Genetic modifiers of muscular dystrophy act on sarcolemmal resealing and recovery from injury
[['Muscular dystrophies are inherited diseases that cause progressive muscle wasting [1] .', 'Many muscular dystrophies are caused by mutations in genes encoding for components of the dystrophin glycoprotein complex , which anchors the actin cytoskeleton of myofibers to their cell membrane , the sarcolemma .', 'Loss of function mutations in the DMD gene cause Duchenne muscular dystrophy ( DMD ) , while mutations in the SGCG gene , which encodes the dystrophin associated protein γ-sarcoglycan , cause limb-girdle muscular dystrophy type 2C ( LGMD 2C ) [2 , 3] .', 'Disruption of the dystrophin complex results in loss of membrane integrity , leading to chronic injury and necrosis of myofibers [4] .', 'Detrimental remodeling , with replacement by fibrofatty tissue , leads to ongoing , progressive impairment of muscle function [1] .', 'This pathological process begins with disruption of the sarcolemma , and mechanisms to enhance sarcolemmal repair may provide insight in possible therapeutic targets for treating muscular dystrophy .', 'Disease progression in the muscular dystrophies is variable even in the presence of the same primary mutation , suggesting that secondary genetic variants , or genetic modifiers , can considerably impact the outcome of muscle wasting [5] .', 'The effect of modifiers is evident in murine models of muscular dystrophy , where the same genetic mutation results in significantly different outcomes dependent on the genetic background of the mouse strain [6] .', 'Dystrophin deficiency is modeled in mice by the mdx mutation , a premature stop codon in exon 23 of the dystrophin gene , while γ-sarcoglycan deficiency is modeled by mice lacking exon 2 of the Sgcg gene [3 , 7] .', 'mdx and Sgcg mutations have been shown to cause muscular dystrophy with strain-dependent variable pathology , which is severe in the DBA/2J genetic background , intermediate in the C57/Bl6-Bl10 strains , and more mild in the 129T2/SvEmsJ ( 129T2 ) background [6 , 8 , 9] .', 'Identification of genetic modifiers and their mechanisms of action is a useful approach to refine prognosis and potentially discover novel therapeutic targets .', 'Several candidate modifiers act as extracellular agonists of signaling cascades , including osteopontin , encoded by the SPP1 gene , and latent TGFβ binding protein 4 ( LTBP4 ) .', 'Osteopontin is a secreted glycoprotein that signals through integrin and CD44 receptors [10] .', 'SPP1 expression is highly upregulated in affected muscles of humans and animals with muscular dystrophy [11–19] .', 'Genetic loss of Spp1 in mdx mice correlates with greater strength , less fibrosis and milder pathology , as compared to control mdx littermates [13] .', 'Moreover , Spp1 ablation has been linked to a shift in macrophage polarization towards a regenerative phenotype in mdx muscles [20] .', 'In humans with DMD , a single nucleotide polymorphism ( SNP ) in the SPP1 promoter ( GG/TG ) correlates with increased grip strength and later loss of ambulation compared to patients with the more prevalent SNP ( TT ) , especially in DMD individuals who are steroid treated [19 , 21 , 22] .', 'Some genetic cohort studies have not shown this same effect [23 , 24] .', 'The manner in which the SPP1 SNP affects osteopontin expression with disease progression is complex , and it is unclear in which SPP1-expressing cell type ( s ) this modifier SNP acts .', 'Ltbp4 was identified as genetic modifier of several pathologic traits in the Sgcg mouse model , including sarcolemmal damage and fibrosis [25] .', 'Latent TGFβ binding protein ( LTBP4 ) is an extracellular protein that binds TGFβ , releasing it upon proteolysis of its hinge region [26] .', 'The LTBP4 modifier also correlates with differential outcomes in humans with muscular dystrophy [21 , 23 , 24] .', 'In mice , the “risk” Ltbp4 allele encodes a shorter hinge region that is more susceptible to proteolysis , and this risk allele is found in the DBA/2J strain correlating with more severe muscular dystrophy .', 'In contrast , most mouse strains including 129T2 and C57 substrains have the protective Ltbp4 allele encoding a longer hinge region that is more resistant to proteolytic cleavage .', 'Overexpression of the protective Ltbp4 allele in the mdx mouse reduces fibrosis and promotes muscle growth [26] .', 'However , the specific molecular