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.

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.

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.

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 Ca(2+)-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.

Sodium channel myotonia and paramyotonia congenita are caused by gain-of-function mutations in the skeletal muscle voltage-gated sodium channel hNav1.4. The first-line drug is the sodium channel blocker mexiletine; however, some patients show side effects or limited responses. We previously showed that two hNav1.4 mutations, p.G1306E and p.P1158L, reduce mexiletine potency in vitro, whereas another sodium channel blocker, flecainide, is less sensitive to mutation-induced gating defects. This observation was successfully translated to p.G1306E and p.P1158L carriers. Thus, the aim of this study was to perform a pharmacological characterization of myotonic Nav1.4 mutations clustered near the fast inactivation gate of the channel. We chose seven mutations (p.V1293I, p.N1297S, p.N1297K, p.F1298C, p.G1306E, p.I1310N, and p.T1313M) from the database of Italian and French networks for muscle channelopathies. Recombinant hNav1.4 mutants were expressed in HEK293T cells for functional and pharmacological characterization using the patch-clamp technique. All the studied mutations impair the kinetics and/or voltage dependence of fast inactivation, which is likely the main mechanism responsible for myotonia. The severity of myotonia is well-correlated to the enhancement of window currents generated by the intersection of the activation and fast inactivation voltage dependence. Five of the six mutants displaying a significant positive shift of fast inactivation voltage dependence reduced mexiletine inhibition in an experimental condition mimicking myotonia. In contrast, none of the mutations impairs flecainide block nor does p.T1313M impair propafenone block, indicating that class Ic antiarrhythmics may constitute a valuable alternative. Our study suggests that mutation-driven therapy would be beneficial to myotonic patients, greatly improving their quality of life.

Episodic ataxia type 1 (EA1) is a human dominant neurological syndrome characterized by continuous myokymia, episodic attacks of ataxic gait and spastic contractions of skeletal muscles that can be triggered by emotional stress and fatigue. This rare disease is caused by missense mutations in the KCNA1 gene coding for the neuronal voltage gated potassium channel Kv1.1, which contributes to nerve cell excitability in the cerebellum, hippocampus, cortex and peripheral nervous system. We identified a novel KCNA1 mutation, E283K, in an Italian proband presenting with paroxysmal ataxia and myokymia aggravated by painful contractures and metabolic dysfunctions. The E283K mutation is located in the S3-S4 extracellular linker belonging to the voltage sensor domain of Kv channels. In order to test whether the E283K mutation affects Kv1.1 biophysical properties we transfected HEK293 cells with WT or mutant cDNAs alone or in a 1:1 combination, and recorded relative potassium currents in the whole-cell configuration of patch-clamp. Mutant E283K channels display voltage-dependent activation shifted by 10mV toward positive potentials and kinetics of activation slowed by ~2 fold compared to WT channels. Potassium currents resulting from heteromeric WT/E283K channels show voltage-dependent gating and kinetics of activation intermediate between WT and mutant homomeric channels. Based on homology modeling studies of the mutant E283K, we propose a molecular explanation for the reduced voltage sensitivity and slow channel opening. Overall, our results suggest that the replacement of a negatively charged residue with a positively charged lysine at position 283 in Kv1.1 causes a drop of potassium current that likely accounts for EA-1 symptoms in the heterozygous carrier.

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 Bartter's 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.

In 2019-2020, the SARS-CoV-2 pandemic has shocked the world and most health care systems, and a "second wave" of the viral spread is ongoing in Europe and in Italy too. While, at the initial outbreak, the treatment of patients had focused on the respiratory symptoms, many diverse clinical manifestations of the disease have to date been reported. However, the complete course of the disease has not yet been fully clarified. In particular, several reports from the real-world clinical practice have highlighted the noxious effects of SARS-CoV-2 on skeletal muscles. In this brief review, we summarized the main current findings about muscular and neuromuscular damages that may be triggered by the virus or by the drugs used to treat COVID-19. Moreover, we underlined the need of attentive care and vigilance for patients with neuro-muscular disorders, who may be particularly susceptible to infection and at increased risk for severe COVID-19.