Taurine and Skeletal Muscle Disorders

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Neurochemical Research, Vol. 29, No. 1, January 2004 (© 2004), pp. 135–142

Taurine and Skeletal Muscle Disorders* Diana Conte Camerino,1,4 Domenico Tricarico,1 Sabata Pierno,1 Jean-François Desaphy,1 Antonella Liantonio,1 Michael Pusch,2 Rosa Burdi,1 Claudia Camerino,3 Bodvael Fraysse,1 and Annamaria De Luca1 (Accepted July 22, 2003)

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.

KEY WORDS: Taurine; skeletal muscle; inherited and acquired myopathies; therapeutic potential.

INTRODUCTION

tent of taurine can play a role in tissue malfunction. In turn, taurine supplementation can be used for pharmacological control of functions in which this amino acid plays a modulatory role. Studies from our laboratory and others have shown that in both excitable and nonexcitable tissues taurine modulates cell function through its effects on ion channel activity. Thus taurine exerts an osmoregulatory action by leaving cells exposed to hypotonic stimulus through different channels permeable to taurine and anions (2,3). In excitable tissues, taurine has a recognized modulatory role on different ion channels, thus controlling membrane excitability and consequently tissue function. In heart, taurine modulates the activity of L-type calcium channels and delayed rectifier potassium channels in a manner that strictly depends upon [Ca2⫹]i and [Ca2⫹]o (4). In addition, taurine inhibits cardiac Na⫹ currents, stimulates T-type Ca2⫹ currents important for automaticity,

Taurine is a sulphonic amino acid present in high concentrations in the free form in many excitable and nonexcitable tissues. Its widespread distribution suggests an involvement in various physiological functions, ranging from osmoregulation to neurotransmission (1). Various evidence supports the view that alteration in tissue con*Special issue dedicated to Dr. Herminia Pasantes-Morales. 1 Unit of Pharmacology, Department of Pharmacobiology, University of Bari, Italy. 2 Institute of Biophysics, CNR Genova, Italy. 3 Department of Human Anatomy and Histology, University of Bari, Italy. 4 Address reprint requests to: Sezione di Farmacologia, Dipartimento Farmacobiologico, Via Orabona 4, Campus, 70125 Bari, Italy. Tel and fax: 39-0805442801; E-mail: [email protected]

135 0364-3190/04/0100–0135/0 © 2004 Plenum Publishing Corporation

136 inhibits ATP-dependent K⫹ channels (KATP), and modulates the activity of ion exchangers. Consequently, taurine has been claimed to maintain constant the levels of intracellular Ca2⫹, thus exerting cardioprotective actions in arrhythmias, ischemia, and heart failure (4). The electrophysiological evidence collected in our laboratories during the last few years has shown that taurine modulates the activity of ion channels also in skeletal muscle fibers (5). In fact, taurine increases chloride channel conductance and modulates gating and kinetic of the voltage-dependent sodium channel, with the overall effect of stabilizing the sarcolemma. Also it modulates the activity of various types of potassium channels and in particular of those able to couple the metabolic state of striated fibers to electrical activity, such as ATP-dependent (KATP) or Ca2⫹-activated (KCa2⫹) potassium channels. Finally, taurine controls intracellular calcium homeostasis, by modulating calcium handling mechanisms and consequently excitation-contraction coupling (for review see [5]). In light of the above considerations, in the present work we review the pathophysiological conditions of skeletal muscle in which taurine can be involved (see Table I for overview). Particular attention is given to the therapeutic implication of taurine in myopathic conditions in which ion channels are involved either primarily as a result of gene-based mutation (channelopathies) or secondarily, in the frame of a genetic disease or of a metabolic dysfunction.

