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Interactions between muscle and the environment

One of the key unanswered questions regarding the variable age of onset and rate of progression of muscular dystrophies is whether the environment influences these factors. One roadblock has been that the basic biology of how many environmental factors interact with muscle is unknown. We have been addressing this question in the zebrafish model. (1) Although muscle pain and weakness are symptoms of Influenza A and B viruses, the mechanisms  mediating influenza-induced muscle damage were not known. We characterized muscle damage and assayed innate immune/inflammatory markers in response to IAV infection in vivo. We also showed that IAV infection greatly exacerbates the extend of fiber damage in zebrafish modeling Duchenne Muscular Dystrophy. Thus, these results showed an important gene-environment interaction between the pro-inflammatory innate immune response and the DMD gene in skeletal muscle. (2) Alchoholic myopathies are characterized by neuromusculoskeletal symptoms such as compromised movement and weakness. The effects of EtOH exposure on skeletal muscle during the growth period that follows primary muscle development are not well understood. We found that this period of development is highly sensitive to EtOH, which triggers a pro-inflammatory response. EtOH damaged muscle end attachments and caused membrane instability. EtOH also exacerbates muscle damage in the zebrafish model of Duchenne Muscular Dystrophy. Thus, our results suggest that EtOH exposure has pleiotropic deleterious effects on skeletal muscle. (3) Whereas a great deal is known about the structural and functional plasticity of healthy skeletal muscle, far less is understood about plasticity and adaptation in diseased muscle. Identifying the basic mechanisms by which activity impacts muscle health in the context of muscle disease is a crucial first step towards identifying potential therapies. We identified a neuromuscular electrical stimulation (NMES) paradigm that improves neuromuscular structure, function, and lifespan. We showed that NMES differently affects gene expression in WT versus dmd mutants, indicating that it is critical to study the impacts of activity on diseased muscle in addition to WT muscle. Finally, we showed that eNMES acts via Itga7 and HO signaling. Taken together, our data not only established a model system for neuromuscular plasticity in healthy versus diseased muscle; but also clearly elucidated beneficial effects of NMES.

  1. Coffey EC, Pasquarella ME, Goody MF, Henry CA. Ethanol Exposure Causes Muscle Degeneration in Zebrafish. J Dev Biol. 2018 Mar 9;6(1) PubMed Central PMCID: PMC5875561.

  2. Goody M, Jurczyszak D, Kim C, Henry C. Influenza A Virus Infection Damages Zebrafish Skeletal Muscle and Exacerbates Disease in Zebrafish Modeling Duchenne Muscular Dystrophy. PLoS Curr. 2017 Oct 25;9 PubMed Central PMCID: PMC5693338.

  3. Kilroy EA, Ignacz AC, Brann KL, Schaffer CE, Varney D, Alrowaished SS, Silknitter KJ, Miner JN, Almaghasilah A, Spellen TL, Lewis AD, Tilbury K, King BL, Kelley JB, Henry, CA. Beneficial impacts of neuromuscular electrical stimulation on muscle structure and function in the zebrafish model of Duchenne Muscular Dystrophy. 2022 ELife, in press.

Novel Mechanisms of Laminin Expression

​Basement membranes (BMs) are specialized sheets of extracellular matrix. BMs are critical for animal development and homeostasis. In skeletal muscle, adhesion of muscle fibers to the BM is necessary for muscle development, regeneration, and homeostasis. This requirement for muscle-BM adhesion is highlighted by the genetic basis for congenital muscular dystrophies; these are progressive, debilitating diseases without cures. Many dystrophies, including Duchenne, Becker, and Merosin-deficient muscular dystrophies, result from mutations that disrupt adhesion of muscle fibers to their surrounding BM. Mechanisms that mediate BM assembly during muscle development and repair are not well understood. The protein laminin is a central component of BMs. We identified a novel Integrin signaling pathway that increases muscle-BM adhesion. This pathway involves Nicotinamide Riboside Kinase 2b (Nrk2b). Nrk2b is expressed specifically in skeletal muscle and generates NAD+. We showed that addition of either exogenous NAD+ or a vitamin precursor to NAD+ is sufficient to correct dystrophic phenotypes in zebrafish lacking either Dystroglycan or Integrin alpha7. This work was recommended by F1000, highlighted by a PLoS Biology Synopsis, highlighted in a PLoS Biologue, and highlighted in Science Daily. As mechanical failure in cell adhesion between muscle fibers and their surrounding BM underlies the etiology of many different dystrophies, the Nrk2b pathway may have therapeutic utility for multiple dystrophies.

