Tuberculosis (TB), caused by infection with Mycobacterium tuberculosis, is a major cause of morbidity and mortality worldwide. Efforts to control it are hampered by difficulties with diagnosis, ...prevention and treatment. Most people infected with M. tuberculosis remain asymptomatic, termed latent TB, with a 10% lifetime risk of developing active TB disease. Current tests, however, cannot identify which individuals will develop disease. The immune response to M. tuberculosis is complex and incompletely characterized, hindering development of new diagnostics, therapies and vaccines. Here we identify a whole-blood 393 transcript signature for active TB in intermediate and high-burden settings, correlating with radiological extent of disease and reverting to that of healthy controls after treatment. A subset of patients with latent TB had signatures similar to those in patients with active TB. We also identify a specific 86-transcript signature that discriminates active TB from other inflammatory and infectious diseases. Modular and pathway analysis revealed that the TB signature was dominated by a neutrophil-driven interferon (IFN)-inducible gene profile, consisting of both IFN- and type I IFN- signalling. Comparison with transcriptional signatures in purified cells and flow cytometric analysis suggest that this TB signature reflects changes in cellular composition and altered gene expression. Although an IFN-inducible signature was also observed in whole blood of patients with systemic lupus erythematosus (SLE), their complete modular signature differed from TB, with increased abundance of plasma cell transcripts. Our studies demonstrate a hitherto underappreciated role of type I IFN- signalling in the pathogenesis of TB, which has implications for vaccine and therapeutic development. Our study also provides a broad range of transcriptional biomarkers with potential as diagnostic and prognostic tools to combat the TB epidemic.
Dysferlin is an integral membrane protein of the transverse tubules of skeletal muscle that is mutated or absent in limb girdle muscular dystrophy 2B and Miyoshi myopathy. Here we examine the role of ...dysferlin's seven C2 domains, C2A through C2G, in membrane repair and Ca2+ release, as well as in targeting dysferlin to the transverse tubules of skeletal muscle. We report that deletion of either domain C2A or C2B inhibits membrane repair completely, whereas deletion of C2C, C2D, C2E, C2F or C2G causes partial loss of membrane repair that is exacerbated in the absence of extracellular Ca2+. Deletion of C2C, C2D, C2E, C2F or C2G also causes significant changes in Ca2+ release, measured as the amplitude of the Ca2+ transient before or after hypo‐osmotic shock and the appearance of Ca2+ waves. Most deletants accumulate in endoplasmic reticulum. Only the C2A domain can be deleted without affecting dysferlin trafficking to transverse tubules, but Dysf‐ΔC2A fails to support normal Ca2+ signalling after hypo‐osmotic shock. Our data suggest that (i) every C2 domain contributes to repair; (ii) all C2 domains except C2B regulate Ca2+ signalling; (iii) transverse tubule localization is insufficient for normal Ca2+ signalling; and (iv) Ca2+ dependence of repair is mediated by C2C through C2G. Thus, dysferlin's C2 domains have distinct functions in Ca2+ signalling and sarcolemmal membrane repair and may play distinct roles in skeletal muscle.
Key points
Dysferlin, a transmembrane protein containing seven C2 domains, C2A through C2G, concentrates in transverse tubules of skeletal muscle, where it stabilizes voltage‐induced Ca2+ transients and participates in sarcolemmal membrane repair.
Each of dysferlin's C2 domains except C2B regulate Ca2+ signalling.
Localization of dysferlin variants to the transverse tubules is not sufficient to support normal Ca2+ signalling or membrane repair.
Each of dysferlin's C2 domains contributes to sarcolemmal membrane repair.
The Ca2+ dependence of membrane repair is mediated by C2C through C2G.
Dysferlin's C2 domains therefore have distinct functions in Ca2+ signalling and sarcolemmal membrane repair.
figure legend The C2 domains of dysferlin play different roles in 5 different activities of the protein: targeting to the transverse tubules, supporting membrane repair, supporting the amplitude of the Ca2+ transient, supporting the amplitude of the Ca2+ transient after injury by hypoosmotic shock (OSI), and suppressing Ca2+ waves after OSI. The relative contributions of each domain to these activities are indicated here. Solid lines: notable contribution; dashed lines, moderate contribution; dotted lines, minimal contribution. The absence of a line indicates that no contribution was detected.
