Fluvastatin delays propagation of viral infection in isolated rat FDB myofibers but does not affect exocytic membrane trafficking.

We have utilized the enveloped viral model to study the effect of fluvastatin on membrane trafficking in isolated rat myofibers. Our immunofluorescence studies constantly showed that infections in myofibers, which were treated with fluvastatin prior and during the infection with either vesicular stomatitis virus (VSV) or influenza A virus, propagated more slowly than in control myofibers without drug treatment. Experiments with a virus expressing Dad1 tagged with green fluorescent protein (GFP-Dad1) showed that fluvastatin did not affect its distribution within the ER/SR network and immunofluorescence staining for GM130 did not show any marked effect on the structure of the Golgi components. Furthermore, fluvastatin did not inhibit trafficking of the chimeric transport marker VSV temperature sensitive G protein (tsG-GFP) from the ER to the Golgi. We next subjected VSV infected myofibers for pulse-chase labeling experiments and found that fluvastatin did not slow down the ER-to-Golgi trafficking or Golgi to plasma membrane trafficking of the viral glycoprotein. These studies show that fluvastatin inhibited the propagation of viral infection in skeletal myofibers but no adverse effect on the exocytic trafficking could be demonstrated. These results suggest that other effects of statins rather than inhibition of ER-to-Golgi trafficking might be behind the myotoxic effects of the statins.

CD8 T-cell priming upon mRNA vaccination is restricted to bone-marrow-derived antigen-presenting cells and may involve antigen transfer from myocytes.

Self-amplifying mRNAs (SAM(®) ) are a novel class of nucleic acid vaccines, delivered by a non-viral delivery system. They are effective at eliciting potent and protective immune responses and are being developed as a platform technology with potential to be used for a broad range of targets. However, their mechanism of action has not been fully elucidated. To date, no evidence of in vivo transduction of professional antigen-presenting cells (APCs) by SAM vector has been reported, while the antigen expression has been shown to occur mostly in the muscle fibres. Here we show that bone-marrow-derived APCs rather than muscle cells are responsible for induction of MHC class-I restricted CD8 T cells in vivo, but direct transfection of APCs by SAM vectors is not required. Based on all our in vivo and in vitro data we propose that upon SAM vaccination the antigen is expressed within muscle cells and then transferred to APCs, suggesting cross-priming as the prevalent mechanism for priming the CD8 T-cell response by SAM vaccines.

Chicken and duck myotubes are highly susceptible and permissive to influenza virus infection.

UNLABELLED: Skeletal muscle, at 30 to 40% of body mass, is the most abundant soft tissue in the body. Besides its primary function in movement and posture, skeletal muscle is a significant innate immune organ with the capacity to produce cytokines and chemokines and respond to proinflammatory cytokines. Little is known about the role of skeletal muscle during systemic influenza A virus infection in any host and particularly avian species. Here we used primary chicken and duck multinucleated myotubes to examine their susceptibility and innate immune response to influenza virus infections. Both chicken and duck myotubes expressed avian and human sialic acid receptors and were readily susceptible to low-pathogenicity (H2N3 A/mallard duck/England/7277/06) and high-pathogenicity (H5N1 A/turkey/England/50-92/91 and H5N1 A/turkey/Turkey/1/05) avian and human H1N1 (A/USSR/77) influenza viruses. Both avian host species produced comparable levels of progeny H5N1 A/turkey/Turkey/1/05 virus. Notably, the rapid accumulation of viral nucleoprotein and matrix (M) gene RNA in chicken and duck myotubes was accompanied by extensive cytopathic damage with marked myotube apoptosis (widespread microscopic blebs, caspase 3/7 activation, and annexin V binding at the plasma membrane). Infected chicken myotubes produced significantly higher levels of proinflammatory cytokines than did the corresponding duck cells. Additionally, in chicken myotubes infected with H5N1 viruses, the induction of interferon beta (IFN-ß) and IFN-inducible genes, including the melanoma differentiation-associated protein 5 (MDA-5) gene, was relatively weak compared to infection with the corresponding H2N3 virus. Our findings highlight that avian skeletal muscle fibers are capable of productive influenza virus replication and are a potential tissue source of infection. IMPORTANCE: Infection with high-pathogenicity H5N1 viruses in ducks is often asymptomatic, and skeletal muscle from such birds could be a source of infection of humans and animals. Little is known about the ability of influenza A viruses to replicate in avian skeletal muscle fibers. We show here that cultured chicken and duck myotubes were highly susceptible to infection with both low- and high-pathogenicity avian influenza viruses. Infected myotubes of both avian species displayed rapid virus accumulation, apoptosis, and extensive cellular damage. Our results indicate that avian skeletal muscle fibers of chicken and duck could be significant contributors to progeny production of highly pathogenic H5N1 viruses.

