Primary Human Myoblasts Research Articles

Distinct roles of NFATc1 and NFATc4 in human primary myoblast differentiation and in the maintenance of reserve cells.

Ca2+ signaling plays a key role during human myoblast differentiation. Among Ca2+-sensitive pathways, calcineurin is essential for myoblast differentiation and muscle regeneration. Nuclear factor of activated T-cell (NFAT) transcription factors are the major calcineurin targets. We investigated the expression and the role of each NFAT gene during human primary myoblast differentiation. We found that three NFAT isoforms are present, NFATc1, NFATc3 and NFATc4. Importantly, while their mRNA expression increases during differentiation, NFATc1 is more highly expressed in myotubes, whilst NFATc4 is specifically maintained in reserve cells. NFATc3 is present in both cell types, although no specific role during myoblast differentiation was observed. Knockdown of either NFATc1 or NFATc4 affects the differentiation process similarly, by decreasing the expression of late differentiation markers, but impairs myotube formation differently. Whereas NFATc1 knockdown strongly reduced the number and the surface area of myotubes, NFATc4 knockdown increased the surface area of myotubes and reduced the pool of reserve cells. We conclude that NFAT genes have specific roles in myotube formation and in the maintenance of the reserve cell pool during human postnatal myogenesis.

FHL1B Interacts with Lamin A/C andEmerin at the Nuclear Lamina andisMisregulated in Emery-Dreifuss Muscular Dystrophy.

Emery-Dreifuss muscular dystrophy (EDMD) is associated with mutations in EMD and LMNA genes, encoding for the nuclear envelope proteins emerin and lamin A/C, indicating that EDMD is anuclear envelope disease. We recently reported mutations in FHL1 gene in X-linked EDMD. FHL1 encodes FHL1A, and the two minor isoforms FHL1B and FHL1C. So far, none have been described at the nuclear envelope. To gain insight into the pathophysiology of EDMD, we focused our attention on the poorly characterized FHL1B isoform. The amount and the localisation of FHL1B were evaluated in control and diseased human primary myoblasts using immunofluorescence and western blotting. We found that in addition to acytoplasmic localization, this isoform strongly accumulated at the nuclear envelope of primary human myoblasts, like but independently of lamin A/C and emerin. During myoblast differentiation, we observed amajor reduction of FHL1B protein expression, especially in the nucleus. Interestingly, we found elevated FHL1B expression level in myoblasts from an FHL1-related EDMD patient where the FHL1 mutation only affects FHL1A, as well as in myoblasts from an LMNA-related EDMD patient. Altogether, the specific localization of FHL1B and its modulation in disease-patient's myoblasts confirmed FHL1-related EDMD as anuclear envelope disease.

Silk fibroin scaffolds with muscle-like elasticity support in vitro differentiation of human skeletal muscle cells.

Human adult skeletal muscle has a limited ability to regenerate after injury and therapeutic options for volumetric muscle loss are few. Technologies to enhance regeneration of tissues generally rely upon bioscaffolds to mimic aspects of the tissue extracellular matrix (ECM). In the present study, silk fibroins from four Lepidoptera (silkworm) species engineered into three‐dimensional scaffolds were examined for their ability to support the differentiation of primary human skeletal muscle myoblasts. Human skeletal muscle myoblasts (HSMMs) adhered, spread and deposited extensive ECM on all the scaffolds, but immunofluorescence and quantitative polymerase chain reaction analysis of gene expression revealed that myotube formation occurred differently on the various scaffolds. Bombyx mori fibroin scaffolds supported formation of long, well‐aligned myotubes, whereas on Antheraea mylitta fibroin scaffolds the myotubes were thicker and shorter. Myotubes were oriented in two perpendicular layers on Antheraea assamensis scaffolds, and scaffolds of Philosamia/Samia ricini (S. ricini) fibroin poorly supported myotube formation. These differences were not caused by fibroin composition per se, as HSMMs adhered to, proliferated on and formed striated myotubes on all four fibroins presented as two‐dimensional fibroin films. The Young's modulus of A. mylitta and B. mori scaffolds mimicked that of normal skeletal muscle, but A. assamensis and S. ricini scaffolds were more flexible. The present study demonstrates that although myoblasts deposit matrix onto fibroin scaffolds and create a permissive environment for cell proliferation, a scaffold elasticity resembling that of normal muscle is required for optimal myotube length, alignment, and maturation. © 2016 The Authors Journal of Tissue Engineering and Regenerative Medicine Published by John Wiley & Sons Ltd. StartCopTextStartCopText© 2016 The Authors Journal of Tissue Engineering and Regenerative Medicine Published by John Wiley & Sons Ltd.

