Laminin-111 protein therapy enhances muscle regeneration and repair in the GRMD dog model of Duchenne muscular dystrophy

Abstract

Duchenne muscular dystrophy (DMD) is a devastating X-linked disease affecting ~1 in 5000 males. DMD patients exhibit progressive muscle degeneration and weakness, leading to loss of ambulation and premature death from cardiopulmonary failure. We previously reported that mouse Laminin-111 (msLam-111) protein could reduce muscle pathology and improve muscle function in the mdx mouse model for DMD. In this study, we examined the ability of msLam-111 to prevent muscle disease progression in the golden retriever muscular dystrophy (GRMD) dog model of DMD. The msLam-111 protein was injected into the cranial tibial muscle compartment of GRMD dogs and muscle strength and pathology were assessed. The results showed that msLam-111 treatment increased muscle fiber regeneration and repair with improved muscle strength and reduced muscle fibrosis in the GRMD model. Together, these findings support the idea that Laminin-111 could serve as a novel protein therapy for the treatment of DMD.

Introduction

Duchenne muscular dystrophy (DMD) is a devastating neuromuscular disease affecting 1 in 5000 boys worldwide (1,2). DMD patients exhibit progressive muscle degeneration and weakness, leading to loss of ambulation and premature death from cardiopulmonary failure (3). DMD is caused by mutations in the dystrophin gene, which encodes a 427 kDa protein. Dystrophin protein is a member of the dystrophin glycoprotein complex (DGC), which acts as a scaffold between the actin cytoskeleton and laminin in the extracellular matrix to stabilize the sarcolemma (4). Mutations in the dystrophin gene lead to a loss of structural continuity between the extracellular matrix and the contractile machinery in skeletal muscle fibers (5,6). In DMD, sarcolemma rupture during muscle fiber contraction causes repeated rounds of muscle degeneration and regeneration (5). Although exon skipping drugs (Eteplirsen, Sarepta, Cambridge, Massachusetts) and corticosteroids/glucocorticords are currently FDA-approved therapeutic options for DMD, they are either limited to a small patient population or have adverse side effects, creating the need for additional treatment options (7,8). Recent advancements in gene and cell-based therapeutics for DMD include dystrophin gene restoration by exon skipping in DMD patient induced pluripotent stem cells (9), CRISPR-Cas9 gene editing in mdx mice and a DMD dog model (10) and micro-dystrophin gene delivery via Adeno-associated Virus (AAV) (now in clinical trials ClinicalTrials.gov: NCT03375164; NCT03368742; NCT03362502) (11,12). While these are promising therapeutic approaches, limitations of viral delivery, immune response or off-target activity in patients have yet to be fully assessed.

Table 1

In vivo tibiotarsal torque/force measurements in GRMD dog PBS versus Laminin-111-treated limbs

Test	GRMD		Kornegay (2014)
	PBS(n = 5)	Lam-111 (n = 5)	P-value	GRMD (n = 10)	Wild-type(n = 8)
Body mass (kg)	7.16 ± 1.12			7.36 ± 1.29	8.46 ± 0.63
Absolute tetanic extension torque (NM)	1.89 ± 0.558	1.78 ± 0.379	0.23	1.42 ± 0.23	2.55 ± 0.37
Absolute tetanic extension force (N)	16.7 ± 4.71	16.8 ± 2.43	0.56	12.73 ± 2.12	22.56 ± 2.76
Body-mass corrected tetanic extension torque (NM/kg)	0.265 ± 0.071	0.271 ± 0.084	0.62	0.20 ± 0.05	0.30 ± 0.04
Body-mass corrected tetanic extension force (N/kg)	2.35 ± 0.630	2.39 ± 0.780	0.67	1.80 ± 0.52	2.68 ± 0.38
Absolute tetanic flexion torque (NM)	0.342 ± 0.085	0.449 ± 0.109	0.030	0.43 ± 0.14	1.08 ± 0.14
Absolute tetanic flexion force (N)	3.03 ± 0.747	4.06 ± 1.00	0.033	3.85 ± 1.13	9.51 ± 1.07
Body-mass corrected tetanic flexion torque (NM/kg)	0.0493 ± 0.015	0.0660 ± 0.025	0.046	0.06 ± 0.02	0.13 ± 0.01
Body-mass corrected tetanic flexion force (N/kg)	0.436 ± 0.138	0.581 ± 0.224	0.048	0.53 ± 0.14	1.12 ± 0.10
ECD (%; 1–10)	35.6 ± 27.43	22.8 ± 16.10	0.395	13.97 ± 10.52	13.68 ± 3.97
ECD (%; 1–30)	56.00 ± 19.24	62.2 ± 21.0	0.698	36.51 ± 11.49	26.8 ± 5.92

