Genetic modifiers of muscular dystrophy act on sarcolemmal resealing and recovery from injury.

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 of muscular dystrophy act on sarcolemmal resealing and recovery from injury

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

Excess osteopontin and TGFβ activation inhibits <i>Anxa1</i> and <i>Anxa6</i> gene expression.

<p><b>A)</b> Model depicting the feed-forward loop in which osteopontin promotes TGFβ, which in turn promotes <i>Spp1</i>/osteopontin expression. <i>Slug/Snail</i> expression, downstream of TGFβ activation, results in <i>Anxa1/Anxa6</i> gene repression. <b>B)</b> Diagram of putative E-box elements upstream of transcriptional start site (TSS) of <i>Anxa1</i> and <i>Anxa6</i> genes in the murine genome. <b>C)</b> qPCR analysis of C2C12 cells after treatment with recombinant osteopontin (rOPN) or rOPN plus the TGFβ inhibitor SB431452. rOPN increased <i>Slug/Snail</i> expression, and this was reversed in the presence of SB431452. <b>D)</b> Correspondingly, ChIP-qPCR showed that the E-box occupancy of the <i>Anxa1</i> and <i>Anxa6</i> promoters by the SLUG/SNAIL protein complex was increased by rOPN, and this was reversed in the presence of SB431452. <b>E)</b> <i>Anxa1 and Anxa6</i> promoter occupancy by SLUG/SNAIL was decreased in the myofiber fraction of <i>mdx/Spp1</i>^<i>-/-</i> muscle compared to control <i>mdx</i> muscle (<i>gastrocnemius</i>). <b>F)</b> PF573228, a chemical inhibitor of OPN-driven signaling leading to <i>Mzf1-Tgfb1</i> axis activation, ablated rOPN-induced upregulation of <i>Slug</i>, <i>Snail</i>, <i>Mzf1</i>, and <i>Tgfb1</i>. <b>G)</b> Accordingly, PF573228 blunted rOPN-dependent increase in SLUG/SNAIL occupancy on <i>Anxa1/6</i> E-boxes in C2C12 myoblasts. <b>H)</b> <i>Mzf1</i>, transcriptional regulator linking the OPN and TGFβ signaling cascades, was downregulated in both hindlimb (<i>quadriceps</i>) and respiratory (<i>diaphragm</i>) muscles of <i>mdx/Spp1</i>^<i>-/-</i> compared to <i>mdx</i>. Histograms, single values & avg±sem; n = 8 independent replicates/group for C2C12 myoblast analyses, n = 4 mice/group for <i>mdx</i>/<i>Spp1</i>^<i>-/-</i> mice analyses; (C-D; E-G) *, P<0.05 vs vehicle, 1way ANOVA + Bonferroni; (D) *, P<0.05 vs <i>mdx</i> control, unpaired t-test with Welch’s correction.</p

Genetic modifiers of muscular dystrophy act on sarcolemmal resealing and recovery from injury

<div><p>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 <i>SPP1</i> gene and latent TGFβ binding protein 4 (<i>LTBP4</i>). 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 <i>Anxa1</i> and <i>Anxa6</i>, genes encoding key components of the muscle sarcolemma resealing process. Genetic manipulation of <i>Ltbp4</i> in dystrophic muscle also directly modulated sarcolemmal resealing, and <i>Ltbp4</i> alleles acted in concert with <i>Anxa6</i>, 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.</p></div

<i>Spp1</i> ablation in <i>mdx</i> muscle is associated with decreased TGFβ gene expression and signaling.

