Biomedical subjects

Jean-François Desaphy

Publications and source records attributed to Jean-François Desaphy.

11 recordsLinked to original sources

PubMed2005

Fiber type-related changes in rat skeletal muscle calcium homeostasis during aging and restoration by growth hormone.

The mechanisms by which aging induces muscle impairment are not well understood yet. We studied the impact of aging on Ca2+ homeostasis in the slow-twitch soleus and the fast-twitch extensor digitorum longus (EDL) muscles of aged rats by using the fura-2 fluorescent probe. In both muscles aging increases the resting cytosolic calcium concentration ([Ca2+]i). This effect was independent on calcium influx since a reduced resting permeability of sarcolemma to divalent cations was observed in aged muscles likely due to a reduced activity of leak channels. Importantly the effects of aging on resting [Ca2+]i, fiber diameter, mechanical threshold and sarcolemmal resting conductances were less pronounced in the soleus muscle, suggesting that muscle impairment may be less dependent on [Ca2+]i in the slow-twitch muscle. The treatment of aged rats with growth hormone restored the resting [Ca2+]i toward adult values in both muscles. Thus, an increase of resting [Ca2+]i may contribute to muscle weakness associated with aging and may be considered for developing new therapeutic strategies in the elderly.

Aging↗

PubMed2005

Recovery of the soleus muscle after short- and long-term disuse induced by hindlimb unloading: effects on the electrical properties and myosin heavy chain profile.

The hindlimb unloading (HU) rat is a model of muscle disuse characterized by atrophy and slow-to-fast phenotype transition of the postural muscles, such as the soleus. We previously found that the resting sarcolemmal chloride conductance (gCl) that is typically lower in slow-twitch myofibers than in fast ones increased in soleus fibers following 1 to 3 weeks of HU in accord with the slow-to-fast transition of myosin heavy chain (MHC) isoforms. Nevertheless, the gCl already raised after a 3-day HU, whereas no change in MHC expression was detected. The present work evaluates the ability of soleus muscle to recover on return to normal load after a short (3 days) or long (2 weeks) disuse period. The changes observed after a 2-week HU were slowly reversible, since 3-4 weeks of reloading were needed to completely recover gCl, fiber diameter, MHC expression pattern, as well as the mechanical threshold Rheobase, an index of calcium homeostasis. After 3-day HU, the gCl increased homogeneously in most of the soleus muscle fibers and gCl recovery was rapidly completed after 4-day reloading. These results suggest different induction mechanisms for gCl augmentation after the short and long HU periods, as well as a possible role for gCl in the slow muscle adaptation to disuse.

Adaptation, Physiological↗

PubMed2004

New potent mexiletine and tocainide analogues evaluated in vivo and in vitro as antimyotonic agents on the myotonic ADR mouse.

The antimyotonic activity of chiral derivatives of mexiletine and tocainide, selected as potent use-dependent blockers of skeletal muscle sodium channels, was evaluated in vivo acutely in myotonic ADR mice. The compounds had either aromatic (Me4 and Me6) or branched isopropyl groups (Me5 and To1) on the asymmetric centre, or had this latter one methylene apart from the amino group (Me2). Therapeutic doses of mexiletine (5-10 mg/kg) and tocainide (7-20 mg/kg) significantly reduced the long time of righting reflex (TRR), typical of ADR mice. Me4, Me5 and Me6 were 2-fold more potent than mexiletine. To1 fully normalised the TRR at 7 mg/kg. The electromyographic analysis confirmed a muscle-based activity for drug effectiveness on TRR. All the compounds reduced the myotonic hyperexcitability of intercostal muscle fibres when tested in vitro by current-clamp recordings, with a potency correlated with their action on sodium channels. On stimulus-evoked firing, the isopropyl analogues were 2-4-fold more potent than parent compounds, while the aromatic analogues were about 10-fold more potent than mexiletine. Patch-clamp recordings confirmed a normal-like pharmacological sensitivity of sodium channels of native ADR muscle fibres. Finally, the in vivo antimyotonic activity is due to the block of sodium channels and divergences with in vitro potency can be related to structure-based changes in drug pharmacokinetics.

Action Potentials↗

PubMed2004

Taurine and skeletal muscle disorders.

