Donatella Biral

Polymyositis, dermatomyositis and malignancy: A further intriguing link

The association between malignancy and autoimmune myositis has been largely described and confirmed by numerous epidemiological studies. The temporal relationship between the two pathologic conditions can vary: malignancy may occur before, at the same time or following the diagnosis of myositis. Beside these observations, the molecular mechanisms underlying this association are still unknown, even though it has been demonstrated a possible antigenic similarity between regenerating myoblasts and some cancer cell populations. To better identify peculiar histopathologic features common to cancer and myositis, we screened muscle biopsies from patients affected with polymyositis, dermatomyositis, myositis in association to cancer, and from patients affected with newly diagnosed cancer, but without myositis. Similarly to the histopatologic features that were observed in the muscle from myositis patients, especially in those with cancer associated myositis, in patients affected with malignancy at the clinical onset of disease we observed early sign of myopathy, characterized by internally nucleated and regenerating myofibers, most of them expressing the neural cell adhesion molecule. The hypothesis that in a particular subset of individuals genetically predisposed to autoimmunity, an initial subclinical tumor-induced myopathy may result in an autoimmune myositis, represents a further intriguing link behind the association of these two conditions.

Home-based functional electrical stimulation rescues permanently denervated muscles in paraplegic patients with complete lower motor neuron lesion

Background. Spinal cord injury causes muscle wasting and loss of function, which are especially severe after complete and permanent damage to lower motor neurons. In a previous cross-sectional study, long-standing denervated muscles were rescued by home-based functional electrical stimulation (h-bFES) training. Objective. To confirm results by a 2-year longitudinal prospective study of 25 patients with complete conus/cauda equina lesions. Methods. Denervated leg muscles were stimulated by h-bFES using a custom-designed stimulator and large surface electrodes. Muscle mass, force, and structure were determined before and after 2 years of h-bFES using computed tomography, measurements of knee torque during stimulation, and muscle biopsies analyzed by histology and electron microscopy. Results. Twenty of 25 patients completed the 2-year h-bFES program, which resulted in (a) a 35% cross-sectional increase in area of the quadriceps muscle from 28.2 ± 8.1 to 38.1 ± 12.7 cm 2 (P < .001), a 75% increase in mean diameter of muscle fibers from 16.6 ± 14.3 to 29.1 ± 23.3 μm (P < .001), and improvements of the ultrastructural organization of contractile material; and (b) a 1187% increase in force output during electrical stimulation from 0.8 ± 1.3 to 10.3 ± 8.1 N m (P < .001). The recovery of quadriceps force was sufficient to allow 25% of the subjects to perform FES-assisted stand-up exercises. Conclusions. Home-based FES of denervated muscle is an effective home therapy that results in rescue of muscle mass and tetanic contractility. Important immediate benefits for the patients are the improved cosmetic appearance of lower extremities and the enhanced cushioning effect for seating.

Ecto-ATPase activity of alpha-sarcoglycan (adhalin).

α-Sarcoglycan is a component of the sarcoglycan complex of dystrophin-associated proteins. Mutations of any of the sarcoglycan genes cause specific forms of muscular dystrophies, collectively termed sarcoglycanopathies. Importantly, a deficiency of any specific sarcoglycan affects the expression of the others. Thus, it appears that the lack of sarcoglycans deprives the muscle cell of an essential, yet unknown function. In the present study, we provide evidence for an ecto-ATPase activity of α-sarcoglycan. α-Sarcoglycan binds ATP in a Mg2+-dependent and Ca2+-independent manner. The binding is inhibited by 3′-O-(4-benzoyl)benzoyl ATP and ADP. Sequence analysis reveals the existence of a consensus site for nucleotide binding in the extracellular domain of the protein. An antibody against this sequence inhibits the binding of ATP. A dystrophin·dystrophin-associated protein preparation demonstrates a Mg-ATPase activity that is inhibited by the antibody but not by inhibitors of endo-ATPases. In addition, we demonstrate the presence in the sarcolemmal membrane of a P2X-type purinergic receptor. These data suggest that α-sarcoglycan may modulate the activity of P2X receptors by buffering the extracellular ATP concentration. The absence of α-sarcoglycan in sarcoglycanopathies leaves elevated the concentration of extracellular ATP and the persistent activation of P2X receptors, leading to intracellular Ca2+ overload and muscle fiber death.

