Pharmacotherapy of the Lipid-Lowering Drugs: Update on Efficacy and Risk.

Lipid-lowering drugs are widely used for the prevention and cure of cardiovascular diseases (CVD) [...].

Cardiovascular, neurological, and pulmonary events following vaccination with the BNT162b2, ChAdOx1 nCoV-19, and Ad26.COV2.S vaccines: An analysis of European data.

The ChAdOx1 nCoV-19 (ChA) (AstraZeneca) and Ad26.COV2.S (AD26) (Janssen) vaccines are virus-based coronavirus disease 2019 (COVID-19) vaccines used worldwide. In spring 2021, venous blood clots and thrombocytopenia were described in some vaccine recipients. We evaluated the frequency of severe adverse events (SAEs) documented in the EudraVigilance European database in young adult (18-64 years old) and older (≥65 years old) vaccine recipients up to 23 June 2021 and related them to coagulation disorders and arterial, cardiac, and nervous system events. Comparison between the frequency of SAEs and SAE-related deaths in ChA and AD26 vs. BNT162b2 COVID-19 (BNT) (Pfizer/BioNTech) vaccine recipients demonstrated: 1) ChA and AD26 recipients than BNT recipients had higher frequencies of not only SAEs caused by venous blood clots and hemorrhage, but also thromboembolic disease and arterial events, including myocardial infarction and stroke; 2) a corresponding higher frequency of SAE-related deaths. The frequency was higher in both young adults and older adults. Comparison between the frequency of SAEs and SAE-related deaths in AD26 vs. ChA recipients demonstrated in AD26 recipients: 1) lower frequency of thrombocytopenia; 2) lower frequency of SAEs in young adult recipients; 3) higher frequency of SAEs in older recipients. Interestingly, most of the venous thrombotic SAEs associated with ChA and AD26 vaccines were not associated with thrombocytopenia, suggesting that TTS (thrombosis with thrombocytopenia syndrome) is not the only type of thrombosis observed following virus-based vaccines. In conclusion, both virus-based COVID-19 vaccines show more SAEs than BNT, but the frequency of the SAE type in the different age groups differs, suggesting that the mechanisms responsible of SAEs overlap only partly.

BCAAs and Di-Alanine supplementation in the prevention of skeletal muscle atrophy: preclinical evaluation in a murine model of hind limb unloading.

Skeletal muscle atrophy occurs in response to various pathophysiological stimuli, including disuse, aging, and neuromuscular disorders, mainly due to an imbalance of anabolic/catabolic signaling. Branched Chain Amino Acids (BCAAs: leucine, isoleucine, valine) supplements can be beneficial for counteracting muscle atrophy, in virtue of their reported anabolic properties. Here, we carried out a proof-of-concept study to assess the in vivo/ex vivo effects of a 4-week treatment with BCAAs on disuse-induced atrophy, in a murine model of hind limb unloading (HU). BCAAs were formulated in drinking water, alone, or plus two equivalents of L-Alanine (2 ALA) or the dipeptide L-Alanyl-L-Alanine (Di-ALA), to boost BCAAs bioavailability. HU mice were characterized by reduction of body mass, decrease of soleus - SOL - muscle mass and total protein, alteration of postural muscles architecture and fiber size, dysregulation of atrophy-related genes (Atrogin-1, MuRF-1, mTOR, Mstn). In parallel, we provided new robust readouts in the HU murine model, such as impaired in vivo isometric torque and ex vivo SOL muscle contractility and elasticity, as well as altered immune response. An acute pharmacokinetic study confirmed that L-ALA, also as dipeptide, enhanced plasma exposure of BCAAs. Globally, the most sensitive parameters to BCAAs action were muscle atrophy and myofiber cross-sectional area, muscle force and compliance to stress, protein synthesis via mTOR and innate immunity, with the new BCAAs + Di-ALA formulation being the most effective treatment. Our results support the working hypothesis and highlight the importance of developing innovative formulations to optimize BCAAs biodistribution.

Statin-Induced Myopathy: Translational Studies from Preclinical to Clinical Evidence.