Taurine is abundantly present in skeletal muscle. We give evidence that this amino acid exerts both short-term and long-term actions in the control of ion channel function and calcium homeostasis in striated fibers. Short-term actions can be estimated as the ability of this amino acid to acutely modulate both ion channel gating and the function of the structures involved in calcium handling. Long-term effects can be disclosed in situations of tissue taurine depletion and are likely related to the ability of the intracellular taurine to control transducing pathways as well as homeostatic and osmotic equilibrium in the tissue. The two activities are strictly linked because the intracellular level of taurine modulates the sensitivity of skeletal muscle to the exogenous application of taurine. Myopathies in which ion channels are directly or indirectly involved, as well as inherited or acquired pathologies characterized by metabolic alterations and change in calcium homeostasis, are often correlated with change in muscle taurine concentration and consequently with an enhanced therapeutic activity of this amino acid. We discuss both in vivo and in vitro evidence that taurine, through its ability to control sarcolemmal excitability and muscle contractility, can prove beneficial effects in many muscle dysfunctions.

Aging↗

PubMed2003

Different flecainide sensitivity of hNav1.4 channels and myotonic mutants explained by state-dependent block.

Flecainide, a class IC antiarrhythmic, was shown to improve myotonia caused by sodium channel mutations in situations where the class IB antiarrhythmic drug mexiletine was less efficient. Yet little is known about molecular interactions between flecainide and human skeletal muscle sodium (hNa(v)1.4) channels. Whole-cell sodium currents (I(Na)) were recorded in tsA201 cells expressing wild-type (WT) and mutant hNa(v)1.4 channels (R1448C, paramyotonia congenita; G1306E, potassium-aggravated myotonia). At a holding potential (HP) of -120 mV, flecainide use-dependently blocked WT and G1306E I(Na) equally but was more potent on R1448C channels. For WT, the extent of block depended on a holding voltage more negative than the activation threshold, being greater at -90 mV as compared to -120 and -180 mV. This behaviour was exacerbated by the R1448C mutation since block at -120 mV was greater than that at -180 mV. Thus flecainide can bind to inactivated sodium channels in the absence of channel opening. Nevertheless, all the channels showed the same closed-state affinity constant (K(R) approximately 480 microM) and the same inactivated-state affinity constant (K(I) approximately 18 microM). Simulations according to the modulated receptor hypothesis mimic the voltage-dependent block of WT and mutant channels by flecainide and mexiletine. All the results suggest similar blocking mechanisms for the two drugs. Yet, since flecainide exerts use-dependent block at lower frequency than mexiletine, it may exhibit greater benefit in all myotonic syndromes. Moreover, flecainide blocks hNa(v)1.4 channel mutants with a rightward shift of availability voltage dependence more specifically than mexiletine, owing to a lower K(R)/K(I) ratio. This study offers a pharmacogenetic strategy to better address treatment in individual myotonic patients.

Dose-Response Relationship, Drug↗

PubMed2003

Decrease in resting calcium and calcium entry associated with slow-to-fast transition in unloaded rat soleus muscle.

Using fura-2 and the manganese quenching technique, we show here that sarcolemmal permeability to cations (SP-Ca) of slow-twitch muscles is greater than that of fast-twitch ones. This appears to be related to a higher expression and/or activity of stretch-activated channels, whereas leak channel activities are similar. During hindlimb suspension (HU), we found highly correlated decreases in SPCa and resting calcium of soleus muscle toward values of extensor digitorum longus (EDL) muscle. This was significant as soon as 3 days of suspension, contrary to soleus muscle caffeine sensitivity and responsiveness that were not modified after this HU period. After 14 days of HU, SP-Ca, resting calcium, and caffeine response of soleus muscle became similar to that normally observed in EDL muscle. These results demonstrate that the correlated decreases in SP-Ca and resting calcium precede most functional changes due to HU. Given the known shortening of HU soleus muscle, we proposed that this could induce a decrease of SP-Ca and a consequent reduction of resting calcium. According to the crucial role of resting cytosolic free calcium in the maintenance and the adaptation of muscle phenotype, our results suggest that slow-to-fast transition of HU soleus muscle is calcium dependent.

Animals↗

PubMed2003

Inhibition of skeletal muscle sodium currents by mexiletine analogues: specific hydrophobic interactions rather than lipophilia per se account for drug therapeutic profile.

