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/lp/ou_press/a-promotive-effect-for-halofuginone-on-membrane-repair-and-14mpaXb29h
- Publisher
- Oxford University Press
- Copyright
- © The Author(s) 2018. Published by Oxford University Press. All rights reserved. For permissions, please email: journals.permissions@oup.com
- ISSN
- 0964-6906
- eISSN
- 1460-2083
- DOI
- 10.1093/hmg/ddy185
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- See Article on Publisher Site
Abstract
Abstract In the absence of dysferlin, skeletal muscle cells fail to reseal properly after injury, resulting in slow progress of the dysferlinopathy muscular dystrophy (MD). Halofuginone, a leading agent in preventing fibrosis in MDs, was tested for its effects on membrane resealing post-injury. A hypo-osmotic shock assay on myotubes derived from wild-type (Wt) and dysferlin-null (dysf−/−) mice revealed that pre-treatment with halofuginone reduces the percentage of membrane-ruptured myotubes only in dysf−/− myotubes. In laser-induced injury of isolated myofibers, halofuginone decreased the amount of FM1–43 at the injury site of dysf−/− myofibers while having no effect on Wt myofibers. These results implicate halofuginone in ameliorating muscle-cell membrane integrity in dysf−/− mice. Halofuginone increased lysosome scattering across the cytosol of dysf−/− primary myoblasts, in a protein kinase/extracellular signal-regulated protein kinase and phosphoinositide 3 kinase/Akt-dependent manner, in agreement with an elevation in lysosomal exocytotic activity in these cells. A spatial- and age-dependent synaptotagmin-7 (Syt-7) expression pattern was shown in dysf−/− versus Wt mice, suggesting that these pattern alterations are related to the disease progress and that sytnaptotagmin-7 may be compensating for the lack of dysferlin at least with regard to membrane resealing post-injury. While halofuginone did not affect patch-repair-complex key proteins, it further enhanced Syt-7 levels and its spread across the cytosol in dysf−/− myofibers and muscle tissue, and increased its co-localization with lysosomes. Together, the data imply a novel role for halofuginone in membrane-resealing events with Syt-7 possibly taking part in these events. Introduction The muscle tissue is exposed to constant mechanical stress, causing damage to the sarcolemma (i.e. the myofiber cytoplasmic membrane). This common situation forces the tissue to maintain quick and efficient repair systems for both large-scale and microscopic contraction-induced sarcolemmal injuries (1). Large-scale injuries are resolved by self-regeneration processes (i.e. assembly of new myofibers) in which the tissue progenitor cells—the satellite cells—play a major role. These usually quiescent cells lying between the basal membrane and the sarcolemma can be induced to enter the cell cycle when regeneration and/or repair is needed (2). In muscular dystrophies (MDs), these events are impaired, causing loss of muscle mass due to repetitive cycles of degeneration (disassembly of severely damaged myofibers) and regeneration, inflammation and, ultimately, increased amounts of fat and connective tissue (i.e. fibrosis) (3,4). Dysferlinopathies are a group of autosomal recessive limb-girdle type 2B MDs caused by mutations to the dysferlin-encoding DYSF gene (5). In contrast to other MDs, such as Duchenne MD (DMD)—the most common MD caused by mutations in the dystrophin gene with very severe skeletal and heart muscle pathology and short life span (6–8)—the dysferlinopathy phenotype appears only during the patient’s third or fourth decade of life (9,10). Though considered a non-lethal disease, once the phenotype appears, it progresses quite rapidly with a severe decline in the patient’s quality of life (11). Dysferlin is a 230 kDa transmembrane protein that is part of the ferlin family of proteins and is highly abundant in the heart and skeletal muscles (1,10,12). It plays a key role in the patch-repair process of microscopic tears of the sarcolemma; in response to post-injury Ca2+ influx, dysferlin initiates the activity of the other lysosome-trafficking and membrane-repair proteins that are part of the patch-repair protein complex. These proteins, such as caveolin 3 (Cav3), Annexins A1 and A2, AHNAK and Tripartite motif-containing protein 72 (TRIM72) mediate the fusion of membrane-bound vesicles, mainly lysosomes, to the injury site (10,13,14). Dysferlin’s role in the initiation of lysosome-trafficking and fusion to the sarcolemma has been shown in studies in which DYSF-null myofibers demonstrated a deficiency in sarcolemmal Ca2+-dependent resealing post-injury (1) and accumulation of lysosomes near the nucleus (15). Furthermore, silencing dysferlin C2A, the protein’s main Ca2+-binding domain, almost completely blocked lysosome fusion to the plasma membrane of bovine coronary arterial endothelia (16). A recent report has placed transforming growth factor β (TGFβ) as a promotor of reactive oxygen species (ROS) levels in dysf−/− myofibers, leading to increased membrane fragility, and has shown that direct inhibition of the TGFβ signaling pathway improves dysf−/− myofiber membrane integrity post-laser wounding (17). Many studies held on non-skeletal-muscle models point toward synaptotagmins in general and Syt-7 particularly, as mediators of lysosome-trafficking and exocytosis (18–21). Synaptotagmins are composed of a small intravesicular sequence, a single transmembrane region and a large cytoplasmic sequence with two C2-domains (22). The synaptotagmin-7 (Syt-7) isoform senses Ca2+ and mediates vesicular exocytosis and fusion in many different cell types (23–26) as well as mediating membrane resealing via lysosomal exocytosis in human fibroblasts (27). Interestingly, the Syt-7 knockout mouse shows normal growth and development but develops an inflammatory myopathy with elevated creatine kinase and muscle weakness with defective lysosome-mediated membrane repair and autoimmune symptoms (28). Halofuginone is a synthetic analogue of the alkaloid febrifugine found in the Dichroa febrifuga plant. Halofuginone has been shown to act as an anti-fibrotic reagent in numerous diseases (29–32). It inhibits fibroblast conversion to myofibroblasts by inhibiting Smad3 phosphorylation, downstream of TGFβ, thereby preventing Smad3 penetration into the nucleus and transcription of the collagen type І-encoding gene (33–36). Halofuginone’s mechanism of action as an inhibitor of Smad3 phosphorylation can be explained, at least in part, by its promotive effect on the phosphorylation levels of Akt and mitogen-activated protein kinase/extracellular signal-regulated protein kinase (MAPK/ERK), and by its enhancement of their association with the non-phosphorylated form of Smad3 (34). Halofuginone has also been reported to inhibit Th17 cell differentiation via association with and reduction of prolyl-tRNA synthetase activity and activation of the amino acid starvation response (37,38). In MDs, halofuginone has been reported to inhibit fibrosis in mouse models for DMD, congenital MD (CMD) and dysferlinopathy, regardless of the cause of the disease (39–41). It reduces tissue inflammation and myofibroblast infiltration, which are typical symptoms of MDs. Some of these effects have recently been explained by elevation of utrophin and reduction of moesin in mouse models for DMD and CMD (42,43). In addition, halofuginone promotes a reduction in the number of centrally nucleated (i.e. regenerating) myofibers and increases the mean myofiber diameter (39–41). Moreover, reports on an increase in myofiber-attached satellite cell proliferation, myotube fusion in vitro and a reduction in the number of apoptotic nuclei and pro-apoptotic markers in response to halofuginone in mouse models of DMD and dysferlinopathies, suggest a direct effect of halofuginone on muscle cells (34,41,44,45). These promotive effects of halofuginone led us to the hypothesis that it can improve membrane integrity and local fusion events, such as resealing post-microscopic injury of dysf−/− myofibers. In this study, we evaluated these effects in intact as well as post-injury muscle cells using various injury protocols. In addition, the study addressed possible mediators of halofuginones’ actions in membrane repair. Results Pretreatment with halofuginone improves myotube membrane integrity during hypo-osmotic shock Dysferlin has been reported to be required for maintenance of sarcolemmal structural integrity as an initiator of patch-repair complex activity (10,46,47). Therefore, we looked at halofuginone’s effect on cytoplasmic membrane integrity in dysf−/− myotubes. Myotube membrane integrity was examined by applying hypo-osmotic shock, which has previously been reported to rapidly induce cell swelling and increase membrane tension, thereby creating mechanical stress at the cell membrane (48–50). Cultured wild-type (termed, Wt) and dysf−/− myotubes were pre-treated with or without 10 nM halofuginone, after which they underwent hypo-osmotic shock (Fig. 1A). The membrane-rupture events were evaluated in the multi-nucleated cells (i.e. myotubes) with the cell-permeable calcein-AM dye, which only fluoresces inside intact cells. Propidium iodide (PI)—a nucleic dye that lacks the ability to penetrate intact membranes—was added to the hypo-osmotic shock medium as a second indicator for membrane-rupture events (Fig. 1A). In general, fewer and thinner myotubes were depicted in the dysf−/− culture than in the Wt one (Fig. 1B). Calcein-AM fluorescence levels (green) remained high in the non-treated and treated Wt myotubes, indicating intact myotubes, while the non-treated dysf−/− myotubes presented a notable reduction in calcein-AM staining and an increase in the fluorescence levels of PI (red), indicating myotubes that underwent membrane rupture (Fig. 1B). In contrast, in the halofuginone-treated dysf−/− cell culture, the calcein-AM levels remained high in the myotubes. For quantitative analysis, myotubes were counted only when containing two or more nuclei, as indicated by 4', 6-diamidino-2-phenylindole (DAPI) staining. The signal ratio of the calcein-AM fluorescence levels at 5 min was calculated with a threshold for cell-membrane-rupture events of 0.5 (48). The osmotic shock raptured a relatively small percentage of the Wt myotubes and more than 80% remained intact (Fig. 1C, white and striped bars, respectively), while in more than 30% of the dysf−/− myotubes membrane rupturing was observed (Fig.1C, white bars). Halofuginone treatment did not affect the membrane rupture of the Wt myotubes. In contrast, the percentage of membrane-rupture-resistant myotubes out of the total number of myotubes was significantly higher in the halofuginone-treated versus the non-treated dysf−/− myotubes (Fig. 1C, striped bars). Moreover, the percentage of membrane-ruptured myotubes was ∼6-fold lower in the halofuginone-treated dysf−/− myotubes than in their non-treated counterparts, similar to the percentage of the membrane-ruptured Wt myotubes (Fig. 1C, white bars). Figure 1. View largeDownload slide Hypo-osmotic shock in myotubes of Wt and dysf−/− mouse primary muscle cells. (A) Timeline scheme of the experiment. Wt and dysf−/− primary myoblasts were induced to differentiate followed by treatment with 10 nM halofuginone (Halo) or not (non-treated) for 24 h, after which myotubes were treated as described in A. (B) Representative depiction of myotubes stained with calcein-AM (calcein, green) and PI (red). T=0, time zero; T=5, 5 min. Scale bar, 50 μm. The signal ratio of calcein-AM fluorescence levels at 5 min to those at time zero was calculated with a threshold for membrane-rupture events of 0.5. (C) Cumulative percentage of membrane-rupture-resistant myotubes (calcein signal ratio ɦgt;0.5; striped bars) and membrane-ruptured myotubes (calcein signal ratio ≤0.5; white bars) out of total number of myotubes. Values are expressed as means±SE of three independent experiments. Data with asterisks differ significantly within treatments (P <0.05). Figure 1. View largeDownload slide Hypo-osmotic shock in myotubes of Wt and dysf−/− mouse primary muscle cells. (A) Timeline scheme of the experiment. Wt and dysf−/− primary myoblasts were induced to differentiate followed by treatment with 10 nM halofuginone (Halo) or not (non-treated) for 24 h, after which myotubes were treated as described in A. (B) Representative depiction of myotubes stained with calcein-AM (calcein, green) and PI (red). T=0, time zero; T=5, 5 min. Scale bar, 50 μm. The signal ratio of calcein-AM fluorescence levels at 5 min to those at time zero was calculated with a threshold for membrane-rupture events of 0.5. (C) Cumulative percentage of membrane-rupture-resistant myotubes (calcein signal ratio ɦgt;0.5; striped bars) and membrane-ruptured myotubes (calcein signal ratio ≤0.5; white bars) out of total number of myotubes. Values are expressed as means±SE of three independent experiments. Data with asterisks differ significantly within treatments (P <0.05). Treatment with halofuginone improves myofiber membrane integrity post-laser wounding To further investigate halofuginone’s effect on membrane integrity, a laser-wounding assay was performed in freshly isolated myofibers from the gastrocnemius of Wt and dysf−/− mice. The myofibers were injured in the presence of FM1–43 (2.5 µM), with or without halofuginone, according to the laser-wounding assay (1). FM1–43 becomes fluorescent upon association with phospholipids but cannot penetrate the double layer of the membrane; hence, it will bind the inner-layer phospholipids only upon injury (51). Non-treated single Wt and dysf−/− myofibers showed immediate accumulation of FM1–43, which was concentrated at the injury site, and by contracting and bending inward at the injury site (Fig. 2A, left panels; Supplementary Material, SV1A and C). Addition of halofuginone to the Wt myofibers had no apparent effect on the injury site (Fig. 2A, upper right panels; Supplementary Material, SV1B). In contrast, halofuginone-treated dysf−/− myofibers did not bend during the experiment period (Fig. 2A, lower right panels; Supplementary Material, SV1D). Calculation of the fluorescence intensity at each time point relative to time zero, thereby capturing its cumulative rank, in myofibers treated or non-treated with halofuginone is shown in Figure 2B and C for Wt and dysf−/− myofibers, respectively. The increase in FM1–43 fluorescence intensity in the non-treated Wt myofibers after 5 min reached a level which was twice as high as time zero (Fig. 2B). Similar increase in FM1–43 fluorescence intensity was observed in the halofuginone-treated Wt myofibers with no significant difference versus the non-treated myofibers (Fig. 2B). In non-treated dysf−/− myofibers, FM1–43 fluorescence intensity reached a level that was 1.5 times higher than that of the non-treated Wt myofibers, suggesting a delayed ability of dysf−/− myofibers in maintaining membrane integrity (1,51). The increase in intensity was significantly lower in the halofuginone-treated