As the complexities of dystrophic pathology have been elucidated over the last few years, it has become increasingly clear that primary monogenetic defects result in multiple secondary pathologies capable of autonomously driving disease progression. Consequently, single-mode therapies fail to comprehensively ameliorate all aspects of pathology. Lama2-related muscular dystrophy (MDC1A) is a devastating congenital muscular dystrophy caused by mutations in the LAMA2 gene that results in multi-faceted secondary pathologies that include inflammation, fibrosis, apoptosis, and necrosis leading to severe muscle weakness and minimal postnatal growth. This study sought to implement a novel combinatorial treatment utilizing losartan, previously shown to ameliorate fibrosis and inflammation in conjunction with transgenic IGF-1 overexpression to improve postnatal growth. We found that dual-therapy rescued inflammation and fibrosis, improved weight gain, and led to remarkable restoration of muscle architecture and locomotory function in DyW mice (mouse model of MDC1A). We further showed using murine growth hormone that postnatal intervention with both therapies also yielded impressive amelioration of dystrophic pathology. Our results suggest for the first time that a combinatorial anti-fibrotic and pro-myogenic therapy could be the foundation of future therapies to a population of afflicted children in serious need.
Knowledge of the underlying complexity of dystrophic pathology has greatly expanded in the last few years. Recent advances have elucidated that secondary pathomechanisms downstream of the primary genetic defects can become autonomous disease drivers in their own right. As such, generating therapies targeted at arresting and/or reversing these secondary pathologies can have a tremendous impact on quality of life and has subsequently become a focus of therapeutic muscle research (1–5). While many single-mode therapies have shown efficacy in various models of muscular dystrophy, the evolving, multi-faceted pathology of these diseases makes it challenging to identify a single drug to treat all aspects of pathology simultaneously. Diseases with complicated pathomechanisms such as muscular dystrophy may require combinatorial therapies to most effectively ameliorate pathology. While combinatorial therapy is already widely employed for other diseases such as AIDS (6), cancer (7), and heart disease (8), it has only recently started to gain resonance in muscle research (9). We and others have conducted proof of concept studies showing combinatorial therapy to be effective in models of muscular dystrophy including laminin-deficient congenital muscular dystrophy (MDC1A) (9,10).
MDC1A, also known as Lama2-related muscular dystrophy, is an autosomal recessive disorder caused by mutations in the LAMA2 gene encoding for the alpha 2 chain of the muscle- and Schwann cell-specific extracellular matrix (ECM) protein laminin-211. Because this protein serves as the vital link between the sarcolemma and ECM, loss of Lama2 results in impaired myofibre anchoring, structural instability, and a multitude of dysregulated signalling pathways that lead to failed regeneration, inflammation, fibrosis, and apoptosis (11,12). Children with this disease present at/soon after birth with muscle weakness, hypotonia, and atrophy; many never achieve independent ambulation, and most die in their teen years due to respiratory insufficiency or failure to thrive (13).
Other than palliative care there is currently no FDA-approved therapy for MDC1A, but multiple single-mode therapies have produced quantifiable improvements in preclinical studies. Of these, the angiotensin II Type 1 receptor blocker (ARB) has been shown to ameliorate fibrotic as well as inflammatory pathology in multiple mouse models of MDC1A (1,3,14). However, it only has marginal effects on overall body and muscle weight (1,14). Conversely, our group has shown that muscle-specific transgenic overexpression of IGF-1 positively impacts body/muscle weight in the DyW mouse model of MDC1A but did not lead to any noticeable amelioration of the inflammatory or fibrotic pathology (15). Because inflammation, fibrosis, and a severe lack of postnatal growth are all major disease drivers, it is impossible to achieve maximal therapeutic benefit without addressing all aspects of pathology.