DISCUSSION Taurine and Excitability-Related Muscle Disorders It has been long claimed that taurine exerts a stabilizing effect on the sarcolemma, reducing muscle fiber excitability. For this action taurine has been considered as therapeutically valuable in the treatment of myotonic syndromes, inherited disorders of skeletal muscle characterized by hyperexcitability, which in turn leads to delayed relaxation, spasms, and cramps. Myotonic syndromes are nondystrophic skeletal muscle channelopathies, caused by mutations in the genes coding for skeletal muscle isoforms of either chloride or sodium channels (6). Chloride channel myotonias comprise both dominant and recessive forms of myotonia congenita in which the mutated gene codifies for ClC-1 protein, the main chloride channel expressed in skeletal muscle. The mutations lead to a loss of function, so that channel gating is impaired in the normal physiological membrane potential range. The result is a severe reduction of the large chloride conductance (gCl) of resting sarcolemma, which normally exerts a shunting action on the depolarization-driven potassium

Conte Camerino et al. accumulation in transverse tubules, thus allowing repolarization and relaxation (7). Sodium channel mutations are responsible for sodium channel myotonia, periodic paralyses, and paramyotonia congenita. The mutated channels show defects of the inactivation processes that lead to an abnormal and disease-causing persistent Na⫹ current and consequently to altered excitability (8). Skeletal muscle ClC-1 channel is a target of taurine action. In fact, in vitro application of millimolar concentrations of taurine to extensor digitorum longus (EDL) muscle decreases membrane excitability through a concentration-dependent increase of gCl, supporting the proposed beneficial effects of the amino acid in conditions characterized by hyperexcitability (9,10). This effect is due to the ability of taurine to directly modulate ClC-1 channel activity through the interaction with a low-affinity site. In fact, analogues of taurine, characterized for having an increased distance between the two charged heads and/or a more distributed positive charge for the replacement of the amino group with aza-cyclo moieties, as well as compounds able to inhibit in the micromolar range the high-affinity transporter, show a decreased potency in enhancing gCl (11). We have recently given electrophysiological evidence of the direct action of taurine on skeletal muscle Cl⫺ channels. Two microelectrode voltage-clamp recordings showed that in vitro application of 20 mM taurine enhances by 100% the Cl⫺ currents sustained by human ClC-1 channel heterologously expressed in Xenopus oocytes. In parallel, taurine shifts the channel activation toward more negative potentials, an effect that likely accounts for the increase in resting gCl observed in native fibers during current-clamp recordings (5,12). We have described previously the effect of taurine in some forms of myotonias induced in the rat by pharmacological agents able to decrease chloride conductance. We observed that taurine increases gCl lowered in vivo by 20,25-diazacholesterol that acts indirectly on the channel, but it is unable to antagonize the block of gCl brought about by direct channel blockers, such as the anthracene-9-carboxylic acid (10). This observation would lead to minimize the therapeutic potential of taurine in chloride channel–related myotonic syndromes, because the direct impairment of ClC-1 gating can hamper the taurine action. Nonetheless, the antimyotonic activity of taurine is sustained by its peculiar effects on voltage-gated sodium channels, also in consideration of the fact that drugs able to depress sarcolemmal excitability through the block of voltage-gated Na⫹ channels (i.e., with a local anesthetic-like action) are clinically useful in the above-described channelopathies (13). We have investigated the effect of taurine on Na⫹

Taurine and Skeletal Muscle Disorders

137

Table I. Skeletal Muscle Disorders with Primary and/or Secondary Involvement of Ion Channels and Therapeutic Potential of Taurine Pathology

Channel involvement

Symptoms

Therapeutic potential of taurine

Myotonia congenita (both recessive and dominant forms) Recessive sodium-channel myotonias Paramyotonia congenita Peridiodic paralyses

Primary inherited channelopathies due to loss-of-function mutations of ClC-1 chloride channel or gain-of-function mutations of SkM2 channel

Hyperexcitability and impaired muscle relaxation

Ischemia and reperfusion injury

Secondary activation of potassium channels

Hyperkalemia, muscle dysfunction

Aging

Secondary alteration in function and expression of several ion channels

Atrophy, weakness, sarcopenia, degeneration, altered excitation–contraction coupling

Disuse

Primary and/or secondary alteration in ion channel function and expression Direct or indirect alteration in chloride channel and in voltage-insensitive calcium selective channels (Leak/TRPC)

Atrophy, change in metabolism, slow-to-fast transition

To reduce membrane hyperexcitability through: 1. Opening of chloride channel and increase in gCl mediated by both short- and long-term actions 2. Modulation of generation and propagation of action potential by blocking sodium channel with a local anesthetic–like mechanism To counteract hyperkalemia by inhibiting KATP and KCa2⫹ channels; to prevent ischemia-induced taurine loss To counteract the decrease in taurine content and the consequent reduction in chloride channel function and the alteration in calcium ion homeostasis To counteract disuse-induced taurine loss