<p><b>A)</b> Heat-map of qPCR analysis of gene expression, normalized to control muscles, showed downregulation of genes associated with the TGFβ pathway and atrophic remodeling in <i>mdx</i>/<i>Spp1</i>^<i>-/-</i> dystrophic muscle (<i>tibialis anterior</i>). All genes shown in the heatmap were significantly different (*). <b>B)</b> Percentage of phosphorylated-Smad3-positive nuclei within myofibers (myonuclei; arrowheads) was significantly decreased in <i>quadriceps</i> (representative immunofluorescence panels) and <i>diaphragm</i> muscles of <i>mdx/Spp1</i>^<i>-/-</i> mice when compared to <i>mdx</i> mice. Box plots, Tukey distribution; heat-map, z-test average values of fold change to control (ctrl); n = 4 mice/group; *, P<0.05 vs <i>mdx</i> control, 1way ANOVA + Bonferroni.</p

The <i>DBA/2J</i> genetic background is associated with increased sarcolemmal damage and higher levels of OPN and TGFβ pathway genes.

<p>The DBA/2J background exacerbates the muscular dystrophy phenotype [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007070#pgen.1007070.ref006" target="_blank">6</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007070#pgen.1007070.ref009" target="_blank">9</a>]. <b>A)</b> The extent of sarcolemmal damage was significantly higher in WT <i>DBA/2J</i> myofibers, as compared to age-matched WT <i>129T2</i> myofibers, both over time and at end-point (arrows). <b>B)</b> Repair cap formation, monitored by GFP-tagged ANXA1, appeared significantly delayed in <i>DBA/2J</i> myofibers (arrowheads), with a smaller cap diameter at end-point (arrowheads). The time series shows stacked consecutive images of the injured site at 10° orientation to reveal the extent of dye accumulation or repair cap formation. FM4-64 (red) and ANXA1-GFP (green) pictures were acquired simultaneously. <b>C)</b> qPCR analysis of cardiotoxin-injured <i>tibialis anterior</i> muscle tissue at 3 days post-injury from <i>129T2</i> and <i>DBA/2J</i> mice versus strain-matched uninjured littermates showed that injury-associated upregulation of <i>Tgfb1</i>, <i>Slug</i>, <i>Snail</i>, <i>Spp1</i>, and <i>Mzf1</i> was significantly higher in the <i>DBA/2J</i> strain than in the <i>129T2</i> strain (dotted line: strain-matched control values, = 1). Marked line plots, avg±sem; box plots, Tukey distribution; histograms, single values & avg±sem; n = 5 mice/group; #, P<0.05 vs vehicle, 2way ANOVA + Bonferroni; *, P<0.05 vs 129T2, unpaired t-test with Welch’s correction.</p

<i>Spp1</i> ablation in <i>mdx</i> muscle resulted in fewer disrupted myofibers in vivo.

<p><b>A)</b> Three hours after systemic EBD delivery, the number of EBD-positive myofibers (red appearance) was significantly less in <i>mdx/Spp1</i>^<i>-/-</i> TA muscles than in control <i>mdx</i> muscles (left, representative immunofluorescence panels; right, quantitation). <b>B)</b> Cardiotoxin injection was performed in the contralateral muscles, increasing the number of EBD-positive fibers. After cardiotoxin injury, there were fewer EBD-positive myofibers in <i>mdx/Spp1</i>^<i>-/-</i> compared to <i>mdx</i> muscles (left, representative immunofluorescence panels; right, quantitation). Box plots, Tukey distribution; n = 4 mice/group; *, P<0.05 vs <i>mdx</i> control, unpaired t-test with Welch’s correction.</p

Relative contribution of <i>Anxa6</i> and <i>Ltbp4</i> alleles on sarcolemmal repair in wildtype muscle.