Statins are the most prescribed and effective drugs to treat cardiovascular diseases (CVD). Nevertheless, these drugs can be responsible for skeletal muscle toxicity which leads to reduced compliance. The discontinuation of therapy increases the incidence of CVD. Thus, it is essential to assess the risk. In fact, many studies have been performed at preclinical and clinical level to investigate pathophysiological mechanisms and clinical implications of statin myotoxicity. Consequently, new toxicological aspects and new biomarkers have arisen. Indeed, these drugs may affect gene transcription and ion transport and contribute to muscle function impairment. Identifying a marker of toxicity is important to prevent or to cure statin induced myopathy while assuring the right therapy for hypercholesterolemia and counteracting CVD. In this review we focused on the mechanisms of muscle damage discovered in preclinical and clinical studies and highlighted the pathological situations in which statin therapy should be avoided. In this context, preventive or substitutive therapies should also be evaluated.

Ryanodine channel complex stabilizer compound S48168/ARM210 as a disease modifier in dystrophin-deficient mdx mice: proof-of-concept study and independent validation of efficacy.

Muscle fibers lacking dystrophin undergo a long-term alteration of Ca2+ homeostasis, partially caused by a leaky Ca2+ release ryanodine (RyR) channel. S48168/ARM210, an RyR calcium release channel stabilizer (a Rycal compound), is expected to enhance the rebinding of calstabin to the RyR channel complex and possibly alleviate the pathologic Ca2+ leakage in dystrophin-deficient skeletal and cardiac muscle. This study systematically investigated the effect of S48168/ARM210 on the phenotype of mdx mice by means of a first proof-of-concept, short (4 wk), phase 1 treatment, followed by a 12-wk treatment (phase 2) performed in parallel by 2 independent laboratories. The mdx mice were treated with S48168/ARM210 at two different concentrations (50 or 10 mg/kg/d) in their drinking water for 4 and 12 wk, respectively. The mice were subjected to treadmill sessions twice per week (12 m/min for 30 min) to unmask the mild disease. This testing was followed by in vivo forelimb and hindlimb grip strength and fatigability measurement, ex vivo extensor digitorum longus (EDL) and diaphragm (DIA) force contraction measurement and histologic and biochemical analysis. The treatments resulted in functional (grip strength, ex vivo force production in DIA and EDL muscles) as well as histologic improvement after 4 and 12 wk, with no adverse effects. Furthermore, levels of cellular biomarkers of calcium homeostasis increased. Therefore, these data suggest that S48168/ARM210 may be a safe therapeutic option, at the dose levels tested, for the treatment of Duchenne muscular dystrophy (DMD).-Capogrosso, R. F., Mantuano, P., Uaesoontrachoon, K., Cozzoli, A., Giustino, A., Dow, T., Srinivassane, S., Filipovic, M., Bell, C., Vandermeulen, J., Massari, A. M., De Bellis, M., Conte, E., Pierno, S., Camerino, G. M., Liantonio, A., Nagaraju, K., De Luca, A. Ryanodine channel complex stabilizer compound S48168/ARM210 as a disease modifier in dystrophin-deficient mdx mice: proof-of-concept study and independent validation of efficacy.

Statin-induced myotoxicity is exacerbated by aging: A biophysical and molecular biology study in rats treated with atorvastatin.

Statin-induced skeletal muscle damage in rats is associated to the reduction of the resting sarcolemmal chloride conductance (gCl) and ClC-1 chloride channel expression. These drugs also affect the ClC-1 regulation by increasing protein kinase C (PKC) activity, which phosphorylate and close the channel. Also the intracellular resting calcium (restCa) level is increased. Similar alterations are observed in skeletal muscles of aged rats, suggesting a higher risk of statin myotoxicity. To verify this hypothesis, we performed a 4-5-weeks atorvastatin treatment of 24-months-old rats to evaluate the ClC-1 channel function by the two-intracellular microelectrodes technique as well as transcript and protein expression of different genes sensitive to statins by quantitative real-time-PCR and western blot analysis. The restCa was measured using FURA-2 imaging, and histological analysis of muscle sections was performed. The results show a marked reduction of resting gCl, in agreement with the reduced ClC-1 mRNA and protein expression in atorvastatin-treated aged rats, with respect to treated adult animals. The observed changes in myocyte-enhancer factor-2 (MEF2) expression may be involved in ClC-1 expression changes. The activity of PKC was also increased and further modulate the gCl in treated aged rats. In parallel, a marked reduction of the expression of glycolytic and mitochondrial enzymes demonstrates an impairment of muscle metabolism. No worsening of restCa or histological features was found in statin-treated aged animals. These findings suggest that a strong reduction of gCl and alteration of muscle metabolism coupled to muscle atrophy may contribute to the increased risk of statin-induced myopathy in the elderly.