Duchenne muscular dystrophy and related myopathies

channels of native muscle fibers by patch clamp recordings in the cell-attached configuration. Na⫹ currents have been elicited with depolarizing test pulses to various membrane potentials from the holding potential of ⫺110 mV. The in vitro application of 10 mM taurine enhanced, up to two-fold, the Na⫹ transients elicited by threshold depolarizing test pulses (test pulse to ⫺70/⫺50 mV), whereas it reduced the current at more depolarized test pulse potentials. The effect of channel block, calculated at the current peak (elicited between ⫺30/⫺20 mV) at this concentration, amounted to about 50%. Also, taurine shifted the activation curve of about 10 mV toward more negative potentials, an effect that likely accounts for the enhancing effect of Na⫹ currents at threshold membrane potentials. In parallel, taurine left-shifted the steady-state inactivation curve by 7.6 mV, indicating that taurine reduces channel availability to opening by stabilizing the blocked channels in the inactivated state. The double effect of both channel activation and inactivation is similar to that already

Progressive muscle degeneration and weakness; muscle fiber loss for necrosis and apoptosis; sarcolemmal instability; altered calcium homeostasis

To ameliorate muscle performance; to counteract taurine loss and to modulate calcium availability for contraction To counteract contractioninduced ischemic reaction To contrast degenerationinduced decrease in gCl

observed in cardiac Na⫹ currents (14,15). The anesthetic-like action exerted by taurine on skeletal muscle Na⫹ channels is likely mediated by the amino group, which is generally recognized as the pharmacophore moiety of Na⫹ channel blockers (16) and can account for a beneficial effect of exogenous taurine for reducing abnormal excitability of myotonic muscle fibers. In particular, taurine can exert a greater therapeutic action in those myotonic states related to sodium channel mutations due to its dual ability to open chloride channels and to block sodium channels. Unfortunately, no animal models of sodium channel myotonias are available to test this hypothesis, although the relative safety of taurine does not impede a direct use in patients affected by sodium channel myotonias. A similar dual mechanism on both chloride and sodium channels could have been responsible for the claimed therapeutic potential of taurine in patients affected by myotonic dystrophy, a myotonic state not directly related to alteration in ion channel function (17).

138 Taurine and Altered Muscle Metabolism Metabolic abnormalities in skeletal muscle are responsible for the impairment of muscle function during prolonged period of ischemia (⬎3 h) and ischemia followed by a short-term reperfusion (⬎30 min). Ischemic phenomena in skeletal muscle can occur as a consequence of trauma, compression of limbs, or in case of peripheral arterial dysfunction (PAD), plaque formation in the peripheral arteries (18,19). Multiple events have been described as occurring in ischemic striated fibers. An impaired phosphorylation potential can result from a decrease in both ATP/ADP ratio and creatine phosphate (CP) content, while Ca2⫹ homeostasis is altered with an increase of intracellular Ca2⫹ transients and/or inhibition of reuptake process into the sarcoplasmic reticulum (SR). Mitochondrial function is also reduced. Furthermore, an overproduction of free radical species is often observed during metabolic stress. This is believed to be the molecular basis of the alteration of membrane fluidity and permeability found in these conditions. Similar metabolic abnormalities can also take place in skeletal muscle secondary to genetic disorders associated with mutations of ion channels or cytoskeletal proteins. During ischemia-reperfusion injury, the opening of ATP-sensitive K⫹ channels (KATP) is known to be involved in the cytoprotective effect of the preconditioning mechanisms (20). In this respect we have shown that a prolonged ischemia of skeletal muscle is accompanied by an increased activity of KATP channels. This would work to prevent the influx of Ca2⫹ ions and preserve the ATP content of the muscle, while the reperfusion period is associated with a decreased activity of KATP channels and a significant drop in ATP content (21,22). During reperfusion, an enhanced activity of the Ca2⫹-activated K⫹ channel ( K Ca2 ⫹ ) is rather observed. The overactivation of this channel type is considered a sensor of cellular damage, being generally observed in conditions of sarcolemmal depolarization and elevation of intracellular Ca2⫹ content such as occurs following trains of action potentials or in certain disease states. We found that the abnormal activation of the muscle K Ca2 ⫹ channel, that is in turn responsible for the hyperkalemia observed during the reperfusion, is related to an overproduction of nitric oxide–reactive molecules with oxidative stress (23). The ischemia and, particularly, the reperfusion period are therefore associated with loss of intracellular K⫹ ions, enzymes such creatine kinase (CK), and amino acids, which are often used as markers of disease progression. One of the amino acids released by skeletal muscle during ischemia-reperfusion processes is taurine

