Diana Conte

Adaptation of Mouse Skeletal Muscle to Long-Term Microgravity in the MDS Mission

The effect of microgravity on skeletal muscles has so far been examined in rat and mice only after short-term (5–20 day) spaceflights. The mice drawer system (MDS) program, sponsored by Italian Space Agency, for the first time aimed to investigate the consequences of long-term (91 days) exposure to microgravity in mice within the International Space Station. Muscle atrophy was present indistinctly in all fiber types of the slow-twitch soleus muscle, but was only slightly greater than that observed after 20 days of spaceflight. Myosin heavy chain analysis indicated a concomitant slow-to-fast transition of soleus. In addition, spaceflight induced translocation of sarcolemmal nitric oxide synthase-1 (NOS1) into the cytosol in soleus but not in the fast-twitch extensor digitorum longus (EDL) muscle. Most of the sarcolemmal ion channel subunits were up-regulated, more in soleus than EDL, whereas Ca2+-activated K+ channels were down-regulated, consistent with the phenotype transition. Gene expression of the atrophy-related ubiquitin-ligases was up-regulated in both spaceflown soleus and EDL muscles, whereas autophagy genes were in the control range. Muscle-specific IGF-1 and interleukin-6 were down-regulated in soleus but up-regulated in EDL. Also, various stress-related genes were up-regulated in spaceflown EDL, not in soleus. Altogether, these results suggest that EDL muscle may resist to microgravity-induced atrophy by activating compensatory and protective pathways. Our study shows the extended sensitivity of antigravity soleus muscle after prolonged exposition to microgravity, suggests possible mechanisms accounting for the resistance of EDL, and individuates some molecular targets for the development of countermeasures.

Therapeutic Approaches to Genetic Ion Channelopathies and Perspectives in Drug Discovery

In the human genome more than 400 genes encode ion channels, which are transmembrane proteins mediating ion fluxes across membranes. Being expressed in all cell types, they are involved in almost all physiological processes, including sense perception, neurotransmission, muscle contraction, secretion, immune response, cell proliferation, and differentiation. Due to the widespread tissue distribution of ion channels and their physiological functions, mutations in genes encoding ion channel subunits, or their interacting proteins, are responsible for inherited ion channelopathies. These diseases can range from common to very rare disorders and their severity can be mild, disabling, or life-threatening. In spite of this, ion channels are the primary target of only about 5% of the marketed drugs suggesting their potential in drug discovery. The current review summarizes the therapeutic management of the principal ion channelopathies of central and peripheral nervous system, heart, kidney, bone, skeletal muscle and pancreas, resulting from mutations in calcium, sodium, potassium, and chloride ion channels. For most channelopathies the therapy is mainly empirical and symptomatic, often limited by lack of efficacy and tolerability for a significant number of patients. Other channelopathies can exploit ion channel targeted drugs, such as marketed sodium channel blockers. Developing new and more specific therapeutic approaches is therefore required. To this aim, a major advancement in the pharmacotherapy of channelopathies has been the discovery that ion channel mutations lead to change in biophysics that can in turn specifically modify the sensitivity to drugs: this opens the way to a pharmacogenetics strategy, allowing the development of a personalized therapy with increased efficacy and reduced side effects. In addition, the identification of disease modifiers in ion channelopathies appears an alternative strategy to discover novel druggable targets.

A large cohort of myotonia congenita probands: novel mutations and a high-frequency mutation region in exons 4 and 5 of the CLCN1 gene

Myotonia congenita is a genetic disease characterized by impaired muscle relaxation after forceful contraction (myotonia) and caused by mutations in the chloride channel voltage-sensitive 1 (CLCN1) gene, encoding the voltage-gated chloride channel of skeletal muscle (ClC-1). In a large cohort of clinically diagnosed unrelated probands, we identified 75 different CLCN1 mutations in 106 individuals, among which 29 were novel mutations and 46 had already been reported. Despite the newly described mutations being scattered throughout the gene, in our patients, mutations were mostly found in exons 4 and 5. Most of the novel mutations located in the region comprising the intramembrane helices are involved in the ion-conducting pathway and predicted to affect channel function. We report for the first time that two mutations, inherited on the same allele as a heterozygous trait, abrogate disease expression, although when inherited singularly they were pathogenic. Such a mode of inheritance might explain the incomplete penetrance reported for autosomal dominant mutations in particular families.