effects of protective and deleterious Ltbp4 alleles on sarcolemmal resealing and repair are still unknown .', 'Intriguingly , it has been shown that the Spp1 promoter is susceptible to TGFβ-driven transcriptional activation [27 , 28] .', 'In mesenchymal cells , osteopontin signaling activates Tgfb1 transcription via the myeloid zinc finger 1 ( MZF1 ) transcriptional factor [29 , 30] .', 'These data support a potential interaction between these two modifiers , Spp1 and Ltbp4 , in muscular dystrophy .', 'Examining the combinatorial effects of genetic modifiers requires a large population , which is challenging for a rare human disorder like DMD .', 'Here we asked whether genetic manipulation of osteopontin and Ltbp4 impacts sarcolemmal resealing and repair .', 'To specifically address this question , we relied on optimized conditions of sarcolemmal micro-injury , and real-time detection of sarcolemmal damage and repair cap formation in isolated live myofibers [31 , 32] .', 'Laser-mediated injury results in larger sarcolemmal damage in dystrophic myofibers than in wildtype myofibers using age- and background-matched conditions [33] .', 'We found that both osteopontin and Ltbp4 modify sarcolemmal repair in wildtype and dystrophic myofibers .', 'Furthermore , we documented how osteopontin and the deleterious Ltbp4 isoform converge in a feed-forward TGFβ signaling loop that correlated with transcriptional repression of annexin genes and impaired sarcolemmal repair .', 'In addition , we dissected the relative impact of the different alleles of Ltbp4 and a third modifier Anxa6 on resealing and repair of injured sarcolemma in background-strain matched conditions .', 'These results indicate that osteopontin and LTBP4 regulate a TGFβ signaling loop to modify sarcolemmal repair in normal and dystrophic muscle .'], ['To gain insight in the role of osteopontin ( OPN ) in myofiber repair , we assessed the effect of recombinant osteopontin ( rOPN ) on sarcolemmal repair of isolated myofibers [32] .', 'We tested the repair capacity after laser-mediated sarcolemmal injury and quantified two parameters in real time:', '( i ) accumulation of the FM4-64 dye at the injury site , marking the extent of damage;', '( ii ) formation of the repair complex monitoring GFP-tagged annexin A1 ( ANXA1-GFP ) protein [31] .', 'DBA/2J wildtype ( WT ) mice were injected intramuscularly with 10μg rOPN at 1 , 3 and 5 days after electroporation with the plasmid encoding ANXA1-GFP .', 'The activity of injected rOPN was confirmed by monitoring expression of Mmp2 and Mzf1 , factors known to be downstream of OPN [30 , 34] .', 'Mmp2 and Mzf1 were increased 48 hours after rOPN injection while vehicle injection did not stimulate this response ( S1A Fig ) .', 'Seven days after electroporation , live myofibers from treated and vehicle injected control muscles were subjected to laser injury .', 'FM4-64 accumulation , which marks the area of injury , was greater in rOPN injected muscle at 240 seconds after laser injury , as compared to vehicle-treated myofibers ( Fig 1A ) .', 'Moreover , the onset of the annexin A1 repair cap formation was significantly slower in rOPN-injected muscle than control myofibers , resulting in a significantly smaller annexin A1 cap size at end-point ( Fig 1B ) .', 'Thus , intramuscular injection of DBA/2J WT muscle with rOPN exacerbated sarcolemmal injury and slowed formation of the annexin-containing repair complex .', 'Genetic ablation of Spp1 in mdx mice results in decreased fibrosis and muscle pathophysiology in mdx mice [13] .', 'We asked whether Spp1 loss had a measurable effect on sarcolemmal repair of mdx myofibers .', 'To this end , we compared myofibers from 20 week-old mdx ( Spp1+/+; control animals ) and age-matched Spp1-deficient mdx ( mdx/Spp1-/- ) littermates for the extent of sarcolemmal damage after laser injury .', 'Myofibers from both mice groups were electroporated with the ANXA1-GFP-encoding plasmid prior to harvest and laser-injury assay .', 'Spp1-deficient dystrophic myofibers showed significantly reduced FM4-64 accumulation at the injury site over time , and a reduction of injury area at end-point when compared to control myofibers ( Fig 2A ) .', 'We monitored annexin A1 repair cap appearance at the site of injury and observed faster rate of cap appearance in mdx/Spp1-/- compared to mdx control myofibers , resulting in a larger cap size at analysis end-point ( Fig 2B ) .', 