Myotonic dystrophy protein kinase is critical for nuclear envelope integrity

Myotonic dystrophy 1 (DM1) is a multisystemic disease caused by a triplet nucleotide repeat expansion in the 3' untranslated region of the gene coding for myotonic dystrophy protein kinase (DMPK). DMPK is a nuclear envelope (NE) protein that promotes myogenic gene expression in skeletal myoblasts. Muscular dystrophy research has revealed the NE to be a key determinant of nuclear structure, gene regulation, and muscle function. To investigate the role of DMPK in NE stability, we analyzed DMPK expression in epithelial and myoblast cells. We found that DMPK localizes to the NE and coimmunoprecipitates with Lamin-A/C. Overexpression of DMPK in HeLa cells or C2C12 myoblasts disrupts Lamin-A/C and Lamin-B1 localization and causes nuclear fragmentation. Depletion of DMPK also disrupts NE lamina, showing that DMPK is required for NE stability. Our data demonstrate for the first time that DMPK is a critical component of the NE. These novel findings suggest that reduced DMPK may contribute to NE instability, a common mechanism of skeletal muscle wasting in muscular dystrophies.     

Mutations in the Drosophila alphaPS2 integrin subunit uncover new features of adhesion site assembly

The Drosophila alphaPS2betaPS integrin is required for diverse development events, including muscle attachment. We characterized six unusual mutations in the alphaPS2 gene that cause a subset of the null phenotype. One mutation changes a residue in alphaPS2 that is equivalent to the residue in alphaV that contacts the arginine of RGD. This change severely reduced alphaPS2betaPS affinity for soluble ligand, abolished the ability of the integrin to recruit laminin to muscle attachment sites in the embryo and caused detachment of integrins and talin from the ECM. Three mutations that alter different parts of the alphaPS2 beta-propeller, plus a fourth that eliminated a late phase of alphaPS2 expression, all led to a strong decrease in alphaPS2betaPS at muscle ends, but, surprisingly, normal levels of talin were recruited. Thus, although talin recruitment requires alphaPS2betaPS, talin levels are not simply specified by the amount of integrin at the adhesive junction. These mutations caused detachment of talin and actin from integrins, suggesting that the integrin-talin link is weaker than the ECM-integrin link.     

Comparing the local dynamic stability of trunk movements between varsity athletes with and without non-specific low back pain

The local dynamic stability of trunk movements, quantified using the maximum Lyapunov exponent (λ_max), can provide important information on the neuromuscular control of spine stability during movement tasks. Although previous research has displayed the promise of this technique, all studies were completed with healthy participants. Therefore the goal of this study was to compare the dynamic stability of spine kinematics and trunk muscle activations, as well as antagonistic muscle co-contraction, between athletes with and without low back pain (LBP). Twenty interuniversity varsity athletes (10 LBP, 10 healthy controls) were recruited to participate in the study. Each participant completed a repetitive trunk flexion task at 15 cycles per minute, both symmetrically and asymmetrically, while trunk kinematics and muscular activity (EMG) were monitored. The local dynamic stability of low back EMG was significantly higher (lower λ_max) in healthy individuals (p=0.002), whereas the dynamic stability of kinematics, the dynamic stability of full trunk system EMG, and the amount of antagonistic co-contraction were significantly higher when moving asymmetrically (p<0.05 for all variables). Although non-significant, kinematic and trunk system EMG stability also tended to be impaired in LBP participants, whereas they also tended to co-contract their antagonist muscles more. This study provides evidence that Lyapunov analyses of kinematic and muscle activation data can provide insight into the neuromuscular control of spine stability in back pain participants. Future research will repeat these protocols in patients with higher levels of pain, with hopes of developing a tool to assess impairment and treatment effectiveness in clinical and workplace settings.     