Database search of spontaneous reports and pharmacological investigations on the sulfonylureas and glinides-induced atrophy in skeletal muscle

The ATP‐sensitive K+ (KATP) channel is an emerging pathway in the skeletal muscle atrophy which is a comorbidity condition in diabetes. The “in vitro” effects of the sulfonylureas and glinides were evaluated on the protein content/muscle weight, fibers viability, mitochondrial succinic dehydrogenases (SDH) activity, and channel currents in oxidative soleus (SOL), glycolitic/oxidative flexor digitorum brevis (FDB), and glycolitic extensor digitorum longus (EDL) muscle fibers of mice using biochemical and cell‐counting Kit‐8 assay, image analysis, and patch‐clamp techniques. The sulfonylureas were: tolbutamide, glibenclamide, and glimepiride; the glinides were: repaglinide and nateglinide. Food and Drug Administration‐Adverse Effects Reporting System (FDA‐AERS) database searching of atrophy‐related signals associated with the use of these drugs in humans has been performed. The drugs after 24 h of incubation time reduced the protein content/muscle weight and fibers viability more effectively in FDB and SOL than in the EDL. The order of efficacy of the drugs in reducing the protein content in FDB was: repaglinide (EC50 = 5.21 × 10−6) ≥ glibenclamide(EC50 = 8.84 × 10−6) > glimepiride(EC50 = 2.93 × 10−5) > tolbutamide(EC50 = 1.07 × 10−4) > nateglinide(EC50 = 1.61 × 10−4) and it was: repaglinide(7.15 × 10−5) ≥ glibenclamide(EC50 = 9.10 × 10−5) > nateglinide(EC50 = 1.80 × 10−4) ≥ tolbutamide(EC50 = 2.19 × 10−4) > glimepiride(EC50=–) in SOL. The drug‐induced atrophy can be explained by the KATP channel block and by the enhancement of the mitochondrial SDH activity. In an 8‐month period, muscle atrophy was found in 0.27% of the glibenclamide reports in humans and in 0.022% of the other not sulfonylureas and glinides drugs. No reports of atrophy were found for the other sulfonylureas and glinides in the FDA‐AERS. Glibenclamide induces atrophy in animal experiments and in human patients. Glimepiride shows less potential for inducing atrophy.

Pharmacovigilance database search discloses ClC-K channels as a novel target of the AT1 receptor blockers valsartan and olmesartan

Human ClC‐K chloride channels are highly attractive targets for drug discovery as they have a variety of important physiological functions and are associated with genetic disorders. These channels are crucial in the kidney as they control chloride reabsorption and water diuresis. In addition, loss‐of‐function mutations of CLCNKB and BSND genes cause Bartters syndrome (BS), whereas CLCNKA and CLCNKB gain‐of‐function polymorphisms predispose to a rare form of salt sensitive hypertension. Both disorders lack a personalized therapy that is in most cases only symptomatic. The aim of this study was to identify novel ClC‐K ligands from drugs already on the market, by exploiting the pharmacological side activity of drug molecules available from the FDA Adverse Effects Reporting System database.

Growth hormone secretagogues prevent dysregulation of skeletal muscle calcium homeostasis in a rat model of cisplatin‐induced cachexia

Cachexia is a wasting condition associated with cancer types and, at the same time, is a serious and dose‐limiting side effect of cancer chemotherapy. Skeletal muscle loss is one of the main characteristics of cachexia that significantly contributes to the functional muscle impairment. Calcium‐dependent signaling pathways are believed to play an important role in skeletal muscle decline observed in cachexia, but whether intracellular calcium homeostasis is affected in this situation remains uncertain. Growth hormone secretagogues (GHS), a family of synthetic agonists of ghrelin receptor (GHS‐R1a), are being developed as a therapeutic option for cancer cachexia syndrome; however, the exact mechanism by which GHS interfere with skeletal muscle is not fully understood.

