Thrombospondin expression in myofibers stabilizes muscle membranes

Skeletal muscle is highly sensitive to mutations in genes that participate in membrane stability and cellular attachment, which often leads to muscular dystrophy. Here we show that Thrombospondin-4 (Thbs4) regulates skeletal muscle integrity and its susceptibility to muscular dystrophy through organization of membrane attachment complexes. Loss of the Thbs4 gene causes spontaneous dystrophic changes with aging and accelerates disease in 2 mouse models of muscular dystrophy, while overexpression of mouse Thbs4 is protective and mitigates dystrophic disease. In the myofiber, Thbs4 selectively enhances vesicular trafficking of dystrophin-glycoprotein and integrin attachment complexes to stabilize the sarcolemma. In agreement, muscle-specific overexpression of Drosophila Tsp or mouse Thbs4 rescues a Drosophila model of muscular dystrophy with augmented membrane residence of βPS integrin. This functional conservation emphasizes the fundamental importance of Thbs’ as regulators of cellular attachment and membrane stability and identifies Thbs4 as a potential therapeutic target for muscular dystrophy. DOI: http://dx.doi.org/10.7554/eLife.17589.001


Introduction
Muscle degenerative diseases such as muscular dystrophy (MD) are most commonly caused by mutations in genes that are part of the dystrophin-glycoprotein (DGC) complex or the integrin complex of proteins (Grounds et al., 2005;McNally and Pytel, 2007). In addition, proper post-translational processing and trafficking of these complexes to the sarcolemma are essential to form a molecular attachment network between the myofilament proteins within the myofibers and the basal lamina and extracellular matrix (ECM) outside the cell (Goddeeris et al., 2013;Liu et al., 2012;Xu et al., 2009). This attachment network provides critical structural support to the plasma membrane (sarcolemma) to withstand contractile forces (Burr and Molkentin, 2015;Grounds et al., 2005;Gumerson and Michele, 2011;Lapidos et al., 2004). When this attachment network is deficient in MD, membrane ruptures occur leading to intracellular calcium influx that causes myofiber necrosis, an inflammatory response, fibrosis and fatty tissue replacement, and ultimately muscle functional loss and death (Burr and Molkentin, 2015;Gumerson and Michele, 2011;Lapidos et al., 2004). Skeletal muscle is perhaps the most sensitive of all tissues to genetic mutations in genes that impact cellular attachment complexes or membrane repair capacity, in part because of the dynamic changes in length that occurs in each myofiber during contraction (Burr and Molkentin, 2015;Grounds et al., 2005;McNally and Pytel, 2007).
Thrombospondins (Thbs) comprise a family of 5 genes in mammals that encode secreted matricellular proteins involved in diverse biologic processes (Adams and Lawler, 2011 ;Schellings et al., 2009). The thrombospondin family consists of two subgroups based on their sequence conservation and oligomeric structure. Thbs3, Thbs4 and Thbs5 form pentamers and are the most similar to Thbs genes found in lower organisms (Adams and Lawler, 2011). Thbs1 and Thbs2 form trimers and have evolved additional domains such as a type 1 repeat important for transforming growth factor-b binding and a region that affects angiogenesis (Adams and Lawler, 2011;Schellings et al., 2009). Drosophila contains a single thrombospondin gene (Tsp) that forms pentamers, and when deficient causes developmental lethality due to disruption in muscle and tendon attachment within the body wall segments of the embryo (Adams and Lawler, 2011;Subramanian et al., 2007).
While traditionally characterized as a secreted ECM or matricellular protein over the past 3 decades, (Adams and Lawler, 2011;Schellings et al., 2009) Thbs can also function within the cell, and in some systems this appears to be their primary role (Ambily et al., 2014;Baek et al., 2013;Brody et al., 2016;Duquette et al., 2014;Lynch et al., 2012;McKeown-Longo et al., 1984;Posey et al., 2014). For example, Thbs4 was recently shown to have a critical cardioprotective function from within the endoplasmic reticulum (ER) where it mediates an adaptive ER stress response (Brody et al., 2016;Lynch et al., 2012). The traditional ER stress response involves sensing of calcium and unfolded or damaged proteins within the ER through the calcium binding chaperone protein BiP (GRP78), which binds/regulates at least 3 distinct stress response pathways initiated by either PKR-like ER kinase (PERK), inositol-requiring enzyme 1a (IRE1a) or activating transcription factor 6 (ATF6), each resident within the ER membrane (Glembotski, 2007;Mori, 2009). These 3 ER stress response mediators initiate a cascade of signaling that alters protein synthesis and other features of cellular adaptation to stress or protein unfolding and aggregation (Glembotski, 2007;Mori, 2009). Here, Thbs4 directly binds the ER luminal domain of ATF6a to promote its shuttling to eLife digest Muscle cells, also known as myofibers, need to be robust in order to withstand the physical stresses of contracting and relaxing. As a result, the cell surface membrane that surrounds myofibers is more strongly anchored to its surroundings than that of other cells. Muscular dystrophies are a group of muscle-wasting disorders that usually arise when this surface membrane becomes less stable. For example, mutations that affect a protein called dystrophin-glycoprotein or integrin protein complexes can cause muscular dystrophy since these proteins normally keep the membrane anchored and stable when the muscle contracts and relaxes.
When myofibers in mammals become injured, as is the case during muscular dystrophy, they produce more proteins called thrombospondins -with thrombospondin-4 being the most common. However, until now it was not clear what these proteins did in muscle cells.
Vanhoutte et al. hypothesized that thrombospondin-4 may protect injured myofibers and tested their theory by first deleting the gene for thrombospondin-4 from mutant mice that were predisposed to develop muscular dystrophy. This worsened the muscle wasting in the mutant mice, and furthermore, deleting the gene for thrombospondin-4 also caused otherwise normal mice to develop muscular dystrophy in their old age. Conversely, when Vanhoutte et al. artificially increased the levels of thrombospondin-4 in the myofibers, it protected the mice against muscular dystrophy. Additional experiments conducted in fruit flies demonstrated that the protective effects of thrombospondin are conserved or similar in insects too. Lastly, biochemical experiments in mouse and rat cells showed that thrombospondin-4 aids dystrophin-glycoproteins and integrins in getting to the cell surface membrane, increasing its stability.
Overall these findings provide a clearer picture of the molecular underpinnings of muscular dystrophies. In the future, more experiments will have to focus on exactly how thrombospondins stabilize and direct dystrophin-glycoproteins and integrins to the cell surface membrane.
the Golgi and then nucleus, thereby inducing genes underlying adaptive aspects of the ER stress response (Brody et al., 2016;Lynch et al., 2012).
Thbs proteins move through the secretory pathway where they appear to facilitate secretion of ECM proteins or perhaps chaperone protein complexes to the cell membrane (Adams and Lawler, 2011). Once secreted, Thbs proteins transiently or permanently reside in the ECM where they interact with fibronectin, collagens and proteoglycans (Adams and Lawler, 2011;Frolova et al., 2014Frolova et al., , 2012Hauser et al., 1995;Sö dersten et al., 2006). Thbs proteins are also recycled back into the cell through the low-density receptor-related protein (LRP) (Wang et al., 2004). One critical feature of the Thbs family is that each member is induced following injury events or in response to processes requiring tissue growth, healing and remodeling. Interestingly, Thbs4 is largely restricted to cardiac and skeletal muscle where its expression is induced with injury or disease (Adams and Lawler, 2011;Chen et al., 2000;Frolova et al., 2014Frolova et al., , 2012Hauser et al., 1995;Lynch et al., 2012;Schellings et al., 2009;Sö dersten et al., 2006). In addition, markers of ER stress are upregulated during progression of skeletal muscle disease and MD (Lavery et al., 2008;Moorwood and Barton, 2014).
Here we observed that in response to MD in skeletal muscle, Thbs4 mRNA and protein are induced. Overexpression of Thbs4 in skeletal muscle of transgenic (Tg) mice protected against MD, while mice lacking Thbs4 (Thbs4 -/-) showed signs of spontaneous MD with aging. Mechanistically, Thbs4 directs a membrane attachment intracellular vesicular trafficking network that promotes greater stability of the DGC and integrin complexes at the sarcolemma of skeletal muscle fibers. This function of Thbs is conserved in Drosophila as overexpression of either mouse Thbs4 or Drosophila Tsp in muscle rescues MD that occurs in Drosophila deficient in its d-sarcoglycan-related gene (Allikian et al., 2007).