Terminal differentiation of cardiac and skeletal myocytes induces permissivity to AAV transduction by relieving inhibition imposed by DNA damage response proteins.

Gene therapy vectors based on the adeno-associated virus (AAV) are extremely efficient for gene transfer into post-mitotic cells of heart, muscle, brain, and retina. The reason for their exquisite tropism for these cells has long remained elusive. Here, we show that upon terminal differentiation, cardiac and skeletal myocytes downregulate proteins of the DNA damage response (DDR) and that this markedly induces permissivity to AAV transduction. We observed that expression of members of the MRN complex (Mre11, Rad50, Nbs1), which bind the incoming AAV genomes, faded in cardiomyocytes at ~2 weeks after birth, as well as upon myoblast differentiation in vitro; in both cases, withdrawal of the cells from the cell cycle coincided with increased AAV permissivity. Treatment of proliferating cells with short-interfering RNAs (siRNAs) against the MRN proteins, or with microRNA-24, which is normally upregulated upon terminal differentiation and negatively controls the Nbs1 levels, significantly increased permissivity to AAV transduction. Consistently, delivery of these small RNAs to the juvenile liver concomitant with AAV markedly improved in vivo hepatocyte transduction. Collectively, these findings support the conclusion that cellular DDR proteins inhibit AAV transduction and that terminal cell differentiation relieves this restriction.

Gene expression profile in human skeletal muscle cells infected with human adenovirus type 36.

Human adenovirus type-36 (HAdV-36) is a specific pathogen that may lead to increased adiposity and obesity. In order to evaluate the effects of HAdV-36 on gene transcription, a microarray analysis of muscle cells infected with HAdV-36 was performed. Gene expression profile was determined by microarray analysis in cultured human skeletal muscle cells with or without HAdV-36 infection. Quantitative real-time PCR (qPCR) assay was performed in selected 35 genes to verify the results of the microarray analysis. A total of 13,060 unique genes were detected in the HAdV-36 infected muscle cells infected with HAdV-36. Among them, 1,004 genes were significantly altered by using a cut-off point at fold change ≥1.5 and P value <0.05. Most of the principal 100 altered genes were involved in development, immune response, signal transduction, transcriptional regulation as well as carbohydrate, lipid and protein metabolism. Thirty-two genes (91.4%) from the 35 selected genes were confirmed by qPCR assay. In addition, HAdV-36 altered 252 genes that are associated with cancer. The study showed HAdV-36 infection upregulated host cell antiviral defense. HAdV-36 also induces changes in gene expression related to cellular signaling pathways of signal transduction, transcriptional regulation as well as carbohydrate, lipid and protein metabolism. However, it remains to be investigated if HAdV-36 infection could lead to oncogenesis.

Prolonged myalgia in Sindbis virus infection: case description and in vitro infection of myotubes and myoblasts.

BACKGROUND: Sindbis virus (SINV) is a mosquito-borne alphavirus found in Eurasia, Africa, and Oceania. Clinical SINV infection is characterized by febrile rash and arthritis and sometimes prolonged arthralgia and myalgia. The pathophysiological mechanisms of musculoskeletal and rheumatic disease caused by SINV are inadequately understood. METHODS: We studied the muscle pathology of SINV infection ex vivo by examining a unique muscle biopsy obtained from a patient with chronic myalgia and arthralgia 6 months after acute SINV infection and assessed potential genetic predisposing factors by determining the human leukocyte antigen (HLA) and complement factor C4 genes and proteins. In addition, we performed in vitro SINV infections of primary human myoblasts and myotubes. RESULTS: In the muscle biopsy we found evidence of muscle regeneration due to previous necrotic lesions likely caused by earlier SINV infection. We showed that human myoblasts and myotubes were susceptible in vitro for SINV infection as the cells became immunoreactive for viral antigens and cytopathic effect was observed. The patient was homozygous for HLA-B*35 alleles and heterozygous for HLA-DRB1*01 and HLA-DRB1*03 alleles and had total deficiency of C4B protein. CONCLUSIONS: This study provides new insights concerning pathological processes leading to chronic symptoms in SINV infection and demonstrates for the first time the susceptibility of human myogenic cells to SINV infection.