Mutations in the dysferlin gene are the cause of Limb-girdle Muscular Dystrophy type 2B and Miyoshi Myopathy. The dysferlin protein has been implicated in sarcolemmal resealing, leading to the idea ...that the pathophysiology of dysferlin deficiencies is due to a deficit in membrane repair. Here, we show using two different approaches that fulfilling membrane repair as asseyed by laser wounding assay is not sufficient for alleviating the dysferlin deficient pathology. First, we generated a transgenic mouse overexpressing myoferlin to test the hypothesis that myoferlin, which is homologous to dysferlin, can compensate for the absence of dysferlin. The myoferlin overexpressors show no skeletal muscle abnormalities, and crossing them with a dysferlin-deficient model rescues the membrane fusion defect present in dysferlin-deficient mice in vitro. However, myoferlin overexpression does not correct muscle histology in vivo. Second, we report that AAV-mediated transfer of a minidysferlin, previously shown to correct the membrane repair deficit in vitro, also fails to improve muscle histology. Furthermore, neither myoferlin nor the minidysferlin prevented myofiber degeneration following eccentric exercise. Our data suggest that the pathogenicity of dysferlin deficiency is not solely related to impairment in sarcolemmal repair and highlight the care needed in selecting assays to assess potential therapies for dysferlinopathies.
Dysferlin-null A/J myofibers generate abnormal Ca
2+
transients that are slightly reduced in amplitude compared to controls. These are further reduced in amplitude by hypoosmotic shock and often ...appear as Ca
2+
waves (Lukyanenko et al., J. Physiol., 2017). Ca
2+
waves are typically associated with Ca
2+
-induced Ca
2+
release, or CICR, which can be myopathic. We tested the ability of a permeable Ca
2+
chelator, BAPTA-AM, to inhibit CICR in injured dysferlin-null fibers and found that 10–50 nM BAPTA-AM suppressed all Ca
2+
waves. The same concentrations of BAPTA-AM increased the amplitude of the Ca
2+
transient in A/J fibers to wild type levels and protected transients against the loss of amplitude after hypoosmotic shock, as also seen in wild type fibers. Incubation with 10 nM BAPTA-AM led to intracellular BAPTA concentrations of ∼60 nM, as estimated with its fluorescent analog, Fluo-4AM. This should be sufficient to restore intracellular Ca
2+
to levels seen in wild type muscle. Fluo-4AM was ∼10-fold less effective than BAPTA-AM, however, consistent with its lower affinity for Ca
2+
. EGTA, which has an affinity for Ca
2+
similar to BAPTA, but with much slower kinetics of binding, was even less potent when introduced as the -AM derivative. By contrast, a dysferlin variant with GCaMP6f
u
in place of its C2A domain accumulated at triad junctions, like wild type dysferlin, and suppressed all abnormal Ca
2+
signaling. GCaMP6f
u
introduced as a Venus chimera did not accumulate at junctions and failed to suppress abnormal Ca
2+
signaling. Our results suggest that leak of Ca
2+
into the triad junctional cleft underlies dysregulation of Ca
2+
signaling in dysferlin-null myofibers, and that dysferlin’s C2A domain suppresses abnormal Ca
2+
signaling and protects muscle against injury by binding Ca
2+
in the cleft.
Departments of Biochemistry and Molecular Biology and of Physiology, University of Maryland School of Medicine, Baltimore, Maryland
Myofibrillogenesis in striated muscles is a highly complex process ...that depends on the coordinated assembly and integration of a large number of contractile, cytoskeletal, and signaling proteins into regular arrays, the sarcomeres. It is also associated with the stereotypical assembly of the sarcoplasmic reticulum and the transverse tubules around each sarcomere. Three giant, muscle-specific proteins, titin (3–4 MDa), nebulin (600–800 kDa), and obscurin ( 720–900 kDa), have been proposed to play important roles in the assembly and stabilization of sarcomeres. There is a large amount of data showing that each of these molecules interacts with several to many different protein ligands, regulating their activity and localizing them to particular sites within or surrounding sarcomeres. Consistent with this, mutations in each of these proteins have been linked to skeletal and cardiac myopathies or to muscular dystrophies. The evidence that any of them plays a role as a "molecular template," "molecular blueprint," or "molecular ruler" is less definitive, however. Here we review the structure and function of titin, nebulin, and obscurin, with the literature supporting a role for them as scaffolding molecules and the contradictory evidence regarding their roles as molecular guides in sarcomerogenesis.