Functional KCa1.1 channels are crucial for regulating the proliferation, migration and differentiation of human primary skeletal myoblasts.

Myoblasts are mononucleated precursors of myofibers; they persist in mature skeletal muscles for growth and regeneration post injury. During myotonic dystrophy type 1 (DM1), a complex autosomal-dominant neuromuscular disease, the differentiation of skeletal myoblasts into functional myotubes is impaired, resulting in muscle wasting and weakness. The mechanisms leading to this altered differentiation are not fully understood. Here, we demonstrate that the calcium- and voltage-dependent potassium channel, KCa1.1 (BK, Slo1, KCNMA1), regulates myoblast proliferation, migration, and fusion. We also show a loss of plasma membrane expression of the pore-forming α subunit of KCa1.1 in DM1 myoblasts. Inhibiting the function of KCa1.1 in healthy myoblasts induced an increase in cytosolic calcium levels and altered nuclear factor kappa B (NFκB) levels without affecting cell survival. In these normal cells, KCa1.1 block resulted in enhanced proliferation and decreased matrix metalloproteinase secretion, migration, and myotube fusion, phenotypes all observed in DM1 myoblasts and associated with disease pathogenesis. In contrast, introducing functional KCa1.1 α-subunits into DM1 myoblasts normalized their proliferation and rescued expression of the late myogenic marker Mef2. Our results identify KCa1.1 channels as crucial regulators of skeletal myogenesis and suggest these channels as novel therapeutic targets in DM1.

The immunoproteasomes are key to regulate myokines and MHC class I expression in idiopathic inflammatory myopathies

Idiopathic inflammatory myopathies (IIMs) are diseases with muscle weakness, morphologically characterized by inflammatory infiltration and increased expression of MHC class I molecule on myofibers. Immunoproteasome, as a proteolytic complex that shapes the repertoire of antigenic peptides, has been previously demonstrated to be over-expressed in IIMs at mRNA level. In this study, we investigated the expression and the function of the immunoproteasome in IIMs in more detail. As shown by immunofluorescence staining, expression of relevant players of the immunoproteasome was detectable in the inflamed skeletal muscle tissue from IIM patients. In fact, two subunits of the immunoproteasome, β1i or β5i were upregulated in sporadic inclusion body myositis, immune-mediated necrotizing myopathies and dermatomyositis muscle biopsies and co-localized with the MHC class I expressing myofibers. Double immunofluorescence revealed that both myofibers and muscle infiltrating cells, including CD8+ T-cells and CD68 + macrophages in IIMs expressed β1i or β5i. In addition, we have also investigated the role of the immunoproteasome in myoblasts during in vitro inflammatory conditions. Using human primary myoblasts cultures we found that pro-inflammatory cytokines, TNF-α or IFN-γ upregulate β1i or β5i. Selective inhibition or depletion of β5i amplified the TNF-α or IFN-γ mediated expression of cytokines/chemokines (myokines) in myoblasts. Furthermore, we demonstrated that specific inhibitors of β1i or β5i reduced the cell surface expression of MHC class I in myoblasts induced by IFN-γ. Taken together, our data suggest that the immunoproteasome is involved in pathologic MHC class I expression and maintenance of myokine production in IIMs. Thus, induction of the immunoproteasome was identified as a pathomechanism underlying inflammation in IIMs.