Thbs4 augments adaptive ER stress signaling in skeletal muscle and mitigates MD
In agreement with previous findings, Thbs4 RNA is induced in muscle biopsies from human patients with Becker MD, Duchenne MD, and limb-girdle MD (LGMD) ( Figure 1A; Figure 1-figure supplement 1A) (Chen et al., 2000). We next turned to 2 different mouse models of MD. One due to deletion of the d-sarcoglycan (Sgcd) gene to model LGMD2F, and a second due to defective dystrophin expression resulting from the mdx mutation that models Duchenne MD (Durbeej and Campbell, 2002). Thbs4 protein is induced in skeletal muscle of each mouse model at six weeks and three months of age, along with induction of an ER stress response associated with greater cleaved ATF6a-N (nuclear form) and increased total BiP levels ( Figure  To model the known increase in Thbs4 protein that occurs in dystrophic skeletal muscle we generated Tg mice with Thbs4 protein overexpression specific to skeletal muscle ( Figure 1D). High levels of Thbs4 protein overexpression were observed in fast-twitch containing muscles such as the quadriceps, gastrocnemius, with intermediate levels in the diaphragm and very low levels in the soleus, while the heart lacked expression ( Figure 1D). Tissue Immunofluorescent analysis revealed that Thbs4 protein was undetectable in uninjured skeletal muscle while the transgene produced abundant expression that co-localized with calreticulin to a vesicular network on the periphery of the myofibers of paraffin-embedded quadriceps and was also clearly inside of collagen I staining that marks the ECM of cryo-embedded quadriceps ( Figure 1E). Furthermore, although Thbs4 protein localization appeared slightly different between paraffin-and cryo-embedded skeletal muscle of the Sgcd -/mouse, induction of endogenous Thbs4 again showed localization within the vesicular network inside the myofibers, and only within limited regions outside of myofibers where fibrotic tissue deposition was prominent ( Figure 1E).
In agreement with the above observations of Thbs subcellular localization, biochemical analysis revealed that Thbs4 was high in the type of glycosylation that typifies ER resident proteins, but it also contains glycosylation that is observed on proteins that transit through the Golgi in route to be deposited in the ECM (Figure 1-figure supplement 2A Immunogold Thbs4 H Thbs4 Calreticulin g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g

Thbs4
Collagen I * Thbs4 Tg WT Figure 1. Thbs4 is induced in dystrophic skeletal muscle and its overexpression augments ER activity and vesicle content. (A) Thbs4 mRNA levels in human skeletal muscle biopsies from normal or patients with Becker MD (BMD; n = 5), Duchenne MD (DMD; n = 10) or 2 different types of limb-girdle MD (LGMD; n = 10 for both). *p<0.05 vs. normal (n = 18) by Student's t test. Data are presented as mean ± SEM. Full analysis including all 11 human muscle diseases is shown in Figure 1-figure supplement 1A. (B,C) Western blot for the expression of Thbs4, ATF6a-N (50 kDa, nuclear) and BiP in the quadriceps of WT, Sgcd -/and mdx mice at six weeks (w) and three months (mo) of age (n = 4 biological replicates). (D) Schematic diagram showing the skeletal muscle-specific transgene to overexpress Thbs4 and (lower) Western blots for Thbs4 or gapdh control from WT and Tg mice at 6 w of age from Quad, quadriceps; Gas, gastrocnemius; Sol, soleus; Diaph, diaphragm; and heart (n = 2 biological replicates). (E) Upper micrographs represent coimmunofluorescent labeling of intracellular Thbs4 (green) with calreticulin (red) on paraffin embedded quadriceps (Quad.) of WT, Thbs4-Tg and Sgcd -/mice at 3 mo of age (scale bar = 20 mm). Arrowheads indicate co-localization of Thbs4 with calreticulin in intracellular vesicles in the myofibers. Lower micrographs represent co-immunofluorescent labeling of Thbs4 (green) with collagen I (red) on cryo-embedded Quad of WT, Thbs4-Tg and Sgcd -/mice at 3 mo of age (scale bar = 50 mm). Arrowheads indicate co-localization of Thbs4 with collagen I in the extracellular milieu; the star marks a myofiber with both intra-and extracellular Thbs4 labeling from a diseased muscle. Nuclei are visualized in blue. Representative images of 4 mice per genotype are shown. (F) Western blot analysis of Thbs4 and the ER-stress proteins ATF6a-N (50 kDa, nuclear), BiP, PDI, calreticulin (Calret.), and Armet in 6w old WT, Thbs4-Tg and Sgcd -/quadriceps (n = 4 biological replicates). (G) Transmission electron microscopy in WT versus Thbs4 Tg quadriceps at 3 mo of age showing a massive expansion of intramyofibrillar and subsarcolemmal ER and associated vesicles with Thbs4 overexpression (arrowheads, scale bar = 2 mm). Representative images of 2 mice per genotype are shown. (H) Immunogold electron microscopy shows that Thbs4 (6 nm gold particles; yellow arrows) robustly localizes to the expanded sub-sarcolemmal vesicular compartment in Thbs4-Tg quadriceps, compared to endogenously expressed Thbs4 in WT quadriceps. Representative images of 2 mice are shown. Scale bar = 50 nm DOI: 10.7554/eLife.17589.003 The following figure supplements are available for figure 1: myotubes and at least in the case of myoblasts is transported to rab7-positive late endosomes, potentially explaining why Thbs4 protein is low or undectable in the ECM of healthy Thbs4 Tg muscles (Figure 1-figure supplement 3). Similar to the results observed with collagen I, co-labeling with another ECM/matricellular protein periostin again showed that Thbs4 could co-localize to the ECM region in Sgcd -/diseased myofibers, although under non-diseased conditions overexpressed Thbs4 was again only appreciably observed intracellularly under the sarcolemma within a peripheral vesicular pattern ( Figure 1E; Figure 1-figure supplement 2C). Hence, although our observations do not exclude the possibility that non-muscle cells might also express Thbs4, our data collectively identify the myofiber as an important cellular source of Thbs4 expression, secretion and re-uptake.
Careful analysis of other markers of the ER compartment and ER stress showed that Thbs4 overexpression in skeletal muscle induced a profile very similar to diseased skeletal muscle in Sgcd -/mice, with increased levels of nuclear ATF6a, BiP, protein disulfide isomerase (PDI), calreticulin and Armet, as compared to WT muscle ( Figure 1F; Figure 1-figure supplement 1D). Remarkably, transmission electron microscopy and immunogold detection revealed that Thbs4 overexpression in skeletal muscle caused a dramatic induction of sub-sarcolemmal and intramyofibrillar ER and post-ER vesicles that contained Thbs4 protein ( Figure 1G,H; Figure 1-figure supplement 2D). These Thbs4-dependent vesicles were highly uniform in size and more electron dense compared with similar vesicles in subsarcolemmal regions from WT muscle. Future studies will investigate the nature of these Thbs4-expanded vesicles and their composition based on known variables (Malhotra and Erlmann, 2015;Paczkowski et al., 2015).
To determine if Thbs4 induction in MD was adaptive or maladaptive we first crossed the Thbs4 Tg into both the Sgcd -/and mdx backgrounds. Importantly, Thbs4 overexpression itself in skeletal muscle caused no histopathology or functional defects compared to WT mice at three and 12 months of age ( More importantly, Thbs4 overexpression significantly reduced multiple histopathological hallmarks of dystrophic disease, including elevated serum creatine kinase (CK) levels, reduced myofiber degeneration/regeneration cycles as marked by reduced centrally nucleated myofibers, reduced fibrotic remodeling and less functional decline in skeletal muscle at both three and 12 months of age in both Sgcd -/and mdx mice, compared with each dystrophic model alone ( The sarcolemma of dystrophic myofibers are weak and frequently rupture, which can be assessed in vivo by Evans blue dye (EBD) uptake into muscle fibers after systemic injection (Goonasekera et al., 2011;Lapidos et al., 2004). Here, the percentage of myofibers with ruptured membranes after forced treadmill running was significantly reduced in Sgcd -/and mdx mice that contained the Thbs4 Tg, compared with Sgcd -/and mdx mice alone, while no EBD uptake was observed in WT or Tg muscle ( Figure  This dramatic protection from MD observed in 2 mouse models of this disease with Tg-mediated overexpression of Thbs4 led us to investigate whether overexpression of Thbs4 would be sufficient to reduce acute dystrophic disease for the first time using a gene therapy-related approach to bypass possible developmental effects of overexpression. Hence, here we performed a study in Sgcd -/mice with an adeno-associated virus serotype-9 (AAV9)-Thbs4 vector, which was injected into the gastrocnemius of three day-old neonates, followed by harvesting at six weeks of age to assess histopathology ( Figure 2-figure supplement 4A). Littermates injected with an eGFP expressing AAV9 were used as a control. In agreement with our previous findings, this approach also resulted in abundant expression of either the control eGFP protein or a 5-fold increase in Thbs4 in the muscle

Loss of Thbs4 predisposes to MD
The induction of Thbs4 that normally occurs in skeletal muscle with dystrophic disease was further shown to be an adaptive and physiologic mechanism through the analysis of mice lacking the Thbs4 gene. Here we crossed Sgcd -/and mdx mice into the Thbs4 null background and performed a full analysis of pathogenesis at three months of age. Thbs4 -/mice alone at three months of age showed minimal or no pathological changes in skeletal muscle, although combinatorial Thbs4 -/-Sgcd -/or Thbs4 -/mdx mice showed a significant worsening of MD, including greater histopathology, greater serum CK levels and reduced treadmill running performance, compared with single null Sgcd and mdx mice (Figure 3A-C; Figure 3-figure supplement 1A-C). Sgcd -/-Thbs4 -/mice also showed significantly greater EBD uptake in skeletal muscle after forced treadmill running versus Sgcd -/mice alone ( Figure 3D,E).
The observation that loss of the Thbs4 gene makes dystrophic pathology significantly worse in both Sgcd -/and mdx mice suggests that induction of this gene product plays an important protective role, and we reasoned that with aging loss of Thbs4 might eventually become pathologic to muscle given that low levels of continuous expression are present. Indeed, we observed that by six months and one year of age, Thbs4 -/mice showed a significant reduction in treadmill running capacity and increased serum CK levels compared to WT muscle ( Figure 3F,G). Furthermore, by one year of age Thbs4 -/muscle had increased signs of ongoing myofiber degeneration/regeneration, as marked by centrally nucleated myofibers, as well as noticeable histopathological and ultrastructural changes and greater EBD uptake compared to WT control muscle ( Figure 3H-J). Collectively, these results suggest that Thbs4 induction with dystrophic disease and low levels of expression during aging produce an adaptive physiologic response that protects skeletal muscle.