Natural history of GRMD and wild-type measurements are reported for comparison. N=Newtons; Nm = Newton meters; ECD = Eccentric contraction decrement.

Several biologics have been shown to increase sarcolemma stability and/or muscle regeneration and may be complementary to gene and cell-based therapeutics (13). One such biologic is Laminin-111. Laminins are heterotrimeric glycoproteins consisting of α, β and γ chains located in the extracellular matrix in most tissues. Laminin-111 is present in the basal lamina during embryonic development and has significant structural homology to the Laminin-211/221 isoforms, the major component of the adult muscle basal lamina (14–16). Beyond structural roles, these laminin isoforms play important roles in muscle growth, signaling, development and repair (17–20). Exogenous treatment with Laminin-111 protein using in vitro and in vivo models has demonstrated that laminin-111 promotes muscle regeneration and stem cell support capacity (21–24).

In addition to the DGC, two additional protein complexes also link laminin in the extracellular matrix to the actin cytoskeleton in muscle. These are the α7β1 integrin and utrophin glycoprotein complexes (UGC) (25). Elevated levels of these laminin-binding complexes in DMD animal models have been shown to ameliorate or slow dystrophic disease progression (26–29). Previous studies have shown treatment of the mdx mouse model with exogenous Laminin-111 increased α7β1 integrin and UGC in skeletal muscle. Laminin-111-treated mdx mice showed reduced muscle fiber damage and fibrosis, and increased levels of muscle fiber regeneration (23,30–32).

Figure 1

msLam-111 persists in the extracellular matrix (ECM) of CT muscle of GRMD after intramuscular treatment. (A) GRMD dogs were treated biweekly with intramuscular injections of 20 mg/kg msLam-111 into the cranial tibial compartment from 2 weeks to 12 weeks of age. Saline was injected into the contralateral compartment. At 14 or 15 weeks, in vivo force studies and necropsy were performed. (B) Intramuscular injections targeted muscles in the cranial tibial compartment. Dosage of msLam-111 was calculated by extrapolation from natural history studies of age-matched GRMD muscles. (C) Detection of Laminin-α1 (green) in msLam-111 versus PBS treated cranial tibial compartment muscles. (D) Relative levels of Lam-111-IgGs in GRMD sera of rMs-Lam111-treated dogs are increased ~1000 to 2000-fold compared to untreated GRMD.

Here, we examined the efficacy of Laminin-111 treatment in the golden retriever muscular dystrophy (GRMD) canine model of DMD. This model displays severe myopathy that more closely recapitulates disease progression observed in DMD patients (33). During the first 3 months of age, the GRMD dog shows progressive tibiotarsal flexor muscle weakness (34), but overall presents with relatively mild myopathy (32). During the next 3 months, affected dogs develop tibiotarsal extensor weakness (34), which progresses into severe myopathy including postural changes. As the disease progresses, GRMD animals show respiratory (35) and cardiac dysfunction (36). Similarly, DMD patients from 0 to 5 years of age, corresponding to the first 3 months in GRMD (37), show mild symptoms of myopathy. They later progress to advanced myopathy from 5 to 10 years of age (3–6 months in GRMD), often leading to loss of ambulation (38). Our results show that early Laminin-111 treatment improves muscle regeneration and tibiotarsal flexor muscle strength at 3 months of age in the GRMD dog. Together with previous studies, our data support the idea that Laminin-111 represents a novel therapeutic candidate for the treatment of DMD.