Promoting differentiation of cultured myoblasts using biomimetic surfaces that present alpha-laminin-2 peptides.

Traditionally, muscle cell lines are cultured on glass coverslips and differentiated to investigate myoblast fusion and differentiation. Efficient differentiation of myoblasts produces a dense network of myotubes with the correct organisation for contraction. Here we have tested the ability of artificially generated, precisely controlled peptide surfaces to enhance the efficiency of myoblast differentiation. We focused on specific short peptides from α-laminin-2 (IKVSV, VQLRNGFPYFSY and GLLFYMARINHA) as well as residues 15–155 from FGF1. We tested if these peptides in isolation, and/or in combination promoted muscle differentiation in culture, by promoting fusion and/or by improving sarcomere organisation. The majority of these peptides promoted fusion and differentiation in two different mouse myogenic cell lines and in primary human myoblasts. The additive effects of all four peptides gave the best results for both mouse cell lines tested, while primary human cell cultures differentiated equally well on most peptide surfaces tested. These data show that a mixture of short biomimetic peptides can reliably promote differentiation in mouse and human myoblasts.

Selective release of muscle-specific, extracellular microRNAs during myogenic differentiation.

MyomiRs are muscle-specific microRNAs (miRNAs) that regulate myoblast proliferation and differentiation. Extracellular myomiRs (ex-myomiRs) are highly enriched in the serum of Duchenne Muscular Dystrophy (DMD) patients and dystrophic mouse models and consequently have potential as disease biomarkers. The biological significance of miRNAs present in the extracellular space is not currently well understood. Here we demonstrate that ex-myomiR levels are elevated in perinatal muscle development, during the regenerative phase that follows exercise-induced myoinjury, and concomitant with myoblast differentiation in culture. Whereas ex-myomiRs are progressively and specifically released by differentiating human primary myoblasts and C2C12 cultures, chemical induction of apoptosis in C2C12 cells results in indiscriminate miRNA release. The selective release of myomiRs as a consequence of cellular differentiation argues against the idea that they are solely waste products of muscle breakdown, and suggests they may serve a biological function in specific physiological contexts. Ex-myomiRs in culture supernatant and serum are predominantly non-vesicular, and their release is independent of ceramide-mediated vesicle secretion. Furthermore, ex-myomiRs levels are reduced in aged dystrophic mice, likely as a consequence of chronic muscle wasting. In conclusion, we show that myomiR release accompanies periods of myogenic differentiation in cell culture and in vivo. Serum myomiR abundance is therefore a function of the regenerative/degenerative status of the muscle, overall muscle mass, and tissue expression levels. These findings have implications for the use of ex-myomiRs as biomarkers for DMD disease progression and monitoring response to therapy.

104. A Gene Deleted High Capacity Adenoviral Vector for Efficient Delivery of a Multiplex DMD Specific CRISPR/Cas9 Machinery