The present study tested whether combining IGF-1 overexpression with losartan treatment could comprehensively ameliorate pathology in DyW mice. Indeed, we found that when DyW mice overexpressing muscle-specific IGF-1 were treated with losartan, they showed markedly increased total body/muscle weight along a significant amelioration of inflammation and fibrosis, and increased locomotory behaviour. We further demonstrated the therapeutic potential of this anti-fibrotic and pro-myogenic combinatorial treatment by treating DyW mice with losartan and daily injections of murine Growth Hormone which is known to stimulate endogenous IGF-1 production (16).
To our knowledge, this is the first time an anti-fibrotic and pro-myogenic combinatorial treatment has been tested and provides a potential therapy regimen not only for MDC1A but also an array of dystrophies and other neuromuscular diseases in which inflammation, fibrosis, and diminished muscle growth comprise the underlying pathology.
Combinatorial treatment increases overall body and muscle weight as well as locomotory behaviour
We have previously shown that DyW mice are substantially smaller than their WT counterparts and that IGF-1 overexpression promotes increased postnatal growth (12,15). In contrast, mice treated with losartan do no show improvements in muscle mass or body weight (3,14). However, growth curves from postnatal weeks three through seven demonstrate that mice receiving losartan in the IGF1 transgenic background are significantly bigger than untreated DyW mice as well as mice receiving single-mode therapies (P < 0.05, two-way ANOVA) (Fig. 1A) . In line with the total bodyweight gain, dual-treatment also resulted in significant weight gains in tibialis anterior (TA), gastrocnemius-soleus (GS), and quadriceps (QD) hind limb muscle mass compared to losartan alone (P < 0.05, two-way ANOVA) (Fig. 1B).
In order to indirectly assess muscle function, two separate muscle performance assays were conducted. Mouse locomotory behavior was evaluated with the number of stand-ups per 5 min and a hind limb retraction test was used as an indirect measure of muscle strength. Losartan treatment results in better stand-up capability than a more modest improvement by IGF-1 overexpression (Fig. 1C and D). Combinatorial treatment leads to significantly more stand-ups than IGF-1 overexpression, but comparable to that of losartan treatment alone (P < 0.01, one-way ANOVA). Dual-treatment also resulted in marginal but still significant increases in time to hind limb retraction compared to either single-mode therapy (P < 0.001, one-way ANOVA).
Combinatorial Treatment Improves Muscle Morphology and Enhances Myogenesis
Dual-treatment with IGF-1 overexpression and losartan led to remarkable improvement in overall muscle morphology compared to either single-mode therapy (Fig. 2A). H&E staining of dual-treated mice shows less interstitial space and fewer infiltrating cells with rescued muscle architecture very similar to WT. Morphometric analysis of TA muscles shows that at 7-weeks, losartan by itself did not impact total fibre numbers over untreated DyW mice (Fig. 2B). In contrast, IGF-1 overexpression alone significantly increases total fibre numbers, however dual-treatment does not result in any further increase. Analyses of fibre size distribution using the minimum Feret diameter show that losartan alone shifts muscle distribution towards smaller fibres (Fig. 2C). Transgenic IGF-1 overexpression in single-mode and dual-treatment caused a rightward shift in fibre distribution with an increased number of large fibres (Fig. 2C).
Next, we assessed genes involved in myogenesis. There was no increase in early differentiation markers such as myoD (data not shown), yet dual-treatment did result in upregulation of the late-differentiation marker myogenin over untreated (P < 0.05, one-way ANOVA) and losartan-treated DyW mice (P < 0.001, one-way ANOVA) (Fig. 2D). We also looked at p21 expression because of the role it plays with myogenin to facilitate cell cycle withdrawal and terminal myocyte differentiation (17). We found that p21 was significantly upregulated in the dual-treatment group over losartan treatment alone (Fig. 2E) (P < 0.05, one-way ANOVA) though no different than untreated DyW mice. Additionally, we found that late-stage myogenesis genes were also upregulated in the combinatorial therapy group in comparison to single-mode treatment. Mef2c was significantly upregulated (P < 0.05, one-way ANOVA) over both single-mode treatments and mrf4 was significantly upregulated (P < 0.05, one-way ANOVA) over losartan alone (Fig. S1).
