Comparative Studies of Hind Limb and Diaphragm Muscles of mdx Mice

Histological, immunocytochemical and immunochemical characteristics of diaphragm muscle from mdx and control mice (14-days to 12-months of age) were compared to those of their hind limb muscles. In contrast to mdx hind limb muscles regeneration after necrosis, which was evident after one month of age, does not restore the diaphragm muscle structure. In contrast also to mdx hind limb muscles in the diaphragm there was a significant extracellular matrix proliferation, which was manifested starting from 6 months of age of the animals by increased intensity of fibronectin and collagen IV immunostaining. In immunocytochemical studies with anti-C terminus.and anti-N terminal domain antibodies no staining of dystrophin was found in the mdx diaphragm, in hind limb mdx muscles rare positively stained fibres were observed starting from 3 months of age of the animals. The Western blotting technique in normal mice diaphragm and hind limb muscles revealed dystrophin bands of equal intensity with anti-mid rod and anti-C terminus antibodies. In mdx samples the staining with anti-C terminus antibodies was evidently weaker. The anti-mid rod antibodies detected dystrophin in the mdx diaphragm between the 3

Cardiovascular magnetic resonance findings in a case of Danon disease

Danon disease is a rare X-linked dominant lysosomal glycogen storage disease that can lead to severe ventricular hypertrophy and heart failure. We report a case of Danon disease with cardiac involvement evaluated with cardiovascular magnetic resonance, including late gadolinium enhancement and perfusion studies

GFP-LC3 labels organised smooth endoplasmic reticulum membranes independently of autophagy

Disruption of autophagy leads to accumulation of intracellular multilamellar inclusions morphologically similar to organised smooth endoplasmic reticulum (OSER) membranes. However, the relation of these membranous compartments to autophagy is unknown. The purpose of this study was to test whether OSER plays a role in the autophagic protein degradation pathway. Here, GFP-LC3 is shown to localise to the OSER membranes induced by calnexin expression both in transiently transfected HEK293 cells and in mouse embryo fibroblasts. In contrast to GFP-LC3, endogenous LC3 is excluded from these membranes under normal conditions as well as after cell starvation. Furthermore, YFP-Atg5, a protein essential for autophagy and known to reside on autophagic membranes, is excluded from the calnexin-positive inclusion structures. In cells devoid of Atg5, a protein essential for autophagy and known to reside on autophagic membranes, colocalisation of calnexin with GFP-LC3 within the multilamellar bodies is preserved. I show that calnexin, a protein enriched in the OSER, is not subject to autophagic or lysosomal degradation. Finally, GFP-LC3 targeting to these membranes is independent of its processing and insensitive to drugs modulating autophagic and lysosomal protein degradation. These observations are inconsistent with a role of autophagic/lysosomal degradation in clearance of multilamellar bodies comprising OSER. Furthermore, GFP-LC3, a fusion protein widely used as a marker for autophagic vesicles and pre-autophagic compartments, may be trapped in this compartment and this artefact must be taken into account if the construct is used to visualise autophagic membranes. J. Cell. Biochem. 107: 86–95, 2009. © 2009 Wiley-Liss, Inc

Emerin Caps the Pointed End of Actin Filaments: Evidence for an Actin Cortical Network at the Nuclear Inner Membrane

X-linked Emery-Dreifuss muscular dystrophy is caused by loss of emerin, a LEM-domain protein of the nuclear inner membrane. To better understand emerin function, we used affinity chromatography to purify emerin-binding proteins from nuclear extracts of HeLa cells. Complexes that included actin, αII-spectrin and additional proteins, bound specifically to emerin. Actin polymerization assays in the presence or absence of gelsolin or capping protein showed that emerin binds and stabilizes the pointed end of actin filaments, increasing the actin polymerization rate 4- to 12-fold. We propose that emerin contributes to the formation of an actin-based cortical network at the nuclear inner membrane, conceptually analogous to the actin cortical network at the plasma membrane. Thus, in addition to disrupting transcription factors that bind emerin, loss of emerin may destabilize nuclear envelope architecture by weakening a nuclear actin network

Further Characterisation of the Molecular Signature of Quiescent and Activated Mouse Muscle Satellite Cells

Satellite cells are the resident stem cells of adult skeletal muscle. To date though, there is a paucity of native markers that can be used to easily identify quiescent satellite cells, with Pax7 probably being the best that is currently available. Here we have further characterized a number of recently described satellite cell markers, and also describe novel ones. Caveolin-1, integrin α7 and the calcitonin receptor proved reliable markers for quiescent satellite cells, being expressed by all satellite cells identified with Pax7. These three markers remained expressed as satellite cells were activated and underwent proliferation. The nuclear envelope proteins lamin A/C and emerin, mutations in which underlie Emery-Dreifuss muscular dystrophy, were also expressed in both quiescent and proliferating satellite cells. Conversely, Jagged-1, a Notch ligand, was not expressed in quiescent satellite cells but was induced upon activation. These findings further contribute to defining the molecular signature of muscle satellite cells