Interleukin-19 (IL-19) induces heme oxygenase-1 (HO-1) expression and decreases reactive oxygen species in human vascular smooth muscle cells

Heme oxygenase-1 (HO-1) has potent anti-inflammatory activity and recognized vascular protective effects. We have recently described the expression and vascular protective effects of an anti-inflammatory interleukin (IL-19), in vascular smooth muscle cells (VSMC) and injured arteries. The objective of this study was to link the anti-inflammatory effects of IL-19 with HO-1 expression in resident vascular cells. IL-19 induced HO-1 mRNA and protein in cultured human VSMC, as assayed by quantitative RT-PCR, immunoblot, and ELISA. IL-19 does not induce HO-1 mRNA or protein in human endothelial cells. IL-19 activates STAT3 in VSMC, and IL-19-induced HO-1 expression is significantly reduced by transfection of VSMC with STAT3 siRNA or mutation of the consensus STAT binding site in the HO-1 promoter. IL-19 treatment can significantly reduce ROS-induced apoptosis, as assayed by Annexin V flow cytometry. IL-19 significantly reduced ROS concentrations in cultured VSMC. The IL-19-induced reduction in ROS concentration is attenuated when HO-1 is reduced by siRNA, indicating that the IL-19-driven decrease in ROS is mediated by HO-1 expression. IL-19 reduces vascular ROS in vivo in mice treated with TNFα. This points to IL-19 as a potential therapeutic for vascular inflammatory diseases and a link for two previously unassociated protective processes: Th2 cytokine-induced anti-inflammation and ROS reduction.     

Mitochondrial impairment observed in fibroblasts from South African Parkinson’s disease patients with parkin mutations

Parkinson’s disease (PD), defined as a neurodegenerative disorder, is characterized by the loss of dopaminergic neurons in the substantia nigra in the midbrain. Loss-of-function mutations in the parkin gene are a major cause of autosomal recessive, early-onset PD. Parkin has been implicated in the maintenance of healthy mitochondria, although previous studies show conflicting findings regarding mitochondrial abnormalities in fibroblasts from patients harboring parkin-null mutations. The aim of the present study was to determine whether South African PD patients with parkin mutations exhibit evidence for mitochondrial dysfunction. Fibroblasts were cultured from skin biopsies obtained from three patients with homozygous parkin-null mutations, two heterozygous mutation carriers and two wild-type controls. Muscle biopsies were obtained from two of the patients. The muscle fibers showed subtle abnormalities such as slightly swollen mitochondria in focal areas of the fibers and some folding of the sarcolemma. Although no differences in the degree of mitochondrial network branching were found in the fibroblasts, ultrastructural abnormalities were observed including the presence of electron-dense vacuoles. Moreover, decreased ATP levels which are consistent with mitochondrial dysfunction were observed in the patients’ fibroblasts compared to controls. Remarkably, these defects did not manifest in one patient, which may be due to possible compensatory mechanisms. These results suggest that parkin-null patients exhibit features of mitochondrial dysfunction. Involvement of mitochondria as a key role player in PD pathogenesis will have important implications for the design of new and more effective therapies.     

Involution of the caudal musculature during metamorphosis in the ascidian,Botryllus schlosseri

Summary The caudal musculature of the free-swimming tadpole of the ascidian, B. schlosseri consists of cylindrical mononucleated cells connected in longitudinal rows flanking the axial notochord. During resorption of the larval tail, which is apparently induced by the contraction of the epidermis, muscle cells are dissociated and pushed into the body cavity where most of them are rapidly engulfed by phagocytes. In the initial stages of tail withdrawal muscle cells display surface alterations due to the disruption of intercellular junctions and disarrangement of myofibrils. Extensive degenerative changes, with shrinkage of mitochondria and disintegration of the contractile material are subsequently observed. Lysosomes and autophagic vacuoles are rarely seen and appear to play a secondary role in the degradation of the muscle cells, which occurs predominantly within the phagocytes. Myofilaments and myofibrils have never been observed within autophagic vacuoles. Clumps of muscle fragments and degenerated phagocytes undergo eventual dissolution in the blood lacunae, concomitantly with the differentiation of the young oozooid.This investigation was supported in part by a grant from the Muscular Dystrophy Associations of America and by CNR contract No. 7100396/04115542 from the Istituto di Biologia del Mare, Venice. We gratefully acknowledge the skillful assistance of Mr. G. Gallian, Mr. M. Fabbri and Mr. G. Tognon. We also thank the staff of the Stazione Idrobiologica at Chioggia for collecting the colonies.     