The T-tubule membrane ATP-operated P2X4 receptor influences contractility of skeletal muscle

Evidence indicates that extracellular ATP may have relevant functions in skeletal muscle, even though the physiological role and distribution of specific signaling pathway elements are not well known. The present work shows that P2X4 receptor, an extracellular ATP‐regulated cell membrane channel permeable to Ca2+, is expressed in several tissues of the rat, including skeletal muscle. A specific antibody detected a protein band of ~60 kDa. Immunofluorescence demonstrated that P2X4 has an intracellular localization, and confocal analysis revealed that the receptor colocalizes with the T‐tubule membrane DHP receptor. Considering that the natural agonist of P2X4 is ATP, we explored if changes of extracellular ATP levels could occur in contracting skeletal muscle to regulate the channel. In vitro experiments showed that substantial ATP is released and rapidly hydrolyzed after electrical stimulation of rat muscle fibers. Results show that the presence of ATP‐degrading enzymes (hexokinase/apyrase), inhibitors of P2X receptors or Ca2+‐free conditions, all abolished the progressive twitch tension potentiation produced in soleus muscle by low‐frequency (0.05 Hz) stimulation. These data reveal that ATP‐mediated Ca2+ entry, most likely through P2X4 receptor, may play an important role in modulating the contractility of skeletal muscle.

Loss of dystrophin and some dystrophin-associated proteins with concomitant signs of apoptosis in rat leg muscle overworked in extension.

Abstract This study investigated the basis for the high severity of damage to skeletal muscle due to eccentric exercise, i.e., to muscles generating force while lengthened. Fast and slow rat leg muscles maintained in an extended position were examined after 2–24 h of continuous stimulation. The treatment caused the injury to some regions of both muscles. Within the better preserved parts of the muscles, i.e., those without signs of necrotic processes, dystrophin, spectrin, and some of the dystrophin-associated proteins (β-dystroglycan, α-sarcoglycan, and γ-sarcoglycan) disappeared from sarcolemma of many fibers. The reduction or loss of dystrophin from the sarcolemma was more evident than that of other proteins examined, with sarcoglycans apparently being the most preserved. Several muscle fibers devoid of dystrophin contained apoptotic nuclei. Simultaneously, Bax, Bcl-2 and caspase-3 proteins appeared in many fibers. Our results indicate that a normal muscle overworking in an extended position undergoes the loss of several membrane skeletal proteins because of the excessive stress to the membrane cytoskeleton, which can lead to fiber death by either apoptosis or necrosis. This experimental model may represent a good model for mimicking the pathogenetic events in several muscular dystrophies.

The Novel Skeletal Muscle Sarcoplasmic Reticulum JP-45 Protein MOLECULAR CLONING, TISSUE DISTRIBUTION, DEVELOPMENTAL EXPRESSION, AND INTERACTION WITH α1.1 SUBUNIT OF THE VOLTAGE-GATED CALCIUM CHANNEL

JP-45 is a novel integral protein constituent of the skeletal muscle sarcoplasmic reticulum junctional face membrane. We identified its primary structure from a cDNA clone isolated from a mouse skeletal muscle cDNA library. Mouse skeletal muscle JP-45 displays over 86 and 50% identity with two hypothetical NCBI data base protein sequences from mouse tongue and human muscle, respectively. JP-45 is predicted to have a cytoplasmic domain, a single transmembrane segment followed by an intralumenal domain enriched in positively charged amino acids. Northern and Western blot analyses reveal that the protein is mainly expressed in skeletal muscle. The mRNA encoding JP-45 appears in 17-day-old mouse embryos; expression of the protein peaks during the second month of postnatal development and then decreases ∼3-fold during aging. Double immunofluorescence of adult skeletal muscle fibers demonstrates that JP-45 co-localizes with the sarcoplasmic reticulum calcium release channel. Co-immunoprecipitation experiments with a monoclonal antibody against JP-45 show that JP-45 interacts with the α1.1 subunit voltage-gated calcium channel and calsequestrin. These results are consistent with the localization of JP-45 in the junctional sarcoplasmic reticulum and with its involvement in the molecular mechanism underlying skeletal muscle excitation-contraction coupling.

Coexistence of two calsequestrin isoforms in rabbit slow-twitch skeletal muscle fibers

The cardiac and skeletal muscle isoforms of calsequestrin (CS), the low affinity, high capacity Ca2+ binding protein localized in the lumen of sarcoplasmic reticulum, are the products of two different genes (Fliegel, L., Leberer, E., Green, N.M. and MacLennan, D.H. (1982) FEBS Lett. 242, 297–300), and can be both purified from slow‐twitch skeletal muscle of the rabbit (Damiani, E., Volpe, P. and Margreth, A. (1990) J. Muscle Res. Cell Motil. 11, 522–530). Here we show that both CS isoforms coexist in slow‐twitch muscle fibers as indicated by indirect immunofluorescent staining of cryosections with affinity‐purified antibodies specific for each CS isoform.