Injection of a recombinant AAV serotype 2 into canine skeletal muscles evokes strong immune responses against transgene products.

Using murine models, we have previously demonstrated that recombinant adeno-associated virus (rAAV)-mediated microdystrophin gene transfer is a promising approach to treatment of Duchenne muscular dystrophy (DMD). To examine further therapeutic effects and the safety issue of rAAV-mediated microdystrophin gene transfer using larger animal models, such as dystrophic dog models, we first investigated transduction efficiency of rAAV in wild-type canine muscle cells, and found that rAAV2 encoding beta-galactosidase effectively transduces canine primary myotubes in vitro. Subsequent rAAV2 transfer into skeletal muscles of normal dogs, however, resulted in low and transient expression of beta-galactosidase together with intense cellular infiltrations in vivo, where cellular and humoral immune responses were remarkably activated. In contrast, rAAV2 expressing no transgene elicited no cellular infiltrations. Co-administration of immunosuppressants, cyclosporine and mycophenolate mofetil could partially improve rAAV2 transduction. Collectively, these results suggest that immune responses against the transgene product caused cellular infiltration and eliminated transduced myofibers in dogs. Furthermore, in vitro interferon-gamma release assay showed that canine splenocytes respond to immunogens or mitogens more susceptibly than murine ones. Our results emphasize the importance to scrutinize the immune responses to AAV vectors in larger animal models before applying rAAV-mediated gene therapy to DMD patients.

Adenovirus vectors based on human adenovirus type 19a have high potential for human muscle-directed gene therapy.

Until recently, adenovirus-based gene therapy has been almost exclusively based on human adenovirus serotype 5 (Ad5). The aim of this study was to systematically compare the efficiency of transduction of primary muscle cells from various species by two adenoviral vectors from subgroups C and D. Transduction of a panel of myoblasts demonstrated a striking specificity of an Ad19a-based replication-defective E1-deleted vector (Ad19aEGFP) for human cells, whereas the Ad5-based vector had high affinity for nonhuman primate myoblasts. Transgene expression correlated well with cell-associated vector genomes. Up to 6.59% of the initially applied Ad19aEGFP vector particles were taken up by human myoblasts, as compared with 0.1% of the corresponding Ad5 vector. Remarkably, Ad19aEGFP but not Ad5EGFP efficiently transduced differentiated human myotubes, an in vitro model for skeletal muscle transduction. Uptake of Ad19aEGFP vector particles in human myotubes was 12-fold more efficient than that of Ad5EGFP. Moreover, both vectors demonstrated an early block at the level of vector uptake in mouse myoblasts and rat L6 cells. Investigation of the underlying mechanism for binding and uptake of the two vectors by human myoblasts showed high susceptibility for Ad19a to neuraminidase and wheat germ agglutinin (WGA) lectin, whereas Ad5-mediated transduction was dependent on binding to the coxsackie-adenovirus receptor (CAR) and sensitive to soluble RGD peptide and heparin. Our study offers insights into species-dependent factors that determine Ad tropism and, moreover, provides a basis for application of the novel Ad19a-based vector for gene transfer into human skeletal muscle.

Muscle wasting induced by HTLV-1 tax-1 protein: an in vitro and in vivo study.