In striated fibers, the activity of mexiletine (Mex)-like sodium channel blockers is strongly modulated by the part of the molecule nearby the asymmetric carbon atom. A lipophilic aromatic phenyl group at this levels, as in 2-(2,6-dimethylphenoxy)-1-phenylethanamine (Me4), markedly increases drug potency, while an increased distance between the stereogenic center and the pharmacophore amino group, as in 3-(2,6-dimethylphenoxy)-2-methylpropan-1-amine (Me2), enhances the use-dependent behavior. In order to better evaluate the role of lipophilicity in drug potency in relation to the structural determinants for a specific binding, lipophilic analogs of Me2 and Me4 were synthesized. Compounds 3-[(2,6-dimethylphenyl)thio]-2-methylpropan-1-amine and 2-[(2,6-dimethylphenyl)thio]-1-phenylethanamine were obtained by isosteric substitution of the oxygen atom with sulfur, while the introduction of a chlorine atom in 4- position of the aryloxy ring lead to 3-(4-chloro-2,6-dimethylphenoxy)-2-methylpropan-1-amine and 2-(4-chloro-2,6-dimethylphenoxy)-1-phenylethanamine. The compounds were tested on nearly maximal Na(+) currents elicited with depolarizing steps at 0.3 Hz (tonic block) and 2-10 Hz (use-dependent block) by means of vaseline-gap voltage-clamp method on single frog muscle fibers.The augmented lipophilicity largely increase drug potency in Me2 analogues, the thio and chlorinated compounds being 20- and 10-fold more potent in producing the tonic block, respectively. However, both compounds showed a 2-fold lower use-dependent behavior vs. the high use-dependent Me2. Surprisingly, the same increase in lipophilicity brought about by the same substitutions, in the already high lipophilic and potent Me4 failed to further improve the potency, although both new analogs were more stereoselective than Me4. No correlation was found between logP and potency of all analogs tested. All compounds acted as inactivated channel blockers. In conclusion, lipophilicity differently influences drug profile based on the molecular determinants controlling drug-receptor interaction.

Animals↗

PubMed2003

Growth hormone secretagogues modulate the electrical and contractile properties of rat skeletal muscle through a ghrelin-specific receptor.

(1) Growth hormone secretagogues (GHS) exhibit potent growth hormone (GH)-releasing activity through the activation of a pituitary receptor. Here, we consider the possibility that GHS can target a specific receptor in rat skeletal muscle and have a role in the control of muscle function. (2) By means of the intracellular microelectrode technique, we found that in vitro application of hexarelin and L-163,255 dose dependently reduced resting chloride (gCl) and potassium (gK) conductances in rat skeletal muscle. These effects were prevented by the GHS-receptor antagonist [D-Lys-3]-GHRP-6, and by either phospholipase C or protein kinase C (PKC) inhibitors. Ghrelin, a natural ligand of GHS receptors, also induced a reduction of muscle gCl and gK, which was antagonised by [D-Lys-3]-GHRP-6. (3) Both GHS shifted the mechanical threshold for the contraction of muscle fibres towards more negative voltages. Accordingly, by means of FURA-2 fluorescent measurements, we demonstrated that L-163,255 induced a resting [Ca(2+)](i) increase, which was reversible and not blocked by nifedipine or removal of external Ca(2+). (4) Ageing is a condition characterised by a deficit of GH secretion, which in turn modifies the electrical and contractile properties of skeletal muscle. In contrast to GH, chronic treatment of aged rats with hexarelin or L-163,255 failed to restore the electrical and contractile muscle properties. Moreover, the two GHS applied in vitro were able to antagonise the beneficial effect on gCl and gK obtained through chronic treatment of aged animals with GH. (5) Thus, skeletal muscle expresses a specific GHS receptor able to decrease gCl and gK through a PKC-mediated intracellular pathway. This peripheral action may account for the lack of restoration of skeletal muscle function in long-term GHS-treated aged animals.

Aging↗

PubMed2003

Different ability of clenbuterol and salbutamol to block sodium channels predicts their therapeutic use in muscle excitability disorders.

