A. De Luca

Chronic hypoxia up‐regulates α1H T‐type channels and low‐threshold catecholamine secretion in rat chromaffin cells

α1H T‐type channels recruited by β1‐adrenergic stimulation in rat chromaffin cells (RCCs) are coupled to fast exocytosis with the same Ca2+ dependence of high‐threshold Ca2+ channels. Here we show that RCCs exposed to chronic hypoxia (CH) for 12–18 h in 3% O2 express comparable densities of functional T‐type channels that depolarize the resting cells and contribute to low‐voltage exocytosis. Following chronic hypoxia, most RCCs exhibited T‐type Ca2+ channels already available at −50 mV with the same gating, pharmacological and molecular features as the α1H isoform. Chronic hypoxia had no effects on cell size and high‐threshold Ca2+ current density and was mimicked by overnight incubation with the iron‐chelating agent desferrioxamine (DFX), suggesting the involvement of hypoxia‐inducible factors (HIFs). T‐type channel recruitment occurred independently of PKA activation and the presence of extracellular Ca2+. Hypoxia‐recruited T‐type channels were partially open at rest (T‐type ‘window‐current’) and contributed to raising the resting potential to more positive values. Their block by 50 μm Ni2+ caused a 5–8 mV hyperpolarization. The secretory response associated with T‐type channels could be detected following mild cell depolarizations, either by capacitance increases induced by step depolarizations or by amperometric current spikes induced by increased [KCl]. In the latter case, exocytotic bursts could be evoked even with 2–4 mm KCl and spike frequency was drastically reduced by 50 μm Ni2+. Chronic hypoxia did not alter the shape of spikes, suggesting that hypoxia‐recruited T‐type channels increase the number of secreted vesicles at low voltages, without altering the mechanism of catecholamine release and the quantal content of released molecules.

Role of tumour necrosis factor α, but not of cyclo-oxygenase-2-derived eicosanoids, on functional and morphological indices of dystrophic progression in mdx mice : a pharmacological approach

The role of tumour necrosis factor (TNF)‐α or cyclo‐oxygenase‐2 (COX‐2) eicosanoids in dystrophinopathies has been evaluated by chronically treating (4–8u2003weeks) adult dystrophic mdx mice with the anti‐TNF‐α etanercept (0.5u2003mg/kg) or the COX‐2 inhibitor meloxicam (0.2u2003mg/kg). Throughout the treatment period the mdx mice underwent a protocol of exercise on treadmill in order to worsen the pathology progression; gastrocnemious muscles from exercised mdx mice showed an intense staining for TNF‐α by immunohistochemistry. In vivo, etanercept, but not meloxicam, contrasted the exercise‐induced forelimb force drop. Electrophysiological recordings ex vivo, showed that etanercept counteracted the decrease in chloride channel function (gCl), a functional index of myofibre damage, in both diaphragm and extensor digitorum longus (EDL) muscle, meloxicam being effective only in EDL muscle. None of the drugs ameliorated calcium homeostasis detected by electrophysiology and/or spectrofluorimetry. Etanercept, more than meloxicam, effectively reduced plasma creatine kinase (CK). Etanercept‐treated muscles showed a reduction of connective tissue area and of pro‐fibrotic cytokine TGF‐β1 vs. untreated ones; however, the histological profile was weakly ameliorated. In order to better evaluate the impact of etanercept treatment on histology, a 4‐week treatment was performed on 2‐week‐old mdx mice, so to match the first spontaneous degeneration cycle. The histology profile of gastrocnemious was significantly improved with a reduction of degenerating area; however, CK levels were only slightly lower. The present results support a key role of TNF‐α, but not of COX‐2 products, in different phases of dystrophic progression. Anti‐TNF‐α drugs may be useful in combined therapies for Duchenne patients.

Effects of chronic treatment with statins and fenofibrate on rat skeletal muscle: a biochemical, histological and electrophysiological study

Skeletal muscle injury by hypolipidemic drugs is not fully understood. An extensive analysis of the effect of chronic treatment with fluvastatin (5 mgkg‐1 and 20 mgkg‐1), atorvastatin (10 mgkg‐1) and fenofibrate (60 mgkg‐1) on rat skeletal muscle was undertaken.