Besides tropical spastic paraparesis/human T-cell leukemia virus type-1 (HTLV-1)-associated myelopathy, the human retrovirus HTLV-1 causes inflammatory disorders such as myositis. Although the pathogenesis of HTLV-1-associated myositis is primarily unknown, a direct effect of cytokines or viral proteins in myocytotoxicity is suspected. We have developed an in vitro cell culture model to study the interactions between primary human muscle cells and HTLV-1 chronically infected cells. When HTLV-1-infected cell lines were added to differentiated muscle cultures, cytopathic changes such as fiber shrinking were observed as early as 1 day after contact. This was accompanied by alterations in desmin and vimentin organization, occurring in the absence of muscle cell infection but with Tax-1 present in myotubes. Cytopathic changes were also observed when infected culture supernatants were added to the muscle cells. Fiber atrophy and cytoskeletal disorganization were confirmed in muscle biopsies from two HTLV-1-infected patients with myositis. Transduction of cultured muscle cells with a lentiviral vector containing the HTLV-1 Tax gene reproduced such effects in vitro. The present data indicate that the myocytotoxicity that is observed in HTLV-1-associated myopathies can be due to a direct effect of the Tax-1 protein expressed in infected inflammatory cells, in the absence of muscle cell infection.

Recombinant adeno-associated virus vectors for gene therapy.

Recombinant adeno-associated virus (rAAV) vectors are based on a non-pathogenic human parvovirus (AAV) that is unique in its ability to persist in human cells without causing any pathologic effects. Studies of the potential barriers to rAAV-mediated transduction of relatively resistant cells has led to an understanding of the mechanisms of cell attachment and entry, cytoplasmic translocation, nuclear entry, conversion to active double-stranded DNA, activation of transcription and establishment of persistent molecular forms. Each of these areas is individually discussed, as are recent applications in vivo in preclinical models and clinical trials.

Asymmetric flaccid paralysis: a neuromuscular presentation of West Nile virus infection.

The neuromuscular aspects of West Nile virus (WNV) infection have not been characterized in detail. We have studied a group of six patients with proven WNV infection. All cases presented with acute, severe, asymmetric, or monolimb weakness, with minimal or no sensory disturbance after a mild flu-like prodrome. Four cases also had facial weakness. Three of our cases had no encephalitic signs or symptoms despite cerebrospinal fluid pleocytosis. Electrophysiological studies showed severe denervation in paralyzed limb muscles, suggesting either motor neuron or multiple ventral nerve root damage. This localization is supported further by the finding of abnormal signal intensity confined to the anterior horns on a lumbar spine magnetic resonance imaging. Muscle biopsies from three patients showed scattered necrotic fibers, implicating mild direct or indirect muscle damage from the WNV infection. In summary, we describe a group of patients with acute segmental flaccid paralysis with minimal or no encephalitic or sensory signs. We have localized the abnormality to either the spinal motor neurons or their ventral nerve roots. It will be important for physicians to consider WNV infection in patients with acute asymmetric paralysis with or without encephalitic symptoms.

Targeting functional subtypes of spinal motoneurons and skeletal muscle fibers in vivo by intramuscular injection of adenoviral and adeno-associated viral vectors.

We report that functional subtypes of spinal motoneurons and skeletal muscle fibers can be selectively transduced using replication-defective adenoviral (ADV) or adeno-associated (AAV) viral vectors. After intramuscular injection in adult rodents, ADV vectors transduced both fast-twitch and slow-twitch skeletal muscle fibers. Intramuscular injection of ADV vectors also caused transduction of spinal motoneurons and dorsal root ganglion cells. However, only neurons innervating the injected muscle were transduced, as shown by co-injection of a retrograde axonal tracer. In adult male rats it is therefore possible to transduce fast or slow spinal motoneurons and muscle fibers selectively since in these animals, the extensor digitorum longus and soleus muscles contain almost exclusively fast or slow motor units, respectively. In rats, AAV vectors transduced muscle fibers in the predominantly fast extensor digitorum longus but not in the predominantly slow soleus muscle. We did not observe any transduction of spinal motoneurons following intramuscular injection of AAV vectors. These results show that physiologically and clinically important subpopulations of cells in the neuromuscular system can be selectively transduced by viral vectors.

Post-mitotic, differentiated myotubes efficiently produce retroviral vector from hybrid adeno-retrovirus templates.