  1. Bailey EC, Alrowaished SS, Kilroy EA, Crooks ES, Drinkert DM, Karunasiri CM, Belanger JJ, Khalil A, Kelley JB, Henry CA. NAD+ improves neuromuscular development in a zebrafish model of FKRP-associated dystroglycanopathy. Skelet Muscle. 2019 Aug 7;9(1):21. PubMed Central PMCID: PMC6685180.

  2. Goody MF, Henry CA. A need for NAD+ in muscle development, homeostasis, and aging. Skelet Muscle. 2018 Mar 7;8(1):9. PubMed Central PMCID: PMC5840929.

  3. Goody MF, Kelly MW, Reynolds CJ, Khalil A, Crawford BD, Henry CA. NAD+ biosynthesis ameliorates a zebrafish model of muscular dystrophy. PLoS Biol. 2012;10(10):e1001409. PubMed Central PMCID: PMC3479101.

  4. Goody MF, Kelly MW, Lessard KN, Khalil A, Henry CA. Nrk2b-mediated NAD+ production regulates cell adhesion and is required for muscle morphogenesis in vivo: Nrk2b and NAD+ in muscle morphogenesis. Dev Biol. 2010 Aug 15;344(2):809-26. PubMed Central PMCID: PMC2917104.

Extracellular matrix dynamics at the myotendinous junction

Remodeling of the ECM regulates adhesion and signaling between cells and their microenvironment. ECM remodeling is critical for embryonic development and regeneration. However, mechanisms underlying ECM remodeling in vivo remain elusive. We were the first to show that Fn is dynamically regulated at the myotendinous junction during muscle development: Fn is degraded as fast-twitch muscle fibers elongate and attach to the MTJ. Whether Fn played a role in muscle development was a central question. Answering this question was not trivial because Fn is required for formation of somites, which give rise to axial skeletal muscle. We used multiple morphometric assays and mutant analysis to show that Fn plays a critical role in MTJ maintenance. Next, we delved into molecular mechanisms of Fn regulation in vivo. We showed that normal laminin organization acts as a “checkpoint” for Fn downregulation. Furthermore, laminin signaling modulates both the expression of fn1b and localization of Matrix metalloproteinase 11 (Mmp11) to MTJs. We found that Mmp11 is both necessary and sufficient for Fn downregulation. Taken together, this study identified a new mechanism in the myomatrix that regulates the remodeling process during morphogenesis in vivo, and that may be profitably targeted in the many pathological conditions in which Fn is dysregulated, to improve muscle tissue structure.

  1. Jenkins MH, Alrowaished SS, Goody MF, Crawford BD, Henry CA. Laminin and Matrix metalloproteinase 11 regulate Fibronectin levels in the zebrafish myotendinous junction. Skelet Muscle. 2016;6:18. PubMed Central PMCID: PMC4852425.

  2. Snow CJ, Henry CA. Dynamic formation of microenvironments at the myotendinous junction correlates with muscle fiber morphogenesis in zebrafish. Gene Expr Patterns. 2009 Jan;9(1):37-42. PubMed Central PMCID: PMC2655214.

  3. Snow CJ, Peterson MT, Khalil A, Henry CA. Muscle development is disrupted in zebrafish embryos deficient for fibronectin. Dev Dyn. 2008 Sep;237(9):2542-53. PubMed Central PMCID: PMC2572006.

  4. Henry CA, McNulty IM, Durst WA, Munchel SE, Amacher SL. Interactions between muscle fibers and segment boundaries in zebrafish. Dev Biol. 2005 Nov 15;287(2):346-60. PubMed PMID: 16225858.

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