dysf−/− myofibers than that in the non-treated ones and after 5 min reached a level, which was approximately half the level of the non-treated dysf−/− myofibers (Fig. 2C). The effect of laser injury on FM1–43 accumulation in dysf−/− myofibers was also tested in Ca2+-free medium; this effect was attenuated in the halofuginone-treated dysf−/− myofibers and resembled that of the non-treated myofibers (data not shown). Figure 2. View largeDownload slide Halofuginone reduces FM1–43 accumulation post-laser injury at the injury sight of single dysf−/− myofibers. Freshly isolated myofibers from the gastrocnemius muscle of 6-week-old Wt or dysf−/− mice were treated, or not (non-treated), with 10 nM halofuginone (Halo) for 24 h. Wounding was achieved with a 5 s UV pulse at 80% power of a 405-diode laser in the presence of FM1–43 and the myofibers were monitored for 5 min under a confocal microscope at 1 s intervals. (A) Micrographs of single Wt (upper panel) and dysf−/− (lower panel) myofibers treated, or not, with halofuginone at time zero (T =0) and at 5 min (T=5 min). FM1–43 becomes fluorescent upon attachment to phospholipids. Arrows indicate the point of the laser wound and the accumulation of FM1–43 at the injury site (red). (B) Intensity of FM1–43 fluorescence of Wt (B) and dysf−/− (C) single myofibers was calculated as In=Im tx-IbtxImt0-Ibt0, where In = normalized intensity, Im= measured intensity at time x (tx) or time zero (t0), Ib =mean background intensity at tx or t0 (mean of the single myofiber background intensity and the total image background intensity). Values are means±SE of eight independent myofibers in a representative experiment out of three independent experiments. Asterisks represent a significant difference between treatments (P < 0.001). Figure 2. View largeDownload slide Halofuginone reduces FM1–43 accumulation post-laser injury at the injury sight of single dysf−/− myofibers. Freshly isolated myofibers from the gastrocnemius muscle of 6-week-old Wt or dysf−/− mice were treated, or not (non-treated), with 10 nM halofuginone (Halo) for 24 h. Wounding was achieved with a 5 s UV pulse at 80% power of a 405-diode laser in the presence of FM1–43 and the myofibers were monitored for 5 min under a confocal microscope at 1 s intervals. (A) Micrographs of single Wt (upper panel) and dysf−/− (lower panel) myofibers treated, or not, with halofuginone at time zero (T =0) and at 5 min (T=5 min). FM1–43 becomes fluorescent upon attachment to phospholipids. Arrows indicate the point of the laser wound and the accumulation of FM1–43 at the injury site (red). (B) Intensity of FM1–43 fluorescence of Wt (B) and dysf−/− (C) single myofibers was calculated as In=Im tx-IbtxImt0-Ibt0, where In = normalized intensity, Im= measured intensity at time x (tx) or time zero (t0), Ib =mean background intensity at tx or t0 (mean of the single myofiber background intensity and the total image background intensity). Values are means±SE of eight independent myofibers in a representative experiment out of three independent experiments. Asterisks represent a significant difference between treatments (P < 0.001). Lysosome scattering across dysf−/− myoblasts is enhanced by halofuginone via the MAPK/ERK and PI3K/akt pathways Lysosome fusion to the cell membrane is a major part of the patch-repair mechanism post-injury. However, Demonbreun et al. (15) have shown that dysf−/− myoblasts display a deficit in the trafficking of lysosomal-associated membrane protein 2B (LAMP2B)-positive vesicles (i.e. lysosomes) across the cytoplasm: they remain close to the nucleus and the Golgi apparatus. Here, we addressed the possibility that halofuginone might also have a role in the membrane-repair process. We first tested the effect of halofuginone on lysosome scattering in primary myoblasts derived from Wt, dysf−/−, and mdx mice as an additional MD mouse model. The myoblasts were treated with or without 10 nM halofuginone for various intervals and then immunostained for the lysosomal marker LAMP2B (15,52). In both non-treated Wt and mdx myoblasts, the lysosomes were similarly scattered across the entire cell cytoplasm after 24 h in culture (Fig. 3A, upper left panels). Halofuginone-treated myoblasts from both mouse lines demonstrated no apparent change in their scattering pattern compared with non-treated myoblasts (Fig. 3A, upper right panels). In the dysf−/− myoblasts, lysosomes of non-treated cells remained close to the nucleus in a circular shape (Fig. 3A, lower left panel, see insert), although the shape of the cells remained normal (Supplementary Material, Fig. S1). However, in the halofuginone-treated cells, the lysosomes scattered across the cell cytoplasm, resembling the lysosome-scattering pattern in Wt and mdx myoblasts (Fig. 3A, lower right panel). The percentage of myoblasts with the scattered lysosome appearance was calculated out of the total number of myoblasts. In Wt and mdx myoblasts, this number was about 60% with no significant change after halofuginone treatment (Fig. 3B). However, in a time-course measurement in dysf−/− myoblasts, while levels of non-treated myoblasts remained at ∼30% throughout the entire period, treatment with halofuginone significantly increased the percentage of dysf−/− myoblasts with scattered lysosomes in a time-dependent manner, to ∼45 and 60% after 18 and 24 h of treatment, respectively (Fig. 3C). Figure 3. View largeDownload slide Halofuginone enhances lysosome scattering in dysf−/− myoblasts. Myoblasts from 5-week-old Wt, mdx and dysf−/− mice were treated with 10 nM halofuginone (Halo) for 24 h or in a time-course manner. The cells were immunostained with antibody against LAMP2B and with DAPI for nuclei. (A) Lysosome scattering in Wt, mdx and dysf−/− myoblasts treated, or not (non-treated), with halofuginone for 24 h. Scale bar, 20 μm. The insert presents a higher magnification of non-treated dysf−/− myoblast. Arrows point to LAMP2B-positive lysosomes that have accumulated around the nucleus. Bar, 5 μm. Quantitation analysis of the percentage of myoblasts with scattered appearance of lysosomes out of the total number of myoblasts in Wt and mdx myoblasts after 24 h treatment (B), and in dysf−/− myoblast cultures in a time-course experiment (C). Results are means±SE of three independent experiments. Asterisks represent significant difference between treatments at each time point (P<0.05). dysf−/− myoblasts were treated as in (A) with or without the addition of 20 µM UO126 (UO) (D) or 25 µM Ly294002 (Ly) (E) for 24 h, followed by immunostaining for LAMP2B and DAPI. The percentage of myoblasts with scattered appearance was calculated as described in (B). Results are means±SE of three independent experiments. Different letters above bars represent statistically significant differences between all treatments (P< 0.05). Figure 3. View largeDownload slide Halofuginone enhances lysosome scattering in dysf−/− myoblasts. Myoblasts from 5-week-old Wt, mdx and dysf−/− mice were treated with 10 nM halofuginone (Halo) for 24 h or in a time-course manner. The cells were immunostained with antibody against LAMP2B and with DAPI for nuclei. (A) Lysosome scattering in Wt, mdx and dysf−/− myoblasts treated, or not (non-treated), with halofuginone for 24 h. Scale bar, 20 μm. The insert presents a higher magnification of non-treated dysf−/− myoblast. Arrows point to LAMP2B-positive lysosomes that have accumulated around the nucleus. Bar, 5 μm. Quantitation analysis of the percentage of myoblasts with scattered appearance of lysosomes out