Taurine: the appeal of a safe amino acid for skeletal muscle disorders.

Taurine is a natural amino acid present as free form in many mammalian tissues and in particular in skeletal muscle. Taurine exerts many physiological functions, including membrane stabilization, osmoregulation and cytoprotective effects, antioxidant and anti-inflammatory actions as well as modulation of intracellular calcium concentration and ion channel function. In addition taurine may control muscle metabolism and gene expression, through yet unclear mechanisms. This review summarizes the effects of taurine on specific muscle targets and pathways as well as its therapeutic potential to restore skeletal muscle function and performance in various pathological conditions. Evidences support the link between alteration of intracellular taurine level in skeletal muscle and different pathophysiological conditions, such as disuse-induced muscle atrophy, muscular dystrophy and/or senescence, reinforcing the interest towards its exogenous supplementation. In addition, taurine treatment can be beneficial to reduce sarcolemmal hyper-excitability in myotonia-related syndromes. Although further studies are necessary to fill the gaps between animals and humans, the benefit of the amino acid appears to be due to its multiple actions on cellular functions while toxicity seems relatively low. Human clinical trials using taurine in various pathologies such as diabetes, cardiovascular and neurological disorders have been performed and may represent a guide-line for designing specific studies in patients of neuromuscular diseases.

Calcium homeostasis is altered in skeletal muscle of spontaneously hypertensive rats: cytofluorimetric and gene expression analysis.

Hypertension is often associated with skeletal muscle pathological conditions related to function and metabolism. The mechanisms underlying the development of these pathological conditions remain undefined. Because calcium homeostasis is a biomarker of muscle function, we assessed whether it is altered in hypertensive muscles. We measured resting intracellular calcium and store-operated calcium entry (SOCE) in fast- and slow-twitch muscle fibers from normotensive Wistar-Kyoto rats and spontaneously hypertensive rats (SHRs) by cytofluorimetric technique and determined the expression of SOCE gene machinery by real-time PCR. Hypertension caused a phenotype-dependent dysregulation of calcium homeostasis; the resting intracellular calcium of extensor digitorum longus and soleus muscles of SHRs were differently altered with respect to the related muscle of normotensive animals. In addition, soleus muscles of SHR showed reduced activity of the sarcoplasmic reticulum and decreased sarcolemmal calcium permeability at rest and after SOCE activation. Accordingly, we found an alteration of the expression levels of some SOCE components, such as stromal interaction molecule 1, calcium release-activated calcium modulator 1, and transient receptor potential canonical 1. The hypertension-induced alterations of calcium homeostasis in the soleus muscle of SHRs occurred with changes of some functional outcomes as excitability and resting chloride conductance. We provide suitable targets for therapeutic interventions aimed at counterbalancing muscle performance decline in hypertension, and propose the reported calcium-dependent parameters as indexes to predict how the antihypertensive drugs could influence muscle function.

Angiotensin II modulates mouse skeletal muscle resting conductance to chloride and potassium ions and calcium homeostasis via the AT1 receptor and NADPH oxidase.