'Furthermore , expression levels of endogenous Anxa1 and Anxa6 , encoding the repair cap proteins annexins A1 and A6 , were significantly upregulated in muscle of mdx/Spp1-/- mice , as compared to littermate control animals ( S2A Fig ) .', 'Moreover , improved sarcolemmal repair in isolated fibers was also reflected in in vivo findings in muscle .', 'Spp1 deficiency correlated with lower levels of fibrosis , quantified as hydroxyproline content , in both quadriceps and diaphragm muscles , as well as with reduced levels of circulating creatine kinase , a marker of striated muscle damage ( S2B and S2C Fig ) , consistent with previous characterization of mdx/Spp1-/- mice [13] [20] .', 'In addition to Spp1’s role in dystrophic muscle remodeling , osteopontin exacerbates injury in toxin-injured wildtype muscles [35] .', 'To assess Spp1’s role in chronic and acute injury of dystrophic muscle , the tibialis anterior ( TA ) muscles of mdx and mdx/Spp1-/- mice were injected with cardiotoxin along the muscle axis , immediately after systemic delivery of Evans Blue Dye ( EBD ) .', 'Each mouse received cardiotoxin in one muscle , while the contralateral remained uninjected .', 'After three hours , muscles were harvested and injury extent was quantified as EBD-positive myofibers per muscle using serial cross-sections .', 'There were fewer EBD-positive myofibers in mdx/Spp1-/- muscles than in mdx muscles , both under basal conditions and after toxin-induced injury ( Fig 3A and 3B ) .', 'Thus , these data suggest that sarcolemmal repair is enhanced and muscle injury is reduced after genetic ablation of osteopontin .', 'The Spp1 promoter is responsive to TGFβ signaling , and the TGFβ1 ligand increases Spp1 levels [27 , 28] .', 'We therefore asked whether TGFβ signaling was altered in mdx/Spp1-/- muscles .', 'To this end , we compared mdx/Spp1-/- and control mdx muscles for expression levels of TGFβ pathway genes and for enrichment of myonuclei positive for phosphorylated SMAD3 ( pSMAD3 ) , a known effector of active TGFβ signaling [36] .', 'Quantitative PCR ( qPCR ) analysis of TA muscles showed that ligands , receptors and downstream factors of the TGFβ pathway , including transcriptional repressors Slug and Snail [37 , 38] , were significantly downregulated in mdx/Spp1-/- mice as compared to mdx ( Fig 4A ) .', 'Immunofluorescence microscopy ( IFM ) of quadriceps and diaphragm muscles showed that the relative ratio of pSMAD3+ myonuclei was significantly reduced in both muscles of mdx/Spp1-/- mice compared to mdx control animals ( Fig 4B ) .', 'Thus , genetic loss of Spp1 correlated with decreased TGFβ signaling in multiple mdx muscles .', 'In non-muscle mesenchymal cells , OPN has been shown to stimulate TGFβ1 upregulation via integrin signaling and the transcriptional factor Mzf1 [29 , 30] .', 'We hypothesized a feed-forward model , in which OPN sustains TGFβ signaling in muscle and its downstream transcriptional factors Slug and Snail , which bind E-box DNA elements of target genes ( Fig 5A ) [39] .', 'Through sequence alignment , we identified a predicted E-box element ( GTCGAC motif ) [39] upstream of the transcriptional start site of Anxa1 ( -7382bp ) and Anxa6 genes ( -3504bp ) ( Fig 5B ) .', 'We tested this pathway in C2C12 myoblasts as well as in the myofiber fraction from mdx muscles using qPCR and chromatin immunoprecipitation ( ChIP ) .', 'C2C12 cells were exposed to 1μg/ml rOPN , and after 48 hours of rOPN treatment , both Anxa1 and Anxa6 were downregulated , while Slug and Snail were upregulated , as compared to vehicle-treated cells .', 'These responses were reversed when C2C12 cells were co-treated with rOPN along with 10μM SB431542 , a chemical compound specifically inhibiting TGFβ signaling activation ( Fig 5C ) [40] .', 'ChIP-qPCR analysis showed that occupancy of Anxa1 and Anxa6 E-box elements by the SLUG/SNAIL transcriptional complex was increased after rOPN treatment , but decreased in the presence of TGFβ inhibitor ( Fig 5D ) .', 'In the myofiber fractions from mdx/Spp1-/- muscle where Anxa1 and Anxa6 expression were increased , these promoters had reduced occupancy of the E-box elements when compared to myofibers from mdx littermates ( Fig 5E ) .', 'When C2C12 cells were co-treated with rOPN and 10μM PF573228 , a chemical inhibitor of the OPN-driven signaling leading to Mzf1-Tgfb1 axis activation [41] , there was blunting of rOPN-associated upregulation of Mzf1 , Tgfb1 , Slug , and Snail ( Fig 5F ) .', 'Consistent with this , SLUG/SNAIL occupancy of Anxa1/6 E-box elements reverted to vehicle-like levels when cells were co-treated with rOPN and PF573228 ( Fig 5G ) .', 'Finally , expression of Mzf1 was significantly lower in Spp1-deficient hindlimb and respiratory muscles than in control mdx muscles ( Fig 5H ) .', 'Thus , excess OPN is associated with repression of Anxa1 and Anxa6 , and increased occupancy of their putative E-box elements by the transcriptional repressor complex SLUG/SNAIL .', 'Repression by SLUG/SNAIL was associated with increased Mzf1 and Tgfb1 levels .', 'Moreover , OPN-associated events were reversed in the presence of a chemical TGFβ inhibitor and were blunted by a chemical inhibitor of OPN signaling , thus supporting the hypothesis of a feed-forward circuitry that involves osteopontin and TGFβ pathways suppressing Anxa1 and Anxa6 and impairing sarcolemmal repair .', 'In Sgcg ( γ-sarcoglycan ) or DMD ( mdx ) mice , the DBA/2J background exacerbates the dystrophic phenotype [6 , 9] .', 'We sought to determine whether the difference in global outcomes associated with those genetic backgrounds was correlated with the efficiency of sarcolemma repair .', 'To exclude effects associated with dystrophic remodeling , we tested WT mice from 129T2 and DBA/2J background using laser-induced sarcolemmal injury .', 'DBA/2J myofibers showed more severe sarcolemmal damage than 129T2 myofibers ( Fig 6A ) .', 'Annexin A1 cap formation , seen as ANXA1-GFP , appeared more slowly in DBA/2J than in 129T2 myofibers and resulted in smaller cap size at end-point ( Fig 6B ) .', 'Although many genetic loci contribute to the DBA/2J background effect , DBA/2J mice feature the Ltbp4 risk allele , which is estimated to contribute to at least 40% of the variance of the muscular dystrophy phenotype in Sgcg mice [42] .', 'RNA sequencing of WT and Sgcg muscle from the severe DBA/2J background was associated with a marked increase in Spp1 expression compared expression in the 129T2 background ( S3 Fig ) .', 'We monitored gene expression changes relevant to the TGFβ pathway three days after cardiotoxin-mediated injury to the TA muscles of age-matched 129T2 or DBA/2J in order to assess the effect of genetic background .', 'After muscle injury , the DBA/2J background was associated with increased expression of Tgfb1 , Slug , Snail , Spp1 , and Mzf1 compared to injured 129T2 muscle ( Fig 6C ) .', 'Thus , the DBA/2J genetic background associated with increased sarcolemmal damage , and higher levels of osteopontin and TGFβ pathway genes .', 'Muscle specific overexpression of the protective Ltbp4 allele ( L4mild ) in mdx mice reduces fibrosis and increases muscle size [43] .', 'Conversely , expression of the risk human LTBP4 allele ( hL4severe ) exacerbates dystrophic remodeling in mdx mice [44] .', 'We tested sarcolemmal damage and repair cap formation in myofibers from transgenic age-matched mdx mice overexpressing either the L4mild ( mdx/L4mild ) , or the hL4severe isoform ( mdx/hL4severe ) at 20 weeks of age .', 'After laser-mediated sarcolemmal injury , the extent of sarcolemmal injury was smaller in myofibers of mdx/L4mild mice than from mdx control animals .', 'Conversely , sarcolemmal damage was increased in mdx/hL4severe myofibers as compared to control muscles ( Fig 7A ) .', 'Repair cap formation , monitored through GFP-labelled annexin A1 ( ANXA1-GFP ) , appeared faster in mdx/L4mild and slower in mdx/hL4severe myofibers than in mdx control myofibers .', 'These trends associated with a bigger cap size in mdx/L4mild and a smaller cap size in mdx/hL4severe , when compared to mdx repair caps at end-point ( Fig 7B ) .', 'Thus , the protective isoform with less TGFβ release associated with reduced sarcolemmal damage and faster cap formation , while the proteolysis-prone , risk allele with higher TGFβ release associated with increased damage and delayed repair cap assembly .', 'Anxa6 , was also identified as a modifier of muscular dystrophy by its action on sarcolemmal repair itself [45] , and the DBA/2J background harbors the risk allele for Anxa6 .', 'In order to discriminate the effects elicited by the Anxa6 and Ltbp4 genetic modifiers on sarcolemmal repair , we generated WT mice in the 129T2 background strain carrying the four homozygous combinations of mild and severe isoforms of Anxa6 and Ltbp4 ( A6mild/L4mild; A6severe/L4mild; A6mild/L4severe; A6severe/L4severe ) .', 'We assessed sarcolemmal damage and annexin A1 cap formation after laser injury in myofibers from age-matched mice from the four cohorts .', 'Muscles homozygous for either A6severe or L4severe had increased sarcolemmal damage , when compared to A6mild/L4mild myofibers .', 'Moreover , the effect of the two severe isoforms appeared additive on the sarcolemmal damage phenotype , as A6severe/L4severe myofibers presented the highest levels of FM4-64 accumulation across the groups ( Fig 8A ) .', 'Repair cap formation was also regulated by the additive effects of the severe isoforms .', 'The A6severe isoform associated with smaller cap size at end-point , while the L4severe isoform associated with a delay in initial onset of formation , when compared to the A6mild/L4mild genotype .', 'Similarly , the A6severe/L4severe correlated with onset delay and smaller size of the ANXA1 repair cap at end-point ( Fig 8B ) .', 'Thus , the polymorphism affecting LTBP4 function modifies sarcolemmal damage and repair cap formation in WT myofibers , and its effects are additive with respect to the Anxa6 polymorphism .', 'We next subjected mice from all four allele combinations to intra-muscular injury with cardiotoxin , targeting both TA and gastrocnemius muscles .', 'To quantify the number of injured myofibers , mice were intra-peritoneally injected with EBD immediately before intramuscular toxin injection .', 'Three hours after dye delivery , the number of dye-positive myofibers in injured muscles was comparably higher in mice with one severe allele , and the highest in mice with both severe alleles , with respect to A6mild/L4mild mice ( Fig 9A ) .', 'Seven days after injury , the injury area at muscle mid-point and fibrotic scarring followed similar trends , as quantified by histologic analyses ( Fig 9B and 9C ) .', 'Serum creatine kinase ( CK ) at 24 hours after injury was higher in the presence of either A6severe , or L4severe alleles , and the highest in mice with both severe alleles , as compared to A6mild/L4mild mice ( Fig 9D ) .', 'Seven days after injury , muscle tissue was analyzed for gene expression trends .', 'Transcriptional levels of Slug and Snail were significantly upregulated in the presence of the L4severe allele regardless of the A6 allele status .', 'Expression of Anxa1 and Anxa6 was also downregulated in the presence of L4severe isoforms ( Fig 9E ) .', 'Thus , Anxa6 and Ltbp4 isoforms modify the extent of damage in acute muscle injury in an additive fashion , and the severe isoform of Ltbp4 , characteristic of the DBA/2J genetic background , correlated with upregulation of Slug and Snail , and downregulation of Anxa1 and Anxa6 .'], ['Genetic modifiers of muscular dystrophy influence outcome through multiple pathways .', 'However , their combinatorial effects , and specifically whether they are additive , synergistic or even opposing in action is challenging to address at a human population level in a rare disorder .', 'Here we utilized a surrogate endpoint , sarcolemmal repair , to begin to assess how osteopontin and LTBP4 modify myofiber repair through a convergent TGFβ pathway circuitry .', 'Furthermore , we showed that upregulated OPN/TGFβ results in transcriptional repression of annexin genes , and this provides one possible means by which the OPN/TGFβ pathway contributes to impaired sarcolemmal resealing .', 'Given the broad gene expression effects of the OPN/TGFβ pathway , we expect that multiple genes mediate the in vivo effect in muscular dystrophy .', 'In vivo , in the absence of osteopontin , mdx muscle had fewer disrupted myofibers , which likely reflected this feed forward loop altering sarcolemmal stability , repair , and even muscle regeneration .', 'Osteopontin and TGFβ signaling constitute a common marker of dystrophic muscle when compared to healthy controls [11–19 , 46–49] .', 'Conversely , genetic manipulation to stifle both cascades reduces dystrophic pathology [13 , 36 , 43 , 50] .', 'To date , these signaling pathways have been mainly associated with activation and tissue remodeling by immune cell infiltrates and resident fibroblasts [10 , 51] .', 'These results indicate that osteopontin and TGFβ pathways act synergistically to directly influence the ability of myofibers to repair after sarcolemmal injury .', 'There is an expected crosstalk among myofibers , fibroblasts , and immune cells during both acute and chronic injury , which further modifies dystrophic features .', 'We found that the presence of either osteopontin or the deleterious form of Ltbp4 resulted in annexin gene repression and increased sarcolemmal damage .', 'A limitation of the sarcolemmal repair assays used for this study is the use of electroporation to express GFP-tagged annexins in the presence of native annexins .', 'To address this , we relied on electroporation of the same construct ( ANXA1-GFP ) in all different genetic contexts , and the results observed in the sarcolemmal repair assay paralleled the results seen when examining muscle injury in vivo , in both dystrophic and WT settings .', 'Importantly , sarcolemmal repair assays were conducted in the presence of Ca2+ , as annexin repair cap formation is known to be Ca2+-dependent [32] .', 'Further analyses in Ca2+-free settings , in combination with finer characterization of membrane composition , may discriminate Spp1- and Ltbp4-dependent effects on repair capacity and membrane mechanical stability , as recently investigated for the repair protein MG53 [52] .', 'In both genetic settings , either Spp1 ablation or the deleterious Ltbp4 alleles , transcriptional regulation of Anxa1 and Anxa6 inversely correlated with expression levels of Slug/Snail and occupancy of the TGFβ-related SLUG/SNAIL repressive complex on the E-box elements upstream of Anxa1 and Anxa6 .', 'These findings corroborate the hypothesis that both genetic modifiers contribute to a self-reinforcing , TGFβ-reliant loop in muscle , consistent with traditional observations of a feed-forward regulation of TGFβ signaling [37 , 38] .', 'The deleterious VTTT haplotype in the LTBP4 gene correlated with more rapid disease progression in DMD patients , while the rs28357094 polymorphism in the SPP1 promoter has been reported as a disease determinant in some studies , but not in others [19 , 21 , 22 , 24 , 53] .', 'However , it is still unclear whether the combination of deleterious polymorphisms at both loci significantly associates with worsened disease outcome .', 'Given the rare nature of DMD , assembling sufficient cohorts for these genetic studies in humans may not be possible .', 'Genome-wide screening for disease traits identified both Ltbp4 and Anxa6 as sarcolemmal damage modifying genes .', 'The DBA/2J mouse strain carries both risk alleles and produces enhanced muscular dystrophy pathology in mice [25 , 45] .', 'Anxa6 alleles regulate annexin repair cap size .', 'However , LTBP4 content determines the efficiency of cap formation after injury , with the deleterious Ltbp4 alleles associated with delayed cap formation .', 'These detrimental effects were additive in the presence of both alleles , and translated in additive effects on the in vivo response to muscle injury .', 'In addition to the transcriptional regulation of annexin genes , it is possible that LTBP4-mediated TGFβ overload impacts sarcolemmal repair through post-transcriptional regulation and/or protein interactions .', 'Ltbp4 genotype also appears to be a modifier of the extent of injury , which may reflect enhanced stability of the myofiber prior to injury .', 'The protective LTBP4 allele in humans has been associated with delayed loss of ambulation in humans with DMD , and this effect was greater in the presence of glucocorticoid steroid regimen in those study cohorts [24] .', 'Interestingly , steroid-associated additive beneficial effects were also reported for the SPP1 polymorphism in DMD patients [21] .', 'We recently reported that glucocorticoid steroids , such as prednisone and deflazacort , improve sarcolemmal repair and annexin cap formation in normal and dystrophic muscle [54] .', 'It has been suggested that glucocorticoid steroids act in muscular dystrophy by synchronizing an asynchronous repair milieu in muscular dystrophy [55] .', 'These data suggest that genetic modifiers beyond SPP1/OPN may contribute to this disorganized repair process and support the development of agents to resynchronize this process .', 'In summary , we now show that both osteopontin and LTBP4 directly modify sarcolemmal repair in myofibers of normal and dystrophic muscles .', 'In the model , excess osteopontin and deleterious LTBP4 converge to sustain TGFβ-mediated gene expression changes , including the repression of annexins , and likely , many other repair genes .', 'These findings indicate a direct role of those genetic modifiers in myofiber damage