of the total number of myoblasts in Wt and mdx myoblasts after 24 h treatment (B), and in dysf−/− myoblast cultures in a time-course experiment (C). Results are means±SE of three independent experiments. Asterisks represent significant difference between treatments at each time point (P<0.05). dysf−/− myoblasts were treated as in (A) with or without the addition of 20 µM UO126 (UO) (D) or 25 µM Ly294002 (Ly) (E) for 24 h, followed by immunostaining for LAMP2B and DAPI. The percentage of myoblasts with scattered appearance was calculated as described in (B). Results are means±SE of three independent experiments. Different letters above bars represent statistically significant differences between all treatments (P< 0.05). Next, we evaluated whether halofuginone’s effect on lysosome scattering in dysf−/− myoblasts is mediated via the MAPK/ERK- or phosphoinositide 3 kinase (PI3K)/Akt-signaling pathways. The dysf−/− myoblasts were treated with or without halofuginone and UO126 (20 µM) or Ly294002 (Ly; 25 µM), pharmacological inhibitors of MAPK/ERK- and PI3K/Akt-signaling pathways, respectively (53), for 24 h; lysosome scattering was evaluated as described for Figure 3A. Addition of inhibitors alone to non-treated cells had no effect on lysosome scattering (Fig. 3D and E). Halofuginone addition alone increased the percentage of myoblasts with lysosomal scattering out of the total number of myoblasts by almost 2-fold. However, this percentage dropped significantly with the combined treatment of UO126 and halofuginone (Fig. 3D). Moreover, the combined treatment of halofuginone with Ly abolished the halofuginone-increased percentage of cells with scattered lysosomes, back to non-treated levels (Fig. 3E). In cells that were treated in parallel with either inhibitor for 1 h, the phosphorylation of MAPK/ERK (P-MAPK/ERK) or Akt (P-Akt) was abolished by UO126 and Ly treatments, respectively, as compared to non-treated cells (Supplementary Material, Fig. S2). Cells treated with halofuginone alone showed enhanced levels of P-Akt compared to the combined treatment with Ly (Supplementary Material, Fig. S2B). In cells treated with halofuginone and UO126 or Ly, the levels of P-MAPK/ERK or P-Akt, respectively, dropped even lower than in the non-treated cells (Supplementary Material, Fig. S2A and B, respectively), indicating halofuginone’s effect on their phosphorylation levels (34). Halofuginone promotes lysosome exocytosis to the cytoplasmic membrane in dysf−/− myoblasts Following halofuginone’s promoting effect over lysosome scattering in dysf−/− myoblasts we were interested in its effect on lysosome fusion to the cytoplasmic membrane (i.e. lysosome exocytosis). This effect was evaluated by a quenching assay of the FM1–43 dye (16,54–56). Intact, uninjured Wt and dysf−/− primary myoblasts were treated or not with halofuginone for 24 h followed by a 2 h incubation with FM1–43 (8 µM). During this 2 h-period FM1–43 is endocytosed by endosomes, which will then fuse to lysosomes in an attempt to degrade the dye, hence resulting in lysosomal loading of FM1–43 (16). Bromophenol blue (BPB), a membrane-impermeant quencher of FM1–43, was then added to the medium and the cells were monitored for additional 20 min. Thus, the decrease in fluorescence could be used as an indicator of exocytosis of the FM1–43-marked lysosomes to the cytoplasmic membrane (16). At time zero, FM1–43 dye was noticeable in the Wt and dysf−/− myoblasts, demonstrating its loading onto the lysosomes. At 20 min, a reduction in FM1–43 fluorescence was noticeable in the Wt myoblasts (Fig. 4A). In contrast, in the dysf−/− myoblasts, most of the dye concentrated around the nucleus and the fluorescence intensity remained strong. In the halofuginone-treated dysf−/− myoblasts, a noticeable reduction in the FM1–43 fluorescence levels was observed. The fluorescence levels were calculated as the ratio between 20 min and time zero. By the end of the monitoring period the FM1–43 levels in the Wt myoblasts reached 50% of their time zero levels, while in the non-treated dysf−/− myoblasts they remained significantly higher at 75% of their time zero levels. In the halofuginone-treated dysf−/− myoblasts, FM1–43 fluorescence levels reached 40% of their time zero levels comparable to the Wt levels (Fig. 4B). Figure 4. View largeDownload slide Halofuginone-treatment enhances lysosome exocytosis to the cytoplasmic membrane of dysf−/− myoblasts. Primary myoblasts of 5-week-old Wt and dysf−/− mice were treated or not with halofuginone for 24 h followed by 2 h incubation with FM1–43. Bromophenol blue was then added to the medium and FM1–43 fluorescence levels in the cells were monitored for 20 min. (A) Representative confocal microscope images show Wt and dysf−/− myoblasts immediately after adding BPB to the medium (T=0, upper panels) and at the end of the monitoring period (T=20 min; lower panels). Scale bar, 20 μm. (B) Intensity of FM1–43 fluorescence of Wt and dysf−/− myoblasts was calculated as the ratio t20t0 calculated out of 5 pictures containing ∼100 myoblast per picture, representing 5 independent experiments. Results are presented as means±SE (n=5). Asterisks represent significant difference between treatments (P<0.05). Figure 4. View largeDownload slide Halofuginone-treatment enhances lysosome exocytosis to the cytoplasmic membrane of dysf−/− myoblasts. Primary myoblasts of 5-week-old Wt and dysf−/− mice were treated or not with halofuginone for 24 h followed by 2 h incubation with FM1–43. Bromophenol blue was then added to the medium and FM1–43 fluorescence levels in the cells were monitored for 20 min. (A) Representative confocal microscope images show Wt and dysf−/− myoblasts immediately after adding BPB to the medium (T=0, upper panels) and at the end of the monitoring period (T=20 min; lower panels). Scale bar, 20 μm. (B) Intensity of FM1–43 fluorescence of Wt and dysf−/− myoblasts was calculated as the ratio t20t0 calculated out of 5 pictures containing ∼100 myoblast per picture, representing 5 independent experiments. Results are presented as means±SE (n=5). Asterisks represent significant difference between treatments (P<0.05). Halofuginone promotes syt-7 levels in isolated dysf−/− myofibers and in vivo To further decipher the role of halofuginone in membrane resealing and lysosomal fusion to the dysf−/− cell membrane, we evaluated the expression levels of a battery of proteins known to be involved in the patch-repair complex that is normally initiated by dysferlin (10,13,14,57). A western blot analysis with antibodies against Cav3, A1 and TRIM72, in lysates derived from Wt and dysf−/− single myofibers, revealed that these protein levels are unchanged by treatment with halofuginone (Supplementary Material, Fig. S3A–C), implying that additional proteins mediate halofuginones’ effects on these events. One such candidate, at least in the absence of dysferlin, might be Syt-7. This Ca2+-sensing protein mediates lysosome-trafficking and exocytosis in various cell types (23–28) and was shown to be expressed in skeletal muscle and to play a role in membrane repair (28). In addition, halofuginone’s promotive effect on membrane resealing post-laser injury was attenuated in the presence of Ca2+-free medium (data not shown), implying a requirement for Ca2+ for halofuginones’ effects. Here, we evaluated Syt-7 expression in single-myofiber cultures derived from Wt and dystrophic muscles. Freshly prepared myofibers from 6-week-old Wt, mdx and dysf−/− mice were immediately treated, or not, with 10 nM