Altered Tnnt3 characterizes selective weakness of fast fibers in mice overexpressing FSHD region gene 1 (FRG1).

Facioscapulohumeral muscular dystrophy (FSHD), a common hereditary myopathy, is characterized by atrophy and weakness of selective muscle groups. FSHD is considered an autosomal dominant disease with incomplete penetrance and unpredictable variability of clinical expression within families. Mice overexpressing FRG1 (FSHD region gene 1), a candidate gene for this disease, develop a progressive myopathy with features of the human disorder. Here, we show that in FRG1-overexpressing mice, fast muscles, which are the most affected by the dystrophic process, display anomalous fast skeletal troponin T (fTnT) isoform, resulting from the aberrant splicing of the Tnnt3 mRNA that precedes the appearance of dystrophic signs. We determine that muscles of FRG1 mice develop less strength due to impaired contractile properties of fast-twitch fibers associated with an anomalous MyHC-actin ratio and a reduced sensitivity to Ca(2+). We demonstrate that the decrease of Ca(2+) sensitivity of fast-twitch fibers depends on the anomalous troponin complex and can be rescued by the substitution with the wild-type proteins. Finally, we find that the presence of aberrant splicing isoforms of TNNT3 characterizes dystrophic muscles in FSHD patients. Collectively, our results suggest that anomalous TNNT3 profile correlates with the muscle impairment in both humans and mice. On the basis of these results, we propose that aberrant fTnT represents a biological marker of muscle phenotype severity and disease progression.

S1P2 receptor promotes mouse skeletal muscle regeneration.

Sphingosine 1-phosphate is a bioactive lipid that modulates skeletal muscle growth through its interaction with specific receptors localized in the cell membrane of muscle fibers and satellite cells. This study analyzes the role of S1P(2) receptor during in vivo regeneration of soleus muscle in two models of S1P(2) deficiency: the S1P(2)-null mouse and wild-type mice systemically treated with the S1P(2) receptor antagonist JTE-013. To stimulate regeneration, muscle degeneration was induced by injecting into soleus muscle the myotoxic drug notexin. Both ablation of S1P(2) receptor and its functional inactivation delayed regeneration of soleus muscle. The exogenous supplementation of S1P or its removal, by a specific antibody, two conditions known to stimulate or inhibit, respectively, soleus muscle regeneration, were without effects when the S1P(2) receptor was absent or inactive. The delayed regeneration was associated with a lower level of myogenin, a muscle differentiation marker, and reduced phosphorylation of Akt, a key marker of muscle growth. Consistently, silencing of S1P(2) receptor abrogated the pro-myogenic action of S1P in satellite cells, paralleled by low levels of the myogenic transcription factor myogenin. The study indicates that S1P(2) receptor plays a key role in the early phases of muscle regeneration by sustaining differentiation and growth of new-forming myofibers.

Sphingosine 1-phosphate signaling is involved in skeletal muscle regeneration.

Sphingosine 1-phosphate (S1P) is a bioactive lipid known to control cell growth that was recently shown to act as a trophic factor for skeletal muscle, reducing the progress of denervation atrophy. The aim of this work was to investigate whether S1P is involved in skeletal muscle fiber recovery (regeneration) after myotoxic injury induced by bupivacaine. The postnatal ability of skeletal muscle to grow and regenerate is dependent on resident stem cells called satellite cells. Immunofluorescence analysis demonstrated that S1P-specific receptors S1P(1) and S1P(3) are expressed by quiescent satellite cells. Soleus muscles undergoing regeneration following injury induced by intramuscular injection of bupivacaine exhibited enhanced expression of S1P(1) receptor, while S1P(3) expression progressively decreased to adult levels. S1P(2) receptor was absent in quiescent cells but was transiently expressed in the early regenerating phases only. Administration of S1P (50 microM) at the moment of myotoxic injury caused a significant increase of the mean cross-sectional area of regenerating fibers in both rat and mouse. In separate experiments designed to test the trophic effects of S1P, neutralization of endogenous circulating S1P by intraperitoneal administration of anti-S1P antibody attenuated fiber growth. Use of selective modulators of S1P receptors indicated that S1P(1) receptor negatively and S1P(3) receptor positively modulate the early phases of regeneration, whereas S1P(2) receptor appears to be less important. The present results show that S1P signaling participates in the regenerative processes of skeletal muscle.

Post-transcriptional silencing of the Drosophila homolog of human ZASP: a molecular and functional analysis.

In humans, mutations in ZASP (the gene for Z-band alternatively spliced PDZ-motif protein) are associated with dilated cardiomyopathy and left ventricular non-compaction. In particular, mutations in or around the Zasp motif seem to be related to myofibrillar myopathy. Thus, "zaspopathies" include symptoms such as Z-line disgregation, proximal and distal muscle weakness, cardiomyopathies, and peripheral neuropathies. In order to understand the role of ZASP in muscle structure and function, we have performed a molecular characterization of the Drosophila ortholog of human ZASP and a functional analysis following the post-transcriptional silencing of the Drosophila gene. Transcriptional analysis of dzasp has revealed six additional exons, with respect to the known 16, and multiple splice variants. We have produced transgenic lines harboring constructs that, through the use of the UAS/Gal4 binary system, have enabled us to drive dsRNA interference of dzasp in a tissue-specific manner. Knockdown individuals show locomotor defects associated with alterations of muscle structure and ultrastructure, consistent with a role of dzasp specifically in the maintenance of muscular integrity.