Results

Previous studies have shown that intramuscular and systemically delivered Laminin-111 protein treatments prevented dystrophic pathology in the mdx mouse model for DMD (30). Although this work is highly promising, the mdx mouse model fails to completely recapitulate DMD patient disease progression (39). Based on previously reported flexion and extension force measurements, tibiotarsal muscles of GRMD dogs are weaker than those of wild-type dogs at 3 months of age and throughout their first year of life (34,37,40) (Table 1). Therefore, we assessed the therapeutic potential of the Laminin-111 protein by performing early intramuscular injections targeted to the cranial tibial compartment, i.e. cranial tibialis (CT), long digital extensor (LDE) and the peroneus longus (PL) muscles. To mitigate the effects of the reported high phenotypic variation between dogs and maintain a low n-number, each dog essentially served as its own control by comparing results from Laminin-111 and saline injected limbs, with results compared at 3 months of age (end of study) (37). In other words, the muscles in one limb were treated with Laminin-111 intramuscularly and compared to the contralateral Phosphate Buffer Saline (PBS)-treated limb in five different GRMD dogs. The cranial tibial compartments were treated intramuscularly biweekly with either saline (PBS) or natural Engelbreth-Holm-Swarm mouse Laminin-111 (msLam-111) protein diluted in saline at a dose of 20 mg/kg of anticipated muscle weight from 2 weeks to 3 months of age (Fig. 1A). To ensure maximum dispersion, msLam-111 or PBS injections were spaced in a grid pattern within the cranial tibial compartment (Fig. 1B) (see Materials and Methods). We hypothesized that treatment into this compartment would improve tibiotarsal flexion muscle force compared to the PBS-treated GRMD contralateral limbs.

Laminin-111 persists in the CT muscle for 2–3 weeks after treatment

Previous work showed that msLam-111 persisted in the mdx mouse muscle beyond 4 weeks after treatment (27). To determine if the exogenous msLam-111 persisted in the muscle after 2–3 weeks post last treatment, cryosections of the CT, LDE and PL muscles were subjected to immunofluorescence to detect laminin-α1 chain. Results showed positive but uneven staining against laminin-α1 surrounding myofibers of the CT muscle in msLam-111-treated limbs. The LDE and PL muscles showed little or no laminin-α1 as detected by immunofluorescence (Fig. 1C,Supplementary Material, Fig. 1).

Figure 2

CLN are significantly reduced in msLam-111-treated CT. (A) The CT muscle showed a significant reduction in CLN after treatment with msLam-111 compared to PBS (N = 5; P-value of 0.008). The LDE (B), PL (C), MG (D), CS (E) and VL (F) muscles show no changes in CLN.

We next used an enzyme-linked immunoabsorbent assay (ELISA) to determine if intramuscular injection of msLam-111 protein in the GRMD dogs produced an immune response. Our data revealed the presence of IgG against msLam-111 in dog sera 24 h post second and fifth treatment (Fig. 1D). This may indicate that treatment with the mouse biologic had elicited an acute immune response in the dogs. These results indicate that even in the presence of an immune response, the msLam-111 persisted in skeletal muscle after 2 to 3 weeks post last treatment, suggesting that laminin-111 had successfully integrated into the basal lamina of CT muscle.

Laminin-111 treatment reduced central myonucleation in the GRMD dog model

Next, we assessed histopathology of the CT, LDE and PL muscles within the cranial tibial compartment, as well as muscles outside of the compartment including the medial head of the gastrocnemius (MG), cranial sartorius (CS), and vastus lateralis (VL). Our results show the treated CT muscle exhibited a significant decrease in centrally located nuclei (CLN; 7.4%), a marker of on-going repair caused by disease/injury, versus the PBS-injected CT (11.5%; P-value of 0.008) (Fig. 2A). No significant changes were observed in CLN’s for msLam-111 relative to PBS-treated limbs in the LDE (17.8% and 20.7%, respectively) and PL (17.8% and 20.3%) (Fig. 2B and C). As expected, we also did not see differences in muscles outside the cranial tibial compartment for treated and untreated limbs: MG (14.0% and 16.6%), CS (13.4% and 10.5%) or VL (10.8% and 6.0%; Fig. 2D–F).