halofuginone for 24 h. Western blot analysis for Syt-7 expression in the myofibers’ protein lysates revealed that although Syt-7 is expressed in myofibers of all three mouse lines, halofuginone treatment increases its levels only in the dysf−/− myofibers, by ∼2-fold compared to their non-treated counterparts; it had no effect on Wt and mdx single myofibers (Fig. 5A). Based on these results, we followed Syt-7 protein expression in muscles of Wt mice in parallel to dysf−/− mice injected, or not, with halofuginone during disease development for up to 12 months. In the pre-treated 4-week-old mice, Syt-7 was only observed in few myofibers and was expressed in a condensed polar manner underneath the cell membrane in both Wt and dysf−/− tissue sections (Fig. 5Ba–d, respectively). No change in Syt-7 expression pattern was observed in 5-month-old or 12-month-old muscles of Wt mice (Fig. 5Be, f, k and l, respectively). In the muscle sections of 5-month-old non-treated dysf−/− mice, Syt-7 was observed in a polar manner underneath the myofiber membrane, in a pattern similar to that of the Wt myofibers from the parallel age group (Fig. 5Bg and h). However, in the 5-month-old halofuginone-treated dysf−/− mice, Syt-7 spread into the cytoplasm of the myofibers while retaining its polar-type expression (Fig. 5Bi and j), and appeared at higher expression levels than in its counterpart non-treated dysf−/− section (compare Fig. 5Bh and j). In the muscle sections of the 12-month-old non-treated dysf−/− mice, in which the dystrophic phenotype is widely spread throughout the skeletal muscles (58), Syt-7 expression appeared more scattered than in the 5-month-old non-treated dysf−/− sections (compare Fig. 5Bm, n, g and h, respectively). This scattering phenomenon was even more pronounced in the 12-month-old halofuginone-treated muscle sections, where Syt-7 was widely expressed across most of the myofiber cytoplasm with an increase in its abundance throughout the tissue compared to the non-treated mice (Fig. 5Bo and p). Figure 5. View largeDownload slide (A) Halofuginone increases synaptotagmin-7 (Syt-7) levels in dysf−/− single myofibers. Freshly prepared single myofibers from 6-week-old Wt, mdx and dysf−/− gastrocnemius were treated, or not, with 10 nM halofuginone for 24 h. Syt-7 protein levels were analyzed in the myofiber protein lysates by western blot with an antibody against Syt-7. Syt-7 protein levels were analyzed by densitometry and normalized to YY1 levels. Values are means±SE of three independent experiments and are presented in arbitrary units (AU). Asterisk represents significant difference within treatments (P<0.05). (B) Syt-7 expression pattern in the dysf−/− mouse is altered in an age-dependent manner and is affected by halofuginone. Dysf−/− mice were injected intraperitoneally with halofuginone (7.5 µg/mouse, 3 times a week) or saline either between the ages of 4 weeks to 5 months, or 9–12 months. Paraffin-embedded sections were prepared from quadriceps and distal muscles and immunostained with an antibody against Syt-7 (red) and DAPI for nuclei (blue). Muscle sections of Wt present a condensed appearance of Syt-7 in a polar manner below the cytoplasmic membrane, regardless of age (a and b, e and f, k and l). Syt-7 expression in the dysf−/− muscle sections of 4-week- (c and d), 5-month- (g and h) and 12-month-old mice (m and n). Syt-7 expression in muscle sections derived from halofuginone-treated dysf−/− mice at 5 months (i and j) and 12 months (o and p) of age. Scale bars, 50 μm (a, c, e, g, i, k, m and o), 100 μm (b, d, f, h, j, l, n and p). Figure 5. View largeDownload slide (A) Halofuginone increases synaptotagmin-7 (Syt-7) levels in dysf−/− single myofibers. Freshly prepared single myofibers from 6-week-old Wt, mdx and dysf−/− gastrocnemius were treated, or not, with 10 nM halofuginone for 24 h. Syt-7 protein levels were analyzed in the myofiber protein lysates by western blot with an antibody against Syt-7. Syt-7 protein levels were analyzed by densitometry and normalized to YY1 levels. Values are means±SE of three independent experiments and are presented in arbitrary units (AU). Asterisk represents significant difference within treatments (P<0.05). (B) Syt-7 expression pattern in the dysf−/− mouse is altered in an age-dependent manner and is affected by halofuginone. Dysf−/− mice were injected intraperitoneally with halofuginone (7.5 µg/mouse, 3 times a week) or saline either between the ages of 4 weeks to 5 months, or 9–12 months. Paraffin-embedded sections were prepared from quadriceps and distal muscles and immunostained with an antibody against Syt-7 (red) and DAPI for nuclei (blue). Muscle sections of Wt present a condensed appearance of Syt-7 in a polar manner below the cytoplasmic membrane, regardless of age (a and b, e and f, k and l). Syt-7 expression in the dysf−/− muscle sections of 4-week- (c and d), 5-month- (g and h) and 12-month-old mice (m and n). Syt-7 expression in muscle sections derived from halofuginone-treated dysf−/− mice at 5 months (i and j) and 12 months (o and p) of age. Scale bars, 50 μm (a, c, e, g, i, k, m and o), 100 μm (b, d, f, h, j, l, n and p). The effect of halofuginone on Syt-7 localization was further evaluated by immunofluorescence staining for Syt-7 in freshly prepared myofibers derived from Wt and dysf−/− mice. In the non-treated Wt myofibers, Syt-7 expression was concentrated on one side of the myofiber, underneath the cell membrane (Fig. 6Aa; Supplementary Material, SV2A), and remained there in the presence of halofuginone (Supplementary Material, SV2B). In the non-treated dysf−/− myofibers, the Syt-7 expression pattern resembled that of the Wt (Fig. 6Ab; Supplementary Material, SV2C). In contrast, in dysf−/− myofibers treated with halofuginone, Syt-7 was expressed in a scattered manner across the myofiber with stronger fluorescence levels than those in the untreated Wt or dysf−/− myofibers (Fig. 6Ac; Supplementary Material, SV2D). Figure 6. View largeDownload slide (A) Halofuginone alters the expression pattern of Syt-7 in single dysf−/− myofibers. Wt (a) and dysf−/− single myofibers were untreated (b) or treated (c) with halofuginone as described in Figure 5A and immunostained with antibody against Syt-7 and DAPI for nuclei. Dashed lines represent the myofiber edges according to the image acquired under bright field of the confocal microscope. Scale bar, 20 μm. (B) Syt-7 co-localizes with LAMP2B-positive lysosomes in single myofibers. Single myofibers derived from 6-week-old Wt and dysf−/− mice were treated as described in (A) and double-immunostained with antibodies against LAMP2B (red) and Syt-7 (green). The pictures represent a 3D computerized model of Syt-7 and LAMP2B co-localization in the Wt (a) and dysf−/− non-treated (b) and treated (c) single myofibers. Insets show the association of LAMP2B-positive lysosomes and Syt-7 in the myofibers. Note that the green signal of Syt-7 is embedded within the red signal of the LAMP2B-positive lysosomes. Scale bars, 0.5 μm (a), 2 μm (b) and 1 μm (c). Figure 6. View largeDownload slide (A) Halofuginone alters the expression pattern of Syt-7 in single dysf−/− myofibers. Wt (a) and dysf−/− single myofibers were untreated (b) or treated (c) with halofuginone as described in Figure 5A and immunostained with antibody against Syt-7 and DAPI for nuclei. Dashed lines represent the myofiber edges according to the image acquired under bright field of the confocal microscope. Scale bar, 20 μm. (B) Syt-7 co-localizes with LAMP2B-positive lysosomes