Blockers of Skeletal Muscle Nav1.4 Channels: From Therapy of Myotonic Syndrome to Molecular Determinants of Pharmacological Action and Back.

The voltage-gated sodium channels represent an important target for drug discovery since a large number of physiological processes are regulated by these channels. In several excitability disorders, including epilepsy, cardiac arrhythmias, chronic pain, and non-dystrophic myotonia, blockers of voltage-gated sodium channels are clinically used. Myotonia is a skeletal muscle condition characterized by the over-excitability of the sarcolemma, resulting in delayed relaxation after contraction and muscle stiffness. The therapeutic management of this disorder relies on mexiletine and other sodium channel blockers, which are not selective for the Nav1.4 skeletal muscle sodium channel isoform. Hence, the importance of deepening the knowledge of molecular requirements for developing more potent and use-dependent drugs acting on Nav1.4. Here, we review the available treatment options for non-dystrophic myotonia and the structure-activity relationship studies performed in our laboratory with a focus on new compounds with potential antimyotonic activity.

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.

Assessment of resveratrol, apocynin and taurine on mechanical-metabolic uncoupling and oxidative stress in a mouse model of duchenne muscular dystrophy: A comparison with the gold standard, α-methyl prednisolone.

Antioxidants have a great potential as adjuvant therapeutics in patients with Duchenne muscular dystrophy, although systematic comparisons at pre-clinical level are limited. The present study is a head-to-head assessment, in the exercised mdx mouse model of DMD, of natural compounds, resveratrol and apocynin, and of the amino acid taurine, in comparison with the gold standard α-methyl prednisolone (PDN). The rationale was to target the overproduction of reactive oxygen species (ROS) via disease-related pathways that are worsened by mechanical-metabolic impairment such as inflammation and over-activity of NADPH oxidase (NOX) (taurine and apocynin, respectively) or the failing ROS detoxification mechanisms via sirtuin-1 (SIRT1)-peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α) (resveratrol). Resveratrol (100mg/kg i.p. 5days/week), apocynin (38mg/kg/day per os), taurine (1g/kg/day per os), and PDN (1mg/kg i.p., 5days/week) were administered for 4-5 weeks to mdx mice in parallel with a standard protocol of treadmill exercise and the outcome was evaluated with a multidisciplinary approach in vivo and ex vivo on pathology-related end-points and biomarkers of oxidative stress. Resveratrol≥taurine>apocynin enhanced in vivo mouse force similarly to PDN. All the compounds reduced the production of superoxide anion, assessed by dihydroethidium staining, with apocynin being as effective as PDN, and ameliorated electrophysiological biomarkers of oxidative stress. Resveratrol also significantly reduced plasma levels of creatine kinase and lactate dehydrogenase. Force of isolated muscles was little ameliorated. However, the three compounds improved histopathology of gastrocnemius muscle more than PDN. Taurine>apocynin>PDN significantly decreased activated NF-kB positive myofibers. Thus, compounds targeting NOX-ROS or SIRT1/PGC-1α pathways differently modulate clinically relevant DMD-related endpoints according to their mechanism of action. With the caution needed in translational research, the results show that the parallel assessment can help the identification of best adjuvant therapies.

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.

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.

Combined modifications of mexiletine pharmacophores for new lead blockers of Na(v)1.4 channels.

Previously identified potent and/or use-dependent mexiletine (Mex) analogs were used as template for the rational design of new Na(v)-channel blockers. The effects of the novel analogs were tested on sodium currents of native myofibers. Data and molecular modeling show that increasing basicity and optimal alkyl chain length enhance use-dependent block. This was demonstrated by replacing the amino group with a more basic guanidine one while maintaining a proper distance between positive charge and aromatic ring (Me13) or with homologs having the chirality center nearby the amino group or the aromatic ring. Accordingly, a phenyl group on the asymmetric center in the homologated alkyl chain (Me12), leads to a further increase of use-dependent behavior versus the phenyl Mex derivative Me4. A fluorine atom in paraposition and one ortho-methyl group on the xylyloxy ring (Me15) increase potency and stereoselectivity versus Me4. Charge delocalization and greater flexibility of Me15 may increase its affinity for Tyr residues influencing steric drug interaction with the primary Phe residue of the binding site. Me12 and Me15 show limited selectivity against Na(v)-isoforms, possibly due to the highly conserved binding site on Na(v). To our knowledge, the new compounds are the most potent Mex-like Na(v) blockers obtained to date and deserve further investigation.