The reduction in CLN’s with msLam-111 treatment in the CT muscle may indicate that msLam-111 protected the muscle from damage or that there was muscle repair, with the myonucleus migrating back to the periphery of the fiber. The fact that the LDE and PL muscles showed no significant changes in CLN’s from msLam-111 injections compared to PBS treatment, along with absence of msLam-111 observed in these muscles, suggests the intramuscular injections favored delivery of msLam-111 to the proportionally larger CT muscle.

Laminin-111 treatment increased muscle regeneration in GRMD dog model

Laminin-111 protein treatment has been shown to promote an increase in muscle regeneration (23,30,32,41). In order to assess if msLam-111 treatments promoted muscle regeneration in the GRMD model, embryonic myosin heavy chain (eMHC), a marker of recent muscle repair was detected by immunofluorescence in CT cryosections and quantified (Fig. 3A). A 2-fold increase in eMHC-positive myofibers was observed in the ms-Lam-111-treated CT (16.52%) relative to PBS controls (7.53%) (P-value = 0.027) (Fig. 3B). Furthermore, Feret’s diameter of muscle fibers from the msLam-111 and PBS-treated limbs was also assessed, showing a shift toward larger muscle fibers with msLam-111 treatment (Fig. 3C). The Feret’s diameter curves were fit to a non-linear regression reporting an r-square of 0.99 for both PBS and msLam-111 limbs. A Students t-test was performed between both curves giving a P-value <0.0001, indicating a significant increase in myofiber size with treatment.

Figure 3

Laminin-111 treatment promotes muscle regeneration in GRMD CT. (A) eMHC (green) positive fibers were imaged as a measure of regeneration in the CT muscle of GRMD dogs. Wheat germ agglutinin (WGA, red) was used to outline fibers, and DAPI (blue) to visualize nuclei. (B) eMHC-positive fibers were quantified and normalized to total fibers. We observed a significant increase in eMHC-positive fibers in the laminin-111-treated limbs (N = 5; P-value of 0.027). (C) Feret diameters were measured as an indicator of fiber size. We found a shift toward larger fibers in the Lam-111-treated limbs (N = 5; P-value < 0.0001).

These results indicate msLam-111 treatment promoted an increase in muscle regeneration and myofiber size in the GRMD dog model.