Angiotensin II (ANG II) plays a role in muscle wasting and remodeling; however, little evidence shows its direct effects on specific muscle functions. We presently investigated the acute in vitro effects of ANG II on resting ionic conductance and calcium homeostasis of mouse extensor digitorum longus (EDL) muscle fibers, based on previous findings that in vivo inhibition of ANG II counteracts the impairment of macroscopic ClC-1 chloride channel conductance (gCl) in the mdx mouse model of muscular dystrophy. By means of intracellular microelectrode recordings we found that ANG II reduced gCl in the nanomolar range and in a concentration-dependent manner (EC50 = 0.06 µM) meanwhile increasing potassium conductance (gK). Both effects were inhibited by the ANG II receptors type 1 (AT1)-receptor antagonist losartan and the protein kinase C inhibitor chelerythrine; no antagonism was observed with the AT2 antagonist PD123,319. The scavenger of reactive oxygen species (ROS) N-acetyl cysteine and the NADPH-oxidase (NOX) inhibitor apocynin also antagonized ANG II effects on resting ionic conductances; the ANG II-dependent gK increase was blocked by iberiotoxin, an inhibitor of calcium-activated potassium channels. ANG II also lowered the threshold for myofiber and muscle contraction. Both ANG II and the AT1 agonist L162,313 increased the intracellular calcium transients, measured by fura-2, with a two-step pattern. These latter effects were not observed in the presence of losartan and of the phospholipase C inhibitor U73122 and the in absence of extracellular calcium, disclosing a Gq-mediated calcium entry mechanism. The data show for the first time that the AT1-mediated ANG II pathway, also involving NOX and ROS, directly modulates ion channels and calcium homeostasis in adult myofibers.

Protein kinase C theta (PKCθ) modulates the ClC-1 chloride channel activity and skeletal muscle phenotype: a biophysical and gene expression study in mouse models lacking the PKCθ.

In skeletal muscle, the resting chloride conductance (gCl), due to the ClC-1 chloride channel, controls the sarcolemma electrical stability. Indeed, loss-of-function mutations in ClC-1 gene are responsible of myotonia congenita. The ClC-1 channel can be phosphorylated and inactivated by protein kinases C (PKC), but the relative contribution of each PKC isoforms is unknown. Here, we investigated on the role of PKCθ in the regulation of ClC-1 channel expression and activity in fast- and slow-twitch muscles of mouse models lacking PKCθ. Electrophysiological studies showed an increase of gCl in the PKCθ-null mice with respect to wild type. Muscle excitability was reduced accordingly. However, the expression of the ClC-1 channel, evaluated by qRT-PCR, was not modified in PKCθ-null muscles suggesting that PKCθ affects the ClC-1 activity. Pharmacological studies demonstrated that although PKCθ appreciably modulates gCl, other isoforms are still active and concur to this role. The modification of gCl in PKCθ-null muscles has caused adaptation of the expression of phenotype-specific genes, such as calcineurin and myocyte enhancer factor-2, supporting the role of PKCθ also in the settings of muscle phenotype. Importantly, the lack of PKCθ has prevented the aging-related reduction of gCl, suggesting that its modulation may represent a new strategy to contrast the aging process.

Development of Riluzole Analogs with Improved Use-Dependent Inhibition of Skeletal Muscle Sodium Channels.

Several commercially available and newly synthesized riluzole analogs were evaluated in vitro as voltage-gated skeletal muscle sodium-channel blockers. Data obtained from the patch-clamp technique demonstrated that potency is well correlated with lipophilicity and the introduction of a protonatable amino function in the benzothiazole 2-position enhances the use-dependent behavior. The most interesting compound, the 2-piperazine analog of riluzole (14), although slightly less potent than the parent compound in the patch-clamp assay as well as in an in vitro model of myotonia, showed greater use-dependent Nav1.4 blocking activity. Docking studies allowed the identification of the key interactions that 14 makes with the amino acids of the local anesthetic binding site within the pore of the channel. The reported results pave the way for the identification of novel compounds useful in the treatment of cell excitability disorders.

Potential benefits of taurine in the prevention of skeletal muscle impairment induced by disuse in the hindlimb-unloaded rat.