Recent advances in the field of designer nuclease directed genome editing hold great promise to correct underlying mutations leading to Duchenne muscular dystrophy. Especially the CRISPR/Cas9 system provides an easy way to design and to assemble RNA guided nucleases offering the potential to develop personalized treatments to correct the multiple different mutations leading to this fatal disease. Recent studies showed efficient genome editing in a myoblast cell line derived from DMD patients and mdx mice. Nevertheless viral delivery of all required CRISPR/Cas9 components including Cas9 and one or multiple guide RNA (gRNA) expression units has not been fully exploited. Gene deleted high-capacity adenoviral vectors (HCAdVs) offer the packaging capacity to deliver the complete CRISPR/Cas9 machinery including several gRNA expression units using a single viral vector. By using a new toolbox that facilitates customization, cloning and production of CRISPR-HCAdVs, we assembled a HCAdV genome containing a Streptococcus pyogenes Cas9 (spCas9) gene including two guide RNA (gRNA) expression units specific for DMD that have shown efficiency to delete exon 51 in dystrophic human myoblasts. CRISPR-HCAdV was amplified in medium scale using a shortened protocol yielding high titers. Infection of cultured HEK293 cells and primary human myoblasts with purified DMD specific CRISPR-HCAdV at different MOIs resulted in strong locus specific deletion efficiency for DMD exon 51 as shown with locus specific PCR. As a comparison we also designed and produced DMD-specific TALEN encoding HCAdVs which allow delivery of a complete TALEN pair using a single vector. We found that it was more complicated to produce double TALEN-HCAdVs compared to multiplex CRSIPR/Cas9-HCAdV as they require controlled expression of TALEN genes by inducible promotors. Furthermore the TALEN system is not suitable for multiplexing and showed less efficiency in T7E1 assays. Our platform enables cloning and production of gene deleted adenoviral vectors for the delivery of a DMD specific CRISPR/Cas9 system within a short time providing a valuable tool for viral delivery of customized CRISPR/Cas9 for DMD treatment. Additional gRNAs or gRNAs with other specificities can be easily included in the vector allowing personalized molecular design of the gene transfer vector. We expect that this may pave the way towards broader applications of the CRISPR technology for DMD treatment including preclinical and eventually clinical studies.

Uncoupling of oxidative phosphorylation and antioxidants affect fusion of primary human myoblasts in vitro

Uncoupling of oxidative phosphorylation and antioxidants affect fusion of primary human myoblasts in vitro

Depleting extracellular vesicles from fetal bovine serum alters proliferation and differentiation of skeletal muscle cells in vitro.

BackgroundFetal bovine serum (FBS) contains a wide range of growth factors, hormones, vitamins, amino acids, fatty acids and trace elements required for cell growth. It was shown that animal sera contain also extracellular vesicles (EVs) with important biological properties; thus we wondered whether EVs present in FBS would influence muscle cell phenotype.EVs were removed from sera by ultracentrifugation (18 h). C2C12, L6 and human primary myoblasts, were grown either in classical media (CM) or in EVs-depleted media. Differentiation was induced by replacing the culture medium either with CM or EV-depleted media. qRT-PCR of relevant genes and miRNA involved in proliferation, differentiation, energy metabolism and EVs formation and secretion were performed.ResultsGrowth of myoblasts in EV-free media during proliferation produces the most unfavorable situation for proper myotube formation, when considering C212 and human myoblasts. Removing EVs from serum committed myoblasts to differentiate precociously (induction of myogenin and decreased expression of myomiR involved in myogenesis). C2C12 and human myoblasts, grown constantly in EV-depleted media during proliferation and differentiation, formed less myotubes than in CM. They had a reduced level of myogenin and a strong increase in myostatin expression, a negative regulator of muscle cell differentiation that affects myotube size. This situation was not reversed when confluent myoblasts were switched to CM for differentiation. Like C2C12 and human cells, L6 formed less myotubes in EVs-depleted media. However, as they do not express myostatin, L6 myotubes were larger and expressed higher level of CKTM2 compared to myotubes grown in CM suggesting that they had reached a higher level of differentiation.ConclusionsResearchers studying the role of muscle EVs in culture conditions should consider that depleting EVs from serum alters the phenotype of muscle cells. Interestingly, the cross-talk between myoblasts and myotubes during myogenesis (Forterre 2014, PLoS One. 2014 Jan 2;9(1):e84153) can be recapitulate by using FBS-EVs as well. This implies that EVs can transfer specific signals to cells from unrelated species and that part of serum EV composition is evolutionarily conserved (e.g.; myomiR are detected in FBS-EVs). EVs in body fluids could have an unsuspected function during embryogenesis and in regulation of cellular processes such as hypertrophy and hyperplasia.Electronic supplementary materialThe online version of this article (doi:10.1186/s12896-016-0262-0) contains supplementary material, which is available to authorized users.