in single myofibers. Single myofibers derived from 6-week-old Wt and dysf−/− mice were treated as described in (A) and double-immunostained with antibodies against LAMP2B (red) and Syt-7 (green). The pictures represent a 3D computerized model of Syt-7 and LAMP2B co-localization in the Wt (a) and dysf−/− non-treated (b) and treated (c) single myofibers. Insets show the association of LAMP2B-positive lysosomes and Syt-7 in the myofibers. Note that the green signal of Syt-7 is embedded within the red signal of the LAMP2B-positive lysosomes. Scale bars, 0.5 μm (a), 2 μm (b) and 1 μm (c). To assess the association between Syt-7 and LAMP2B-positive vesicles (i.e. lysosomes), double-immunostaining with anti-LAMP2B antibody (red) and anti-Syt-7 antibody (green) was performed in single Wt and dysf−/− myofibers. In all myofibers, part of the Syt-7 protein was ‘embedded’ in the LAMP2B-positive lysosomes, while the other part was observed in a non-associated form as demonstrated in the 3D composite images (Fig. 6B; arrows pointing toward the embedded form of Syt-7). In agreement with the results presented in Figures 3A and C and 5A, halofuginone treatment increased lysosome scattering and Syt-7 expression levels in the dysf−/− myofibers compared to their non-treated counterparts (Fig. 6Bc versus b). Discussion In several MDs, including dysferlinopathy, halofuginone has been reported to improve muscle pathology by reducing fibrosis and by directly affecting muscle-cell apoptosis and proliferation (39–41,44,45). In this study, we demonstrate that halofuginone has a direct impact on membrane integrity and lysosomal scattering and exocytotic processes, and is important for membrane repair, in dysf−/− mouse muscle cells. Moreover, for the first time, we describe a unique spatial- and age-dependent expression of Syt-7 in skeletal muscle during dysferlinopathy development in mice. Its increased levels and co-localization with the lysosomal marker, LAMP2B, imply an involvement of Syt-7 in membrane repair in the absence of dysferlin. Halofuginone treatment of dysf−/− mouse myotubes prior to hypo-osmotic shock wounding reduced the percentage of cells with ruptured membranes, while increasing the percentage of membrane-rupture-resistant cells. As expected, halofuginone had no effect on the Wt myotubes, in agreement with previous reports (34,42–45), emphasizing again that its effects are manifested only in dystrophic muscles (36). Together, the data suggest an ameliorating effect of halofuginone on the integrity and resilience of dysf−/− myotube membranes. This conclusion is further supported by the results of the laser-wounding assay with freshly isolated dysf−/− versus Wt myofibers; the wounding process—as indicated by FM1–43 accumulation—was significantly lower when halofuginone was present prior to the injury, than in the untreated dysf−/− myofibers, while having no significant effect on the Wt myofibers. The Wt and the non-treated dysf−/− myofibers contracted and bent at the injury site. Mechanical and laser-induced wounding of Xenopus oocyte membranes results in contraction at the wound edges, bringing them together to assist in wound closure (59,60). A similar mechanism may be occurring in skeletal muscle myofibers post-injury, hence their contraction post-laser wounding. The fact that halofuginone-treated dysf−/− myofibers did not bend or contract at the injury site, at least during the experimental period, supports our conclusion that halofuginone promotes dysf−/− membrane integrity and myofiber resilience. One possible explanation for the role of halofuginone in promoting membrane integrity, particularly in dysf−/− myofibers, could be its inhibitory effect on the TGFβ/Smad3-signaling pathway. TGFβ has been found to enhance ROS levels in myofibers, resulting in increased membrane fragility; blockage of TGFβ signaling in myofibers of transgenic mice improved myofiber sarcolemmal integrity (17). Moreover, elevated ROS levels have been reported to correlate with advancing dysferlinopathy pathology (61). Halofuginone has been found to inhibit Smad3 phosphorylation in dysf−/− mouse muscles (41), and in dysf−/− single myofibers (data not shown). Taken together with the data presented here, it is conceivable that halofuginone improves sarcolemmal integrity in dysf−/− myofibers, at least in part, via its inhibitory effect on Smad3 phosphorylation downstream of TGFβ. Lysosome fusion to the cytoplasmic membrane is considered a hallmark of membrane resealing post-micro injuries (62). In skeletal muscle, the membrane-resealing process is mediated by the patch-repair protein complex, which is known to be compromised by lack of dysferlin (1,10,14,63). Indeed here, the dysf−/− myoblasts demonstrated markedly lower levels of lysosome scattering as compared to the Wt myoblasts; most dysf−/− myoblasts’ lysosomes remained close to the nucleus, in agreement with previous reports (15). However, halofuginone completely rescued the lysosome scattering in dysf−/− myoblasts in a time-dependent manner, suggesting its positive effect on the lysosomes’ scattering ability in absence of dysferlin. Moreover, the data indicate that the positive effect is MAPK/ERK and PI3K/Akt pathway-dependent. While halofuginone doubled the lysosome scattering, the addition of pharmacological inhibitors for these pathways significantly prevented its positive effect, implying that this effect is transduced via the activation of MAPK/ERK and PI3K/Akt pathways (Fig. 4D and E). These pathways have been reported to mediate skeletal cell adhesion (64) and vesicle docking and fusion to the plasma membrane (65,66), both of which require the lysosomes’ initial scattering across the cell periphery (67). Both, MAPK/ERK and PI3K/Akt pathways have been shown to mediate halofuginones’ effects on cell proliferation and apoptosis in dysf−/− muscle (45). However, in those cases as well in the case of lysosome scattering, the promotive effect of halofuginone on MAPK/ERK and PI3K/Akt signaling pathways could be a direct or indirect one. Interestingly, similar to Wt myoblasts, the lysosome scattering was not affected by halofuginone in mdx myoblasts, suggesting that in this case, halofuginone’s effect may not be related to its canonical effect on the TGFβ/Smad3-signaling pathway. As discussed below, in dysferlin-null muscle cells, other proteins may mediate the beneficial effect of halofuginone on membrane repair. To this point, the data demonstrate that in dysf−/− muscle cells, halofuginone enhances lysosome exocytosis, presumably by promoting their trafficking across the cell toward the fusion site, by actin filaments and microtubules (68). Moreover, based on our previous reports that halofuginone improved mdx and dysf−/− myotube fusion (34,41), and the evidence that halofuginone’s promotive effect on membrane resealing post-laser injury was attenuated in a Ca2+-free medium (data not shown), it is conceivable that halofuginone also promotes lysosomal exocytosis at least in vitro, thereby ameliorating membrane repair post-micro injury. In muscle cells, the membrane-repair process involves the activity of the patch-repair protein complex. However, here, halofuginone failed to affect the levels of the key proteins Cav3, A1 and TRIM72 in dysf−/− myofibers, implying the involvement of additional proteins in mediating its effects on lysosome scattering and fusion to the sarcolemma. Many studies performed with non-skeletal-muscle models have pinpointed synaptotagmins in general, and Syt-7 in particular, as