Thbs4 directly impacts sarcolemma stability of myofibers
To directly evaluate the structural integrity of the sarcolemma we first employed a model of 3 successive lengthening-contraction injury cycles to the tibialis anterior (TA) muscle in a whole leg immobilization preparation ( Figure 4A). Remarkably, overexpression of Thbs4 significantly protected against lengthening-contraction injuries over all 3 bouts compared to WT mice ( Figure 4B). As anticipated, the TA from Sgcd -/mice showed much greater loss of functional recovery compared to WT after lengthening-contraction injury, but the presence of the Thbs4 Tg provided significant protection, achieving a recovery response now similar to WT levels ( Figure 4B). Moreover, loss of Thbs4 resulted in greater injury with all 3 cycles of lengthening-contraction injury, similar to Sgcd -/-( Figure 4C). Interestingly, the passive force of the muscle after simple stretching was greater with the Thbs4 Tg, while it was reduced in Thbs4 -/muscle, although the twitch force itself was not significantly different between any of the groups (Figure 4-figure supplement 1A,B). We also noticed that the tendons were weaker in Thbs4 -/-TA muscle, which were more likely to rupture in the isolated lengthening-contraction injury assay (Figure 4-figure supplement 1C).
Individual myofibers were isolated from the flexor digitalis brevis (FDB) muscle and subjected to laser injury with subsequent measurement of FM1-43 fluorescent dye uptake as a direct measure of membrane stability and repair ( Figure 4D). FDB myofibers from Thbs4 Tg mice showed less dye Membranes of myofibers are shown in green. *p<0.05 vs WT; #p<0.05 vs. Sgcd -/by one-way ANOVA with post hoc Tukey's test. N = 5 and 6 mice for Sgcd -/and Sgcd -/-Thbs4 -/-, respectively. Scale bar = 40 mm. (F,G) Time to fatigue in seconds with forced downhill treadmill running and quantitation of serum CK levels in WT and Thbs4 -/mice at the indicated ages; abbreviations, y = year. n = 6 mice per genotype per age for panel F. For panel G, n = 7 mice per genotype at six weeks of age; n = 5 WT and 6 Thbs4 -/mice at three months of age, n = 5 mice per genotype at six months of age and n = 9 WT and 8 Thbs4 -/mice at one year of age. *p<0.05 vs WT at the same age by Student's t test. (H) H&E histological staining (upper) and transmission electron microscopy (lower) of tissue pathology in Quad at one year of age in WT and Thbs4 -/mice. The H&E scale bar = 25 mm. The electron microscopy scale bar = 2 mm. The arrows show myofibers with central nucleation due to loss of the Thbs4 gene. Representative images of 6 mice per genotype for H&E staining and 2 mice per genotype for electron microscopy. (I) Percentage of myofibers with centrally located nuclei in Thbs4 -/compared to WT Quad at one year of age. *p<0.001 vs WT by Student's t test. n = 6 mice per genotype. (J) Masson's trichrome stained (upper) and EBD uptake (lower) histological images in WT and Thbs4 -/mice in the Diaph at one year of age. The EBD images show membranes in green and fibers with EBD uptake produce red fluorescence. Representative images of 6 mice per genotype studied. Scale bars = 50 mm. All data are represented as mean ± SEM. DOI: 10.7554/eLife.17589.012 The following figure supplement is available for figure 3: uptake compared with WT myofibers, suggesting that Thbs4 overexpression was inherently protective to the membrane ( Figure 4D,E). As expected, FDB myofibers from Sgcd -/mice showed greater dye uptake suggesting greater injury with less efficient repair, while the presence of the Thbs4 Tg was protective in Sgcd -/myofibers, bringing dye uptake levels back to WT ( Figure 4D,E). FDB myofibers from Thbs4 -/mice also showed greater dye uptake compared with WT myofibers after laser injury, while double Thbs4 -/-Sgcd -/myofibers displayed an even greater injury response than either Thbs4 or Sgcd single deletions alone ( Figure 4F). Taken together these results indicate that Thbs4 overexpression provides greater stability to the sarcolemma, while its loss renders the sarcolemma less stable. More importantly, since this assay uses isolated myofibers devoid of ECM attachments, it suggests that part of Thbs4-dependent protection occurs from within the myofiber.

Thbs4 augments intracellular trafficking through ATF6a
To investigate a molecular mechanism whereby Thbs4 might directly regulate the stability of the sarcolemma in skeletal muscle we examined intracellular vesicular trafficking and membrane attachment complex formation. Two important clues that directed our investigation were the known augmentation in the adaptive ER stress response pathway and the dramatic increase in sub-sarcolemmal vesicles observed in skeletal muscle with Thbs4 overexpression. Hence, we instituted two in vitro assays to assess ER-to-Golgi and Golgi-to-sarcolemma vesicular trafficking in response to Thbs4 activity in primary neonatal rat ventricular myocytes ( Figure 5; Figure 5-figure supplement 1A,B, technical issues prevented such studies in myotubes or myofibers). To assess ER-to-Golgi trafficking a red fluorescent protein (RFP)-labeled Golgi resident enzyme, GalNacT2-RFP, was used with and without Thbs4 overexpression by fluorescent recovery after photobleaching (FRAP, Figure 5-figure supplement 1A). In parallel, ATF6a activity was also modulated since Thbs4 is known to directly regulate this ER-stress transcription factor (Brody et al., 2016;Lynch et al., 2012). Here, Thbs4 overexpression significantly accelerated ER-to-Golgi vesicular trafficking, which was fully inhibited by cooverexpression of a dominant negative (dn) ATF6a construct ( Figure 5A). Similarly, Golgi-to-sarcolemmal trafficking rates, measured with VSVG-enhanced green floursecent protein (eGFP) after inverse (i) FRAP, were significantly accelerated upon Thbs4 overexpression, which was again inhibited with ATF6a-dn ( Figure 5B; Figure 5-figure supplement 1B). Furthermore, accelerated trafficking with Thbs4 was mimicked by overexpression of a constitutively nuclear (cn) ATF6a construct ( Figure 5C,D).
To examine this effect of Thbs4 in greater molecular detail we also employed 2 domain-specific Thbs4 constructs and a related oligomeric glycoprotein Nell2 ( Figure 5E-J) (Brody et al., 2016;Kuroda et al., 1999;Lynch et al., 2012). Like Thbs4, Nell2 contains an N-terminal laminin-G like (LamG) domain and an epidermal growth factor (EGF)-like repeat domain but lacks the ATF6a interacting Type III repeat (T3R) and TSP-C domains from Thbs4. Importantly, unlike full-length Thbs4, overexpression of Nell2 or the LamG domain of Thbs4 did not increase ER-to-Golgi or post-Golgi trafficking rates ( Figure 5E,F,I,J). However, overexpression of just the T3R domain of Thbs4, which functions as the ATF6a interacting region, was sufficient to accelerate ER to Golgi and post-Golgi trafficking ( Figure 5G,H). Thbs4 overexpression in skeletal muscle also augmented the levels of trafficking regulatory proteins such as Sar1, Rab24, Rab6, Rab3 and Rab8, which control ER-to-Golgi and post-Golgi vesicular trafficking (Figure 5-figure supplement 1C) (Brandizzi and Barlowe, 2013;Stenmark, 2009). Collectively, these results indicate that Thbs4 accelerates intracellular vesicular trafficking in an ATF6a-dependent manner, thereby suggesting at least one molecular mechanism whereby Thbs4 might enhance membrane stability through greater fluxing of vesicles to the sarcolemma.