Reliable and versatile immortal muscle cell models from healthy and myotonic dystrophy type 1 primary human myoblasts

Primary human skeletal muscle cells (hSkMCs) are invaluable tools for deciphering the basic molecular mechanisms of muscle-related biological processes and pathological alterations. Nevertheless, their use is quite restricted due to poor availability, short life span and variable purity of the cells during in vitro culture. Here, we evaluate a recently published method of hSkMCs immortalization, relying on ectopic expression of cyclin D1 (CCND1), cyclin-dependent kinase 4 (CDK4) and telomerase (TERT) in myoblasts from healthy donors (n=3) and myotonic dystrophy type 1 (DM1) patients (n=2). The efficacy to maintain the myogenic and non-transformed phenotype, as well as the main pathogenetic hallmarks of DM1, has been assessed. Combined expression of the three genes i) maintained the CD56(NCAM)-positive myoblast population and differentiation potential; ii) preserved the non-transformed phenotype and iii) maintained the CTG repeat length, amount of nuclear foci and aberrant alternative splicing in immortal muscle cells. Moreover, immortal hSkMCs displayed attractive additional features such as structural maturation of sarcomeres, persistence of Pax7-positive cells during differentiation and complete disappearance of nuclear foci following (CAG)7 antisense oligonucleotide (ASO) treatment. Overall, the CCND1, CDK4 and TERT immortalization yields versatile, reliable and extremely useful human muscle cell models to investigate the basic molecular features of human muscle cell biology, to elucidate the molecular pathogenetic mechanisms and to test new therapeutic approaches for DM1 in vitro.

Establishment of a Novel Primary Human Skeletal Myoblast Cellular Model for Chikungunya Virus Infection and Pathogenesis.

Chikungunya virus (CHIKV) is a re-emerging arbovirus known to cause chronic myalgia and arthralgia and is now considered endemic in countries across Asia and Africa. The tissue tropism of CHIKV infection in humans remains, however, ill-defined. Due to the fact that myositis is commonly observed in most patients infected with CHIKV, we sought to develop a clinically relevant cellular model to better understand the pathogenesis of CHIKV infection. In this study, primary human skeletal muscle myoblasts (HSMM) were established as a novel human primary cell line that is highly permissive to CHIKV infection, with maximal amounts of infectious virions observed at 16 hours post infection. Genome-wide microarray profiling analyses were subsequently performed to identify and map genes that are differentially expressed upon CHIKV infection. Infection of HSMM cells with CHIKV resulted in altered expressions of host genes involved in skeletal- and muscular-associated disorders, innate immune responses, cellular growth and death, host metabolism and virus replication. Together, this study has shown the establishment of a clinically relevant primary human cell model that paves the way for the further analysis of host factors and their involvement in the various stages of CHIKV replication cycle and viral pathogenesis.

The Tocotrienol-Rich Fraction Is Superior to Tocopherol in Promoting Myogenic Differentiation in the Prevention of Replicative Senescence of Myoblasts.

Aging results in a loss of muscle mass and strength. Myoblasts play an important role in maintaining muscle mass through regenerative processes, which are impaired during aging. Vitamin E potentially ameliorates age-related phenotypes. Hence, this study aimed to determine the effects of the tocotrienol-rich fraction (TRF) and α-tocopherol (ATF) in protecting myoblasts from replicative senescence and promoting myogenic differentiation. Primary human myoblasts were cultured into young and senescent stages and were then treated with TRF or ATF for 24 h, followed by an analysis of cell proliferation, senescence biomarkers, cellular morphology and differentiation. Our data showed that replicative senescence impaired the normal regenerative processes of myoblasts, resulting in changes in cellular morphology, cell proliferation, senescence-associated β-galactosidase (SA-β-gal) expression, myogenic differentiation and myogenic regulatory factors (MRFs) expression. Treatment with both TRF and ATF was beneficial to senescent myoblasts in reclaiming the morphology of young cells, improved cell viability and decreased SA-β-gal expression. However, only TRF treatment increased BrdU incorporation in senescent myoblasts, as well as promoted myogenic differentiation through the modulation of MRFs at the mRNA and protein levels. MYOD1 and MYOG gene expression and myogenin protein expression were modulated in the early phases of myogenic differentiation. In conclusion, the tocotrienol-rich fraction is superior to α-tocopherol in ameliorating replicative senescence-related aberration and promoting differentiation via modulation of MRFs expression, indicating vitamin E potential in modulating replicative senescence of myoblasts.