We have examined the ability of proliferating myoblasts and post-mitotic, differentiated myotubes to produce retroviral vector using hybrid adeno-retroviral vectors as templates. We show that production of retroviral vector from myoblasts peaks 48 h after adenoviral infection at 4.8 x 10(4) cfu/ml and is scarcely detectable by 96 h. Both fully and partially differentiated myotubes were able to generate a sustained increase in the levels of retroviral vector compared with myoblasts peaking 48 h at 1.4 x 10(5) cfu/ml and 1.8 x 10(5) cfu/ml, respectively. Addition of the cell cycle inhibitor aphidicolin (5 microg/ml) had no effect on the production of retroviral vector from fully differentiated myotubes, but resulted in an 80% increase in vector production from partially differentiated myotubes. Thus indicating that retroviral vector production is more efficient in post-mitotic myotubes and is independent of muscle cell cycle progression.

Characterization of heterokaryons between skeletal myoblasts and somatic cells formed by fusion with HVJ (Sendai virus); effects on myogenic differentiation.

In skeletal myogenic differentiation, myoblasts fuse with myogenic cells spontaneously, but do not fuse with non-myogenic cells either in vivo or in vitro, suggesting that the fusion of myoblasts with non-myogenic cells is unsuitable for differentiation. To understand the inevitability of the fusion among myoblasts, we prepared heterokaryons in crosses between quail myoblasts transformed with a temperature-sensitive mutant of Rous sarcoma virus (QM-RSV cells) and rodent non-myogenic cells, such as tumor cells, fibroblasts, or neurogenic cells by HVJ (Sendai virus) and examined how myogenic differentiation was influenced in the prepared heterokaryons, focusing on myogenin expression and myofibril formation as markers of differentiation. When presumptive QM-RSV cells were fused with non-myogenic cells by HVJ and induced to differentiate, both myogenin expression and myofibril formation were suppressed. When myotubes of QM-RSV cells that had already expressed myogenin and formed myofibrils were fused with non-myogenic cells, both myogenin and myofibrils disappeared. Especially, fibrous structures of myofibrils were significantly lost and dots or aggregations of F-actin were formed within 24 hr after formation of heterokaryons. However, the fusion of presumptive or differentiated QM-RSV cells with rodent myoblasts did not disturb myogenin expression or myofibril formation. These results suggest that mutual fusion of myoblasts is indispensable for normal myogenic differentiation irrespective of the species, and that some factors inhibiting myogenic differentiation exist in the cytoplasm of non-myogenic cells, but not in myoblasts.

Activation of the JNK pathway is important for cardiomyocyte death in response to simulated ischemia.

Multiple signaling pathways, including the c-Jun N-terminal kinase (JNK) pathway, are activated in myocardial ischemia and reperfusion (MI/R) and correlate with cell death. However, the role of the JNK pathway in MI/R-induced cell death is poorly understood. In a rabbit model, we found that ischemia followed by reperfusion resulted in JNK activation which could be detected in cytosol as well as in mitochondria. To address the functional role of the JNK activation, we examined the consequences of blockade of JNK activation in isolated cardiomyocytes under conditions of simulated ischemia. The JNK activity was stimulated approximately sixfold by simulated ischemia and reperfusion (simulated MI). When a dominant negative mutant of JNK kinase-2 (dnJNKK2), an upstream regulator of JNK, and JNK-interacting protein-1 (JIP-1) were expressed in myocytes by recombinant adenovirus, the activation of JNK by simulated MI was reduced 53%. Furthermore, the TNFalpha-activated JNK activity in H9c2 cells was completely abolished by dnJNKK2 and JIP-1. In correlation, when dnJNKK2 and JIP-1 were expressed in cardiomyocytes, both constructs significantly reduced cell death after simulated MI compared to vector controls. We conclude that activation of the JNK cascade is important for cardiomyocyte death in response to simulated ischemia.

Cationic lipids and polymers are able to enhance adenoviral infection of cultured mouse myotubes.