Laminin-111 treatment improved limb flexion strength in the GRMD dog model

In vivo tibiotarsal torque/force was measured in all dogs at ~3 months of age after which muscles were removed at necropsy for histopathology assessments. Wet muscle weights for CT, LDE and PL from both limbs were measured post-mortem, normalized to body mass and compared. The treated CT was 47% larger than the PBS-treated muscle (P-value 0.29), while PL and LDE weights were comparable (Fig. 4A and Supplementary Material, Fig. 2).In vivo torque/force measurements were done 2–3 weeks after the final treatment. Tibiotarsal flexion and extension tetanic torque/force and eccentric contraction deficit (ECD) were measured as previously described (34) (Table 1). This time point was selected for muscle strength testing based on the predicted maximum effect msLAM-111 treatment would have on GRMD muscle regeneration and repair (31). Muscles of the cranial tibial compartment contribute primarily to tibiotarsal flexion, so we expected that flexion values would be higher in treated versus control limbs. Limbs treated with msLam-111 generated significantly greater absolute and body-mass-corrected tetanic flexion torque/force (corrected force, 0.581 ± 0.22 N/kg) than contralateral controls treated with PBS (corrected force, 0.436 ± 0.14 N/kg) (P-value of 0.048) (Fig. 4B,Table 1). When compared to the GRMD natural history value of 0.53 ± 0.14 N/kg (40), our results suggest a treatment effect. Tibiotarsal extension tetanic torque/force assesses mainly the gastrocnemius and superficial digital flexor muscles, which are outside of the treated cranial tibial compartment. Thus, as expected, body-mass-corrected tetanic extension torque/force did not differ between msLam-111 (corrected force, 2.39 ± 0.78 N/kg) and PBS (corrected force, 2.35 ± 0.63 N/kg) treated limbs (Fig. 4C,Table 1). Notably, each of these is higher than the 1.80 ± 0.52 N/kg value recorded from tibiotarsal extensors at 3 months of age in a GRMD natural history study (40), suggesting the possibility of a systemic effect from the treatment. Values for the ECD were highly variable between the two treatment groups and did not differ significantly over the first 10 eccentric contractions (PBS 35.6 ± 27.43% versus msLam-111 22.8 ± 16.1%). These values tended to be higher than those of the natural history study reported with similar variability (13.97 ± 10.52%) (40). Subsequent ECD measurements (10–30) showed high variability between limbs (PBS 56.0 ± 19.24% versus msLam-111 62.2 ± 21.0%). Natural history published values were lower, although variable for ECD (36.51 ± 11.49%) (40) (Table 1). Injury due to the multiple injections (up to 40 injection spots per limb) might also explain the lack of ECD improvement. Overall, in vivo muscle force measurement data show that biweekly intramuscular msLam-111 treatments led to ~33% increase in tetanic flexion force production relative to PBS-treated control muscles.

Figure 4

msLam-111 treatment increases in vivo flexion tetany of GRMD muscles and increase in wet muscle weight in CT. At week 14 or 15, tibiotarsal joint tetanic force was increased for flexion (B) but not for extension (C) measured bilaterally and normalized to body mass (N/kg) (N = 5; P-value of 0.048). Wet muscle weight was measured for muscles of the cranial tibial compartment post-necropsy. (A) The treated CT muscle was 47% larger compared to the untreated muscle (P-value 0.29).

Laminin-111 treatment showed a trend toward reduced muscle fibrosis in the GRMD dog model

Next, msLam-111 and PBS-treated GRMD CT muscle biopsies were assessed for fibrosis by Sirius red and hydroxyproline (HOP) quantification. Sirius red staining appeared reduced and the extracellular matrix appeared more continuous within the myofibers in msLam-111-treated CT muscles relative to controls (Supplementary Material, Fig. 3A). Similarly, we observed a trend toward a decrease in HOP content in msLam-111-treated CT muscle biopsies compared to PBS (P-value of 0.107; Supplementary Material, Fig. 3B).

Laminin-111 treatment increased average levels of α7β1 integrin protein in GRMD muscle

The α7β1 integrin and UGC have been shown to be major modifiers of disease progression in DMD. Previous studies in mdx mice have shown that treatment with msLam-111 increases both laminin-binding complexes in skeletal muscle (42). To determine if msLam-111 treatment increased the levels of α7β1 integrin and utrophin complexes in the GRMD dog, western blot analysis was used to detect components of these complexes in the CT muscle 2–3 weeks post last treatment (Supplementary Material, Fig. 4). Our results demonstrate variability between the five dogs in the study with no statistically significant increase in α7A integrin, utrophin, β-dystroglycan or γ-sarcoglycan proteins. However, there was a trend toward a treatment effect, as our results showed a 62% average increase in α7B integrin (P-value of 0.15) and 34% in β1D integrin protein (P-value of 0.36) in msLam-111-treated muscles. Additional studies at 6 months of age might substantiate an increase in the α7β1 integrin complex.