Gene amplification during myogenic differentiation.

Gene amplifications are mostly an attribute of tumor cells and drug resistant cells. Recently, we provided evidence for gene amplifications during differentiation of human and mouse neural progenitor cells. Here, we report gene amplifications in differentiating mouse myoblasts (C2C12 cells) covering a period of 7 days including pre-fusion, fusion and post-fusion stages. After differentiation induction we found an increase in copy numbers of CDK4 gene at day 3, of NUP133 at days 4 and 7, and of MYO18B at day 4. The amplification process was accompanied by gamma-H2AX foci that are indicative of double stand breaks. Amplifications during the differentiating process were also found in primary human myoblasts with the gene CDK4 and NUP133 amplified both in human and mouse myoblasts. Amplifications of NUP133 and CDK4 were also identified in vivo on mouse transversal cryosections at stage E11.5. In the course of myoblast differentiation, we found amplifications in cytoplasm indicative of removal of amplified sequences from the nucleus. The data provide further evidence that amplification is a fundamental mechanism contributing to the differentiation process in mammalians.

Proliferation of Human Primary Myoblasts Is Associated with Altered Energy Metabolism in Dependence on Ageing In Vivo and In Vitro.

Background. Ageing is associated with suppressed regenerative potential of muscle precursor cells due to decrease of satellite cells and suppressive intramuscular milieu on their activation, associated with ageing-related low-grade inflammation. The aim of the study was to characterize the function of oxidative phosphorylation (OXPHOS), glycolysis, adenylate kinase (AK), and creatine kinase (CK) mediated systems in young and older individuals. Materials and Methods. Myoblasts were cultivated from biopsies taken by transcutaneous conchotomy from vastus lateralis muscle in young (20–29 yrs, n = 7) and older (70–79 yrs, n = 7) subjects. Energy metabolism was assessed in passages 2 to 6 by oxygraphy and enzyme analysis. Results. In myoblasts of young and older subjects the rate of OXPHOS decreased during proliferation from passages 2 to 6. The total activities of CK and AK decreased. Myoblasts of passage 2 cultivated from young muscle showed higher rate of OXPHOS and activities of CK and AK compared to myoblasts from older subjects while hexokinase and pyruvate kinase were not affected by ageing. Conclusions. Proliferation of myoblasts in vitro is associated with downregulation of OXPHOS and energy storage and transfer systems. Ageing in vivo exerts an impact on satellite cells which results in altered metabolic profile in favour of the prevalence of glycolytic pathways over mitochondrial OXPHOS of myoblasts.