Gating of myotonic Na channel mutants defines the response to mexiletine and a potent derivative

Background: Myotonia and periodic paralysis caused by sodium channel mutations show variable responses to the anti-myotonic drug mexiletine. Objective: To investigate whether variability among sodium channel mutants results from differences in drug binding affinity or in channel gating. Methods: Whole-cell sodium currents (INa) were recorded in tsA201 cells expressing human wild-type (WT) and mutant skeletal muscle sodium channels (A1156T, hyperkalemic periodic paralysis; R1448C, paramyotonia congenita; G1306E, potassium-aggravated myotonia). Results: At a holding potential (hp) of −120 mV, mexiletine produced a tonic (TB, 0.33 Hz) and a use-dependent (UDB, 10 Hz) block of peak INa with a potency following the order rank R1448C > WT ≈ A1156T > G1306E. Yet, when assayed from an hp of −180 mV, TB and UDB by mexiletine were similar for the four channels. The different midpoints of channel availability curves found for the four channels track the half-maximum inhibitory value (IC50) measured at −120 mV. Thus differences in the partitioning of channels between the closed and fast-inactivated states underlie the different IC50 measured at a given potential. The mexiletine-derivative, Me7 (α-[(2-methylphenoxy)methyl]-benzenemethanamine), behaved similarly but was ∼5 times more potent than mexiletine. Interestingly, the higher drug concentrations ameliorated the abnormally slower decay rate of myotonic INa. Conclusions: These results explain the basis of the apparent difference in block of mutant sodium channels by mexiletine and Me7, opening the way to a more rationale drug use and to design more potent drugs able to correct specifically the biophysical defect of the mutation in individual myotonic patients.

Aging and chloride channel regulation in rat fast-twitch muscle fibres

By the use of pharmacological tools, we tested the hypothesis that age-related alterations in the regulatory pathways of chloride channels might contribute to the lowered chloride conductance (GCl) found in skeletal muscle of aged rats. The restingGCl of extensor digitorum longus (EDL) muscles from adult rats either young (3–4 months old) or aged (29 months old) was measured by means of computerized intracellular microelectrode recordings. In EDL muscle from 3 to 4-month-old rats, 4-β-phorbol 12,13-dibutyrate (4-β-PDB), a direct activator of protein kinase C (PKC), decreasedGCl in a concentration-dependent manner. The same effect was exerted by cholera toxin. The effects of both the phorbol ester and cholera toxin were inhibited by staurosporine, thus indicating that either direct or indirect (via G protein) activation of PKC accounts for the decrease ofGCl. An increase of cytosolic Ca2+ by the ionophore A23187 also significantly decreasedGCl by 25%. In EDL muscles from aged rats, 4-β-PDB was 20-fold more potent in blockingGCl than in muscles from younger controls, and the ionophore blockedGCl by 40%. On the other hand, cholera toxin was ineffective. Our findings support the hypothesis that in fast-twitch muscle the regulation of chloride channels by PKC and Ca2+ is a target of the aging process.

Stereoselective effects of mexiletine enantiomers on sodium currents and excitability characteristics of adult skeletal muscle fibers