mediators of lysosome-trafficking, exocytosis and membrane resealing (18,19,23–25,69,70), making Syt-7 a suitable candidate in compensating for the lack of dysferlin. The data presented here also place Syt-7 as a possible mediator of halofuginones’ effects on these processes in dysferlinopathies. This is due to the followings: (a) like dysferlin, Syt-7 is a Ca2+ sensor localized on the membrane of mature lysosomes (18,27,69). Furthermore, cleavage of dysferlin exon 40a releases a synaptotagmin module for membrane repair (70). (b) Although it presents normal growth and development, the Syt-7-knockout mouse develops enhanced inflammation and fibrosis, elevated creatine kinase and muscle weakness with defective lysosome-mediated membrane repair (28), suggesting a role for Syt-7 in muscle membrane repair. (c) The positive effect of halofuginone on membrane repair was attenuated in Ca2+-free medium (data not shown), implying a Ca2+-sensing protein mediating its effects. Our results show, for the first time, a spatial- and age-dependent expression pattern of Syt-7 protein in dysf−/− versus Wt mice, suggesting that these pattern alterations are related to the disease progression. Injecting halofuginone into dysf−/− mice further enhanced Syt-7 expression levels and its spread across the cytosol in the dysf−/− mouse muscles (Fig. 5B) and myofibers (Fig. 6A). Moreover, while Syt-7 protein was expressed in isolated myofibers derived from young Wt as well as mdx and dysf−/− mice, halofuginone treatment elevated its levels only in dysf−/− myofibers, suggesting a unique effect on Syt-7 levels in dysferlinopathies. Part of Syt-7 co-localized with lysosomes in single myofibers from Wt and dysf−/− mice, supporting at least in part the previously suggested role for Syt-7 in lysosomal exocytosis and muscle membrane resealing post-injury (28). However, halofuginone enhanced Syt-7 expression levels and induced the scattering of both Syt-7 and lysosomes only in dysf−/− myofibers; it is thus conceivable that in these myofibers, halofuginone improves the association of Syt-7 with the lysosomes. In young DMD patients and mdx mice, the expression of utrophin, which shares some homology with dystrophin (71,72), is enhanced and hence, it is believed to partially compensate for the lack of dystrophin functions (72). Our previous studies have shown that halofuginone enhanced utrophin levels in mdx mice in correlation with fibrosis reduction (43). The results of our study imply a parallel phenomenon in dysferlinopathies in which Syt-7 might compensate for the lack of dysferlin at least with regard to membrane resealing post-injury and may partially explain the late onset of this disease. The halofuginones’ promotive effects over Syt-7 expression pattern and levels as well as the association with lysosomes in dysf−/− mice implies halofuginones further contribute to the compensatory effect of Syt-7 both, prior to disease phenotype appearance and during disease development. In summary, the results presented in this paper demonstrate halofuginone’s improvement of membrane integrity and protection of the sarcolemma from microscopic tears in dysferlinopathy muscle cells. This is partially explained by halofuginone’s promotive effect on lysosome-trafficking, which is Akt- and MAPK/ERK-signaling pathway-dependent, and exocytotic events. Moreover, the findings reveal a spatial- and age-dependent pattern of Syt-7 in relation to the disease development, and its association with lysosome scattering, implying Syt-7 in compensating for the lack of dysferlin in membrane resealing post-injury. The surpassed effects of halofuginone on Syt-7 scattering and association with lysosomes may in part explain halofuginone’s action in enhancing membrane repair in dysf−/− mouse myofibers. Materials and Methods Reagents Dulbecco’s modified Eagle’s medium (DMEM), sera and an antibiotic–antimycotic solution were purchased from Biological Industries (Beit-Haemek, Israel). Halofuginone bromhydrate was obtained from Akashi Therapeutics Inc. (Cambridge, MA). Ly and UO126 were purchased from Calbiochem (Darmstadt, Germany). Calcein-AM and FM1–43 were purchased from Molecular Probes (Thermo Fisher Scientific, Waltham, MA). PI and BPB were purchased from Sigma Aldrich (St. Louis, MO). Animals and experimental design Male dysf−/− [mixed 129SvJ and C57/BL/g background (Stock 006830) in which a 12-kb region of the dysf gene containing the last three exons is deleted, removing the transmembrane domain], mdx [C57BL/10ScSn-Dmdmdx/J (Stock 001801), dystrophin-deficient] and Wt C57/Bl/6J mice (Jackson Laboratories, Bar Harbor, ME) were housed in cages under constant photoperiod (12 L:12 D) with free access to food and water. For the in-vivo experiment, two age groups of dysf−/− mice were injected intraperitoneally with either saline or 7.5 μg halofuginone three times a week. One group was injected from 4 weeks to 5 months of age, a period during which early and later on, pronounced dystrophic changes can be found in distal and proximal muscles, respectively (58). The second group was injected between 9 and 12 months of age during which time the dystrophic phenotype is widely spread throughout the skeletal muscles with pronounced elevation of endomysial fibrosis and inflammation (58). Before the initiation of the treatments at 4 weeks of age and at the end of each injection period, mice were sacrificed and biopsies from distal and quadriceps muscles were collected for further analyses. All animal experiments were carried out according to the guidelines of the Volcani Center Institutional Committee for Care and Use of Laboratory Animals (IL-234/10). Mouse cell preparation and immunostaining Primary myoblasts from the hind-leg muscles of 5-week-old Wt and dysf−/− mice were prepared as described previously, with less than 5% of these cells being non myogenic (44,45). Cells were plated at a density of 3 × 105 cell/cm2 on 90-mm diameter Petri dishes and grown in DMEM supplemented with 20% fetal bovine serum (FBS) at 37.5°C with humidified atmosphere and 5% CO2 in air. For immunostaining, cells were plated on glass coverslips at a density of 5 × 104 cell/cm2. Cells were fixed with 2% (w/v) paraformaldehyde in PBS and blocked with 20% (v/v) goat serum (GS) in PBS for 1 h at room temperature followed by overnight incubation at 4°C with LAMP2B antibody (1:600, Abcam, Cambridge, UK). Cy3 donkey anti-rat IgG (1:500, Jackson Laboratories) was used as a secondary antibody. Cell nuclei were stained with DAPI (1:1000). The myoblasts were visualized under a fluorescence microscope equipped with a DP-11 digital camera (Olympus, Hamburg, Germany). Single myofiber preparation and immunostaining Single myofibers were isolated from the gastrocnemius muscle of 6-week-old Wt and dysf−/− mice as described previously (45). Briefly, gastrocnemius muscles were incubated for 2 h in 0.28% (w/v) collagenase type I in DMEM. The collagenase-treated muscles were then transferred to 10% (v/v) horse serum (HS) in DMEM for titration with a wide-mouth pipette. At the end of the preparation, myofibers were washed with 10% HS in DMEM and transferred to 60-mm diameter Petri dishes for further analyses. For immunostaining, the myofibers were fixed with 4% paraformaldehyde in PBS (w/v), washed with 0.5% (v/v) Triton-X100 and 1% (v/v) Tween-20 in PBS, and blocked with 20% GS and 1% (w/v) bovine serum albumin (BSA) in 0.05% Tween–PBS for 1 h at room temperature. This was followed by overnight incubation at 4°C with polyclonal antibodies against