Thbs4 stabilizes sarcolemmal attachment complexes
To more definitively investigate if greater vesicular trafficking rates associated with Thbs4 activity might augment the residency of membrane attachment complex proteins, we generated membrane- WT . Thbs4 regulates skeletal muscle sarcolemma stability. (A) Schematic representing the first of 3 consecutive lengthening contraction-induced muscle injury cycles using an in situ tibialis anterior (TA) muscle preparation. Briefly, an isometric contraction was performed to determine baseline force generation, followed by 2 consecutive eccentric contractions and finally another isometric contraction (= injury cycle 1). The force deficit shown in panels B and C was calculated between the first and second isometric contraction in between the two lengthening-contractions, which was repeated 3 Figure 4 continued on next page specific protein preparations for Western blotting, as well as performed immunohistochemistry on skeletal muscle for direct visualization of the membranes in cross-section. As previously observed, loss of d-sarcoglycan in skeletal muscle of Sgcd -/mice resulted in the near complete absence of the other sarcoglycans at the membrane (as they all form a complex; Figure 6A) (Durbeej and Campbell, 2002). Interestingly, Thbs4 Tg alone displayed increased sarcolemmal levels of b-dystroglycan, dystrophin and b1D-integrin ( Figure 6A). More importantly, the Thbs4 Tg in the Sgcd deficient background resulted in greater membrane localization of a-, b-, and g-sarcoglycan and b-dystroglycan, as well as in dystrophin, utrophin and b1D-integrin ( Figure 6A). More quantitative assessment of this effect by Western blotting of membrane-specific protein extracts showed that even the Thbs4 Tg alone gave increased membrane levels of utrophin, a-and b-dystroglycan, as well as b1D-, a7 and a5-integrins compared to WT controls ( Figure 6B; Figure 6-figure supplement 1A,B; red boxes), which likely explains our earlier observations whereby Thbs4 Tg skeletal muscle was protected from lengthening-contraction injury and laser injury in isolated myofibers versus WT. The Thbs4 Tg also augmented the membrane residency of these same proteins in the Sgcd -/and mdx background, as well as increased membrane levels of a-sarcoglycan and b-sarcoglycan in skeletal muscle of Sgcd -/mice ( Figure 6B; Figure 6-figure supplement 1A,B, for replicate samples). Importantly, previous work has shown that overexpression of DGC components augment the assembly of the entire complex and are inherently protective to MD (Allikian et al., 2004;Grounds et al., 2005;Gumerson and Michele, 2011;Tinsley et al., 1998). Furthermore, we observed that expression of b1-and a7-integrin from total cytoplasmic protein extracts was not increased, suggesting that Thbs4 overexpression directly augmented membrane trafficking and localization of these critical attachment proteins to the surface ( Figure 6B, lower panel). Finally, we also observed that loss of Thbs4 in skeletal muscle partially reduced membrane residency of a-and b-dystroglycan, b1D-and a7-integrins ( Figure 6C, burgundy boxes). Collectively, these observations solidify a mechanism whereby Thbs4 regulates membrane stability in skeletal muscle by augmenting the trafficking of membrane attachment protein complexes to the sarcolemma. As previously reported with other Thbs proteins (Adams and Lawler, 2011), we observed that Thbs4 could directly bind intracellular b1D-integrin, but not a7-integrin, in primary neonatal rat cardiomyocytes ( Figure 6-figure supplement 1C). In addition, both Thbs4 and a5-integrin localized to b1D-integrin-positive intracellular vesicles in WT, Thbs4 Tg and Sgcd -/quadriceps muscle, placing Thbs4 within the same vesicles and intracellular compartment as the attachment proteins themselves ( Figure 6-figure supplement 1D,E).

ATF6a drives vesicular expansion in skeletal muscle but does not protect against MD
Given the results presented to this point, we hypothesized that ATF6a induced ER and post-ER vesicular expansion was responsible for increased intracellular trafficking of membrane attachment protein complexes to the sarcolemma and hence, protection from MD. Indeed, ATF6a was previously shown to expand the ER and post-ER vesicular compartment when activated or overexpressed (Brody et al., 2016;Lynch et al., 2012). Thus, to directly test this hypothesis we generated skeletal (B,C) Reduction in isometric force generation as a percentage of baseline force after each lengthening contraction injury cycle in the indicated genotypes of mice shown in the legend. *p<0.05 vs WT; # p<0.05 vs WT and Thbs4-Tg; § P<0.05 vs Sgcd -/and Thbs4-Tg by one-way ANOVA with post hoc Tukey's test. n = 6 mice for WT, Thbs4 Tg and Sgcd -/-Thbs4 Tg and n = 10 mice for Sgcd -/for panel B. n = 6 mice for WT, n = 10 mice for Sgcd -/and n = 5 mice for Thbs4 -/for panel C. (D) Representative images before and after laser injury and influx of FM1-43 dye (green fluorescence) in FDB myofibers in the presence of 1.25 mM Ca 2+ isolated from indicated genotypes. The white tag in each image is the position for the laser injury. Scale bars = 10 mm. (E,F) Quantitative time course in seconds of FM1-43 fluorescent dye entry in FDB myofibers from the indicated genotypes of mice in the presence of 1.25 mM Ca 2+ . Laser injury occurred at 60 s. n = 6 fibers per animal from 3 animals per genotype for panels D-F; *p<0.05 vs WT; #p<0.05 vs Sgcd -/-; §p<0.05 vs Thbs4 -/by one-way ANOVA with post hoc Tukey's test. Data points for Sgcd -/in panel E and F were derived from a single set of experiments. All data are represented as mean ± SEM. DOI: 10.7554/eLife.17589.014 The following figure supplement is available for figure 4: Utro. Dystro. Dysfer.
Gapdh Sarcolemmal y y y y y y y g g g . Similar to Thbs4 Tg mice, ultrastructural analysis of skeletal muscle from ATF6a Tg mice showed a remarkable expansion of ER and the sub-sarcolemmal vesicular compartment, although these ATF6a-dependent vesicles appeared less dense in comparison to those observed in Thbs4 Tg mice ( Figure 7D versus Figure 1G). Next, ATF6a mice were crossed with both Sgcd -/and mdx mice to directly examine the hypothesis that the adaptive ER stress response and sub-sarcolemmal expansion of vesicles induced by ATF6a was a protective mechanism underlying Thbs4 action. However, ATF6a overexpression in the Sgcd -/or mdx dystrophic background provided no protection whatsoever ( Figure  There was no reduction in histopathology or serum CK levels or membrane rupture as assessed with EBD, nor was treadmill running improved by the ATF6a Tg in either the Sgcd -/or mdx backgrounds. More importantly, ATF6a overexpression did not increase the sarcolemmal localization of any of the membrane attachment proteins observed with Thbs4 overexpression in skeletal muscle ( Figure 7K). Thus, while ATF6a overexpression activated an adaptive ER stress response in skeletal muscle with a dramatic induction of intracellular and sub-sarcolemmal vesicles to an even higher level than observed in our Thbs4 overexpressing mice, it did not augment the membrane residency of membrane stabilizing proteins in muscle. Hence, our data indicate that ATF6a is only one part of a more integrated mechanism whereby Thbs4 regulates membrane stability of skeletal muscle.
Taken together, our data so far indicate that increased levels of Thbs4 itself and its trafficking through the secretory pathway are essential to increase membrane attachment protein complexes at the sarcolemma. To test this hypothesis, we took advantage of a previously established adenoviral construct encoding a Thbs4 calcium-binding containing mutant that is retained in the ER (Ad-Thbs4-mCa 2+ ) (Brody et al., 2016). Importantly, the mutant still induces an ATF6a mediated ER-stress response, both in neonatal rat cardiomyocytes and when expressed in the gastrocnemius muscle of early postnatal rat pups (Brody et al., 2016). Utilizing an identical in vivo approach with adenoviral gene transfer into the gastrocnemius of early neonatal rat pups, we compared bgal control with fulllength Thbs4 versus the Thbs4-mCa 2+ for effects on b1 Integrin membrane levels. The data showed that only the secretion competent full-length Thbs4, but not the full-length ER-retained Thbs4 mutant, promoted greater b1 integrin membrane occupancy (Figure 7-figure supplement 3). Hence, Thbs4 must move through the secretory pathway to chaperone at least b1 integrin to the sarcolemma.

Conservation of the Thbs membrane stability mechanism in Drosophila
Our results in mice were reminiscent of data from Drosophila, which have a single Tsp gene that when deficient causes embryonic lethality due to ruptures in tendon/muscle attachments (Subramanian et al., 2007). Moreover, Tsp in Drosophila was also shown to interact with aPS2/bPS integrin (Chanana et al., 2007;Subramanian et al., 2007). Thus to investigate a potential Figure 6 continued biological replicates). Abbreviations: Utro, utrophin; Dystro, dystrophin; Dysfer, dysferlin; a-DG, a-dystroglycan; b-DG, b-dystroglycan; d-SCG, dsarcoglycan; a-SCG, a-sarcoglycan; b-SGC, b-sarcoglycan; b1D-, a7-and a5-integrin. The red boxes show increased protein levels. Also see Figure 6-figure supplement 1 for replicates. (C) Representative immunoblotting for structural components of the DGC-and integrin-associated protein complexes in sarcolemmal preparations from Thbs4 -/and WT quadriceps at four months of age (n = 4 biological replicates). The burgundyboxed areas show reduced protein levels. Ponceau staining of a nonspecific band and dihydropyridine receptor a1 (Cav1.1) were used as loading controls for sarcolemmal protein extracts; Gapdh was used as loading control for total cell protein extracts. DOI: 10.7554/eLife.17589.018 The following figure supplement is available for figure 6:
Calret. Collectively, these results indicate that Thbs proteins underlie an ancient program for membrane stabilization through regulation of intracellular attachment protein complexes and their content at the surface membrane, although ER expansion through ATF6a appears to have evolved phylogenetically after Drosophila.