Conte Camerino et al. (24–26). Whether the loss of taurine is solely a marker of tissue damage, or a cytoprotective mechanism against ischemic insult, is still matter of debate (27,28). A consistent experimental observation in this field is that taurine, at millimolar concentrations, inhibits several types of K⫹ channels, including the KATP channels of heart and skeletal muscles. In our experiments, taurine blocks the skeletal muscle KATP channel by binding in the vicinity of the sulphonylurea receptors of the channel complex and affecting the channel gating (29). We also showed that taurine is capable of blocking the muscle K Ca2 ⫹ channel at positive membrane potentials, reducing the outward K⫹ current through changes of the reversal potentials of the channel to K⫹ ions (30). The blocking effects of taurine are extended also to other voltage-dependent K⫹ channels and the inward rectifier K⫹ channel in other tissues (4). Therefore the depletion of taurine occurring during ischemia and reperfusion could contribute to the early activation of KATP channels with fiber repolarization and salvage of intracellular ATP contents. This is also demonstrated by the fact that the depletion of taurine induced by guanidinoethane sulfonate (GES), a known inhibitor of the high-affinity transporter for taurine, significantly increases the macroscopic resting K⫹ conductance from 280 ⫾ 27 ␮S/cm2 (n ⫽ 15 fibers) in the controls to 508 ⫾ 28 ␮S/cm2 (n ⫽ 38 fibers) in rats treated with GES (31). From a pharmacological point of view, the supplementation of taurine could prove beneficial, because taurine is able to counteract the hyperkalemia observed during the reperfusion period by blocking the K Ca2 ⫹ channel.

Taurine and Disuse- and Aging-Related Muscle Dysfunction Other than exerting a pharmacological control of skeletal muscle excitability through a direct modulation of ion channel function, taurine exerts a long-term physiological control of both muscle contractility and Cl⫺ channel function as a result of its large intracellular concentrations (30). A variety of evidence is available in this regard. A strict relationship exists, during postnatal development, between the muscle content of taurine and the value of gCl (30), while a 50% depletion of taurine in adult rat EDL muscle, obtained with a 4-week chronic treatment with GES, is paralleled by a significant decrease of gCl, a consequent increase in membrane excitability and a change in the excitation–contraction coupling mechanism (31). The alterations observed in GES-treated taurine-depleted muscles are similar to those naturally