Branched-Chain Amino Acids and Di-Alanine Supplementation in Aged Mice: A Translational Study on Sarcopenia.

In age-related sarcopenia, the gradual loss of skeletal muscle mass, function and strength is underpinned by an imbalanced rate of protein synthesis/breakdown. Hence, an adequate protein intake is considered a valuable strategy to mitigate sarcopenia. Here, we investigated the effects of a 12-week oral supplementation with branched-chain amino acids (BCAAs: leucine, isoleucine, and valine) with recognized anabolic properties, in 17-month-old (AGED) C57BL/6J male mice. BCAAs (2:1:1) were formulated in drinking water, alone or plus two L-Alanine equivalents (2ALA) or dipeptide L-Alanyl-L-Alanine (Di-ALA) to boost BCAAs bioavailability. Outcomes were evaluated on in/ex vivo readouts vs. 6-month-old (ADULT) mice. In vivo hind limb plantar flexor torque was improved in AGED mice treated with BCAAs + Di-ALA or 2ALA (recovery score, R.S., towards ADULT: ≥20%), and all mixtures significantly increased hind limb volume. Ex vivo, myofiber cross-sectional areas were higher in gastrocnemius (GC) and soleus (SOL) muscles from treated mice (R.S. ≥ 69%). Contractile indices of isolated muscles were improved by the mixtures, especially in SOL muscle (R.S. ≥ 20%). The latter displayed higher mTOR protein levels in mice supplemented with 2ALA/Di-ALA-enriched mixtures (R.S. ≥ 65%). Overall, these findings support the usefulness of BCAAs-based supplements in sarcopenia, particularly as innovative formulations potentiating BCAAs bioavailability and effects.

Growth hormone secretagogues modulate inflammation and fibrosis in mdx mouse model of Duchenne muscular dystrophy.

Introduction: Growth hormone secretagogues (GHSs) exert multiple actions, being able to activate GHS-receptor 1a, control inflammation and metabolism, to enhance GH/insulin-like growth factor-1 (IGF-1)-mediated myogenesis, and to inhibit angiotensin-converting enzyme. These mechanisms are of interest for potentially targeting multiple steps of pathogenic cascade in Duchenne muscular dystrophy (DMD). Methods: Here, we aimed to provide preclinical evidence for potential benefits of GHSs in DMD, via a multidisciplinary in vivo and ex vivo comparison in mdx mice, of two ad hoc synthesized compounds (EP80317 and JMV2894), with a wide but different profile. 4-week-old mdx mice were treated for 8 weeks with EP80317 or JMV2894 (320 µg/kg/d, s.c.). Results: In vivo, both GHSs increased mice forelimb force (recovery score, RS towards WT: 20% for EP80317 and 32% for JMV2894 at week 8). In parallel, GHSs also reduced diaphragm (DIA) and gastrocnemius (GC) ultrasound echodensity, a fibrosis-related parameter (RS: ranging between 26% and 75%). Ex vivo, both drugs ameliorated DIA isometric force and calcium-related indices (e.g., RS: 40% for tetanic force). Histological analysis highlighted a relevant reduction of fibrosis in GC and DIA muscles of treated mice, paralleled by a decrease in gene expression of TGF-ß1 and Col1a1. Also, decreased levels of pro-inflammatory genes (IL-6, CD68), accompanied by an increment in Sirt-1, PGC-1α and MEF2c expression, were observed in response to treatments, suggesting an overall improvement of myofiber metabolism. No detectable transcript levels of GHS receptor-1a, nor an increase of circulating IGF-1 were found, suggesting the presence of a novel receptor-independent mechanism in skeletal muscle. Preliminary docking studies revealed a potential binding capability of JMV2894 on metalloproteases involved in extracellular matrix remodeling and cytokine production, such as ADAMTS-5 and MMP-9, overactivated in DMD. Discussion: Our results support the interest of GHSs as modulators of pathology progression in mdx mice, disclosing a direct anti-fibrotic action that may prove beneficial to contrast pathological remodeling.

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.

Oxtr/TRPV1 expression and acclimation of skeletal muscle to cold-stress in male mice.