Paving the way for Bartter syndrome type 3 drug discovery: a hope from basic research

Understanding the genetic background and the molecular mechanisms of inherited kidney channelopathies is essential to definition of targeted therapeutic approaches for affected patients. This article is protected by copyright. All rights reserved

Expression of sarcolemmal ion channels in slow and fast-twitch muscles of rodents in simulated and actual microgravity

Abstracts presented at the 39th European Muscle Conference of the European Society for Muscle Researchs presented at the 39th European Muscle Conference of the European Society for Muscle Research Abano Terme, Padova, Italy, September 11–15th, 2010 European Society for Muscle Research 2011 Abstracts for the oral presentationss for the oral presentations Mechanism of muscle contraction Direct evidence for the cross-bridge lever arm mechanism in muscle contraction studied using the gas environmental chamber and site-directed antibodies H. Sugi, H. Minoda, T. Okabe, Y. Inayoshi, T. Miyakawa, M. Tanokura, E. Katayama Department of Physiology, Teikyo University, Tokyo, Japan; Department of Applied Physics, Tokyo Institute of Agriculture and Technology, Tokyo, Japan; Department of Life Sciences, University of Tokyo, Tokyo, Japan; Department of Electron Microscopy, University of Tokyo, Tokyo, Japan During muscle contraction, the cross-bridges in myosin filaments first attach to actin filaments in the form of MADPPi, undergo a conformational change (power stroke) associated with release of Pi and ADP to produce sliding between actin and myosin filaments, and then detach from actin filaments upon binding with next ATP. The detached cross-bridges undergo a reversed conformational change (recovery stroke) associated with reaction, MATP ? MADPPi. It is suggest that the cross-bridge strokes result from rotation of the crossbridge lever arm domain around the converter domain, while the catalytic domain remains rigid. To ascertain the validity of the lever arm mechanism in muscle contraction, we recorded ATP-induced movement at different regions within individual cross-bridges in living myosin filaments, using the gas environmental chamber, with which biological macromolecules can be kept in living state in an electron microscope. Three different regions of the cross-bridge were position-marked with site-directed antibodies; antibody 1 to the distal catalytic region, antibody 2 to the interface between the catalytic and converter domains, and antibody 3 to the boundary between the lever arm domain and myosin subfragment 2. We have found that the average amplitude of ATP-induced movement was 6.14 nm at both the distal catalytic domain and the catalytic-converter domain interface, and 3.77 nm at the lever arm domain-subfragment 2 boundary, providing the first direct evidence for the cross-bridge lever arm mechanism. Muscle force generation examined by laser temperature-jump and ramp shortening

The analysis of myotonia congenita mutations discloses functional clusters of amino acids within the CBS2 domain and the C-terminal peptide of the ClC-1 channel

Myotonia congenita (MC) is a skeletal‐muscle hyperexcitability disorder caused by loss‐of‐function mutations in the ClC‐1 chloride channel. Mutations are scattered over the entire sequence of the channel protein, with more than 30 mutations located in the poorly characterized cytosolic C‐terminal domain. In this study, we characterized, through patch clamp, seven ClC‐1 mutations identified in patients affected by MC of various severities and located in the C‐terminal region. The p.Val829Met, p.Thr832Ile, p.Val851Met, p.Gly859Val, and p.Leu861Pro mutations reside in the CBS2 domain, while p.Pro883Thr and p.Val947Glu are in the C‐terminal peptide. We showed that the functional properties of mutant channels correlated with the clinical phenotypes of affected individuals. In addition, we defined clusters of ClC‐1 mutations within CBS2 and C‐terminal peptide subdomains that share the same functional defect: mutations between 829 and 835 residues and in residue 883 induced an alteration of voltage dependence, mutations between 851 and 859 residues, and in residue 947 induced a reduction of chloride currents, whereas mutations on 861 residue showed no obvious change in ClC‐1 function. This study improves our understanding of the mechanisms underlying MC, sheds light on the role of the C‐terminal region in ClC‐1 function, and provides information to develop new antimyotonic drugs.

Mapping ligand binding pockets in chloride ClC‐1 channels through an integrated in silico and experimental approach using anthracene‐9‐carboxylic acid and niflumic acid

Although chloride channels are involved in several physiological processes and acquired diseases, the availability of compounds selectively targeting CLC proteins is limited. ClC‐1 channels are responsible for sarcolemma repolarization after an action potential in skeletal muscle and have been associated with myotonia congenita and myotonic dystrophy as well as with other muscular physiopathological conditions. To date only a few ClC‐1 blockers have been discovered, such as anthracene‐9‐carboxylic acid (9‐AC) and niflumic acid (NFA), whereas no activator exists. The absence of a ClC‐1 structure and the limited information regarding the binding pockets in CLC channels hamper the identification of improved modulators.