Signaling through the TRAIL receptor DR5/FADD pathway plays a role in the apoptosis associated with skeletal myoblast differentiation

Apoptosis rather than differentiation is a physiological process during myogenesis and muscle regeneration. When cultured myoblasts were induced to differentiate, we detected an increase in caspase 8 activity. Pharmacological inhibition of caspase 8 activity decreased apoptosis. Expression of a dominant-negative mutant of the adapter protein FADD also abrogated apoptosis, implicating a death ligand pathway. Treatment with TRAIL, but not Fas, induced apoptosis in these myoblasts. Accordingly, treatment with a soluble TRAIL decoy receptor or expression of a dominant-negative mutant of the TRAIL receptor DR5 abrogated apoptosis. While TRAIL expression levels remained unaltered in apoptotic myoblasts, DR5 expression levels increased. Finally, we also detected a reduction in FLIP, a death-receptor effector protein and caspase 8 competitive inhibitor, to undetectable levels in apoptotic myoblasts. Thus, our data demonstrate an important role for the TRAIL/DR5/FADD/caspase 8 pathway in the apoptosis associated with skeletal myoblast differentiation. Identifying the functional apoptotic pathways in skeletal myoblasts may prove useful in minimizing the myoblast apoptosis that contributes pathologically to a variety of diseases and in minimizing the apoptosis of transplanted myoblasts to treat these and other disease states

Uncoordinated Transcription and Compromised Muscle Function in the Lmna-Null Mouse Model of Emery-Dreifuss Muscular Dystrophy

LMNA encodes both lamin A and C: major components of the nuclear lamina. Mutations in LMNA underlie a range of tissue-specific degenerative diseases, including those that affect skeletal muscle, such as autosomal-Emery-Dreifuss muscular dystrophy (A-EDMD) and limb girdle muscular dystrophy 1B. Here, we examine the morphology and transcriptional activity of myonuclei, the structure of the myotendinous junction and the muscle contraction dynamics in the lmna-null mouse model of A-EDMD. We found that there were fewer myonuclei in lmna-null mice, of which ∼50% had morphological abnormalities. Assaying transcriptional activity by examining acetylated histone H3 and PABPN1 levels indicated that there was a lack of coordinated transcription between myonuclei lacking lamin A/C. Myonuclei with abnormal morphology and transcriptional activity were distributed along the length of the myofibre, but accumulated at the myotendinous junction. Indeed, in addition to the presence of abnormal myonuclei, the structure of the myotendinous junction was perturbed, with disorganised sarcomeres and reduced interdigitation with the tendon, together with lipid and collagen deposition. Functionally, muscle contraction became severely affected within weeks of birth, with specific force generation dropping as low as ∼65% and ∼27% of control values in the extensor digitorum longus and soleus muscles respectively. These observations illustrate the importance of lamin A/C for correct myonuclear function, which likely acts synergistically with myotendinous junction disorganisation in the development of A-EDMD, and the consequential reduction in force generation and muscle wasting

Immunohistochemistry on a Panel of Emery-Dreifuss Muscular Dystrophy Samples Reveals Nuclear Envelope Proteins as Inconsistent Markers for Pathology

Reports of aberrant distribution for some nuclear envelope proteins in cells expressing a few Emery–Dreifuss muscular dystrophy mutations raised the possibility that such protein redistribution could underlie pathology and/or be diagnostic. However, this disorder is linked to 8 different genes encoding nuclear envelope proteins, raising the question of whether a particular protein is most relevant. Therefore, myoblast/fibroblast cultures from biopsy and tissue sections from a panel of nine Emery–Dreifuss muscular dystrophy patients (4 male, 5 female) including those carrying emerin and FHL1 (X-linked) and several lamin A (autosomal dominant) mutations were stained for the proteins linked to the disorder. As tissue-specific nuclear envelope proteins have been postulated to mediate the tissue-specific pathologies of different nuclear envelopathies, patient samples were also stained for several muscle-specific nuclear membrane proteins. Although linked proteins nesprin 1 and SUN2 and muscle-specific proteins NET5/Samp1 and Tmem214 yielded aberrant distributions in individual patient cells, none exhibited defects through the larger patient panel. Muscle-specific Tmem38A normally appeared in both the nuclear envelope and sarcoplasmic reticulum, but most patient samples exhibited a moderate redistribution favouring the sarcoplasmic reticulum. The absence of striking uniform defects in nuclear envelope protein distribution indicates that such staining will be unavailing for general diagnostics, though it remains possible that specific mutations exhibiting protein distribution defects might reflect a particular clinical variant. These findings further argue that multiple pathways can lead to the generally similar pathologies of this disorder while at the same time the different cellular phenotypes observed possibly may help explain the considerable clinical variation of EDMD