Taurine and Skeletal Muscle Disorders occurring in skeletal muscle of aged subjects. Indeed, by HPLC analysis we found that a significant decrease in the taurine content occurs in skeletal muscle of aged rats, in parallel with a marked decrease in gCl. Although this latter phenomenon is well correlated with a decrease in mRNA for ClC-1, we evaluated the possibility to counteract the aging-induced decrease of gCl with an in vivo taurine treatment (33,34). We found that a 3-month chronic treatment with taurine to aged rats (1 g/kg in drinking water), significantly prevented both the loss of intracellular taurine and the decrease in gCl, supporting the strict link between appropriate tissue levels of the amino acid and the function of the Cl⫺ channel (33). The use of specific pharmacological tools allowed us to propose that taurine exerts long-term physiological control on Cl⫺ channels, likely through an action on the enzymatic biochemical pathways capable of modulating channel function (33,35,36). In fact, the muscle chloride channel is controlled in a negative manner by a phosphorylation pathway involving a Ca2⫹ and phospholipiddependent protein kinase C (PKC) (36). Taurine has been shown to inhibit PKC activity, by reducing both the formation of diacylglycerol and the level of available free Ca2⫹, through the stimulation of its uptake into intracellular stores (37). This latter mechanism can be of importance in skeletal muscle, since taurine stimulates Ca2⫹ uptake by SR (1,38,39). Interestingly, a strict relationship seems to exist between the intracellular taurine levels and the pharmacological ability of exogenous taurine to increase gCl. In fact, higher than normal sensitivity to taurine has been found in gCl in taurine-depleted muscles (GEStreated or aged) (31,35), whereas the in vitro application of taurine on the slow-twitch soleus muscle, which is characterized by higher levels of taurine versus the fast EDL (40), exerts a small increase in gCl, despite this parameter being lower than in EDL muscle (5). These observations, other than supporting the pharmacological use of taurine in conditions of tissue depletion, suggest that other yet unknown Cl⫺ channel–mediated actions of taurine in skeletal muscle can occur in relation to different muscle phenotype-related metabolism. This can play an important role in tissue dysfunction resulting from muscle disuse. This condition is known to be related to a phenotype transition mostly involving the postural slow muscle type into a faster phenotype. By using hindlimb-suspended rats as an animal model of muscle disuse, we have recently found that an increase in chloride channel and a decrease in cytosolic calcium levels toward the values typical of fast twitch muscles are both early events that precede slow-to-fast fiber transition and that are likely pivotal to trigger the trans-

139 ition itself (41). On the basis of the information collected to date between the taurine content and ionic homeostasis in fast twitch muscle fibers, the relationship between the high taurine content in slow muscle fibers with the lower value of chloride conductance and the higher level of cytosolic calcium typical of this phenotype is far from being straightforward. Other addressed experiments are needed to fulfill these points and to better state the role of taurine in muscle plasticity phenomena. However, it is tempting to speculate that taurine may exert in slow twitch muscle fibers an action that is not yet well characterized and that a disuseinduced loss of taurine from slow muscle fibers may be somehow involved in the observed early changes leading to fiber type transition. An interesting hypothesis is related to a phenotype-dependent permeability to taurine aimed at maintaining an osmotic equilibrium in relation to muscle fiber metabolism (2). For instance, aquaporin4 (AQP4), a water channel, is expressed, in a plasticdependent manner, in fast twitch but not in slow twitch muscle fibers, likely in relation to the necessity of the former to rapidly equilibrate water to lower the lactate formed during anaerobic contraction (42,43). Thus a relationship may exist between the expression of AQP4 and muscle taurine content. The verification of these hypotheses may lead to the use of taurine as a therapeutic strategy to prevent disuse-related slow-to-fast phenotype transition and muscle impairment.

Taurine and Calcium-Related Degenerative Myopathies In contractile tissues, the increase in cytosolic Ca2⫹ ions, triggered by a multiple-step mechanism known as excitation–contraction coupling (e–c), is a highly regulated spatial and temporal phenomenon to ensure a proper contraction and relaxation. As already stated, taurine is able to control excitation–contraction coupling in cardiomyocytes through a direct ability to modulate Ca2⫹ channels and therefore the influx of Ca2⫹ that is pivotal for cardiac contraction (4). As a consequence, all other Ca2⫹-handling mechanisms are modulated (40). The e–c coupling of skeletal muscle fiber is almost independent from external Ca2⫹. Taurine controls this function by modulating intracellular Ca2⫹ levels. Intracellular taurine can bind, with a low affinity, to neutral phospholipids and such a binding can allosterically modulate the binding of Ca2⫹ ion to acid phopholipids, which would raise the calcium concentration for the activity of pumps and exchangers (1). Accordingly, taurine has been found to enhance the Ca-ATPase activity, thus increasing