Discussion

Previous studies have demonstrated that msLam-111 treatment in the mdx mouse model of DMD ameliorates dystrophic pathology by stabilizing the skeletal muscle sarcolemma through elevated α7β1 integrin and utrophin protein complex levels (32,41,43). Muscle regeneration was also enhanced by treatment with msLam-111 in mdx mouse muscle (24). However, the mdx mouse does not completely phenocopy the muscle pathology and disease severity observed in DMD patients. The GRMD model is considered the gold standard and has dystrophic physiopathology and cardiomyopathy progression that is more representative of the DMD patient disease progression (37). In this study, we assessed the potential regenerative and repair capacity of msLam-111 in the GRMD canine model of DMD.

Previous studies have shown that a single dose of intraperitoneally delivered msLam-111 persisted up to 4 weeks in mdx mouse muscle (41). Although exogenously delivered msLam-111 protein persisted 2 to 3 weeks after the last treatment, msLam-111 was not uniformly detected in GRMD myofibers. While our technique targeted the CT, LDE and PL muscles in the cranial tibial compartment, it is highly likely that the CT was preferentially targeted due to the intramuscular delivery technique. The CT is the largest muscle, overlaps the LDE muscle and closely borders the tibia. The PL is positioned most caudally away from the tibia. Consistent with this idea, laminin-α1 staining and significant reduction of CLN were only detected in CT of treated limbs.

An increase in the number of eMHC-positive fibers indicates presence of regenerated or repaired fibers induced by msLam-111 treatment. Additionally, the increase in fiber diameter, taken together with the observed reduction in central myonucleation, was interpreted as direct evidence of repair-induced fusion enhancement by the msLam-111 treatment. These results indicate that msLam-111 treatments provided a micro-environment and niche that supported muscle repair and regeneration in the GRMD model.

Using ELISA, we found presence of IgG against msLam-111 in the sera of GRMD dogs, indicating that the treatments elicited an immune response against the biologic. Such an immune response was expected due to the introduction of a foreign mouse protein into canine muscle. This response might have neutralized or reduced the amount of laminin-111 protein available in the muscle and would also account for the variability observed in some of the preclinical outcome measures.

Our results show that treatment of GRMD muscle with msLam-111 improved body-mass-corrected flexion strength relative to the PBS-treated control limb. Flexion in the msLam-111-treated limb was comparable to the natural history controls, while the value in PBS-treated limbs was lower. This may indicate that biweekly injections delayed the progression of tibiotarsal joint weakness over the first 3 months of life. Additionally, tibiotarsal extension forces for the PBS and ms-Lam111-treated limbs used in this study were increased compared to previous reported natural history results from GRMD dogs using these same methods (40). This would be compatible with a systemic effect, although variation among dogs could also have contributed. Considering our histological results, the increase in body-mass-normalized strength appears to be due to improvements in muscle regeneration and repair. Treated CT muscle wet weight appeared to be larger and therefore body-mass-corrected strength increased. These findings suggest that msLam-111 and/or analogous regenerative/repair therapeutics may be valuable tools for rebuilding muscle in dystrophic patients, particularly in combination with emerging DMD therapies.

Although we did not observe a statistically significant increase in α7β1 integrin and UGC, there was a trend toward an increase in α7β1 integrin at 2 to 3 weeks after the final treatment. These results suggest that in a severe model of DMD, msLam-111 activity on stabilizing laminin-binding complexes may be more transient and dosing of the biologic may have to be more frequent in order to maintain efficacy for sarcolemmal stability.

Together, data reported in this study show that Laminin-111 represents a novel therapeutic candidate for DMD patients capable of enhancing muscle repair and regeneration. This is the first report of msLam-111 treatment in a large animal model of DMD. Even though the laminin-111 protein was of mouse origin, dog skeletal muscle showed improvements in strength and histological markers of muscle disease. Transition into the clinic will be largely dependent on the production and use of clinical grade recombinant human laminin-111. Recombinant human Laminin-111 protein would be unlikely to induce an immune response in patients as observed in the present study and, therefore, could further enhance muscle regeneration/repair and strength compared to that observed in the GRMD dog model. Recombinant human laminin-111 has the potential to treat all patients regardless of the mutation and could be combined with other emerging therapeutic approaches for DMD including gene therapy, gene editing or stem cell therapy.