regulation and support their consideration for novel therapeutic avenues for treating dystrophic muscle .'], ['Mice were housed in a specific pathogen free facility in accordance with Institutional Animal Care and Use Committee ( IACUC ) regulations .', 'Euthanasia was performed through carbon dioxide or anesthetic gas inhalation followed by cervical dislocation and removal of the heart .', 'All methods using living animals in this study were performed in ethical accordance with the American Veterinary Medical Association ( AVMA ) and under protocols fully approved by both the Institutional Animal Care and Use Committee ( IACUC ) at Northwestern University Feinberg School of Medicine ( protocol number ISO00000911 ) .', 'Consistent with the approvals stipulated by these protocols , all efforts were made to minimize suffering .', 'mdx and mdx/Spp1-/- littermates from a mixed BL/6-BL/10 background were previously described [13] .', '129T2/SvEmsJ ( 129T2 ) and DBA/2J WT inbred mice were purchased from the Jackson Laboratory ( Bar Harbor , ME; Stock # 002065 and 000671 , respectively ) .', 'Anxa6 and Ltbp4 alleles from DBA/2J background were bred on the 129T2 background by means of initial 129T2 x DBA/2J breeding , followed by seven generations of mating the compound heterozygotes with 129T2 mice .', 'All mice used for experiments with DBA/2J Anxa6 and Ltbp4 alleles were conducted on littermates obtained from mating pairs of compound heterozygotes from the 129T2 background .', 'Mdx mice bearing the BAC-hLTBP4 transgene or the HSA::Ltbp4129 transgene were previously described [43 , 44] .', 'Both mdx transgenic lines were generated and bred on a mixed BL/6-BL/10 genetic background; the control cohort of mice for the experiments was created by pooling transgene-deficient littermates from both transgenic lines .', 'Both females and males were used in 129T2 and DBA/2J mice experiments , while only males in experiments with mdx mice .', 'Age of mice at the time of experiment was 8 weeks , unless otherwise specified .', 'Mice were maintained on a 12 hour light/dark cycle and fed ad libitum .', 'The plasmid encoding human annexin A1 with a carboxy-terminal GFP was obtained from Origene ( Rockville , MD; Cat# RG201569 ) .', 'Myofibers from the flexor digitorum brevis ( FDB ) muscles were electroporated in vivo , as previously described in [56] with modifications described in [31] .', 'Briefly , the footpad was injected with 10μl of hyaluronidase ( 8units ) ( Cat #H4272 , Sigma , St . Louis , MO ) .', 'After 2 hours , the footpad was injected with 20μl of 2μg/μl endotoxin-free plasmid .', 'Electroporation was conducted with following parameters: 20 pulses , 20 ms in duration/each , at 1Hz , at 100 V/cm .', 'Recovery was allowed for seven days after electroporation to avoid electroporation-induced damage and to allow plasmid expression [57] .', 'Individual myofibers were then explanted and isolated as previously detailed [31] .', 'Live myofibers were ablated with a laser as described [31 , 32 , 45] .', 'Briefly , myofibers were dissociated in PBS supplemented with 0 . 2% BSA and 4mg/ml collagenase type II ( Cat # 17101 , Life Technologies , Grand Island , NY ) at 37 degrees in 10% CO2 .', 'Muscle was triturated ( 20–30 pipetting motions through edge-cut 1000μl filter tip ) after 60 and 120 minutes .', 'Fibers were then seeded on MatTek dishes ( Cat # P35G-1 . 5-14-C , MatTek , Ashland MA ) in Ringers solution and , after 30 minutes , prepared for imaging by adding FM 4–64 dye ( T-13320 , Molecular Probes , Grand Island , NY ) to a final concentration of 2 . 5μm .', 'Laser ablation and subsequent real-time imaging were performed at room temperature using a Nikon A1R laser scanning confocal equipped with GaSP detectors through a 60x Apo lambda 1 . 4 NA objective driven by Nikon Elements AR software .', 'A single pixel set as 120 nm ( 0 . 