Discussion
A vast majority of the Thbs literature over the past 3 decades have invoked or interpreted data consistent with a primary extracellular function for these proteins, while only a handful have shown a direct intracellular function (Adams and Lawler, 2011;Ambily et al., 2014;Baek et al., 2013;Brody et al., 2016;Christopherson et al., 2005;Duquette et al., 2014;Frolova et al., 2014Frolova et al., ,2010Frolova et al., , 2012Hauser et al., 1995;Lynch et al., 2012;McKeown-Longo et al., 1984;Posey et al., 2014;Schellings et al., 2009;Sö dersten et al., 2006). Here, we identify a fundamental yet previously unrecognized intracellular role for Thbs4 in skeletal muscle, where it directly augments selective vesicular trafficking and chaperones DGC and integrin attachment complexes to the membrane, leading to greater stability and levels of select complexes at the sarcolemma, and thereby enhancing the mechanical stability of the myofiber (Figure 9). In fact, our findings identify Thbs4 as a crucial component to maintain muscle fiber integrity as loss of Thbs4 results in sarcolemma weakness that causes spontaneous dystrophic changes with aging. Importantly, the Thbs protective effect holds true in both mouse and Drosophila skeletal muscle.
As secreted matricellular proteins, Thbs' are first produced in the ER where they are glycosylated and transit to the Golgi for additional modifications, where after they traverse the remainder of the secretory pathway (Adams and Lawler, 2011). Interestingly, we observed that skeletal muscle-specific overexpression of Thbs4 did not result in accumulation within the ECM, but predominantly produced an intracellular protein localization pattern within the ER and post-ER vesicular network. In fact, we have also since generated cardiac-specific Tg mice overexpressing Thbs1, 2, 3, 4 or 5 in the heart ( [Lynch et al., 2012] and data not shown). In these hearts all five Thbs' reside mainly within the   intracellular vesicular network and ER with only limited detectable protein accumulation outside cardiomyocytes. This is in dramatic contrast to the overexpression of an array of other matricellular proteins, such as periostin, which saturates the ECM when overexpressed (Oka et al., 2007). In contrast, dystrophic skeletal muscle did reveal occasional Thbs4 protein accumulation in fibrotic regions around myofibers, confirming that this protein can reside for a period of time in the ECM. These dynamic localization differences at baseline versus during fibrotic disease could be attributed to Thbs recycling at the cell surface (Adams and Lawler, 2011;Wang et al., 2004). Indeed, we observed rapid up take of recombinant Thbs4 when given exogenously to cultured C2C12 myoblasts or myotubes. It is possible that extracellular Thbs4 reuptake could occur through its designated receptor or through the integrin and DGC complexes. Indeed, other matricellular proteins such as SPARC were shown to be actively taken up back into myofibers through such a process with integrin associated endocytosis, sorting and recycling (Chlenski et al., 2011;De Franceschi et al., 2015;Nakamura et al., 2014). Our data identify ATF6a as a transcriptional regulator of secretory pathway activity in muscle, a function that was previously established for the inositol-requiring enzyme 1a / X-box binding protein (IRE1a/XBP-1) axis of the canonical ER stress response pathway during plasma cell differentiation (Shaffer et al., 2004). Importantly, although enhancement of the ATF6a-mediated adaptive ER stress pathway was sufficient to drive the dramatic expansion of the ER and post-ER vesicular content and increase vesicular trafficking to the membrane, it did not augment membrane residency of DGC and integrin attachment complexes at the sarcolemma, nor was it sufficient to protect against MD. Thus, ATF6a is only part of a more complex intracellular mechanism whereby Thbs4 regulates mechanical stability of the muscle fiber and its sarcolemma, in coordination with greater vesicle formation and trafficking (Figure 9).
Data from both Drosophila and zebrafish show that thrombospondin proteins localize outside of cells within the tendinous junctions (Chanana et al., 2007;Subramanian and Schilling, 2014;Subramanian et al., 2007). In addition, vertebrate Thbs1 and Thbs2 genes have evolved domains that are tailored to affecting processes outside the cell, such as altering transforming growth factorb activity and the angiogenic response (Adams and Lawler, 2011;Bornstein, 2001;Carlson et al., 2008). Hence, the simplest interpretation of our data and that reported in the literature is that Thbs proteins are complex, multifactorial proteins that function both inside and outside the cell. However, our working hypothesis is that Thbs4 appears more tailored to intracellular functionality in cardiac and skeletal muscle (Figure 9), and this same paradigm appears to hold true for the other Thbs family members in other tissues. For example, Thbs1 was previously shown to localize to the intracellular side of membrane attachment sites by immunogold-electron microscopy in endothelial cells (Hiscott et al., 1997). Furthermore, Thbs proteins are known to strongly interact with many different integrin heterodimers, and singular proteomic analysis of the a5b1 integrin complex identified Thbs2 as a core element of its 'interactome' (Adams and Lawler, 2011;Bouvard et al., 2013;De Franceschi et al., 2015;Plow et al., 2000;Schiller et al., 2013). Our ultrastructural and biochemical analyses showed that the Thbs-integrin complex resides within the lumen of vesicles. Although further, in-depth analyses will suggest whether Thbs4 co-regulates the signaling competence of this integrin complex and the exact preassembly stage that is influenced by Thbs4 on the way to the cell surface. Finally, Thbs1 silencing or overexpression in human cancer cells was shown to decrease or enhance integrin protein levels, respectively, in the intracellular and plasma membrane compartment and thereby modulate cellular adhesion (Duquette et al., 2013;John et al., 2010).
Taken together, various lines of evidence indicate that Thbs proteins stabilize integrins and the DGC at membrane attachment complexes of the sarcolemma through an intracellular function. Importantly, this observation holds true in both mouse and Drosophila skeletal muscle where integrin protein content at the cell membrane was increased with Thbs4 overexpression, yet very little Thbs . Model of how Thbs4 functions as an intracellular regulator of muscle cellular attachment and membrane stability. As a matricellular protein, thrombospondin-4 (Thbs4) pentamers are synthesized in the ER lumen and then transported to the Golgi, where after they traverse the secretory pathway to fuse with the plasma membrane for secretion. Thbs4 can then reside within the extracellular matrix (ECM) or be actively endocytosed and returned Figure 9 continued on next page protein was observed outside the cell in either species at baseline. While previously published data showed a large concentration of Drosophila Tsp protein to the myotendinous junction in stage 16 embryos, this is also the very same region where the integrins are highly concentrated in identical foci and the data do not distinguish if Tsp in inside or outside the cell (Subramanian et al., 2007). At earlier embryonic stages (stage 12-13) however, the Tsp protein is intracellular with a diffuse pattern similar to that of integrins that have yet to be deposited at the cell membrane (Subramanian et al., 2007). Moreover, in later stage Drosophila larvae Tsp is no longer detected in the myotendinous junctions or in tendons. Rather, a network of Tsp positive staining is observed within the muscle of larvae (unpublished observations, Talila Volk). However, in mammals Thbs4 can oligomerize with Thbs5 and be deposited in the tendon (Hauser et al., 1995;Sö dersten et al., 2006), loss of Thbs4 -/in mice showed altered ECM composition and weakened muscle-tendons (Frolova et al., 2014), and altered inflammatory responses associated with arteriogenesis (Frolova et al., 2010), whereas Thbs1 and Thbs2 were shown to function from outside the cell in augmenting developmental synaptogenesis (Christopherson et al., 2005). Hence, Thbs proteins are clearly complex regulatory proteins with intra-and extracellular functions.
One aspect of the biology that was not conserved in Drosophila was the ability of Thbs or Tsp overexpression to expand the intracellular vesicular compartment in this lower organism, likely because Drosophila does not rely on an ATF6-like mechanism for the ER stress response, and the Thbs interacting domain within ATF6a is not contained in the Drosophila homologue of this gene (Mori, 2009). Thus, the ability of Thbs proteins to activate ATF6a to augment ER protein production and secretory pathway activity is a later evolutionary adaptation beyond just stabilizing membrane attachment complexes, which in higher organisms more effectively coordinates tissue remodeling and healing through the Thbs proteins.
Taken together, the unique aspects of muscle membrane biology allowed us to uncover a previously unknown and possibly dominant intracellular function of the Thbs proteins that is evolutionarily conserved (Figure 9). This new model for Thbs protein function has many disease ramifications, especially in skeletal muscle, a tissue that is highly sensitive to mutations in genes that cause weaknesses in cellular attachment and membrane stability. Indeed, there is a lack of therapeutic strategies to effectively treat MD and our study suggests that this protein may provide a universal approach to strengthen the sarcolemma in skeletal muscle if employed in a gene therapy approach. Importantly, this protein is already present in muscle and it would not be perceived as a neo-antigen by viral-mediated overexpression, hence it should be well tolerated and best used in patients where the genetic basis of their MD disease is due to a loss of structural support of the sarcolemma (majority of cases).

