Influenza A Virus Infection Damages Zebrafish Skeletal Muscle and Exacerbates Disease in Zebrafish Modeling Duchenne Muscular Dystrophy

Abstract

INTRODUCTION: Both genetic and infectious diseases can result in skeletal muscle degeneration, inflammation, pain, and/or weakness. Duchenne muscular dystrophy (DMD) is the most common congenital muscle disease. DMD causes progressive muscle wasting due to mutations in Dystrophin. Influenza A and B viruses are frequently associated with muscle complications, especially in children. Infections activate an immune response and immunosuppressant drugs reduce DMD symptoms. These data suggest that the immune system may contribute to muscle pathology. However, roles of the immune response in DMD and Influenza muscle complications are not well understood. Zebrafish with dmd mutations are a well-characterized model in which to study the molecular and cellular mechanisms of DMD pathology. We recently showed that zebrafish can be infected by human Influenza A virus (IAV). Thus, the zebrafish is a powerful system with which to ask questions about the etiology and mechanisms of muscle damage due to genetic and/or infectious diseases.METHODS: We infected zebrafish with IAV and assayed muscle tissue structure, sarcolemma integrity, cell-extracellular matrix (ECM) attachment, and molecular and cellular markers of inflammation in response to IAV infection alone or in the context of DMD.RESULTS: We find that IAV-infected zebrafish display mild muscle degeneration with sarcolemma damage and compromised ECM adhesion. An innate immune response is elicited in muscle in IAV-infected zebrafish: NFkB signaling is activated, proinflammatory cytokine expression is upregulated, and neutrophils localize to sites of muscle damage. IAV-infected dmd mutants display more severe muscle damage than would be expected from an additive effect of dmd mutation and IAV infection, suggesting that muscle damage caused by Dystrophin-deficiency and IAV infection is synergistic.DISCUSSION: These data demonstrate the importance of preventing IAV infections in individuals with genetic muscle diseases. Elucidating the mechanisms of immune-mediated muscle damage will not only apply to DMD and IAV, but also to other conditions where the immune system, inflammation, and muscle tissue are known to be affected, such as autoimmune diseases, cancer, and aging. INTRODUCTIONSkeletal muscle is critical for homeostasis because skeletal muscle is required for breathing, posture, locomotion, metabolism, thermoregulation, and the immune response. Muscle tissue is remarkably plastic and can increase or decrease in mass in response to genetic and environmental factors. Muscle degeneration is a serious health issue that can reduce lifespan and quality of life. Muscle wasting can be caused by aging, injury, disuse, medications, genetic mutations, and infectious or inflammatory diseases. Understanding how muscle growth, regeneration, and degeneration are regulated in response to genetic and environmental insults alone and in combination is an important undertaking in order to be able to promote muscle health in cases of sickness and disease.Skeletal muscle damage occurs ...

Ethanol Exposure Causes Muscle Degeneration in Zebrafish

“…Tg(NF-kB:EGFP) zebrafish were continuously exposed to 0 or 2% EtOH from 30 hpf on and then fixed at 3 dpf. Muscle fibers in control Tg(NF-kB:EGFP) zebrafish were long and linear ( Figure 3 A1,A2) and did not express GFP at 72 hpf ( Figure 3 A3–A6, [ 25 ]). EtOH treatment increased NF-kB activity as evidenced by increased GFP expression in muscle fibers ( Figure 3 B3–B6).…”

“…In contrast, the primary cause of muscle damage in laminin a2 mutant zebrafish is failure of adhesion of muscle fibers to the MTJ. We found that human influenza A virus causes both sarcolemmal damage and disrupts muscle fiber adhesion to the MTJ [ 25 ], indicating that these modes of damage are not mutually exclusive.…”

Alternative Experimental Models for Studying Influenza Proteins, Host–Virus Interactions and Anti-Influenza Drugs

“…2 dpf zebrafish were infected with PR8 and muscle fiber damage was observed [244]. Muscle damage seemed to worsen at 48 hpi compared to 24 hpi.…”

“…This effect could be due to pro-inflammatory innate immune signaling cascades being induced via the NFkB pathway (suggested by using NFkB reporter line (NFkB:EGFP) nc ). Using a transgenic fish, Tg(BACmpo:gfp) i114 where GFP expression was controlled by the neutrophil-specific myeloperoxidase (mpo) promoter, it was reported that neutrophils infiltrated muscle tissue and was localized to the unanchored end of retracted fibers after infection [244]. This could be mediated by the p38 MAPK, Rho/ROCK and PKC pathways [244].…”

“…Using a transgenic fish, Tg(BACmpo:gfp) i114 where GFP expression was controlled by the neutrophil-specific myeloperoxidase (mpo) promoter, it was reported that neutrophils infiltrated muscle tissue and was localized to the unanchored end of retracted fibers after infection [244]. This could be mediated by the p38 MAPK, Rho/ROCK and PKC pathways [244]. Finally, using a zebrafish muscular dystrophy model (sapje/dmd), infected zebrafish were found to have severe muscle degeneration in almost every muscle segment, with increased mortality at 4 and 5 dpi compared to the PBS injected mutant zebrafish.…”