Hindlimb unloading (HU) in rats induces severe atrophy and a slow-to-fast phenotype transition in postural slow-twitch muscles, as occurs in human disuse conditions, such as spaceflight or bed rest. In rats, a reduction of soleus muscle weight and a decrease of cross-sectional area (CSA) were observed as signs of atrophy. An increased expression of the fast-isoform of myosin heavy chain (MHC) showed the phenotype transition. In parallel the resting cytosolic calcium concentration (restCa) was decreased and the resting chloride conductance (gCl), which regulates muscle excitability, was increased toward the values of the fast-twitch muscles. Here, we investigated the possible role of taurine, which is known to modulate calcium homeostasis and gCl, in the restoration of muscle impairment due to 14-days-HU. We found elevated taurine content and higher expression of the taurine transporter TauT in the soleus muscle as compared to the fast-twitch extensor digitorum longus (EDL) muscle of control rats. Taurine level was reduced in the HU soleus muscle, although, TauT expression was not modified. Taurine oral supplementation (5 g/kg) fully prevented this loss, and preserved resting gCl and restCa together with the slow MHC phenotype. Taurine supplementation did not prevent the HU-induced drop of muscle weight or fiber CSA, but it restored the expression of MURF-1, an atrophy-related gene, suggesting a possible early protective effect of taurine. In conclusion, taurine prevented the HU-induced phenotypic transition of soleus muscle and might attenuate the atrophic process. These findings argue for the beneficial use of taurine in the treatment of disuse-induced muscle dysfunction.

Therapeutic Targets in Amyotrophic Lateral Sclerosis: Focus on Ion Channels and Skeletal Muscle.

Amyotrophic Lateral Sclerosis is a neurodegenerative disease caused by progressive loss of motor neurons, which severely compromises skeletal muscle function. Evidence shows that muscle may act as a molecular powerhouse, whose final signals generate in patients a progressive loss of voluntary muscle function and weakness leading to paralysis. This pathology is the result of a complex cascade of events that involves a crosstalk among motor neurons, glia, and muscles, and evolves through the action of converging toxic mechanisms. In fact, mitochondrial dysfunction, which leads to oxidative stress, is one of the mechanisms causing cell death. It is a common denominator for the two existing forms of the disease: sporadic and familial. Other factors include excitotoxicity, inflammation, and protein aggregation. Currently, there are limited cures. The only approved drug for therapy is riluzole, that modestly prolongs survival, with edaravone now waiting for new clinical trial aimed to clarify its efficacy. Thus, there is a need of effective treatments to reverse the damage in this devastating pathology. Many drugs have been already tested in clinical trials and are currently under investigation. This review summarizes the already tested drugs aimed at restoring muscle-nerve cross-talk and on new treatment options targeting this tissue.

Metabolic Pathways and Ion Channels Involved in Skeletal Muscle Atrophy: A Starting Point for Potential Therapeutic Strategies.

Skeletal muscle tissue has the important function of supporting and defending the organism. It is the largest apparatus in the human body, and its function is important for contraction and movements. In addition, it is involved in the regulation of protein synthesis and degradation. In fact, inhibition of protein synthesis and/or activation of catabolism determines a pathological condition called muscle atrophy. Muscle atrophy is a reduction in muscle mass resulting in a partial or complete loss of function. It has been established that many physiopathological conditions can cause a reduction in muscle mass. Nevertheless, it is not well known the molecular mechanisms and signaling processes causing this dramatic event. There are multiple concomitant processes involved in muscle atrophy. In fact, the gene transcription of some factors, oxidative stress mechanisms, and the alteration of ion transport through specific ion channels may contribute to muscle function impairment. In this review, we focused on the molecular mechanisms responsible for muscle damage and potential drugs to be used to alleviate this disabling condition.

Thrombotic events with or without thrombocytopenia in recipients of adenovirus-based COVID-19 vaccines.

COVID-19, the severe acute respiratory syndrome, is one of the major emergencies that have affected health care systems. Drugs and oxygen are only partially effective in saving lives in patients with severe COVID-19, and the most important protection from death is vaccination. The widespread use of COVID-19 adenovirus-based vaccines has provided evidence for the occurrence of rare venous thrombotic events including cerebral venous thrombosis and splanchnic venous thrombosis in recipients of Vaxzevria and Jcovden vaccines and the review focus on them. One year ago, thromboses in Vaxzevria recipients have been associated with thrombocytopenia in the presence of antibodies to platelet factor 4 and have been called vaccine-induced immune thrombotic thrombocytopenia (VITT). The incidence of VITT is equal to 9-31 events per one million doses of vaccines as evaluated by health agencies worldwide and is higher in female and young vaccine recipients. More recently, by using the European EudraVigilance database, it has been demonstrated that the incidence of thrombosis in recipients of adenovirus-based vaccines is 5-10 fold higher than that of VITT and 7-12 fold higher than observed in the recipients of Comirnaty, an mRNA-based vaccine, suggesting that adenovirus-based vaccines cause not only VITT but also thrombosis without thrombocytopenia (non-VITT thrombosis). The incidence of the vaccine-dependent non-VITT thrombosis is different in the adenovirus-based vaccines and the VITT/non-VITT incidence ratio depends on the severity of thrombosis and is inversely related to the age of the recipients. The possible causes and clinical implications of non-VITT thrombosis in vaccine recipients are discussed.