The effects of the enantiomers of mexiletine were tested on sodium currents of frog skeletal muscle fibers recorded by means of the three vaseline gap voltage clamp method and compared with the effects produced by tocainide enantiomers. The R-( − ) mexiletine produced a tonic block of the sodium current, elicited by single depolarizing test pulses from the holding potential of − 100 mV to − 20 mV, with an IC50 of 43.9 ± 1 μM, whereas the corresponding S-( + ) enantiomer produced the same effects at about twofold higher concentrations. A similar stereoselectivity was observed with tocainide enantiomers, but at about 5 fold higher concentrations. Both the R-( − ) and S-( + ) enantiomers of mexiletine and tocainide produced a further use-dependent block of sodium currents when the test pulse was applied repetitively at a frequency of 2 Hz. The use dependent behaviour led to a significant lowering of the IC50 values with respect to the tonic block but the eudismic ratios ([IC50S-( + )]/[IC50R( − )]) and the relative potency between mexiletine and tocainide were maintained. All the tested compounds produced a left shift of the steady state inactivation curves (h∞) , suggesting a high-affinity interaction with the inactivated sodium channels. Again a stronger potency of R-( − ) vs. S-( + ) enantiomers and of mexiletine vs. tocainide was observed. The excitability characteristics recorded from the semitendinosus muscle by the two microelectrode technique were modified by the tested drugs in agreement with their ability to block sodium current. Thus a concentration-related increase in the threshold current required to elicit an action potential was observed along with a decrease in the amplitude and a shortening of the latency of action potential and a decrease in the firing capability of the membrane. Again the R-( − ) isomers were more potent than the S-( + ) ones and mexiletine was more effective than tocainide. These data corroborate the presence of a stereospecific site for these drugs on adult skeletal muscle sodium channels. The constant eudismic ratios between the enantiomers during both tonic and use-dependent block suggest that the increase in the apparent affinity of the receptor during statedependent conformational changes of the channel does not enhance its stereospecificity. The decrease in effective concentration upon high frequency stimulation supports the potential usefulness of low doses of R-( − ) mexiletine in the treatment of the abnormal hyperexcitability of the myotonic muscles, with a likely reduction of unwanted side effects.

Statins and fenofibrate affect skeletal muscle chloride conductance in rats by differently impairing ClC-1 channel regulation and expression

Background and purpose:u2002 Statins and fibrates can produce mild to life‐threatening skeletal muscle damage. Resting chloride channel conductance (gCl), carried by the ClC‐1 channel, is reduced in muscles of rats chronically treated with fluvastatin, atorvastatin or fenofibrate, along with increased resting cytosolic calcium in statin‐treated rats. A high gCl, controlled by the Ca2+‐dependent protein kinase C (PKC), maintains sarcolemma electrical stability and its reduction alters muscle function. Here, we investigated how statins and fenofibrate impaired gCl.

Evaluation of potential synergistic action of a combined treatment with alpha-methyl-prednisolone and taurine on the mdx mouse model of Duchenne muscular dystrophy

A. Cozzoli, J.‐F. Rolland, R. F. Capogrosso, V. T. Sblendorio, V. Longo, S. Simonetti, B. Nico and A. De Luca (2011) Neuropathology and Applied Neurobiology37, 243–256u2028Evaluation of potential synergistic action of a combined treatment with alpha‐methyl‐prednisolone and taurine on the mdx mouse model of Duchene muscular dystrophy

Pharmacological block of chloride channels of developing rat skeletal muscle affects the differentiation of specific contractile properties

A specific chloride channel blocker, anthracene 9-carboxylic acid was locally applied for 8–9 days on the extensor digitorum longus muscle of 7–8-day-old rats. The effects of chronic anthracene 9-carboxylic acid treatment on muscle development, were evaluated in vitro on the electrical properties with intracelluar microelectrodes and in vivo on the contractile parameters by recording isometric concentractions. Our data show that the treatment prevented the normal development of chloride conductance so that by 15 days of age it was 45% lower in fibers of the treated muscles when compared to age-related control fibers. Potassium conductance was not significantly changed by the treatment. In vivo the anthracene-9-carboxylic acid-treated muscles were slower to contract and relax; having a 20% slower time to peak twitch force and time of half relaxation. These muscles were also 32% less fatiguable with respect to the controls. Moreover, in most of the treated muscles tetanic contractions during high-frequency stimulation were not maintained. The block of chloride channels in developing striated fibers appears to affect the differentiation of specific properties of fast skeletal muscle such as the speed of contraction.

Evaluation of the pharmacological activity of the major mexiletine metabolites on skeletal muscle sodium currents

Mexiletine (Mex), an orally effective antiarrhythmic agent used to treat ventricular arrhythmias, has also been found to be effective for myotonia and neuropathic pain. It is extensively metabolized in humans but little information exists about the pharmacodynamic properties of its metabolites.