We explored the involvement of oxytocin receptor (Oxtr)/transient-receptor-potential-vanilloid-1 (TRPV1) genes and oxytocin (Oxt) on the adaptation of skeletal muscle to cold stress challenge in mice. Oxtr expression in hypothalamic paraventricular (PVN), supraoptic nuclei (SON), and hippocampus (HIPP) were evaluated by immunohistochemistry in parallel with the measurement of circulating Oxt. The Oxtr and TRPV1 gene expressions in soleus (SOL) and tibialis anterior (TA) muscles were investigated by RT-PCR. Histological studies of the cardiac muscle after cold stress were also performed. Male mice (n = 15) were divided into controls maintained at room temperature (RT = 24°C), exposed to cold stress (CS) at T = 4°C for 6 h , and 5 days. Immunohistochemical studies showed that Oxtr protein expression increased by two-fold (P = 0.01) in PVN and by 1.5-fold (P = 0.0001) in HIPP after 6 h- and 5 days of CS but decreased by 2-fold (P = 0.026) in SON in 5 days. Both Oxtr and TRPV1 gene expression increased after 6 h and 5 days of CS in SOL and TA muscles. Oxtr vs TRPV1 gene expression in SOL and TA muscles evaluated by regression analysis was linearly correlated following CS at 6 h and 5 days but not at control temperature of 24 ± 1°C, supporting the hypothesis of coupling between these genes. The circulating levels of Oxt are unaffected after 6 h of CS but decreased by 0.2-fold (P = 0.0141) after 5 days-CS. This is the first report that Oxtr and TRPV1 expressions are upregulated in response to cold acclimation in skeletal muscle. The up-regulation of Oxtr in PVN and HIPP balances the decrease of circulating Oxt.

ß-Dystroglycan Restoration and Pathology Progression in the Dystrophic mdx Mouse: Outcome and Implication of a Clinically Oriented Study with a Novel Oral Dasatinib Formulation.

ROS-activated cSrc tyrosine kinase (TK) promotes the degradation of ß-dystroglycan (ß-DG), a dystrophin-glycoprotein complex component, which may reinforce damaging signals in Duchenne muscular dystrophy (DMD). Therefore, cSrc-TK represents a promising therapeutic target. In mdx mice, a 4-week subcutaneous treatment with dasatinib (DAS), a pan-Src-TKs inhibitor approved as anti-leukemic agent, increased muscle ß-DG, with minimal amelioration of morphofunctional indices. To address possible dose/pharmacokinetic (PK) issues, a new oral DAS/hydroxypropyl(HP)-ß-cyclodextrin(CD) complex was developed and chronically administered to mdx mice. The aim was to better assess the role of ß-DG in pathology progression, meanwhile confirming DAS mechanism of action over the long-term, along with its efficacy and tolerability. The 4-week old mdx mice underwent a 12-week treatment with DAS/HP-ß-CD10% dissolved in drinking water, at 10 or 20 mg/kg/day. The outcome was evaluated via in vivo/ex vivo disease-relevant readouts. Oral DAS/HP-ß-CD efficiently distributed in mdx mice plasma and tissues in a dose-related fashion. The new DAS formulation confirmed its main upstream mechanism of action, by reducing ß-DG phosphorylation and restoring its levels dose-dependently in both diaphragm and gastrocnemius muscle. However, it modestly improved in vivo neuromuscular function, ex vivo muscle force, and histopathology, although the partial recovery of muscle elasticity and the decrease of CK and LDH plasma levels suggest an increased sarcolemmal stability of dystrophic muscles. Our clinically oriented study supports the interest in this new, pediatric-suitable DAS formulation for proper exposure and safety and for enhancing ß-DG expression. This latter mechanism is, however, not sufficient by itself to impact on pathology progression. In-depth analyses will be dedicated to elucidating the mechanism limiting DAS effectiveness in dystrophic settings, meanwhile assessing its potential synergy with dystrophin-based molecular therapies.

Synthesis and in vitro sodium channel blocking activity evaluation of novel homochiral mexiletine analogs.

New chiral mexiletine analogs were synthesized in their optically active forms and evaluated in vitro as use-dependent blockers of skeletal muscle sodium channels. Tests carried out on sodium currents of single muscle fibers of Rana esculenta demonstrated that all of them exerted a higher use-dependent block than mexiletine. The most potent analog, (S)-3-(2,6-dimethylphenoxy)-1-phenylpropan-1-amine (S)-(5), was six-fold more potent than (R)-Mex in producing a tonic block. As observed with mexiletine, the newly synthesized compounds exhibit modest enantioselective behavior, that is more evident in 3-(2,6-dimethylphenoxy)butan-1-amine (3).