Redox homeostasis, oxidative stress and disuse muscle atrophy.

A pivotal role has been ascribed to oxidative stress in determining the imbalance between protein synthesis and degradation leading to muscle atrophy in many pathological conditions and in disuse. However, a large variability in disuse-induced alteration of redox homeostasis through muscles, models and species emerges from the literature. Whereas the causal role of oxidative stress appears well established in the mechanical ventilation model, findings are less compelling in the hindlimb unloaded mice and very limited in humans. The mere coexistence of muscle atrophy, indirect indexes of increased reactive oxygen species (ROS) production and impairment of antioxidant defence systems, in fact, does not unequivocally support a causal role of oxidative stress in the phenomenon. We hypothesise that in some muscles, models and species only, due to a large redox imbalance, the leading phenomena are activation of proteolysis and massive oxidation of proteins, which would become more susceptible to degradation. In other conditions, due to a lower extent and variable time course of ROS production, different ROS-dependent, but also -independent intracellular pathways might dominate determining the variable extent of atrophy and even dispensable protein oxidation. The ROS production and removal are complex and finely tuned phenomena. They are indeed important intracellular signals and redox balance maintains normal muscle homeostasis and can underlie either positive or negative adaptations to exercise. A precise approach to determine the levels of ROS in living cells in various conditions appears to be of paramount importance to define and support such hypotheses.

Increased sarcolemma chloride conductance as one of the mechanisms of action of carbonic anhydrase inhibitors in muscle excitability disorders.

To get insight into the mechanism of action of carbonic anhydrase inhibitors (CAI) in neuromuscular disorders, we investigated effects of dichlorphenamide (DCP) and acetazolamide (ACTZ) on ClC-1 chloride channels and skeletal muscle excitability. We performed patch-clamp experiments to test drugs on chloride currents in HEK293T cells transfected with hClC-1. Using the two-intracellular microelectrode technique in current-clamp mode, we measured the effects of drugs on the resting chloride conductance and action potential properties of sarcolemma in rat and mouse skeletal muscle fibers. Using BCECF dye fluorometry, we measured the effects of ACTZ on intracellular pH in single rat muscle fibers. Similarly to ACTZ, DCP (100 µM) increased hClC-1 chloride currents in HEK cells, because of the negative shift of the open probability voltage dependence and the slowing of deactivation kinetics. Bendroflumethiazide (BFT, 100 µM), structurally related to DCP but lacking activity on carbonic anhydrase, had little effects on chloride currents. In isolated rat muscle fibers, 50-100 µM of ACTZ or DCP, but not BFT, induced a ~ 20% increase of the resting chloride conductance. ACTZ reduced action potential firing in mouse muscle fibers. ACTZ (100 µM) reduced intracellular pH to 6.8 in rat muscle fibers. These results suggest that carbonic anhydrase inhibitors can reduce muscle excitability by increasing ClC-1 channel activity, probably through intracellular acidification. Such a mechanism may contribute in part to the clinical effects of these drugs in myotonia and other muscle excitability disorders.

Is oxidative stress a cause or consequence of disuse muscle atrophy in mice? A proteomic approach in hindlimb-unloaded mice.

