and diaphragm muscles and did not appear grossly altered 9. Grisel P, Meinhardt A, Lehr HA, Kappenberger L, Barrandon Y, Vassalli G: The by the presence of the MRL background. In contrast, fibrosis was visually MRL mouse repairs both cryogenic and ischemic myocardial infarcts MRL/D2 reduced in the quadriceps and heart of Sgcg compared to Sgcg with scar. Cardiovasc Pathol 2008, 17:14–22. MRL/D2 mice. The diaphragm muscle retained evidence of fibrosis in Sgcg , 10. Robey TE, Murry CE: Absence of regeneration in the MRL/MpJ mouse but the white stripes of fibrosis were smaller, and intact diaphragm heart following infarction or cryoinjury. Cardiovasc Pathol 2008, 17:6–13. muscle was still evident compared to the near total replacement of 11. Oh YS, Thomson LE, Fishbein MC, Berman DS, Sharifi B, Chen PS: Scar diaphragm muscle in Sgcg mice. Figure S2. Shown is staining for formation after ischemic myocardial injury in MRL mice. Cardiovasc Pathol apoptosis with TUNEL and caspase indicating no gross differences 2004, 13:203–206. MRL/D2 between Sgcg and Sgcg muscle. CD3 and MAC1 staining to 12. Harty M, Neff AW, King MW, Mescher AL: Regeneration or scarring: an examine T cell and macrophage infiltrate also did not appear grossly immunologic perspective. Dev Dyn 2003, 226:268–279. altered by the presence of the MRL background. 13. Gourevitch D, Clark L, Chen P, Seitz A, Samulewicz SJ, Heber-Katz E: Matrix metalloproteinase activity correlates with blastema formation in the regenerating MRL mouse ear hole model. Dev Dyn 2003, 226:377–387. Abbreviations 14. Heber-Katz E, Leferovich J, Bedelbaeva K, Gourevitch D, Clark L: The scarless BMP: Bone morphogenetic protein; D2: (DBA/2J); DMD: Duchenne muscular heart and the MRL mouse. Philos Trans R Soc Lond B Biol Sci 2004, dystrophy; HOP: Hydroxyproline; LTBP4: Latent TGFβ-binding protein; 359:785–793. MRL: Murphy Roth Large; QTL: Quantitative trait loci; SNP: Single nucleotide 15. Bedelbaeva K, Gourevitch D, Clark L, Chen P, Leferovich JM, Heber-Katz E: polymorphism; TGFβ: Transforming growth factor β. The MRL mouse heart healing response shows donor dominance in allogeneic fetal liver chimeric mice. Cloning Stem Cells 2004, 6:352–363. Competing interest 16. Heydemann A: The super super-healing MRL mouse strain. Front Biol The authors have no competing interests related to this work. 2012, in press. 17. Spencer MJ, Montecino-Rodriguez E, Dorshkind K, Tidball JG: Helper (CD4 (+)) and cytotoxic (CD8(+)) T cells promote the pathology of dystrophin- Authors’ contributions deficient muscle. Clin Immunol 2001, 98:235–243. AH conducted the phenotypic analysis of muscle. KAS conducted the 18. 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Naviaux RK, Le TP, Bedelbaeva K, Leferovich J, Gourevitch D, Sachadyn P, Zhang XM, Clark L, Heber-Katz E: Retained features of embryonic metabolism in the adult MRL mouse. Mol Genet Metab 2009, 96:133–144. • Convenient online submission • Thorough peer review doi:10.1186/2044-5040-2-26 • No space constraints or color figure charges Cite this article as: Heydemann et al.: The superhealing MRL background improves muscular dystrophy. Skeletal Muscle 2012 2:26. • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit

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Skeletal MuscleSpringer Journals

Published: Dec 5, 2012

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