Muscular sound and force relationship during isometric contraction in man

The contracting muscle generates a low frequency sound detectable at the belly surface, ranging from 11 to 40 Hz. To study the relationship between the muscular sound and the intensity of the contraction a sound myogram (SMG) was recorded by a contact sensor from the biceps brachii of seven young healthy males performing 4-s isometric contractions from 10% to 100% of the maximal voluntary contraction (MVC), in 10% steps. Simultaneously, the electromyogram (EMG) was recorded as an index of muscle activity. SMG and EMG were integrated by conventional methods (iSMG and iEMG). The relationship between iSMG and iEMG vs MVC% is described by parabolic functions up to 80% and 100% MVC respectively. Beyond 80% MVC the iSMG decreases, being about half of its maximal value at 100% MVC. Our results indicate that the motor unit recruitment and firing rate affect the iSMG and iEMG in the same way up to 80% MVC. From 80% to 100% MVC the high motor units' discharge rate and the muscular stiffness together limit the pressure waves generated by the dimensional changes of the active fibres. The muscular sound seems to reflect the intramuscular visco-elastic characteristics and the motor unit activation pattern of a contracting muscle.     

Molting-specific downregulation of C. elegans body-wall muscle attachment sites: The role of RNF-5 E3 ligase

Repeated molting of the cuticula is an integral part of arthropod and nematode development. Shedding of the old cuticle takes place on the surface of hypodermal cells, which are also responsible for secretion and synthesis of a new cuticle. Here, we use the model nematode Caenorhabditis elegans to show that muscle cells, laying beneath and mechanically linked to the hypodermis, play an important role during molting. We followed the molecular composition and distribution of integrin mediated adhesion structures called dense bodies (DB), which indirectly connect muscles to the hypodermis. We found the concentration of two DB proteins (PAT-3/β-integrin and UNC-95) to decrease during the quiescent phase of molting, concomitant with an apparent increase in lateral movement of the DB. We show that levels of the E3-ligase RNF-5 increase specifically during molting, and that RNF-5 acts to ubiquitinate the DB protein UNC-95. Persistent high levels of RNF-5 driven by a heatshock or unc-95 promoter lead to failure of ecdysis, and in non-molting worms to a progressive detachment of the cuticle from the hypodermis. These observations indicate that increased DB dynamics characterizes the lethargus phase of molting in parallel to decreased levels of DB components and that temporal expression of RNF-5 contributes to an efficient molting process.     

Morpholino knockdown of lysyl oxidase impairs zebrafish development, and reflects some aspects of copper metabolism disorders

Lysyl oxidase (LOX), a copper-dependent amine oxidase known in mammals to catalyze the cross-linking of collagen and elastin in the extracellular matrix, is a member of a multigenic family. Eight genes encoding lysyl oxidase isoforms have been identified in zebrafish. Recent studies have revealed a critical role for two zebrafish lysyl oxidases-like in the formation of the notochord. We now present the role of Lox in zebrafish development. lox morpholino-mediated knockdown results in a mildly undulated notochord, truncated anterior-posterior axis, tail bending and smaller head. Analyses of morphants show a complete disorganization of muscle somites and neural defects, in accordance with the lox expression pattern. Lox inhibition also induces pigment defects and pharyngeal arch deformities consistent with neural crest dysfunction. Taken together, these data reveal a role for Lox in early morphogenesis, especially in muscle development and neurogenesis, and resume some aspects of physiopathology of copper metabolism.