Ergogenic Effect of BCAAs and L-Alanine Supplementation: Proof-of-Concept Study in a Murine Model of Physiological Exercise.

BACKGROUND: Branched-chain amino acids (BCAAs: leucine, isoleucine, valine) account for 35% of skeletal muscle essential amino acids (AAs). As such, they must be provided in the diet to support peptide synthesis and inhibit protein breakdown. Although substantial evidence has been collected about the potential usefulness of BCAAs in supporting muscle function and structure, dietary supplements containing BCAAs alone may not be effective in controlling muscle protein turnover, due to the rate-limiting bioavailability of other AAs involved in BCAAs metabolism. METHODS: We aimed to evaluate the in vivo/ex vivo effects of a 4-week treatment with an oral formulation containing BCAAs alone (2:1:1) on muscle function, structure, and metabolism in a murine model of physiological exercise, which was compared to three modified formulations combining BCAAs with increasing concentrations of L-Alanine (ALA), an AA controlling BCAAs catabolism. RESULTS: A preliminary pharmacokinetic study confirmed the ability of ALA to boost up BCAAs bioavailability. After 4 weeks, mix 2 (BCAAs + 2ALA) had the best protective effect on mice force and fatigability, as well as on muscle morphology and metabolic indices. CONCLUSION: Our study corroborates the use of BCAAs + ALA to support muscle health during physiological exercise, underlining how the relative BCAAs/ALA ratio is important to control BCAAs distribution.

Safinamide's potential in treating nondystrophic myotonias: Inhibition of skeletal muscle voltage-gated sodium channels and skeletal muscle hyperexcitability in vitro and in vivo.

The antiarrhythmic sodium-channel blocker mexiletine is used to treat patients with myotonia. However, around 30% of patients do not benefit from mexiletine due to poor tolerability or suboptimal response. Safinamide is an add-on therapy to levodopa for Parkinson's disease. In addition to MAOB inhibition, safinamide inhibits neuronal sodium channels, conferring anticonvulsant activity in models of epilepsy. Here, we investigated the effects of safinamide on skeletal muscle hNav1.4 sodium channels and in models of myotonia, in-vitro and in-vivo. Using patch-clamp, we showed that safinamide reversibly inhibited sodium currents in HEK293T cells transfected with hNav1.4. At the holding potential (hp) of -120 mV, the half-maximum inhibitory concentrations (IC50) were 160 and 33 µM at stimulation frequencies of 0.1 and 10 Hz, respectively. The calculated affinity constants of safinamide were dependent on channel state: 420 µM for closed channels and 9 µM for fast-inactivated channels. The p.F1586C mutation in hNav1.4 greatly impaired safinamide inhibition, suggesting that the drug binds to the local anesthetic receptor site in the channel pore. In a condition mimicking myotonia, i.e. hp. of -90 mV and 50-Hz stimulation, safinamide inhibited INa with an IC50 of 6 µM, being two-fold more potent than mexiletine. Using the two-intracellular microelectrodes current-clamp method, action potential firing was recorded in vitro in rat skeletal muscle fibers in presence of the chloride channel blocker, 9-anthracene carboxylic acid (9-AC), to increase excitability. Safinamide counteracted muscle fiber hyperexcitability with an IC50 of 13 µM. In vivo, oral safinamide was tested in the rat model of myotonia. In this model, intraperitoneal injection of 9-AC greatly increased the time of righting reflex (TRR) due to development of muscle stiffness. Safinamide counteracted 9-AC induced TRR increase with an ED50 of 1.2 mg/kg, which is 7 times lower than that previously determined for mexiletine. In conclusion, safinamide is a potent voltage and frequency dependent blocker of skeletal muscle sodium channels. Accordingly, the drug was able to counteract abnormal muscle hyperexcitability induced by 9-AC, both in vitro and in vivo. Thus, this study suggests that safinamide may have potential in treating myotonia and warrants further preclinical and human studies to fully evaluate this possibility.

Elucidating the Contribution of Skeletal Muscle Ion Channels to Amyotrophic Lateral Sclerosis in search of new therapeutic options.