Mouse models
Skeletal muscle-specific transgenic mice for Thbs4 and ATF6a were generated using the modified human skeletal a-actin (Ska) promoter construct as previously described (Goonasekera et al., 2011;Lynch et al., 2012). Briefly, full-length mouse Thbs4 cDNA was obtained from Open Biosystems Figure 9 continued to the intracellular compartment by a recycling receptor. In addition to its established extracellular functions, combined studies in the heart and skeletal muscle now reveal that while in the ER, Thbs4 can compete with BiP (GRP78) for binding to the ER-resident transcription factor ATF6a, thereby facilitating ATF6a translocation to the Golgi for processing and subsequent shuttling to the nucleus where it regulates expression of ER stress responsive genes that are also part of the unfolded protein response (UPR). ATF6a induction in cardiac and skeletal muscle, or by overexpression in Tg mice (lower panel) causes a dramatic expansion of the ER and post-ER vesicles, as well as increased vesicular trafficking to the membrane. The ability of Thbs4 to induce ATF6a processing and nuclear trafficking also causes this same ER expansion and augmentation of intracellular vesicular trafficking to the membrane. However, Thbs4 uniquely regulates trafficking of selected integrins and dystrophin-associated glycoprotein complexes (DGC) members to the sarcolemma, thereby enhancing the mechanical stability of the myofiber, such as observed in Thbs4 Tg muscle (middle panel). DOI: 10.7554/eLife.17589.026 (Accession number: BC139414) and amplified by PCR and cloned into the BamHI and EcoRV sites of the Ska-promotor expressing vector (forward: 5'-CGCGGATCCATGCCGGCCCCACGCGCG-3', and reverse: 5'-ATCTCAATTATCCAAGCGGTC AAAACTCTGGG-3'). Full-length mouse ATF6a cDNA was obtained from a previously generated pcDNA1-ATF6a plasmid (Lynch et al., 2012). Mouse ATF6a cDNA was amplified by PCR and subsequently cloned by PCR into the KpnI and NotI sites of the Ska-promotor expressing vector (forward: 5'-GGGGTACCATGGAGTCGCCTTTTAGTCC-3', and reverse: 5'-ATAAGAATGCGGCCGCCTACTGCAACGACTCAGGGAT-3'). All constructs were confirmed by DNA sequencing.To make Tg mice, the Ska-plasmid backbone was removed and the Ska-Thbs4 and Ska-ATF6a fragments were gel purified followed by Elutip-D column purification (Schleicher and Schuell Bioscience; Dassel, Germany, Cat. 10462617) for newly fertilized oocyte injection at the Cincinnati Children's Hospital Transgenic Animal and Genome Editing Core Facility. All transgenic mice were produced in the FVB/N background. Mice deficient for Thbs4 (Thbs4 -/-; Strain: B6.129P2-Thbs4tm1Dgen/J) and mdx mice (Strain: C57BL/10ScSn-Dmd mdx /J) were purchased from Jackson Laboratories (Bar Harbor, Maine). Sgcd -/mice were previously described (Hack et al., 2000). Next, Ska-Thbs4-Tg, ska-ATF6a-Tg and Thbs4 -/mice were backcrossed for at least 6 generations into the Sgcd -/background to generate Sgcd -/-Thbs4-Tg mice, Sgcd -/-ATF6a-Tg mice and Sgcd -/-Thbs4 -/mice, as well as their littermate controls. An identical breeding strategy was used to generate mdx Thbs4 -/mice. In addition, males from each transgenic line were crossed to mdx heterozygous females to generate mdx-Tg and mdx non-Tg male littermates and their appropriate controls. All animal experiments were approved by the Institutional Animal Care and Use Committee of the Cincinnati Children's Hospital Medical Center (Protocol# IACUC2013-0013). No human subjects or human tissue was directly used in experiments in this study.

Thbs4 mRNA expression levels in various human muscle diseases
A search of the 'National Center for Biotechnology Information Gene Expression Omnibus (NCBI GEO)' database (Barrett and Edgar, 2006) revealed that Bakay and colleagues recently performed microarray experiments on human muscle biopsies of various muscle diseases (GEO accession GDS1956/204776, [Bakay et al., 2006]). Available data included 11 different muscle diseases, with a total of 121 human muscle biopsy specimens tested on Affymetrix U133A microarrays. Individual Thbs4 mRNA levels from samples with Becker Muscular Dystrophy (BMD, n = 5); Duchenne Muscular Dystrophy (DMD, n = 10); dystrophy due to calpain-3 mutations (LGMD2A, n = 10); and dystrophy due to a paucity of dysferlin (LGMD2B, n = 10) were averaged and compared to those from healthy muscle biopsies (n = 18) using an unpaired two-tailed t-test.

Adeno-associated virus (AAV) serotype-9 production and injection (gene-therapy)
Mouse Thbs4 or an eGFP cDNA was amplified by PCR and inserted into the BamHI and XhoI sites of pAAV-MCS vector. AAV9-CMV-eGFP and AAV9-CMV-Thbs4 were produced using the triple transfection method in HEK293 cells as previously described and stored at À80˚C until commencing the in vivo experiments (Gray et al., 2011;Zincarelli et al., 2008). Next, both left and right gastrocnemius muscles of three-day-old Sgcd -/mice were injected with either AAV9-Thbs4 or AAV9-eGFP (both 1E10 viral particles in 30 ml isotonic saline; [Goonasekera et al., 2011]). Mice were sacrificed at six weeks of age. The left gastrocnemius of each mouse was fixed, processed, paraffin embedded, sectioned, and stained with H&E and Masson's trichrome, whereas the right muscles were snap-frozen in liquid nitrogen for storage in À80˚C. A subset of muscles were embedded in Optimal Cutting Temperature Compound (O.C.T, Tissue-Tek, Sakura Americas, Torrance, CA, Cat #4583), frozen, and 7 mm cryosections were generated to confirm eGFP expression by direct fluorescence (not shown).

Protein preparations and western blotting
Quadriceps, gastrocnemius, soleus, diaphragm and hearts were harvested and immediately frozen in liquid nitrogen for storage at À80˚C. To evaluate ER-stress and Thbs4 protein expression, muscles were homogenized (Fisher Scientific, Waltham, MA, TissueMiser) in ice-cold RIPA buffer containing Halt Protease Inhibitor cocktail (ThermoScientific, Waltham, MA, #78430). Next, samples were sonicated (SP Scientific, Warminster, PA, VirSonic 60, power setting 3 for 3 times 10 s), lysates were cleared by centrifugation at 14,000 rpm for 14 min at 4˚C and stored at À80˚C.
To evaluate glycosylation pattern, quadriceps protein extracts were treated with Endoglycosidase H (Endo H; New England Biolabs Inc., Ipswich, MA, P07P2), peptide N-glycosidase F (PNGase F, New England Biolabs Inc., P0704) or protein deglycosylation mix (New England Biolabs Inc., P6039) prior to SDS-PAGE, according to the manufacturer's instructions. Endo H cleaves high mannose residues at hybrid oligosaccharides present on proteins in the ER, whereas PNGase cleaves both these and more complex oligosaccharides that result from processing in the Golgi (Hewett et al., 2004). Control samples were treated the same way without addition of enzymes.
To evaluate DGC-associated proteins, fresh quadriceps muscle was harvested and crude sarcolemmal isolates were prepared as previously described (Kobayashi et al., 2008). Briefly, freshly harvested quadriceps was homogenized in 7.5x volumes of ice-cold lysis buffer (20 mM Na 4 P 2 O 7 , 20 mM NaH 2 PO 4 , 1 mM MgCl 2 , 0.303 M sucrose, 0.5 mM EDTA, pH 7.1 with 5 mg/ml aprotinin and leupeptin, 0.5 mg/ml pepstatin A, 0.23 mM PMSF, 0.64 mM benzamidine, and 2 mM calpain inhibitor I and calpeptin), then centrifuged 14,000 g for 20 min at 4˚C; the pellet was re-suspended, rehomogenized and both supernatants were centrifuged 30,000 g for 30 min at 4˚C after which the pellet was re-suspended in 100 ml lysis buffer and stored at À80˚C until further use. Extracellular protein fractionation from quadriceps muscle was essentially performed as previously described (Tjondrokoesoemo et al., 2016). In summary, freshly harvested quadriceps muscle was minced, washed with PBS and subjected a 1 hr washing step in 0.5 M NaCl, 10 mM Tris-HCl, 25 mM EDTA (PH 7.5). Next, samples were decellularized overnight in 0.1% SDS, 25 mM EDTA, followed by extracellular matrix extraction with 4 M guanidine hydrochloride, 50 mM C 2 H 3 NaO 2 , 25 mM EDTA (pH 5.8). Finally, proteins were precipitated overnight in 80% EtOH, air dried and treated with protein deglycoylation mix (New England Biolabs Inc., P6039).
b1D-integrin positive intracellular vesicles were isolated from quadriceps using an endoplasmic reticulum isolation kit (Sigma Aldrich, ER0100), according to the manufacturer's instructions. Briefly, tissues were homogenized in isotonic extraction buffer using a 2 ml Dounce homogenizer. Homogenates were cleared by centrifugation at 12,000 g for 15 min at 4˚C. Two mg of vesicles was incubated with for 12 hr at 4˚C with an antibody raised against the cytoplasmic domain of b1D-integrin (Millipore, MAB1900), immunoprecipitated using A/G magnetic beats (ThermoFisher Scientific, #88803) at 4˚C for 1hr, and subsequently subjected to SDS-PAGE.

Forced treadmill running, Evan's Blue Dye (EBD) uptake and serum CK levels
To assess the exercise capacity of mice and sarcolemmal stability, mice were subjected to forced treadmill running in the presence of EBD as previously described (Goonasekera et al., 2011). Briefly, adult mice were intraperitoneally injected with EBD (10 mg/ml; 0.1 ml per 10g body weight) and 24 hr later subjected to forced downhill treadmill running to measure membrane rupture events. For the exercise protocol, exhaustion of the mice was assessed as greater than 10 consecutive seconds on the shock grid without attempting to re-engage running on the treadmill. Mice were then sacrificed and quadriceps, gastrocnemius and diaphragm was embedded in O.C.T. and frozen in liquid nitrogen. Tissue sections were cut at a thickness of 7 mm, air-dried, washed in PBS and stained with wheat germ agglutinin conjugated to FITC (Sigma-Aldrich, green) for 1 hr at RT to visualize the membranes. Images were taken on a Nikon Eclipse Ti-S inverted microscope system equipped with NIS Elements Advanced Research (AR) microscope imaging software (Nikon Instruments Inc. Melville, NY) to determine the percentage of EBD-positive fibers. In addition, blood was taken from a separate cohort of un-exercised mice of each genotype to evaluate their baseline serum CK levels as previously described at the clinical laboratory of Cincinnati Children's Hospital Medical Center by an observer blinded to the genotypes (Kobayashi et al., 2008).