From action potential to contraction: neural control and excitation-contraction coupling in larval muscles of Drosophila.

The neuromuscular system of Drosophila melanogaster has been studied for many years for its relative simplicity and because of the genetic and molecular versatilities. Three main types of striated muscles are present in this dipteran: fibrillar muscles, tubular muscles and supercontractile muscles. The visceral muscles in adult flies and the body wall segmental muscles in embryos and larvae belong to the group of supercontractile muscles. Larval body wall muscles have been the object of detailed studies as a model for neuromuscular junction function but have received much less attention with respect to their mechanical properties and to the control of contraction. In this review we wish to assess available information on the physiology of the Drosophila larval muscular system. Our aim is to establish whether this system has the requisites to be considered a good model in which to perform a functional characterization of Drosophila genes, with a known muscular expression, as well as Drosophila homologs of human genes, the dysfunction of which, is known to be associated with human hereditary muscle pathologies.

High-frequency fatigue of skeletal muscle: role of extracellular Ca(2+).

The present study evaluated whether Ca(2+) entry operates during fatigue of skeletal muscle. The involvement of different skeletal muscle membrane calcium channels and of the Na(+)/Ca(2+) exchanger (NCX) has been examined. The decline of force was analysed in vitro in mouse soleus and EDL muscles submitted to 60 and 110 Hz continuous stimulation, respectively. Stimulation with this high-frequency fatigue (HFF) protocol, in Ca(2+)-free conditions, caused in soleus muscle a dramatic increase of fatigue, while in the presence of high Ca(2+) fatigue was reduced. In EDL muscle, HFF was not affected by external Ca(2+) levels either way, suggesting that external Ca(2+) plays a general protective role only in soleus. Calciseptine, a specific antagonist of the cardiac isoform (alpha1C) of the dihydropyridine receptor, gadolinium, a blocker of both stretch-activated and store-operated Ca(2+) channels, as well as inhibitors of P2X receptors did not affect the development of HFF. Conversely, the Ca(2+) ionophore A23187 increased the protective action of extracellular Ca(2+). KB-R7943, a selective inhibitor of the reverse mode of NCX, produced an effect similar to that of Ca(2+)-free solution. These results indicate that a transmembrane Ca(2+) influx, mainly through NCX, may play a protective role during HFF development in soleus muscle.

Neuromuscular junction in abdominal muscles of Drosophila melanogaster during adulthood and aging.

The neuromuscular junction (NMJ) of Drosophila melanogaster has been established as a productive model for the study of synaptogenesis, synaptic plasticity, vesicle recycling, and other synaptic functions in embryos and larvae. It also has potential for the study of long-term plasticity during adult life and degenerative processes associated with aging. Here we provide a detailed description of the morphology and ultrastructure of the NMJ on abdominal dorsal longitudinal muscles throughout adult life from eclosion to senescence. In contrast to the case in the larva, the predominant type of terminals in these muscles in the adult fly consists of only two or three branches with tightly packed synaptic boutons. We observed qualitative and quantitative changes as mean bouton size increased gradually during adulthood, and the largest boutons were present in the old fly. The length of nerve branches first increased and thereafter decreased gradually during most of adult life. Branch diameter also decreased progressively, but branch number did not change. The subsynaptic reticulum became progressively thinner, and "naked" boutons were found in old flies. Ultrastructural traits gave indications of an age-associated increment in autophagy, larger synaptic vesicles, and impaired endocytosis. We propose that NMJ aging in the fly correlates with impaired endocytosis and membrane dynamics. This view finds a functional correlate in flies carrying a temperature-sensitive mutation in shibire that reversible blocks endocytosis; age significantly reduces the time required for complete paralysis and increases the time of recovery, thus confirming the age-dependent alteration in vesicle dynamics.

Post-transcriptional silencing and functional characterization of the Drosophila melanogaster homolog of human Surf1.

Mutations in Surf1, a human gene involved in the assembly of cytochrome c oxidase (COX), cause Leigh syndrome, the most common infantile mitochondrial encephalopathy, characterized by a specific COX deficiency. We report the generation and characterization of functional knockdown (KD) lines for Surf1 in Drosophila. KD was produced by post-transcriptional silencing employing a transgene encoding a dsRNA fragment of the Drosophila homolog of human Surf1, activated by the UAS transcriptional activator. Two alternative drivers, Actin5C-GAL4 or elav-GAL4, were used to induce silencing ubiquitously or in the CNS, respectively. Actin5C-GAL4 KD produced 100% egg-to-adult lethality. Most individuals died as larvae, which were sluggish and small. The few larvae reaching the pupal stage died as early imagos. Electron microscopy of larval muscles showed severely altered mitochondria. elav-GAL4-driven KD individuals developed to adulthood, although cephalic sections revealed low COX-specific activity. Behavioral and electrophysiological abnormalities were detected, including reduced photoresponsiveness in KD larvae using either driver, reduced locomotor speed in Actin5C-GAL4 KD larvae, and impaired optomotor response as well as abnormal electroretinograms in elav-GAL4 KD flies. These results indicate important functions for SURF1 specifically related to COX activity and suggest a crucial role of mitochondrial energy pathways in organogenesis and CNS development and function.