Intermediate filaments (IFs) play a key role in the integration of structure and function of striated muscle, primarily by mediating mechanochemical links between the contractile apparatus and ...mitochondria, myonuclei, the sarcolemma and potentially the vesicle trafficking apparatus. Linkage of all these membranous structures to the contractile apparatus, mainly through the Z-disks, supports the integration and coordination of growth and energy demands of the working myocyte, not only with force transmission, but also with de novo gene expression, energy production and efficient protein and lipid trafficking and targeting. Desmin, the most abundant and intensively studied muscle intermediate filament protein, is linked to proper costamere organization, myoblast and stem cell fusion and differentiation, nuclear shape and positioning, as well as mitochondrial shape, structure, positioning and function. Similar links have been established for lysosomes and lysosome-related organelles, consistent with the presence of widespread links between IFs and membranous structures and the regulation of their fusion, morphology and stabilization necessary for cell survival.
µ-Crystallin is a NADPH-regulated thyroid hormone binding protein encoded by the
gene in humans. It is primarily expressed in the brain, muscle, prostate, and kidney, where it binds thyroid hormones, ...which regulate metabolism and thermogenesis. It also acts as a ketimine reductase in the lysine degradation pathway when it is not bound to thyroid hormone. Mutations in
can result in non-syndromic deafness, while its aberrant expression, predominantly in the brain but also in other tissues, has been associated with psychiatric, neuromuscular, and inflammatory diseases. CRYM expression is highly variable in human skeletal muscle, with 15% of individuals expressing ≥13 fold more
mRNA than the median level. Ablation of the
gene in murine models results in the hypertrophy of fast twitch muscle fibers and an increase in fat mass of mice fed a high fat diet. Overexpression of
in mice causes a shift in energy utilization away from glycolysis towards an increase in the catabolism of fat via β-oxidation, with commensurate changes of metabolically involved transcripts and proteins. The history, attributes, functions, and diseases associated with
, an important modulator of metabolism, are reviewed.
Xenografts of human skeletal muscle generated in mice can be used to study muscle pathology and to test drugs designed to treat myopathies and muscular dystrophies for their efficacy and specificity ...in human tissue. We previously developed methods to generate mature human skeletal muscles in immunocompromised mice starting with human myogenic precursor cells (hMPCs) from healthy individuals and individuals with facioscapulohumeral muscular dystrophy (FSHD). Here, we examine a series of alternative treatments at each stage in order to optimize engraftment. We show that (i) X-irradiation at 25Gy is optimal in preventing regeneration of murine muscle while supporting robust engraftment and the formation of human fibers without significant murine contamination; (ii) hMPC lines differ in their capacity to engraft; (iii) some hMPC lines yield grafts that respond better to intermittent neuromuscular electrical stimulation (iNMES) than others; (iv) some lines engraft better in male than in female mice; (v) coinjection of hMPCs with laminin, gelatin, Matrigel, or Growdex does not improve engraftment; (vi) BaCl
is an acceptable replacement for cardiotoxin, but other snake venom preparations and toxins, including the major component of cardiotoxin, cytotoxin 5, are not; and (vii) generating grafts in both hindlimbs followed by iNMES of each limb yields more robust grafts than housing mice in cages with running wheels. Our results suggest that replacing cardiotoxin with BaCl
and engrafting both tibialis anterior muscles generates robust grafts of adult human muscle tissue in mice.
Key points
Dysferlin, the protein missing in limb girdle muscular dystrophy 2B and Miyoshi myopathy, concentrates in transverse tubules of skeletal muscle, where it stabilizes voltage‐induced Ca2+ ...transients against loss after osmotic shock injury (OSI).
Local expression of dysferlin in dysferlin‐null myofibres increases transient amplitude to control levels and protects them from loss after OSI.
Inhibitors of ryanodine receptors (RyR1) and L‐type Ca2+ channels protect voltage‐induced Ca2+ transients from loss; thus both proteins play a role in injury in dysferlin's absence. Effects of Ca2+‐free medium and S107, which inhibits SR Ca2+ leak, suggest the SR as the primary source of Ca2+ responsible for the loss of the Ca2+ transient upon injury.
Ca2+ waves were induced by OSI and suppressed by exogenous dysferlin.
We conclude that dysferlin prevents injury‐induced SR Ca2+ leak.