Activation of muscle beta(2)-adrenergic receptors successfully counteracted sarcolemma inexcitability in patients suffering from hyperkalemic periodic paralysis (HPP), a hereditary disease caused by mutations in the gene encoding the skeletal muscle sodium channel. Looking for potential modulation of these channels by beta(2)-adrenergic pathway using patch-clamp technique, we found that clenbuterol blocked sodium currents (I(Na)) in rat skeletal muscle fibers and in tsA201 cells transfected with the human channel isoform, whereas salbutamol did not. The effects of clenbuterol were independent of beta-adrenoceptor stimulation. Instead, clenbuterol structure and physicochemical characteristics as well as I(Na) blocking properties resembled those of local anesthetics, suggesting direct binding to the channels. Similar experiments with the chemically similar beta-antagonists propranolol and nadolol, suggested the presence of two hydroxyl groups on the aromatic moiety of the drugs as a molecular requisite for impeding sodium channel block. Importantly, clenbuterol use-dependently inhibited action potential firing in rat skeletal muscle fibers, owing to beta-adrenoceptor-independent I(Na) block. From a clinical point of view, our study defines the rationale for the safe use of salbutamol in HPP patients, whereas clenbuterol may be more indicated in patients suffering from myotonic syndromes, a condition characterized by sarcolemmal overexcitability, because use-dependent I(Na) block can inhibit abnormal runs of action potentials.

Adrenergic beta-Agonists↗

PubMed2003

Optimal requirements for high affinity and use-dependent block of skeletal muscle sodium channel by N-benzyl analogs of tocainide-like compounds.

Newly synthesized tocainide analogs were tested for their state-dependent affinity and use-dependent behavior on sodium currents (INa) of adult skeletal muscle fibers by means of the Vaseline-gap voltage clamp method. The drugs had the pharmacophore amino group constrained in position alpha [N-(2,6-dimethylphenyl)pyrrolidine-2-carboxamide (To5)] or beta [N-(2,6-dimethylphenyl)pyrrolidine-3-carboxamide (To9)] in a proline-like cycle and/or linked to a lipophilic benzyl moiety as in N-benzyl-tocainide (Benzyl-Toc), 1-benzyl-To5 (Benzyl-To5), and 1-benzyl-To9 (Benzyl-To9). INa were elicited with pulses to -20 mV from different holding potentials (-140, -100, and -70 mV) and stimulation frequencies (2 and 10 Hz). All compounds were voltage-dependent and use-dependent channel blockers. The presence of a proline-like cycle increased the potency; i.e., To5 was 3- and 10-fold more effective than Toc in blocking INa at the holding potential of -140 and -70 mV, respectively. The benzyl group on the amine further enhanced drug effectiveness with the following scale: Benzyl-To9 >/= Benzyl-Toc > Benzyl-To5. At a holding potential of -100 mV and 10-Hz stimulation, Benzyl-To9 blocked INa with a half-maximal concentration of 0.5 microM, being 60 and 400 times more potent than To9 and Toc, respectively. The similar effectiveness of Benzyl-Toc and Benzyl-To9 was paralleled by a similar spatial arrangement by equilibrium geometry modeling. In addition, the latter had a higher pKa value that probably contributed to a slow kinetic during its high use-dependent behavior. Benzyl-To5 had its lowest energy level at a more folded conformation that justifies the less favorable profile among the N-benzylated analogs. The new compounds are the most potent tocainide-like sodium channel blockers so far described and have high therapeutic potentials.

Animals↗

PubMed2002

Change of chloride ion channel conductance is an early event of slow-to-fast fibre type transition during unloading-induced muscle disuse.

Disuse of postural slow-twitch muscles, as it occurs in hypogravity, induces a slow-to-fast myofibre type transition. Nothing is known about the effects of weightlessness on the resting membrane chloride conductance (gCl), which controls sarcolemma excitability and influences fibre type transition during development and adult life. Using the current-clamp method, we observed that rat hindlimb unloading (HU) for 1-3 weeks increased gCl in fibres of the slow-twitch soleus (Sol) muscle toward values found in fast muscle. Northern blot analysis suggested that this effect resulted from an increased ClC-1 chloride channel mRNA level. In the meantime, a 4-fold increase in fibres expressing fast isoforms of the myosin heavy chain (MHC) was observed by immunostaining of muscle sections. Also, Sol muscle function evolved toward a fast phenotype during HU, as demonstrated by the positive shift of the threshold potential for contraction. After 3-days HU, Sol muscle immunostaining and RT-PCR experiments revealed no change in MHC protein and mRNA expression, whereas the gCl was already maximally increased, due to a pharmacologically probed, increased activity of ClC-1 channels. Thus the increase in gCl is an early event in Sol muscle experiencing unloading, suggesting that gCl may play a role in muscle adaptation to modified use. Pharmacological modulation of ClC-1 channels may help to prevent disuse-induced muscle impairment.

Animals↗