<p>In addition to <i>Ltbp4</i>, Anxa6 has also been shown to modify muscular dystrophy in mice [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007070#pgen.1007070.ref025" target="_blank">25</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007070#pgen.1007070.ref045" target="_blank">45</a>]. The deleterious alleles of <i>Ltbp4</i> (L4) or <i>Anxa6</i> (A6) (severe) were compared to those from the protective 129 strain (mild) in the sarcolemma injury assay. <b>A)</b> Doubly homozygous A6^mild/L4^mild myofibers had the least injury while doubly homozygous A6^severe/L4^severe fibers had the greatest injury, marked by FM4-64. Myofibers with mixed homozygous genotypes were intermediate with respect to FM4-64 marked injury. <b>B)</b> A similar pattern was observed for Annexin A1 (ANXA1) repair caps, where doubly homozygous mild alleles of L4 and A6 assembled caps more rapidly and produced larger repair caps than doubly homozygous severe alleles. However, ANXA1 repair caps were smaller with the A6 homozygous severe allele, despite the presence of the mild L4 allele, suggesting that repair cap formation is dominated by the A6 genotype. FM4-64 and ANXA1-GFP images used for the analyses in <b>A-B</b> were acquired simultaneously. Marked line plots, avg±sem; box plots, Tukey distribution; n = 50 myofibers (5 mice)/group; marked line plots: #, P<0.05 vs control (A6^mild/L4^mild), +, P<0.05 vs A6^severe/L4^mild and A6^mild/L4^severe groups, 2way ANOVA + Bonferroni; boxplots: *, P<0.05 vs control (A6^mild/L4^mild), +, P<0.05 vs A6^severe/L4^mild and A6^mild/L4^severe groups, 1way ANOVA + Bonferroni.</p

<i>mdx</i> mice lacking osteopontin (mdx/<i>Spp1</i>^<i>-/-</i><i>)</i> recover from sarcolemmal injury more efficiently.

<p><b>A)</b> The sarcolemmal wounding assay was performed on <i>flexor digitorum brevis</i> myofibers from <i>mdx</i> or <i>mdx/Spp1</i>^<i>-/-</i> animals, after electroporation with the ANXA1-GFP-encoding plasmid. The extent of sarcolemmal damage, monitored through accumulation of FM4-64 dye after laser injury, was significantly reduced in <i>mdx</i>/<i>Spp1</i>^<i>-/-</i> myofibers compared to control dystrophic myofibers. This was quantified over time as dye accumulation at site of injury (left) and area of injury at end-point (right; arrows). The time series (left images) represent stacked consecutive images of the injured site at 10° orientation to reveal the extent of dye accumulation. <b>B)</b> Repair cap onset, monitored by GFP-tagged ANXA1, was significantly faster in <i>mdx/Spp1</i>^<i>-/-</i> myofibers (arrowheads), resulting in a larger cap diameter at end-point (arrowheads). Time series of stacked consecutive images of the injured site at 10° orientation revealed the extent of dye accumulation or repair cap formation. FM4-64 and ANXA1-GFP pictures were acquired simultaneously. Marked line plots, avg±sem; box plots, Tukey distribution; n = 40 myofibers (4 mice)/group; #, P<0.05 vs <i>mdx</i> control, 2way ANOVA + Bonferroni; *, P<0.05 vs <i>mdx</i> control, unpaired t-test with Welch’s correction.</p

Excess osteopontin exacerbates sarcolemmal injury and delays repair cap formation.

<p>The sarcolemmal wounding assay was performed on <i>flexor digitorus brevis</i> muscles from WT muscle in the presence or absence of recombinant osteopontin (rOPN; 10μl at 1μg/μl). <b>A)</b> The extent of sarcolemmal damage, monitored as FM4-64 area, was greater in rOPN-treated myofibers compared to vehicle-treated control, both over time and at end-point (arrows). <b>B)</b> Repair cap formation, monitored by GFP-tagged annexin A1 (GFP-ANXA1) was delayed in rOPN-treated myofibers (arrowheads). Repair cap diameter at end-point was smaller in treated compared to control myofibers (arrowheads). Over time image series represents stacked consecutive images of the injured site at 10° orientation to reveal the extent of dye accumulation or repair cap formation (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007070#pgen.1007070.s002" target="_blank">S1B Fig</a>). FM4-64 and ANXA1-GFP pictures were acquired simultaneously. Marked line plots, avg±sem; box plots, Tukey distribution; n = 50 myofibers (5 mice)/group; #, P<0.05 vs vehicle, 2way ANOVA + Bonferroni; *, P<0.05 vs vehicle, unpaired t-test with Welch’s correction.</p