Lengthening contraction based injury and isometric muscle force measurements
Mice were anesthetized with an intraperitoneal injection of pentabarbitol and placed supine on the muscle testing apparatus (Aurora Scientific, Aurora, ON, Canada). A midline incision running from the ankle to the thigh was created and the skin and fascia was gently removed leaving the tibialis anterior (TA) muscle exposed. The leg was immobilized by securing it in a custom jig (Aurora Scientific) with thumbscrews at the distal femur. A 4-0 nylon suture was tied to the distal TA securing it with a small plastic ring at the muscle tendon junction. The distal tendon was transected and the TA elevated to remove its contact with the tibia, and the muscle was mounted to a servomotor (Aurora Scientific, 305C) using the plastic ring. Two intramuscular electrodes were placed on either side of the peroneal nerve and stimulation voltages and optimal muscle length (L 0 ) were determined and then adjusted to produce maximal isometric force (P 0 ) at a stimulation frequency of 200 Hz. Five consecutive isometric contractions were averaged as a measure of the maximal specific tension. An additional lengthening contraction injury protocol was added in which an isometric contraction was performed to determine baseline force generation, followed by 2 consecutive 20% L 0 lengthening contractions, and finally another isometric contraction performed at L 0. The force deficit was calculated between the first isometric contraction and those following the lengthening injury cycles (See Figure 4A for schematic representation). This contraction injury protocol was repeated 2 more times and the force deficit was calculated relative to the pre-injury isometric contraction. In a subset of experiments passive tension was measured prior to the lengthening contraction protocol. Here the TA was set to L 0 and passively stretched to 5, 10, 15, and 20% of L 0 . Each stretch was held for 2 min before the length was returned to its starting position. Maximal passive tension was recorded at the peak of the stretch. A 2-min rest period occurred between each contraction for all experiments and force values were normalized to the muscle's physiologic cross-sectional area. In some cases we observed tendon breaks during the lengthening contraction protocol. Percentage of tendon breaks, assessed by complete physical rupture of the tendon at the muscle aponeurosis, was recorded.
To assess internalization of rThbs4 by cultured C2C12 myoblasts and myotubes, established approaches were used as previously described (Chlenski et al., 2011;Nakamura et al., 2014). First, cells plated in Ibidi m-slide 8-well dishes were treated with either 1 mg/ml Alexa-488 labeled rThbs4 or equal amounts of Alexa-488 labeled BSA control for the indicated periods. Next, cells were rinsed with sterile PBS and fixed with 4% paraformaldehyde for 10 min. After fixation cells were washed three times with PBS, followed by blocking with 3% normal goat serum/PBS/0.1% Triton for 20 min at room temperature and subsequently incubated with Alexa Fluor-568 labeled phalloidin (Life Sciences, Cat# A12380; 1/100 in blocking buffer) at room temperature to visualize the F-actin cytoskeleton or incubated with anti-Rab7 (late endosomes; Cell Signaling, #9367; 1/100 in blocking buffer) overnight at 4˚C, followed by an Alexa fluor-568 (red) secondary antibody for 45 min (Invitrogen, 1:400 in blocking buffer). In both conditions nuclei were counterstained with DAPI nuclear DNA stain (Invitrogen, 1:10.000), mounted in Ibidi mounting medium for fluorescent microscopy (Ibidi USA, Cat# 50001) and visualized using a Nikon A1 confocal laser microscope system (Nikon Instruments Inc. Melville, NY) as described above. In parallel, cells plated in 6-well plates were treated with either 1 mg/ml Biotin labeled rThbs4. Next, cells were rinsed with PBS, lysates were prepared as described above and intracellular biotin labeled proteins were visualized and quantified by western blot analysis using Streptavidin DyLight 650 conjugate (ThermoFisher Scientific, Cat# 84547) at a 1:1000 dilution on the Odyssey CLx Imaging System (Li-COR Biosciences, Lincoln, NE). All experiments described above were performed in triplicate.

Muscle fiber isolation and laser induced membrane injury
Flexor digitorum brevis (FDB) muscle fibers were isolated from male age-matched mice of each genotype as previously described and plated onto 35 mm glass-bottomed MatTek dishes (MatTek Corp., Ashland MA, P35G-0-10-C) in isotonic Tyrode buffer containing 1.25 mM Ca 2+ (Cai et al., 2009). Membrane damage was induced in the presence of 2.5 mM FM1-43 dye (Molecular Probes, Eugene OR) using a Nikon A1 confocal laser microscope through a Plan Apo 60x H 2 O immersion objective. To induce damage, a 5x5 pixel area of the sarcolemma on the surface of the muscle fiber was irradiated using a UV laser at full power (80 mW, 351/364) for 10 s at t = 60 s (Cai et al., 2009). Images were captured 5 min after irradiation at 5-second intervals (Cai et al., 2009). For each image, fluorescence intensity in an area of about 200 mm 2 directly adjacent to the injury site was measured using ImageJ software. To allow for statistical analysis from different experiments, data are presented as fluorescence intensity relative to the value before injury (DF/F0).

In vivo adenoviral transduction and tissue processing
Experiments were performed as previously described (Brody et al., 2016). Briefly, either purified AdThbs4-Flag, AdThbs4-mCa 2+ -Flag or Adbgal control were injected into the left and right gastrocnemius muscle of individual one-day-old Sprague Dawley rat pups (Envigo, Indianapolis IN, USA), followed by an additional injection 48 hr later (10 8 viral particles for each injection). Rat pups were sacrificed at eight days of age and muscles were embedded in O.C.T. frozen, and 10 mm cryosections were generated. To visualize adenoviral transduction (Flag) and the effects of our constructs on the membrane residency of b1 integrin, tissue sections washed three times with PBS, followed by blocking with 3% normal goat serum/PBS/0.1% Triton for 30 min at room temperature and then incubated with anti-flag and anti-b1 integrin primary antibodies (Cell Signaling, #2368; 1:500 and EMD Millipore, MAB1997, 1:100, respectively, in blocking buffer) overnight at 4˚C. Primary antibodies were detected by applying Alexa Fluor-488 conjugated goat-anti-rabbit and biotinylated antimouse (Vector Laboratories, M.O.M kit, 1:500) followed by Alexa Fluor-568 streptavidin conjugate (Invitrogen,1:200) for 45 min at RT. Sections were mounted in Vectashield Hard Set (Vector Laboratories, H-1400) to prevent photobleaching and imaged as described above. Sarcolemmal localized b1 integrin was quantified as percentage of red area per square surface of adenoviral transduced (Flag-positive) myofibers using ImageJ software.
Life cell quantitative imaging and photobleaching to evaluate ER to Golgi (FRAP) and Golgi to membrane vesicular trafficking (iFRAP) was performed using a Nikon A1 confocal laser microscope system and equipped with Plan Apo 40x oil immersion objective (NA = 1.0), an INU-TIZ-F1 stage top incubator (Tokai hit CO, Ltd, Shizuoka-ken, Japan) and NIS Elements AR microscope imaging software (Nikon Instruments Inc.) as previously described with modifications (Hirschberg et al., 1998;Patterson et al., 2008;Zaal et al., 1999). For ER-to-Golgi protein trafficking experiments, 24 hr after adenoviral infection, Ibidi dishes were infected with CellLight Golgi-RFP Bacmam 2.0 (ThermoFisher Scientific, c10593), a baculovirus containing a fusion construct of human Golgi resident enzyme N-acetylgalactosaminyltransferase and TagRFP (GalNacT2-RFP), according to manufacturer's instructions and incubated overnight. The next day, 100 mg/ml cycloheximide (Sigma-Aldrich, C4859) was added to the NRVMs to block new protein synthesis, 30 min prior to imaging. After acquiring a few baseline images, fluorescence from the Golgi pool of RFP was bleached by irradiating a region of interest (ROI) that encompasses the juxtanuclear Golgi region with a high intensity laser at 561 nm (100% laser power; Figure 5-figure supplement 1A). Next, recovery of GalNacT2-RFP was monitored by time-lapse imaging (5% laser power) at 5-min intervals for 2 hr as a measure of ER to Golgi protein trafficking.
For Golgi to membrane protein trafficking experiments, 24 hr after initial adenoviral infection, NRVMs in MatTek dishes were infected with adenovirus harboring the temperature sensitive VSVG-eGFP and incubated at 40˚C for 24 hr to retain the VSVG-eGFP in the ER (Hirschberg et al., 1998;Patterson et al., 2008). Approximately 60 min prior to imaging, 100 mg/ml cycloheximide was added to the cells. Thirty minutes prior to imaging MatTek dishes were shifted to 32˚C allowing the VSVG-eGFP to traffic to the Golgi. After acquiring a few baseline images, the cargo pool in the Golgi was selectively highlighted by photobleaching VSVG-eGFP from the entire cell excluding the perinucelear Golgi network using a high intensity laser at 488 nm (100% laser power; iFRAP; Figure 5-figure supplement 1B). Then, time-lapse imaging (5% laser power) at 1-minute intervals for 2 hr was performed to monitor export of VSVG-eGFP molecules from the Golgi using a second area that encompasses the Golgi network as a measurement for Golgi to membrane protein trafficking.
The combination of low energy, high attenuation, and the less concentrated excitation laser beam caused by the low NA objective resulted in negligible photobleaching during repetitive imaging in all experiments. As such, control experiments performing either time-lapsed imaging for 2 hr or the above described FRAP experiment in Golgi-RFP expressing cells in the presence of cycloheximide and brefeldin A (Sigma-Aldrich, B5936; 5 mg/ml) and iFRAP experiment in VSVG-eGFP expressing cells in the presence of cycloheximide and AlF (AlCl 3 , 60 mM and NaF, 20 mM; 30 min after shift to 32˚C) showed no difference in Golgi fluorescence intensity (data not shown) (Hirschberg et al., 1998;Patterson et al., 2008). Analysis of FRAP and loss of fluorescence after inverse FRAP (iFRAP) experiments was performed as previously described (Patterson et al., 2008;Zaal et al., 1999). The Golgi fluorescent values were normalized to the average baseline Golgi fluorescence prior to FRAP or to the first data point after iFRAP.