The discovery of pathogenetic mechanisms is essential to identify new therapeutic approaches in Amyotrophic Lateral Sclerosis (ALS). Here we investigated the role of the most important ion channels in skeletal muscle of an ALS animal model (MLC/SOD1G93A) carrying a mutated SOD1 exclusively in this tissue, avoiding motor-neuron involvement. Ion channels are fundamental proteins for muscle function, and also to sustain neuromuscular junction and nerve integrity. By a multivariate statistical analysis, using machine learning algorithms, we identified the discriminant genes in MLC/SOD1G93A mice. Surprisingly, the expression of ClC-1 chloride channel, present only in skeletal muscle, was reduced. Also, the expression of Protein Kinase-C, known to control ClC-1 activity, was increased, causing its inhibition. The functional characterization confirmed the reduction of ClC-1 activity, leading to hyperexcitability and impaired relaxation. The increased expression of ion channel coupled AMPA-receptor may contribute to sustained depolarization and functional impairment. Also, the decreased expression of irisin, a muscle-secreted peptide protecting brain function, may disturb muscle-nerve connection. Interestingly, the in-vitro application of chelerythrine or acetazolamide, restored ClC-1 activity and sarcolemma hyperexcitability in these mice. These findings show that ion channel function impairment in skeletal muscle may lead to motor-neuron increased vulnerability, and opens the possibility to investigate on new compounds as promising therapy.

A long-term treatment with taurine prevents cardiac dysfunction in mdx mice.

Taurine is an amino acid abundantly present in heart and skeletal muscle. Duchenne muscular dystrophy (DMD) is a genetic disorder in which the absence of dystrophin leads to skeletal muscle wasting and heart failure. An altered taurine metabolism has been described in dystrophic animals and short-term taurine administration exerts promising amelioration of early muscular alterations in the mdx mouse model of DMD. To reinforce the therapeutic and nutraceutical taurine potential in DMD, we evaluated the effects of a long-term treatment on cardiac and skeletal muscle function of mdx mice in a later disease stage. Taurine was administered in drinking water (1 g/kg/day) to wt and mdx mice for 6 months, starting at 6 months of age. Ultrasonography evaluation of heart and hind limb was performed, in parallel with in vivo and ex vivo functional tests and biochemical, histological and gene expression analyses. 12-month-old mdx mice showed a significant worsening of left ventricular function parameters (shortening fraction, ejection fraction, stroke volume), which were significantly counteracted by the taurine treatment. In parallel, histologic signs of damage were reduced by taurine along with the expression of proinflammatory myocardial IL-6. Interestingly, no effects were observed on hind limb volume and percentage of vascularization or on in vivo and ex vivo muscle functional parameters, suggesting a tissue-specific action of taurine in relation to the disease phase. A trend toward increase in taurine was found in heart and quadriceps from treated animals, paralleled by a slight decrease in mdx mice plasma. Our study provides evidences that taurine can prevent late heart dysfunction in mdx mice, further corroborating the interest on this amino acid toward clinical trials.

Gentamicin treatment in exercised mdx mice: Identification of dystrophin-sensitive pathways and evaluation of efficacy in work-loaded dystrophic muscle.

Aminoglycosides force read through of premature stop codon mutations and introduce new mutation-specific gene-corrective strategies in Duchenne muscular dystrophy. A chronic treatment with gentamicin (32 mg/kg/daily i.p., 8-12 weeks) was performed in exercised mdx mice with the dual aim to clarify the dependence on dystrophin of the functional, biochemical and histological alterations present in dystrophic muscle and to verify the long term efficiency of small molecule gene-corrective strategies in work-loaded dystrophic muscle. The treatment counteracted the exercise-induced impairment of in vivo forelimb strength after 6-8 weeks. We observed an increase in dystrophin expression level in all the fibers, although lower than that observed in normal fibers, and found a concomitant recovery of aquaporin-4 at sarcolemma. A significant reduction in centronucleated fibers, in the area of necrosis and in the percentage of nuclear factor-kB-positive nuclei was observed in gastrocnemious muscle of treated animals. Plasma creatine kinase was reduced by 70%. Ex vivo, gentamicin restored membrane ionic conductance in mdx diaphragm and limb muscle fibers. No effects were observed on the altered calcium homeostasis and sarcolemmal calcium permeability, detected by electrophysiological and microspectrofluorimetric approaches. Thus, the maintenance of a partial level of dystrophin is sufficient to reinforce sarcolemmal stability, reducing leakiness, inflammation and fiber damage, while correction of altered calcium homeostasis needs greater expression of dystrophin or direct interventions on the channels involved.