Combinatorial Therapy Ameliorates Inflammatory Pathology
Losartan has previously been shown to be anti-inflammatory while IGF-1 overexpression has led to exacerbated inflammation (14,15,18). In dual-treatment, the negative inflammatory effect of IGF-1 is indeed significantly reduced as supported by reduced CD11b gene expression and Mac-1 immunohistochemistry (P < 0.0001, one-way ANOVA) (Fig. 3A–C).
Combinatorial Treatment Rescues Fibrosis and Extra/Matricellular Protein Dysregulation
We have shown previously that untreated DyW mice and IGF-1 overexpressing DyW mice exhibit widespread fibrosis while losartan treatment ameliorates fibrotic pathology (15,18). Picrosirius red staining shows that dual-treatment results in substantially less interstitial collagen staining compared to IGF-1 overexpression, similar to what is seen with losartan treatment alone. Additionally, we have shown that DyW mice exhibit overexpression of extra/matricellular proteins collagen 1A, fibronectin, osteopontin, and periostin. Losartan treatment is able to downregulate these proteins to WT levels (12,14). In contrast, DyW mice overexpressing IGF-1 do not show any attenuation of matricellular protein dysregulation (Fig. 4A–D). Similar to losartan-treated mice, combinatorial treatment reversed transcriptional overexpression of most extra/matricellular proteins to WT levels, suggesting that even when coupled with IGF-1 overexpression, losartan still prevents the upregulation of ECM genes.
Combinatorial Therapy Slows Diaphragm Muscle Loss
Even though one of the main drivers of morbidity and mortality in MDC1A and other muscular dystrophies is respiratory failure, there has been little research into this aspect of pathology. Our study found that morphologically, diaphragms (DP) of untreated mice were significantly thinner (P < 0.0001, one-way ANOVA) than WT mice and exhibited a higher number of infiltrating cells as seen in H&E staining (Fig. 5A). Picrosirius red staining (Fig. 5B) shows increased interstitial collagen staining in untreated mice though not as severe as seen in hind limb muscles at the same time point (Fig. 2A). Each treatment resulted in an increase in DP thickness and overall morphological improvements in terms of infiltrating cells and fibrosis. However, IGF-1 overexpression led to statistically thicker DP than untreated DyW mice (P < 0.05, one-way ANOVA).
Losartan and Growth Hormone Combinatorial Therapy also Results in Significant Pathological Amelioration
In order to further test the therapeutic potential of this pro-myogenic/anti-fibrotic dual therapy, we utilized daily injections of the murine growth hormone (GH) in place of IGF-1 overexpression starting at postnatal week two. GH is upstream of endogenous IGF-1 production (16) and currently is a more readily available growth-promoting drug with a good safety profile in children (19). Dual-treatment with GH and losartan resulted in significant increases in total body weight over losartan-treated DyW mice at all-time points after week 3 postnatal (P < 0.05, two-way ANOVA) (Fig. 6A). GH/losartan treatment also resulted in larger hind limb muscles. Dual therapy resulted in significantly larger TA, GS, and QD (P < 0.05, two-way ANOVA) than untreated DyW mice (Fig. 6B). GH/losartan-treated mice also exhibited significantly greater standups/5 min (P < 0.01, two-way ANOVA) and time to hind limb retraction (P < 0.05, two-way ANOVA) (Fig. 6C and D) compared to untreated DyW mice.