Drosophila husbandry and life-span assay
Male Drosophila of each genotype were collected at one-day posteclosion. Throughout the study, Drosophila were aged at 25˚C with a maximum of 12 flies in 25x95 mm polystyrene vials (Fischer Scientific, AS515) and transferred to new vials containing fresh food every three to four days without the use of anesthesia. Survival was recorded every day until 40 days of age. Kaplan-Meier statistical analysis was performed and significance determined by log-rank (Mantel-Cox) tests.

Drosophila negative geotaxis assay
The negative geotaxis assay was performed as previously described (Allikian et al., 2007). For each genotyped tested, male Drosophila were collected and kept at no more than 12 flies per vial. At 2, 5, 10, 15 and 20 days of age, they were immobilized using CO 2, and 12 groups of the various genotypes were transferred into empty polystyrene vials with a line drawn at 80 mm from the base of the vial. (Fisher Scientific, AS515). Drosophila were allowed to recover for 1 hr before testing. Each vial was assayed by gently tapping the flies down to the bottom of the vial, thereby engaging their negative geotactic response. The number of Drosophila able to climb across the 80-mm against gravity in 10 s was recorded. Four separate trials were performed with a 1-minute resting period in between. Percentage of Drosophila across were averaged and expressed as 'physical performance'. Genotypes were assayed simultaneously to eliminate variability attributed to RT and room humidity.

Drosophila histological analysis and immunohistochemistry
To obtain longitudinal sections of the dorsal median indirect flight muscles, 30-day old Drosophila of each genotype were positioned into mounting collars (Genesee Scientific,San Diego, CA 48-100) and subsequently fixed overnight at 4˚C in Carnoy's solution (6:3:1 ethanol:chlorophorm:acetic acid), dehydrated, and infiltrated with paraffin. Longitudinal sections were cut at 7 mm thickness and stained for H&E to evaluate dystrophic pathology in the dorsal median indirect flight muscle. To obtain cryosections of the dorsal median indirect flight muscles, the same mounting collars were positioned into O.C.T. freezing medium and placed into liquid nitrogen cooled-isopentane. For Thbs4, Tsp and calreticulin immunohistochemistry, paraffin sections were cleared of paraffin in xylene (2 times for 4 min), rehydrated in ethanol (100% EtOH 2 X 4', 95% EtOH 1 X 3', 70%EtOH 1 X 2', H 2 O 1'), incubated for 15 min in PBT (PBS/0.2% Triton X-100) and blocked in PBTB (PBT, 2% BSA) for one hour at RT. The sections were incubated with primary antibody overnight at 4˚C (Thbs4: Santa Cruz, Sc-7657-R, 1:50 dilution in PBTB; Tsp, 1/50 [Subramanian et al., 2007]; calreticulin: Abcam, ab2907; 1:100 dilution in PBTB), rinsed with PBT and incubated with the appropriate secondary antibody (goat-anti-rabbit-Alexa Fluor-488, Invitrogen; 1:400 in PBTB) for 2 hr at RT along with counterstains. Counterstains included membrane marker WGA conjugated to Alexa Fluor-647 (Invitrogen, W32466; 5 mg/ml, Far Red) and/or a nuclear marker DAPI (Invitrogen, D3571; 1 mg/ml in H 2 O). For Drosophila integrin subunit bPS immunostaining, cryosections were post-fixed in ice-cold methanol for 10 min, washed in PBT, blocked for one hour at room temperature with PBTB and incubated with primary antibody overnight at 4˚C (DSHB: CF.6G11; 1:50 in PBTB). Then, tissue was then washed in PBS and subsequently incubated with rabbit-anti-mouse-Alexa Fluor-488 (1:300 in PBTB; Invitrogen) for 2 hr. For each immunostain, consecutive sections were incubated with secondary antibodies alone as a control for specificity (not shown). Images were obtained using a Nikon A1 confocal laser microscope system equipped with 40x H 2 O immersion objective (NA = 1.1) and NIS Elements Advanced Research (AR) microscope imaging software (Nikon Instruments Inc.).

Transmission electron microscopy
Fresh quadriceps was harvested and immediately immersed in relaxing buffer (0.15% sucrose, 5% dextrose, 100 mM KCl in PBS), subsequently fixed overnight (3.5% glutaraldehyde, 0.15% sucrose in 0.1 M sodium cacodylate ph 7.4) and post-fixed in 1% OsO 4 (in water) for 2 hr at RT. Next they were washed, dehydrated and embedded using Epoxy resin. Tissue processing for electron microscopy of the dorsal median inferior flight muscle was performed as previously described (Allikian et al., 2007). Briefly, 25 day-old flies were positioned dorsal side up on a spot of OTC freezing medium, dipped in liquid nitrogen, bisected sagittally using a pre-cooled razor and then fixed overnight in 2.5% glutaraldehyde in NaH 2 PO 4 0.1 M pH 7.4 at 48˚C. Next, they were washed and post-fixed in 2% osmic acid in phosphate buffer for 2 hr at room temperature, dehydrated and embedded using Epon resin. Ultrathin sections of all tissues were counterstained with 1.5% uranyl acetate, 70% ethanol and lead nitrate/Na citrate. Images were obtained using a Hitachi 7600 transmission electron microscope connected to an AMT digital camera.
Sub-sarcolemmal vesicular expansion in myofibers of mouse quadriceps relative to the length of the sarcolemma was determined from 8 to 10 images per myofiber at 2000X of non-overlapping longitudinal regions that were randomly collected. In each image, the size of the vesicular content perpendicular to the sarcolemma -between the sarcolemma and the first sarcomere, and the length of the sarcolemma was determined using ImageJ software. The number of sarcomeric tears per myofiber in Drosophila dorsal median inferior flight muscle was determined by imaging complete myofibers at 1500X from longitudinal sections. Clear disruptions within the sarcomeric structure as indicated in Figure 8G and the associated Figure 8-figure supplement 1G.
For immunogold labeling of Thbs4, mouse quadriceps and Drosophila dorsal median inferior flight muscle were fixed with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) overnight. After 2 buffer washes, samples were post-fixed with 0.1% OsO 4 in the same buffer for 30 min, dehydrated and embedded in hydrophilic acrylic resin following manufacture instruction (L.R. White, #14380; Electron Microscopy Sciences, Hatfield, PA). Ultrathin sections were cut using a Leica Ultra-Cut S or UC6rt ultramicrotome at a thickness of 90 nm and placed on Formvar and carbon coated 200-mesh nickel for immunogold labeling of Thbs4. Briefly, ultrathin sections on grids were first treated with 1% sodium metaperiodate for 60 s to quench OsO 4 . After several washes with distilled water, grids were placed on drops of PBS containing 5% BSA and 0.1% cold-water fish gelatin to block nonspecific binding. Sections were then incubated overnight at 4˚C with goat anti-thrombo-spondin4 polyclonal primary antibody (R&D Systems, AF2390) at a final concentration of 5 mg/ml. Following several washes, sections were incubated in 6 nm colloidal gold particles conjugated rabbit anti-goat (Electron Microscopy Sciences; #25223) at a concentration of 10-20 mg/ml for 2 hr. After additional washes, all ultrathin sections were stained with 5% uranyl acetate for 2 min and 2% lead citrate for 15 min. For each experiment, both non-transgenic and transgenic sections were labeled and consecutive sections were incubated with secondary antibody alone as a control for specificity (not shown). Immunogold labeling was imaged on a JEOL JEM-1400 transmission electron microscope (JEOL Ltd, Japan) equipped with a Gatan US1000 CCD camera (Gatan, Pleasanton, CA).

Statistics
All results are presented as mean ± SEM. All data was normally distributed. Statistical analysis was performed with unpaired two-tailed Student's t test for two independent groups or one-way ANOVA with post hoc Tukey's test for multiple comparisons of 3 or more independent groups, as indicated in the individual figure legends. For survival analysis, Kaplan-Meier statistical analysis was performed and significance was determined by log-rank (Mantel-Cox) tests. All statistics were performed using GraphPad Prism 5.0 for Mac OS X and values were considered statistical significant when p<0.05. No statistical analysis was used to predetermine sample size. The experiments were not randomized and no animals were excluded from analysis. The investigators were not blinded to allocation during experiments and outcome assessment, except for data displayed in Figure 2B Sample size for the mouse and Drosophila experiments were estimated based on previous experiments with similar procedures but also based on past power calculations for appropriate group sizes, and based on this all data reported here were based on adequate sampling. No outlier data were excluded.