INCREASED EXPRESSION OF DYSTROPHIN, BETA -DYSTROGLYCAN AND ADHALIN IN DENERVATED RAT MUSCLES

SummaryTo evaluate a potential regulatory role of the nerve, the distribution and expression of dystrophin, of β-dystroglycan (43DAG) and adhalin (50DAG), two of the dystrophin-associated proteins and utrophin (dystrophin related protein or DRP) were studied in rat muscles after 2 weeks of denervation. We found that dystrophin, β-dystroglycan and adhalin were overexpressed in denervated muscle, whereas utrophin did not increase and was found only in the post-synaptic membrane. The study of the distribution of dystrophin in the sarcolemma of single muscle fibres indicates that the molecular organization of dystrophin was maintained after denervation. Dystrophin in addition of forming a scaffold around the fibre was found around the clusters of AChR that reappeared in the extra-synaptic membrane after denervation. Also β-dystroglycan colocalises at these clusters. These results suggest that the increase in dystrophin, β-dystroglycan and adhalin is correlated with the reappearance of AChRs in the extra synaptic membrane.

Atrophy/hypertrophy cell signaling in muscles of young athletes trained with vibrational-proprioceptive stimulation

Abstract Objective: To compare the effects of isokinetic (ISO-K) and vibrational-proprioceptive (VIB) trainings on muscle mass and strength. Methods: In 29 ISO-K- or VIB-trained young athletes we evaluated: force, muscle fiber morphometry, and gene expression of muscle atrophy/hypertrophy cell signaling. Results: VIB training increased the maximal isometric unilateral leg extension force by 48·1%. ISO-K training improved the force by 24·8%. Both improvements were statistically significant (P0·01). The more functional effectiveness of the VIB training in comparison with the ISO-K training was shown by the statistical significance changes only in VIB group in: rate of force development in time segment 0-50 ms (P<0·001), squat jump (P<0·05) and 30-m acceleration running test (P<0·05). VIB training induced a highly significant increase of mean diameter of fast fiber (+9%, P<0·001), but not of slow muscle fibers (−3%, not significant). No neural cell adhesion molecule-positive (N-CAM+) and embryonic myosin heavy chain-positive (MHC-emb+) myofibers were detected. VIB induced a significant twofold increase (P<0·05) of the skeletal muscle isoform insulin-like growth factor-1 (IGF-1) Ec mRNA. Atrogin-1 and muscle ring finger-1 (MuRF-1) did not change, but myostatin was strongly downregulated after VIB training (P<0·001). Peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) expression increased in post-training groups, but only in VIB reached statistical significance (+228%, P<0·05). Discussion: We demonstrated that both trainings are effective and do not induce muscle damage. Only VIB-trained group showed statistical significance increase of hypertrophy cell signaling pathways (IGF-1Ec and PGC-1α upregulation, and myostatin downregulation) leading to hypertrophy of fast twitch muscle fibers.

Evidence for localization of the myotonic dystrophy protein kinase to the terminal cisternae of the sarcoplasmic reticulum

Myotonic dystrophy is an autosomal dominant multisystem disease primarily affecting skeletal muscle and is characterized by the presence of an amplified trinucleotide repeat in the 3–2; untranslated region of the myotonic dystrophy protein kinase gene. In this study, the subcellular localization of the myotonic dystrophy protein kinase in muscle tissues has been investigated at both morphological and biochemical level, by using antibodies against the myotonic dystrophy protein kinase. Immunofluorescence studies and Western-blot analysis were carried out with antibodies raised against both a synthetic peptide and a recombinant fusion protein fragment specific for the myotonic dystrophy protein kinase. The kinase is localized both to the surface membranes, and within the skeletal fibres in the region of the A-I band boundary. Consistent with the A-I location of the kinase is that Western-blot analysis of purified fractions from sarcoplasmic reticulum show that triads and sarcoplasmic reticulum terminal cisternae are immunoreactive for two myotonic dystrophy protein kinase proteins of different molecular weight (85 and 54 kDa). The relative amount of these two proteins is different in relation to the muscle type, the 85 kDa protein being more evident in skeletal than in cardiac fibres. In addition, immunofluorescence studies of cardiac muscle reveal a heavy concentration of DM-PK localized to the intercalated discs, as well as a weaker reaction in the sarcoplasm. These results taken together suggest that multiple isoforms of the DM-PK may exist and that they may be differentially located in muscle tissues