H&E staining showed that DyW mice treated with both losartan and GH exhibited improved overall muscle architecture with more uniform fibre size and less infiltrating cells over untreated DyW mice (Fig. 7A). We also see fibrotic resolution in GH/losartan-treated mice, similar to losartan/IGF-1 overexpression, when compared to untreated DyW mice (Fig. 7B). In contrast to IGF-1 overexpression and IGF-1tg/losartan dual therapy, GH/losartan did not result in any increase in the total number of fibres (Fig. 7C). One explanation could be that transgenic mice overexpress IGF-1 prenatally during development and therefore could impact myogenesis differently than postnatal therapy. Fibre distribution using the minimum Feret diameter was, however, shifted towards larger fibres in GH/losartan-treated mice (Fig. 7D), analogous to what was seen with IGF-1tg/losartan dual-treatment though less pronounced (Fig. 2C). We also examined if dual-treatment with GH and losartan improved DP muscle size. We found that dual-treatment indeed resulted in healthier DP and attenuation of fibrosis as seen in H&E and Picrosirius red staining respectively compared to untreated DyW mice (Fig. 8A and B) and also resulted in significantly thicker DP than untreated DyW mice (P < 0.05, one-way ANOVA) (Fig. 8C). Lastly, to confirm that GH was indeed upregulating IGF-1 expression, we measured gene expression of IGF-1 and the IGF-1 receptor. Both IGF-1 and IGF-1R transcripts were significantly upregulated by dual-treatment (P < 0.0001, one-way ANOVA) (Fig. S2). Taken together, the data presented here suggest that postnatal dual administration of GH and losartan results in comparable pathology amelioration as transgenic IGF-1 overexpression/losartan dual therapy.
While the genetic defects and associated secondary pathologies of MDC1A and other muscular dystrophies have been well known for years, the role played by each of these secondary pathophysiologies to exacerbate the overall disease progression has proven to be quite intricate. Pre-clinical studies have shown that targeting single pathomechanisms is beneficial, yet it does not achieve sufficient amelioration of pathology; thus, identifying combinatorial treatment regimens to target multiple pathomechanisms simultaneously may be a more effective means of therapy. Using genetic manipulation we have previously shown that simultaneous targeting of two pathomechanisms can lead to a remarkable amelioration of pathology (10). The present study elucidates that concurrent anti-fibrotic and pro-myogenic therapy is able to robustly attenuate dystrophic pathology in the DyW mouse.
The AT1 receptor blocker losartan has been shown to ameliorate fibrotic and inflammatory pathology in various models of degenerative muscle diseases, and its anti-fibrotic effects have been established in different models of MDC1A by three independent labs (1,3,14,18). These results are particularly promising in the context of MDC1A because fibrosis is a very early signature of this disease and losartan is safe for use in children (20). However, as seen in our study, losartan represses myogenic genes and therefore could explain the lack of muscle growth associated with losartan single-mode therapy. It has been shown that Ang II, via the AT1 receptor, is critical to satellite cell activation and chemotaxis during regeneration. Thus, inhibition of the AT1 receptor with losartan inhibits this aspect of muscle regeneration/growth following myotrauma (21). Conversely, we have also shown that transgenic IGF-1 overexpression leads to improved muscle and overall body weight but does not lead to any amelioration of either inflammation or fibrosis (15), and in fact can negatively impact these pathologies in chronic disease states (22,23). The pro-myogenic effects of IGF-1 could be dampened in a chronically inflamed setting (like in DyW muscle) and may be more effective in an environment that is rescued from inflammation and fibrosis. Therefore, pairing the anti-inflammatory and anti-fibrotic effects of losartan with the pro-myogenic effects of IGF-1 could provide a synergistic benefit to ameliorate DyW pathology.
Indeed, IGF-1 overexpression and losartan dual therapy resulted in significant mitigation of inflammation and fibrosis resulting in vastly improved overall growth and muscle morphology. Since dysregulation of matricellular proteins is such an early signature of this disease, we believe it is imperative to attenuate their dysregulation in order restore a matrix that is conducive to proper postnatal growth. Many of these proteins (for example osteopontin, periostin, and fibronectin) can not only promote and exacerbate fibrosis, but also be detrimental to satellite cell (SC) differentiation due to their negative effects on the myomatrix and tissue rigidity. For instance, it has been shown that myoblasts express less myogenin and exhibit lower fusion capacity when plated on a surface of only collagen I (24). Additionally, while a transient increase in fibronectin (FN) is necessary for SC differentiation, chronically elevated FN has been shown to inhibit myogenic differentiation (25). Likewise, while a surge of osteopontin expression is necessary in normal muscle regeneration (26), chronically high expression of OPN results in increased inflammatory cell infiltration, a stiffened matrix, and augmented fibrosis (27). In the same vein, periostin also negatively affects ECM remodeling leading to blunted muscle regeneration (28,29). Thus, it can be conceived that the increased ECM rigidity produced by the dysregulation of these proteins promotes further fibrosis and is detrimental to the ability of SCs to differentiate towards successful myogenesis. Therefore, it can be argued that the restoration of ECM homeostasis with losartan allows for the pro-myogenic effects of IGF-1 to be effective in driving SC-mediated postnatal growth.