Materials and Methods

Animal treatments

All three major muscles in the cranial tibial compartment, the CT, LDE and PL, were targeted with percutaneous intramuscular injections with msLam-111. For the purpose of calculating dosage, the cranial tibial compartment mass was extrapolated from previously reported muscle wet weights in GRMD. Based on prior reports (44,45), the GRMD PL weighs 1.34 ± 0.417 grams at 3 months of age and makes up about 15% of the volume of the three muscles in the cranial tibial compartment, independent of age. So, by extrapolation, we estimated that the three muscles together would weight about 9 grams at 3 months. With even growth over the 2-week to 3-month period, the combined muscle weight gain should be about 1.5 grams every 2 weeks, i.e. 1.5 grams at 2 weeks, 3 grams at 4 weeks, 4.5 grams at 6 weeks, 6 grams at 8 weeks, 7.5 grams at 10 weeks and 9 grams at 12 weeks (3 months). For this study, we gave msLam-111 at 20 mg/kg of muscle weight, beginning with 0.03 mg at 2 weeks. The msLam111 solution was concentrated at 1 mg/ml, so 0.03 ml was needed at 2 weeks. Depending on the size of the dog, we estimated that the cranial tibial compartment would only accommodate a total volume of 0.5 to 0.6 ml at 2 weeks. The 0.03 ml of msLam-111 was ‘topped off’ with PBS to reach the injection volume. We increased the total volume injected by 0.25 ml each 2 weeks so that 2.0 mls was injected at 3 months. The contralateral limb was injected with the same volume of PBS.

A grid pattern was drawn over the approximate location of the three muscles of the cranial tibial compartment with sites ~0.2–0.5 cm apart and 0.05 mls was injected at each site in the grid. The needle was advanced percutaneously at each point in the grid until the tip contacted the tibia, withdrawn slightly to ensure that the needle aperture was within the muscle and then withdrawn further through the muscle as the laminin-111 was injected. The number of injection sites increased as the dogs grew, with 10 at 2 weeks and 40 at 3 months (Fig. 1B). The person that performed the injections and subsequent functional testing was blinded to which compartment had received msLam111 versus vehicle. The team receiving and processing the biopsies were also blinded to treatments. After analysis was completed, the code was revealed.

GRMD in vivo force assessment

For the injections and all tests, dogs were intubated and anesthetized with acepromazine maleate (0.02 mg/kg), butorphanol (0.4 mg/kg) and atropine sulfate (0.04 mg/kg). At 3 months of treatment, tibiotarsal joint force and ECD were measured bilaterally as previously described (34). Studies were completed without knowledge of the treated versus control limbs. Dogs were euthanized for extensive gross pathologic, histopathologic and molecular studies.

Immunofluoresence and histology

Muscle samples from CT, LDE, PL, MG, CS and VL were embedded in optimum cutting temperature (Fisher Scientific, Waltham, Massachussetts) medium. Tissues were cyrosectioned at 10 μm thick and stained with WGA (Vector Laboratories, Burlingame, California, 1:00) for 10 min. Slides were prepared using Vectashield mounting media with DAPI stain (Vector Laboratories). CT slides were also immunostained with anti-eMHC (Developmental Studies Hybridroma Bank (DSHB), Iowa City, Iowa, BF-45, 1:40) or anti-laminin α1 (Mab200, 1:1). Laminin-α1 slides were permeabilized using 0.2% Triton in PBS for 20 min before blocking. All stains were followed by secondary antibody incubation for 1 h using FITC-anti mouse or FITC-anti rabbit antibodies (Jackson laboratories, Sacramento, California, 1:200) and 10 min of WGA (WGA-647 1:100). All slides were imaged using the Olympus Fluoview FV 1000 laser confocal microscope and analyzed using Image J-win32 software. To measure fibrosis CT slides were stained with Sirius Red (Sigma-Aldrich, St. Louis, Missouri) as previously described (23). Images were captured using Axiovision 4.8 software.