0144 μm2 ) was ablated using the 405 nm laser at 100% power for up to 5 seconds .', 'Images were acquired as follows: one image prior to damage ( 0 seconds; reference for relative fluorescence analyses ) , one image right after laser injury ( bleach point ) , 10 images every 2 seconds after injury , and then one image every 10 seconds for up to 240 seconds after injury .', 'Quality control for myofibers selected for laser ablation relied on following parameters .', 'Only myofibers adherent to the MatTek dish from end to end and not contracting during imaging were used .', 'Imaged fibers were required to have intact sarcomeres and unruptured sarcolemma .', 'The region of the myofiber selected for laser injury was required to be linear without visible deformation or peripheral nuclei .', 'ANXA1-GFP fluorescence within the myofiber body at time 0 was required to be between 200 and 2000 relative light units ( RLUs ) , as per ImageJ analyses .', 'Relative fluorescence from an 85μm2 circular region encompassing the lesion area and ANXA1 cap diameter ( perpendicular to myofiber axis ) over time were calculated from images acquired as described above ( FM4-64 and ANXA1 images acquired simultaneously ) , normalizing values to the pre-injury intensity .', 'This method allows inter-group comparisons and reduces variability .', 'Prism Graphpad was used to calculate averages , and values were normalized to the pre-bleach intensity ( F/F0 ) .', 'All measurements were from n = 4 or 5 mice per group ( depending on experiment ) , with ≥10 myofibers per mouse ( total 40–50 per genotype or condition ) .', 'Data analysis was conducted blinded to genotype/treatment group .', 'Stack rendering of time course image sequences was conducted by using the “3D project” built-in feature of Image J with default parameters ( Projection method: Brightest Point; Slice spacing: 1 . 00μm ) .', 'Stacks were then tilted by 10° in order to show extent of sarcolemmal damage or repair cap formation over time .', 'rOPN was purchased as lyophilized powder ( Cat #441-OP; R&D Systems , Minneapolis , MN ) , resuspended as per manufacturer’s instructions , and stored at -80°C .', 'Treatment of FDB myofibers with rOPN was conducted injecting the foo
['Introduction', 'Results', 'Discussion', 'Materials and methods']
['Genetic disruption of the dystrophin complex produces muscular dystrophy characterized by a fragile muscle plasma membrane leading to excessive muscle degeneration .', 'Two genetic modifiers of Duchenne Muscular Dystrophy implicate the transforming growth factor β ( TGFβ ) pathway , osteopontin encoded by the SPP1 gene and latent TGFβ binding protein 4 ( LTBP4 ) .', 'We now evaluated the functional effect of these modifiers in the context of muscle injury and repair to elucidate their mechanisms of action .', 'We found that excess osteopontin exacerbated sarcolemmal injury , and correspondingly , that loss of osteopontin reduced injury extent both in isolated myofibers and in muscle in vivo .', 'We found that ablation of osteopontin was associated with reduced expression of TGFβ and TGFβ-associated pathways .', 'We identified that increased TGFβ resulted in reduced expression of Anxa1 and Anxa6 , genes encoding key components of the muscle sarcolemma resealing process .', 'Genetic manipulation of Ltbp4 in dystrophic muscle also directly modulated sarcolemmal resealing , and Ltbp4 alleles acted in concert with Anxa6 , a distinct modifier of muscular dystrophy .', 'These data provide a model in which a feed forward loop of TGFβ and osteopontin directly impacts the capacity of muscle to recover from injury , and identifies an intersection of genetic modifiers on muscular dystrophy .']
['Genetic modifiers for muscular dystrophy have been identified through transcriptomic and genomic profiling in humans and mouse models .', 'Two modifiers , Ltbp4 and Spp1 , encode extracellular proteins while a third modifier , Anxa6 , specifies a membrane-associated protein .', 'Using a model of muscle injury , we assessed the interaction of these modifiers , identifying a feed forward loop between Ltbp4 and Spp1 that promotes TGFβ signaling .', 'This feed forward loop is expected to contribute to the progressive nature of muscular dystrophy .', 'We also evaluated the interaction between Anxa6 and Ltbp4 , identifying an additive effect of these two genetic modifiers .']
['biotechnology', 'traumatic injury', 'medicine and health sciences', 'engineering and technology', 'lasers', 'animal models', 'muscle proteins', 'model organisms', 'experimental organism systems', 'optical equipment', 'genetic engineering', 'research and analysis methods', 'proteins', 'musculoskeletal injury', 'gene expression', 'muscular dystrophies', 'mouse models', 'critical care and emergency medicine', 'trauma medicine', 'biochemistry', 'signal transduction', 'cell biology', 'equipment', 'neurology', 'tgf-beta signaling cascade', 'genetics', 'biology and life sciences', 'cell signaling', 'signaling cascades']