Dysferlin concentrates in the transverse tubules of skeletal muscle and stabilizes Ca2+ transients when muscle fibres are subjected to osmotic shock injury (OSI). We show here that voltage‐induced Ca2+ transients elicited in dysferlin‐null A/J myofibres were smaller than control A/WySnJ fibres. Regional expression of Venus‐dysferlin chimeras in A/J fibres restored the full amplitude of the Ca2+ transients and protected against OSI. We also show that drugs that target ryanodine receptors (RyR1: dantrolene, tetracaine, S107) and L‐type Ca2+ channels (LTCCs: nifedipine, verapamil, diltiazem) prevented the decrease in Ca2+ transients in A/J fibres following OSI. Diltiazem specifically increased transients by ∼20% in uninjured A/J fibres, restoring them to control values. The fact that both RyR1s and LTCCs were involved in OSI‐induced damage suggests that damage is mediated by increased Ca2+ leak from the sarcoplasmic reticulum (SR) through the RyR1. Congruent with this, injured A/J fibres produced Ca2+ sparks and Ca2+ waves. S107 (a stabilizer of RyR1–FK506 binding protein coupling that reduces Ca2+ leak) or local expression of Venus‐dysferlin prevented OSI‐induced Ca2+ waves. Our data suggest that dysferlin modulates SR Ca2+ release in skeletal muscle, and that in its absence OSI causes increased RyR1‐mediated Ca2+ leak from the SR into the cytoplasm.
Key points
Dysferlin, the protein missing in limb girdle muscular dystrophy 2B and Miyoshi myopathy, concentrates in transverse tubules of skeletal muscle, where it stabilizes voltage‐induced Ca2+ transients against loss after osmotic shock injury (OSI).
Local expression of dysferlin in dysferlin‐null myofibres increases transient amplitude to control levels and protects them from loss after OSI.
Inhibitors of ryanodine receptors (RyR1) and L‐type Ca2+ channels protect voltage‐induced Ca2+ transients from loss; thus both proteins play a role in injury in dysferlin's absence. Effects of Ca2+‐free medium and S107, which inhibits SR Ca2+ leak, suggest the SR as the primary source of Ca2+ responsible for the loss of the Ca2+ transient upon injury.
Ca2+ waves were induced by OSI and suppressed by exogenous dysferlin.
We conclude that dysferlin prevents injury‐induced SR Ca2+ leak.
Dysferlin is an integral membrane protein of the transverse tubules of skeletal muscle that is mutated or absent in limb girdle muscular dystrophy 2B and Miyoshi myopathy. Here we examine the role of ...dysferlin's seven C2 domains, C2A through C2G, in membrane repair and Ca
release, as well as in targeting dysferlin to the transverse tubules of skeletal muscle. We report that deletion of either domain C2A or C2B inhibits membrane repair completely, whereas deletion of C2C, C2D, C2E, C2F or C2G causes partial loss of membrane repair that is exacerbated in the absence of extracellular Ca
. Deletion of C2C, C2D, C2E, C2F or C2G also causes significant changes in Ca
release, measured as the amplitude of the Ca
transient before or after hypo-osmotic shock and the appearance of Ca
waves. Most deletants accumulate in endoplasmic reticulum. Only the C2A domain can be deleted without affecting dysferlin trafficking to transverse tubules, but Dysf-ΔC2A fails to support normal Ca
signalling after hypo-osmotic shock. Our data suggest that (i) every C2 domain contributes to repair; (ii) all C2 domains except C2B regulate Ca
signalling; (iii) transverse tubule localization is insufficient for normal Ca
signalling; and (iv) Ca
dependence of repair is mediated by C2C through C2G. Thus, dysferlin's C2 domains have distinct functions in Ca
signalling and sarcolemmal membrane repair and may play distinct roles in skeletal muscle. KEY POINTS: Dysferlin, a transmembrane protein containing seven C2 domains, C2A through C2G, concentrates in transverse tubules of skeletal muscle, where it stabilizes voltage-induced Ca
transients and participates in sarcolemmal membrane repair. Each of dysferlin's C2 domains except C2B regulate Ca
signalling. Localization of dysferlin variants to the transverse tubules is not sufficient to support normal Ca
signalling or membrane repair. Each of dysferlin's C2 domains contributes to sarcolemmal membrane repair. The Ca
dependence of membrane repair is mediated by C2C through C2G. Dysferlin's C2 domains therefore have distinct functions in Ca
signalling and sarcolemmal membrane repair.
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