Two-dimensional proteomic maps of soleus (Sol), a slow oxidative muscle, and gastrocnemius (Gas), a fast glycolytic muscle of control mice (CTRL), of mice hindlimb unloaded for 14 days (HU mice) and of HU mice treated with trolox (HU-TRO), a selective and potent antioxidant, were compared. The proteomic analysis identified a large number of differentially expressed proteins in a pool of approximately 800 proteins in both muscles. The protein pattern of Sol and Gas adapted very differently to hindlimb unloading. The most interesting adaptations related to the cellular defense systems against oxidative stress and energy metabolism. In HU Sol, the antioxidant defense systems and heat shock proteins were downregulated, and protein oxidation index and lipid peroxidation were higher compared with CTRL Sol. In contrast, in HU Gas the antioxidant defense systems were upregulated, and protein oxidation index and lipid peroxidation were normal. Notably, both Sol and Gas muscles and their muscle fibres were atrophic. Antioxidant administration prevented the impairment of the antioxidant defense systems in Sol and further enhanced them in Gas. Accordingly, it restored normal levels of protein oxidation and lipid peroxidation in Sol. However, muscle and muscle fibre atrophy was not prevented either in Sol or in Gas. A general downsizing of all energy production systems in Sol and a shift towards glycolytic metabolism in Gas were observed. Trolox administration did not prevent metabolic adaptations in either Sol or Gas. The present findings suggest that oxidative stress is not a major determinant of muscle atrophy in HU mice.

Antioxidant treatment of hindlimb-unloaded mouse counteracts fiber type transition but not atrophy of disused muscles.

Oxidative stress was proposed as a trigger of muscle impairment in various muscle diseases. The hindlimb-unloaded (HU) rodent is a model of disuse inducing atrophy and slow-to-fast transition of postural muscles. Here, mice unloaded for 14 days were chronically treated with the selective antioxidant trolox. After HU, atrophy was more pronounced in the slow-twitch soleus muscle (Sol) than in the fast-twitch gastrocnemius and tibialis anterior muscles, and was absent in extensor digitorum longus muscle. In accord with the phenotype transition, HU Sol showed a reduced expression of myosin heavy chain type 2A (MHC-2A) and increase in MHC-2X and MHC-2B isoforms. In parallel, HU Sol displayed an increased sarcolemma chloride conductance related to an increased expression of ClC-1 channels, changes in excitability parameters, a positive shift of the mechanical threshold, and a decrease of the resting cytosolic calcium concentration. Moreover, the level of lipoperoxidation increased proportionally to the degree of atrophy of each muscle type. As expected, trolox treatment fully prevented oxidative stress in HU mice. Atrophy was not prevented but the drug significantly attenuated Sol phenotypic transition and excitability changes. Trolox treatment had no effect on control mice. These results suggest possible benefits of antioxidants in protecting muscle against disuse.

Changes in Expression and Cellular Localization of Rat Skeletal Muscle ClC-1 Chloride Channel in Relation to Age, Myofiber Phenotype and PKC Modulation.

The ClC-1 chloride channel 1 is important for muscle function as it stabilizes resting membrane potential and helps to repolarize the membrane after action potentials. We investigated the contribution of ClC-1 to adaptation of skeletal muscles to needs induced by the different stages of life. We analyzed the ClC-1 gene and protein expression as well as mRNA levels of protein kinase C (PKC) alpha and theta involved in ClC-1 modulation, in soleus (SOL) and extensor digitorum longus (EDL) muscles of rats in all stage of life. The cellular localization of ClC-1 in relation to age was also investigated. Our data show that during muscle development ClC-1 expression differs according to phenotype. In fast-twitch EDL muscles ClC-1 expression increased 10-fold starting at 7 days up to 8 months of life. Conversely, in slow-twitch SOL muscles ClC-1 expression remained constant until 33 days of life and subsequently increased fivefold to reach the adult value. Aging induced a downregulation of gene and protein ClC-1 expression in both muscle types analyzed. The mRNA of PKC-theta revealed the same trend as ClC-1 except in old age, whereas the mRNA of PKC-alpha increased only after 2 months of age. Also, we found that the ClC-1 is localized in both membrane and cytoplasm, in fibers of 12-day-old rats, becoming perfectly localized on the membrane in 2-month-old rats. This study could represent a point of comparison helpful for the identification of accurate pharmacological strategies for all the pathological situations in which ClC-1 protein is altered.