We also tested postnatal therapeutic potential of a dual pro-myogenic and anti-fibrotic approach, and administered daily injections of murine Growth Hormone (GH) in place of transgenic IGF-1 overexpression. We chose to use GH instead of systemic administration of IGF-1 since IGF-1 therapeutics are still only in clinical trials whereas GH has a long record of safe use in children (minimal adverse health events recorded directly related to treatment) (19) and thus potentially offers a more readily accessible route for implementation. We found that this combinatorial therapy also resulted in substantial improvement in overall DyW pathology including increases in overall postnatal growth, locomotory behaviour, and remarkable resolution in muscle morphology. The efficacy of this dual therapy when delivered postnatally is significant because clinical therapies will need to be effective when administered following onset of pathology.
In conclusion, these results provide convincing evidence that a pro-myogenic and anti-fibrotic combinatorial therapy is an efficacious treatment strategy for degenerative muscle diseases like MDC1A. Since both losartan and GH are FDA-approved and safe for use in children, this study offers a proof of concept and a potential path to the clinic for a patient population that currently has no available therapy. One must, however, acknowledge that while this strategy may be more effective to treat aggressive diseases such as dystrophies, the administration of multiple drugs simultaneously could complicate the challenge of life-long therapy. Therefore, future studies could be aimed at determining optimal windows for dual-treatment in order to maximize efficacy while minimizing the time when both therapies are needed concurrently. For example, it is conceivable that growth-inducing therapies, like IGF-1 or GH, may be most useful during the physiological growth phase and less effective outside of that time. This is indeed what is seen with IGF-1 overexpression where growth plateaus outside of week 7 postnatal (15). Determining if such a window exists would serve as another critical step in elucidating a beneficial therapy for a patient population in dire need.
Materials and Methods
All animals were housed at the Laboratory Animal Care Facility – Charles River Campus (LACF-CRC) of Boston University on a 12:12-hr light-dark cycle. Food and water were provided ad libitum. All procedures were performed in accordance to the protocol approved by the IACUC of Boston University (protocol number 13-055). Heterozygous B6.129 Lama2dy-W/+ mice, carrying a targeted mutation in the Lama2 gene, were kindly provided by Dr. Eva Engvall (Burnham Institute, La Jolla, CA, USA). MLC/mIGF-1 mice overexpressing muscle-specific IGF-1 were kindly provided to us by Dr. Elizabeth Barton at the University of Florida. Mice carrying the IGF-1 transgene were mated with LAMA2 +/- heterozygotes to generate DyW-IGF-1tg mice. PCR assays on tail DNA were utilized to genotype animals and animals carrying two mutant alleles (termed DyW) were utilized as experimental DyW animals. Losartan treatment was started at two weeks post-natal and continued until tissue collection at 7 weeks of age. Losartan was dissolved in the drinking water and provided ad libitum (600mg/L, Cozaar®,Merck pharmaceuticals. Drug water was supplemented with 25g/L of sucrose to increase palatability). Typically water consumptions by mice are in the range of 1.5ml/10g/day. Murine Growth Hormone (National Hormone and Peptide Program, Torrance, CA) was administered via daily intraperitoneal injections at a dose of 0.5mg/kg/day.
Muscle locomotory tests
Two different tests were performed to assess locomotory behaviour: the standup test and the tail suspension test. Stand up tests consisted of quantifying the mouse exploratory behavior by counting the number of times the animals stood up on their hind limbs during a period of 5min. For the tail suspension test, each animal was suspended in mid-air by the tail for a period of 10 s, and the time to the first episode of hind leg retraction was recorded; this procedure was repeated three times and the average time to the first episode of retraction for each mouse was calculated.