msLam-111 ELISA

Levels of antibodies against msLam111 were detected and quantified using ELISA. Recombinant Laminin-111 protein was incubated overnight in Immulon 1B (Thermo Scientific, Waltham, Massachusetts) ELISA plates followed by 1 h of 5% BSA blocking. Then, GRMD sera were incubated overnight at a 1:40 dilution. Fluorescein isothiocyanate (FITC) AffiniPure Donkey Anti-Dog (Jackson ImmunoResearch, West Grove, Philadelphia) was used at a 1:250 dilution to detect levels of anti-lam-111 binding.

Western blot assay

Protein samples were extracted from CT tissue using radioimmunoprecipitation assay buffer containing 1:500 dilution of protease inhibitor cocktail. Protein concentration was determined with BCA (Thermo Scientific) protein assay kit. Samples were separated in an SDS-PAGE and transferred onto nitrocellulose membrane. Detection of α7B integrin, β1D integrin, α7A integrin was done as previously described (46). β-dystroglycan H-242 (Santa Cruz, Santa Cruz, California, 1:200), utrophin H-300 (Santa Cruz, 1:100) and δ-sarcoglycan H-55 (Santa Cruz, 1:200) were incubated overnight followed by 1 h incubation with secondary antibodies Alexa Fluor 680 or 800 conjugated goat anti-rabbit (Invitrogen, Carlsbad, California). α-Tubullin (Abcam, Burlingame, California, 1:1000) was used to normalize each protein and blots were imaged using LI-COR imaging system. Protein bands were quantified using Image J-win32.

HOP assay

HOP content was quantified as described (47). GRMD samples were weighed and hydrolyzed in 6 M hydrochloric acid at 110°C overnight. Hydoxyproline standards (0–30ug/ml, Sigma-Aldrich) were prepared in each assay. Ten μl of hydrolysate/standards were combined with 150 μl isopropanol. Then, 72 μl of 1.4% chloramine-T (Sigma-Aldrich) in citrate buffer was added to each sample and allowed to oxidize at room temperature for ten minutes. One ml of Erlich’s reagent (7.5 g p-dimenthylaminobenzaldehyde, Sigma-Aldrich, 25 ml ethanol, 1.7 ml 12 N sulfuric acid) was added and incubated for 30 min at 55°C. Samples were read in triplicate at 560 nm.

Statistical analysis

All data was assessed by pairwise student T-test using GraphPad Prism software. Means were considered statistically significant with ^*P < 0.05, ^**P < 0.01, ^***P < 0.001.

Acknowledgements

The authors would like to thank Dr Madeleine Durbeej for kindly providing anti-laminin-α1 antibody.

Conflict of Interest statement. The University of Nevada, Reno, has a patent on the therapeutic use of laminin-111 and its derivatives. The patent inventors are Dean J. Burkin and Jachinta Rooney. This patent has been licensed to Prothelia Inc., and the University of Nevada, Reno has a small equity share in this company and Joe N. Kornegay is a paid consultant of Solid Biosciences.

Funding

Muscular Dystrophy Association (MDA)23898; Parent Project Muscular Dystrophy (PPMD); National Institutes of Health (NIH)/National Institute of Arthritis and Musculoskeletal and Skin Diseases(NIAMS) (R01AR064338-01A1 to D.J.B.); MDA Development Grant (240684).

Author contributions

D.J.B., J.N.K. conceptualization and study design; J.N.K. methodology; P.B.F., T.M.F., R.D.W. validation, formal analysis; P.B.F., T.M.F., R.D.W., H.J.H., A.M.N., J.N.K. investigation; D.J.B. resources; P.B.F., R.D.W. writing original draft; P.B.F., R.D.W., D.J.B., T.M.F., J.N.K. writing-review and editing; P.B.F., T.M.F., R.D.W. visualization, supervision; P.B.F., D.J.B. project administration; D.J.B. funding acquisition.