Musclin is an activity-stimulated myokine that enhances physical endurance

Exercise remains the most effective way to promote physical and metabolic wellbeing, but molecular mechanisms underlying exercise tolerance and its plasticity are only partially understood. In this study we identify musclin-a peptide with high homology to natriuretic peptides (NP)-as an exercise-responsive myokine that acts to enhance exercise capacity in mice. We use human primary myoblast culture and in vivo murine models to establish that the activity-related production of musclin is driven by Ca(2+)-dependent activation of Akt1 and the release of musclin-encoding gene (Ostn) transcription from forkhead box O1 transcription factor inhibition. Disruption of Ostn and elimination of musclin secretion in mice results in reduced exercise tolerance that can be rescued by treatment with recombinant musclin. Reduced exercise capacity in mice with disrupted musclin signaling is associated with a trend toward lower levels of plasma atrial NP (ANP) and significantly smaller levels of cyclic guanosine monophosphate (cGMP) and peroxisome proliferator-activated receptor gamma coactivator 1-α in skeletal muscles after exposure to exercise. Furthermore, in agreement with the established musclin ability to interact with NP clearance receptors, but not with NP guanyl cyclase-coupled signaling receptors, we demonstrate that musclin enhances cGMP production in cultured myoblasts only when applied together with ANP. Elimination of the activity-related musclin-dependent boost of ANP/cGMP signaling results in significantly lower maximum aerobic capacity, mitochondrial protein content, respiratory complex protein expression, and succinate dehydrogenase activity in skeletal muscles. Together, these data indicate that musclin enhances physical endurance by promoting mitochondrial biogenesis.

The NKG2D-IL-15 signaling pathway contributes to T-cell mediated pathology in inflammatory myopathies.

NKG2D is an activating receptor on T cells, which has been implicated in the pathogenesis of autoimmune diseases. T cells are critically involved in idiopathic inflammatory myopathies (IIM) and have been proposed as specific therapeutic targets. However, the mechanisms underlying T cell-mediated progressive muscle destruction in IIM remain to be elucidated. We here determined the involvement of the NKG2D - IL-15 signaling pathway. Primary human myoblasts expressed NKG2D ligands, which were further upregulated upon inflammatory stimuli. In parallel, shedding of the soluble NKG2D ligand MICA (sMICA) decreased upon inflammation potentially diminishing inhibition of NKG2D signaling. Membrane-related expression of IL-15 by myoblasts induced differentiation of naïve CD8+ T cells into highly activated, cytotoxic CD8+NKG2Dhigh T cells demonstrating NKG2D-dependent lysis of myoblasts in vitro. CD8+NKG2Dhigh T cell frequencies were increased in the peripheral blood of polymyositis (PM) patients and correlated with serum creatinine kinase concentrations, while serum sMICA levels were not significantly changed. In muscle biopsy specimens from PM patients expression of the NKG2D ligand MICA/B was upregulated, IL-15 was expressed by muscle cells, CD68+ macrophages as well as CD4+ T cells, and CD8+NKG2D+ cells were frequently detected within inflammatory infiltrates arguing for a local signaling circuit in the inflammatory muscle milieu. In conclusion, the NKG2D - IL-15 signaling pathway contributes to progressive muscle destruction in IIM potentially opening new therapeutic avenues.

CX3CL1--a macrophage chemoattractant induced by a single bout of exercise in human skeletal muscle.

Monocytes/macrophages (MOs/MΦs) are suggested to be crucial for skeletal muscle repair and remodeling. This has been attributed to their proangiogenic potential, secretion of growth factors, and clearance of tissue debris. Skeletal muscle injury increases the number of MΦs in the tissue, and their importance for muscle regeneration has been supported by studies demonstrating that depletion of MOs/MΦs greatly impairs repair after muscle injury. Whether noninjurious exercise leads to induced expression of chemoattractants for MOs/MΦs is poorly investigated. To this end, we analyzed the expression of CX3CL1 (fractalkine), CCL2 (MCP-1), and CCL22 (MDC) in human skeletal muscle after a bout of exercise, all of which are established MO/MΦ chemotactic factors that are expressed by human myoblasts. Muscle biopsies from the musculus vastus lateralis were obtained up to 24 h after 1 h of cycle exercise in healthy individuals and in age-matched nonexercised controls. CX3CL1 increased at both the mRNA and protein level in human skeletal muscle after one bout of exercise. It was not possible to distinguish changes in CCL2 or CCL22 mRNA levels between biopsy vs. exercise effects, and the expression of CCL22 was very low. CX3CL1 mainly localized to the skeletal muscle endothelium, and it increased in human umbilical vein endothelial cells stimulated with tissue fluid from exercised muscle. CX3CL1 increased the expression of proinflammatory and proangiogenic factors in THP-1 monocytes (a human acute monocytic leukemia cell line) and in human primary myoblasts and myotubes. Altogether, this suggests that CX3CL1 participates in cross-talk mechanisms between endothelium and other muscle tissue cells and may promote a shift in the microenvironment toward a more regenerative milieu.