Serum and muscle tissue collection
Mice were first briefly bled from the submandibular vein to obtain blood samples, which were left to clot for 15 min at room temperature then spun at 2,000 g for 8 min to separate serum. The animals were then euthanized with isoflurane (Webster Veterinary, Devens, MA, USA) before isolating the tibialis anterior (TA), gastrocnemius/soleus complex (GS), quadriceps muscles (QD), and diaphragm (DP). Tissues were weighed and snap frozen in liquid nitrogen for RNA and protein extraction. TA muscles used for histology were embedded in Tissue-Tek OCT Compound (Sakura Finetek USA, Inc., Torrance, CA, USA) and frozen in isopentane (Sigma-Aldrich, St. Louis, MO, USA) chilled in liquid nitrogen. Serial transverse sections (7μm), were prepared using the Leica CM 1850 cryostat (Leica Microsystems, Inc.) and stored at -80 ̊C. Described in detail in (30).
Mid-belly serial transverse sections (7µm) were stained with H&E and Picrosirius red according to the manufacturer's instructions. Briefly, the tissue sections were fixed with acetone for 10 min and then rehydrated by passing through decreasing grades of alcohol and stained with hematoxylin stain 3 (Fisher Scientific, Fair Lawn, NJ, USA) for 1 min, washed, then subsequently stained with Ruben's Eosin-Phloxine Working Solution (Biocare Medical LLC) for 2 min. The sections were dehydrated by passing through the increasing grades of alcohol, cleared with xylene, and mounted using Cytoseal. For Picrosirius Red, tissues were fixed and rehydrated as described above. Sections were then stained with Picrosirius red (American MasterTech Scientific, Inc., Lodi, CA, USA) for 15 min, rinsed with 0.5% acetic acid, dehydrated with three quick passages in 100% ethanol, cleared with xylene for 10 min and cover-slipped with Cytoseal. Imaging was conducted using a Nikon DSFi1 camera head attached to a light microscope (Nikon ECLIPSE 50i) and morphometric analyses were completed using NIS-Elements Basic Research 3.0 software.
Frozen sections were let acclimate to room temperature for 15 min and then fixed/permeablized in acetone at -20 degrees Celsius for 15 min. They were let dry for 15 min and washed 2x2 min in PBS. Sections were blocked for 1 h in PBS containing 5% normal goat serum. Following blocking, slides were incubated in a 1:200 dilution of Mac-1 (anti-CD11b, BD Biosciences Cat #557672) for 45 min and then washed 2X5 min in PBS, incubated in 1 μg/ml DAPI (Sigma-Aldrich; Cat # D 9542) for 5 min and washed one more time for 5 min before being mounted in a 2:1 mixture of PBS:Glycerol and imaged with the same hardware/software mentioned above. Mac-1 quantitation was done by averaging Mac-1 positive cells on three different 40x field views.
RNA from 25 mg liquid nitrogen snap frozen pooled hind limb muscles was extracted with TRIzol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. 1μg RNA was reverse-transcribed with the High Capacity cDNA Reverse Transcription Kit (Applied Biosystem, Foster City, CA, USA). Analysis of gene expression was performed by TaqMan qRT-PCR assays (Applied Biosystems, Foster City, CA, USA) on ABI 7300 Real Time PCR system. 18s ribosomal subunit RNA served as the endogenous control and gene expression was calculated by using the ΔΔCt method.
ANOVA tests were performed using GraphPad Prism 6 software. Data are presented as mean ± standard deviation. The number of animals used for analysis is included in the corresponding figure legend.
We would also like to thank Stefan Girgenrath for valuable comments and advice.
Conflict of Interest statement. None declared.
This work was supported by the research grants awarded to M.G. from Cure CMD, Struggle Against Muscular Dystrophy (SAM) and Muscular Dystrophy Association (218938).