Adenovirus-mediated gene transfer has been used to promote efficient expression of various reporter and therapeutic transgenes such as minidystrophin in skeletal muscle tissue. However, down-regulation of the adenovirus internalisation receptors, alpha(v)/beta3 and alpha(v)beta5, in adult myofibres and in mature cultured myotubes makes them less susceptible to infection than neonatal muscle or cultured myoblasts. It has been reported elsewhere that adenoviral transduction of cells that are normally refractory to infection can be enhanced by complexing virus particles with cationic lipids or cationic polymers. In this study we describe increased levels of adenovirus-mediated transduction of cultured C2C12 myotubes, when the vector is complexed with either of the cationic lipids Lipofectamine or 1,3-dioleoyloxy-2-(6-carboxyspermyl)propylamide (DOSPER) or the cationic polymer polyethylenimine. The presence of polycations allowed a smaller dose of adenovirus vector to be used to attain the same level of infection seen with adenovirus alone, which has important relevance to future in vivo studies. Electron microscopic analysis of adenovirus/polycation complexes showed large aggregates as opposed to single adenovirus particles in the absence of polycations. Finally, by complexing adenovirus particles with polycations, partial protection against the neutralising effect of adenovirus antiserum was observed.

Extended tropism of an adenoviral vector does not circumvent the maturation-dependent transducibility of mouse skeletal muscle.

BACKGROUND: Efficient adenoviral gene delivery to mature skeletal muscle has been hindered by different factors. The low levels of adenoviral attachment receptor (CAR) that have been reported in this tissue may be a limiting factor. Therefore, adenoviral transduction of mature muscle may be improved by extending the tropism of the adenoviral vectors to attachment receptors that are highly expressed in mature myofibers. In this study, we have investigated whether an extended tropism adenoviral vector which additionally attaches to the broadly expressed heparan-containing receptors (AdPK) can bypass the maturation-dependent adenoviral transducibility of mouse skeletal muscle. METHODS: The adenoviral vector AdPK carrying the LacZ gene was evaluated as a gene delivery vehicle in mouse skeletal muscle at different maturities in vitro and in vivo. The viral transduction efficiencies were determined by histochemical and ONPG analysis of the beta-galactosidase activity level. RESULTS: Higher transduction efficiencies were detected in immature muscle from normal mice, and in mature muscle from merosin-deficient dy/dy mice (carrying myofibers with an impaired extracellular matrix) and dystrophin-deficient mdx mice (showing a high level of myoblast activity) when compared to mature muscle from normal mice. CONCLUSION: Despite the enhanced attachment characteristics, the extended tropism adenoviral vector is, similarly to the wild-type adenoviral vector in previous studies, still hindered by both a protective extracellular matrix and the diminished myoblast-mediation in mature muscle.

Muscle maturation: implications for gene therapy.

Skeletal muscle is a promising target tissue for gene therapy, for both muscle and non-muscle disorders. A variety of methods have been studied to transfer genes into skeletal muscle, including retroviral, adenoviral and herpes simplex viral vectors. However, various factors impede muscle-based viral gene therapy. Here, we discuss why some viral vectors cannot efficiently transduce mature muscle fibers, and describe some new approaches to overcome this barrier.

Viral gene delivery to skeletal muscle: insights on maturation-dependent loss of fiber infectivity for adenovirus and herpes simplex type 1 viral vectors.

The mechanisms causing age-dependent loss of muscle fiber infectivity observed in vivo for both adenoviral (Ad) and herpes simplex virus type 1 (HSV-1) gene delivery vectors remain poorly understood. Here we investigate the possible bases for this phenomenon using the novel application of enzymatically isolated, viable, single muscle fibers. We show that maturation-dependent loss of fiber infectivity is recapitulated in single fibers, and, thus, is not solely due to host immune response. Using localized irradiation of muscle in vivo, we show data suggesting that Ad infectivity of differentiated myofibers depends, at least in part, on myoblasts to mediate fiber transduction. On the other hand, infection of single fibers by HSV-1 is not affected by irradiation. Using confocal microscopy, we show that the basal lamina of myogenic cells efficiently infected by HSV-1 is structurally less organized than that of fibers resistant to infection by HSV-1. As well, we show that single myofibers isolated from adult, basal lamina-defective mice (merosin-deficient, dy/dy) are at least 10-fold more susceptible to infection by HSV-1 than are myofibers isolated from control mice. Together, these observations support the hypothesis that the basal lamina acts as a physical barrier to HSV-1 infection of mature muscle.