FHL1 protein isoforms in Emery-Dreifuss muscular dystrophy

Emery-Dreifuss muscular dystrophy (EDMD) is a hereditary muscular disorder characterized by early joint contractures, progressive muscular wasting and weakness of scapuloperoneal distribution, and at adult age, patients develop cardiac abnormalities with a high risk of sudden death[1]. EDMD encompasses both X-linked and autosomal inheritance due to mutations in the genes encoding the nuclear envelope proteins emerin, lamin A/C[2-4]. First mutations in the Four-and-a-half LIM domain 1 gene (FHL1) being responsible for X-linked EDMD were described by Gueneau et al.[5]. The human FHL1 gene encodes three alternatively spliced isoforms, named FHL1A, FHL1B and FHL1C, with FHL1A being the most abundantly expressed protein isoform in striated muscle. There is still little known about the precise localization and functions of the three different FHL1 isoforms in human skeletal muscle. Here, we describe for the first time the subcellular localization of FHL1A, FHL1B, and FHL1C in vitro in differentiating human primary myoblasts. Localization of FHL1 protein isoforms was studied at the myoblast and myotube stages by confocal microscope analysis. Endogenous FHL1B protein localization was detected by an anti-FHL1B specific antibody, while for FHL1A and FHL1C, as no efficient isoform-specific antibodies were available, an anti-Flag antibody was used to follow Flag-tagged FHL1A and Flag-tagged FHL1C protein expression, after lentiviral transduction of human primary myoblasts. Successful transduction was confirmed by western blotting of whole extracts from myoblasts and myotubes using an anti-Flag antibody. In human myoblasts, Flag-FHL1A and Flag-FHL1C showed both a cytoplasmic and a nuclear distribution, while the nuclear staining was more pronounced in Flag-FHL1C transduced myoblasts. Endogenous FHL1B protein gave a moderate cytoplasmic and a strong nuclear staining. During 6- and 12-days of human myoblast differentiation, localization of all three FHL1 protein isoforms shifted from the nucleus to the cytoplasm. In addition, all FHL1 protein isoforms were observed to co-localize with phalloidin-stained actin fibers. Collectively, these results indicate differentiation-related changes in expression and subcellular localization of the human FHL1 protein isoforms.

SiRNA delivery into cultured primary human myoblasts – optimization of electroporation parameters and theoretical analysis

Introduction of genetic material into muscle tissue has been extensively researched, including isolation and in vitro expansion of primary myoblasts as a potential source of cells for skeletal and heart muscle tissue engineering applications. In this study, we optimized the electroporation protocol for introduction of short interfering ribonucleic acid (siRNA) against messenger RNA for Hypoxia Inducible Factor 1α (HIF-1α) into cultured primary human myoblasts. We established optimal pulsing protocol for siRNA electro transfection, and theoretically analyzed the effect of electric field and pulse duration on silencing efficiency and electrophoretic displacement of siRNA. Silencing of HIF-1α was determined with quantitative polymerase chain reaction and Western Blot. The most efficient silencing (71% knockdown) was achieved with 8 × 2 ms pulses, E = 0.6 kV/cm. Viability was determined immediately, 1 h and 48 h after electroporation. In general, there was a trade-off between efficient silencing and preserved viability. Electric field and pulse duration are crucial parameters for silencing, since both increase membrane permeabilization and electrophoretic transfer of siRNA. Short-term viability showed immediate toxicity of pulses due to membrane damage, while indirect effects on cell proliferation were observed after 48 h. Presented results are important for faster optimization of electroporation parameters for ex vivo electrotransfer of short RNA molecules into primary human myoblasts.