LAMP2B (1:600) and/or Syt-7 (1:200; Synaptic Systems, Goettingen, Germany). Cy3 donkey anti-rat IgG (1:500) and 488-Alexa Fluor goat anti-rabbit IgG (1:300, Jackson Laboratories) were used as secondary antibodies. Nuclei were stained with DAPI. The myofibers were visualized under a Leica sp8 inverted laser-scanning confocal microscope (Leica Camera, Wetzlar, Germany), equipped with a 405 nm diode laser, a 488 nm optically pumped semiconductor laser (OPSL), a 558 nm OPSL and an HC PL APO CS2 63X/1.40 oil objective. ‘GREEN’ was excited at 488 nm; ‘RED’ was excited at 558 nm. Hypo-osmotic shock assay Mouse primary myoblasts were plated at a density of 2.4 × 104 cell/cm2 in 12-well plates and induced to differentiate for 3 days in differentiation medium containing 2% HS in DMEM. Cells were then treated for 24 h with 10 nM halofuginone in medium containing 20% FBS in DMEM (growth medium) as previously described (34,41). At the end of the treatment, the wells were washed once in DMEM and incubated with DMEM containing calcein-AM (1:200) and DAPI (1:100) for 20 min. Hypo-osmotic shock was obtained by incubating the cells with 10% (v/v) DMEM in double-distilled water (DDW), approximating 30 mOsm hypo-osmotic shock. PI (15 µg/ml) was added to the hypo-osmotic medium as described (38). Leica sp8 inverted laser-scanning confocal microscope, equipped with a 405 nm diode laser, a 552 nm OPSL, a 488 nm OPSL and an HC PL FLUOTAR 10X/0.30 dry objective was used. ‘RED’ was excited at 493 nm and ‘GREEN’ at 495 nm. Cell fluorescence was followed for 5 min immediately after medium change to hypo-osmotic medium and acquisitions were performed at time intervals of 15 s. Images were analyzed with FIJI (ImageJ) software. Myotubes were selected as cells containing two or more nuclei, as indicated by DAPI staining. Single-nucleated cells were not measured. Myofiber laser-wounding assay The assay was conducted according to Bansal et al. (1). Freshly isolated single myofibers were kept in DMEM supplemented with 10% HS with or without 10 nM halofuginone for 24 h. The myofibers were then washed once in DMEM and mounted in a glass slide chamber (Nunc Lab Tek chambered cover glass, Thermo Fisher Scientific). The membrane was damaged in the presence of FM1–43 dye (2.5 µM) by irradiating a 5 × 5 µm2 area of the myofiber sarcolemma surface at 80% power of the Leica sp8 diode 405 laser (UV) for 5 s (1). Images were captured every second from 3 s before the damage to 5 min post-damage under the Leica sp8 inverted laser-scanning confocal microscope, equipped with a 405 nm diode laser and a 488 nm OPSL, and using the HC PL FLUOTAR 10X/0.30 dry objective. ‘RED’ was excited at 510 nm. Eight single myofibers were used for each treatment and were maintained in 5% CO2, 37°C and 95% relative humidity throughout the experiment. For every image taken, the fluorescence intensity at the site of damage was analyzed with FIJI (ImageJ) software. The fluorescence intensity was normalized according to the equation: In=Imtx-Ibtx/Imt0-Ibt0 where In = normalized intensity, Im = measured intensity at time x (tx) or time zero (t0), Ib = mean background intensity at tx or t0 (mean of the single myofiber background intensity and the total image background intensity); t0 represents the mean fluorescence intensity of three images taken before membrane damage induction and tx is an image taken every second after damage induction (73). FM1–43 fluorescence levels were calculated using FIJI (ImageJ) software. Quenching assay FM1–43 quenching experiments were performed according to Han et al. (16). Briefly, primary myoblasts were plated in a glass slide chamber at a density of 3.5 × 103 cell/chamber and grown in DMEM supplemented with 20% FBS with or without 10 nM halofuginone for 24 h, after which 8 µM FM1–43 was added to the medium for an additional 2 h, allowing its internalization and endocytosis into the lysosomal vesicles. At the end of the incubation, 1 mM BPB was added to the medium and its fluorescence was monitored for 20 min at 1 min intervals under a Leica sp8 inverted laser-scanning confocal microscope equipped with a 488 nm OPSL using the HC PL FLUOTAR 10X/0.30 dry objective. ‘RED’ was excited at 510 nm. Images were captured every minute for a total of 5 min. Cell cultures were maintained in 5% CO2, 37°C and 95% relative humidity. Immunohistochemistry Muscle samples were fixed with 4% paraformaldehyde in PBS at 4°C overnight, dehydrated and embedded in paraffin as previously described (39). Sections (5 µm) were prepared, deparaffinized and rehydrated with ethanol and citrate buffer. Immunohistochemistry was conducted as previously described (42,44). Briefly, the sections were incubated with 10% GS and 2% BSA in 0.05% Tween-20–PBS for 1 h at room temperature followed by overnight incubation at 4°C with Syt-7 antibody (1:200). The secondary antibody was Cy3 goat anti-rabbit IgG (1:200, Jackson Laboratories). Nuclei were then stained with DAPI. Microscope observations and image acquisition were performed with the Leica sp8 inverted laser-scanning confocal microscope, equipped with a 405 nm diode laser, 488 nm OPSL, 552 nm OPSL and the HC PL APO CS2 63X/1.40 oil objective. ‘RED’ was excited at 552 nm. Western blot analysis Western blot analysis was performed as described previously (53). Briefly, equal amounts of protein were resolved by SDS-PAGE and then transferred to nitrocellulose membranes (Bio-Rad Laboratories, Hercules, CA). After blocking, the membranes were incubated with primary antibodies at 4°C overnight. The following primary antibodies were used: rabbit polyclonal anti-Syt-7 (1:1000), anti-Ying-Yang 1 (YY1; 1:500) and mouse monoclonal anti-Cav3 (1:500; Santa Cruz Biotechnology, Dallas, TX), anti-A1 and anti-TRIM72 (1:1000; Abcam), anti-phospho-Akt and anti-phospho-MAPK/ERK (P-Akt and P-MAPK/ERK, respectively; 1:500; Cell Signaling Technology, Danvers, MA). The transcriptional repressor protein YY1 was chosen because its gene expression is not altered in dystrophic mice after halofuginone treatment (74). Statistical analysis The data were subjected to one-way analysis of variance (ANOVA) and to all-pairs Tukey–Kramer HSD test using JMP® software (75). Supplementary Material Supplementary Material is available at HMG online. Acknowledgements The authors thank Ann Bigot and the platform for immortalization from the Centre for research in Myology of the Institut de Myologie in Paris for the immortalized clones, Cédric M. Blouin for assistance with the hypo-osmotic shock assay, Sergei Grigoryan, Einat Zelinger and Avi Jacob for their technical assistance with the confocal microscope and data processing. H.B.-T. is supported by a fellowship from R.H. Smith for excellence. Conflict of Interest statement. None declared. References 1 Bansal D. , Miyake K. , Vogel S.S. , Groh S. , Chen C.C. , Williamson R. , McNeil L.P. , Campbell P.K. ( 2003 ) Defective membrane repair in dysferlin-deficient muscular dystrophy . Nature , 423 , 168 – 172 . Google Scholar CrossRef Search ADS PubMed 2 Zammit P.S. , Partridge T.A. , Yablonka-Reuveni Z. ( 2006 ) The skeletal muscle satellite cell: the stem cell that came in from the cold . J. Histochem. Cytochem ., 54 , 1177 – 1191 . Google Scholar CrossRef Search ADS PubMed 3 Emery A.E. ( 2002 ) The muscular dystrophies . Lancet , 359 , 687 – 695 . Google Scholar CrossRef Search ADS PubMed 4 Wallace G.Q. , McNally E.M. 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Human Molecular Genetics – Oxford University Press
Published: Aug 1, 2018