140 both accumulation and release of Ca2⫹ in sarcoplasmic reticulum (38,39). Conditions of taurine depletion affect e–c coupling of striated fibers, likely through an increase of free Ca2⫹ concentration. The e–c coupling mechanism can be measured overall in voltage-clamp conditions as the threshold of membrane potential for striated fiber contraction in relation to the duration of the depolarizing step applied (mechanical threshold [MT]). This process occurs with a monoexponential time course up to reach an equilibrium that is the rheobase; that is, the voltage at which the speed of Ca2⫹ release equals that of Ca2⫹ reuptake. A taurine depletion produced by chronic treatment with GES, shifts (by about 10 mV) the rheobase voltage toward more negative potentials, indicative of an increased level of cytosolic Ca2⫹. This shift is due to taurine depletion, because the in vitro application of 60 mM taurine, ineffective on control muscle, significantly counteracts the alteration of MT of GES-treated ones (31). Similar observations have been made on skeletal muscle of aged rats, in which a natural decrease in the taurine content occurs in parallel with a shift of the rheobase voltage toward more negative potentials. In agreement with the observations described in the previous paragraph, a chronic in vivo treatment with taurine of aged rats restored the intracellular levels of taurine and counteracted the change of MT typical of this condition (33). Muscular dystrophies are hereditary disorders of skeletal muscle in which the progressive muscle fiber necrosis and degeneration have been related to a loss in cytosolic Ca2⫹ homeostasis. Duchenne muscular dystrophy (DMD) is the most common of these pathologies and is related to a disassembling of the sarcolemmal dystrophin-glycoprotein complex resulting from the genetic lack of cytoskeletal protein dystrophin (45). As a consequence, the sarcolemma becomes weak and the stress of contraction leads to an increased calcium influx. Recent studies suggest the involvement of higher activity of leak/TRP channel types in the observed increase of sarcolemmal permeability to calcium (46; personal unpublished observation). This in turn leads to an increase in intracellular Ca2⫹ levels, which triggers both e–c dysfunction and activation of calcium-dependent proteases, leading to fiber necrosis and degeneration. Sarcolemmal damage can lead to a loss of intracellular components such as taurine. In fact, an increased urinary excretion of taurine and other amino acids has been observed in DMD patients (47). The loss of taurine content can contribute to some pathological features of dystrophic muscle. An NMR analysis aimed at identifying markers of degeneration and regeneration events in dystrophin-less fibers revealed a decrease in the level

Conte Camerino et al. of both taurine and creatine in muscles of dystrophic mdx mouse, the most widely used animal model for DMD, during the active degeneration period of the mouse life span. These alterations are compensated during adulthood, when a spontaneous and successful regeneration takes place in this phenotype (48). Accordingly, we found by HPLC that the level of taurine in hindlimb muscles, heart, and brain of 6-month-old mdx mice are only slightly lower compared to normal controls. Nonetheless, the plasma levels are significantly higher, suggesting that an alteration in taurine turnover does occur in this phenotype (49,50). In agreement with the proposed higher level of Ca2⫹ in the muscle fibers of the mdx mouse, the rheobase voltage of mdx EDL muscle fibers is significantly shifted toward more negative potentials of about 7 mV. The in vitro application of 60 mM taurine significantly restores the alteration of MT observed (49,50), suggesting a potential therapeutic interest of taurine for improving muscle function in the dystrophic condition. Based on the above observations, we have recently tested the ability of in vivo administration of taurine to ameliorate the functional signs of dystrophic muscle degeneration. As a model we used the exercised mdx mouse, which develops a more severe phenotype of muscular dystrophy detectable on various cellular indexes (51). We found that the daily administration of taurine in 1% enriched chow not only prevented the typical exercise-induced loss of muscle strength observed in vivo in the mdx mice, but significantly increased strength over the normal value. Also 6–8 weeks of treatment with taurine significantly ameliorated the e–c coupling mechanism of exercised EDL mdx muscle fibers, because the strength-duration curve of MT was significantly shifted toward the more positive potentials typical of sedentary wild-type muscles. In fact, the rheobase value was ⫺67.9 ⫾ 0.6 mV, about 5 mV more positive than the value recorded in mdx EDL muscle and very close to the value recorded in control EDL fibers. The taurine treatment was also effective in accelerating the steps involved in reaching the rheobase because 1/␶ was changed from 0.11 ⫾ 0.01 s⫺1 to 0.13 ⫾ 0.007 s⫺1, a value similar to that recorded in sedentary wild type. These effects were similar to those previously observed after in vitro application of taurine on mdx EDL muscle (50). The beneficial effect of taurine on MT of dystrophic mice was specific, because a similar treatment with creatine, a substance claimed to improve muscle energetic state and thus calcium reuptake by SR, was totally unable to ameliorate MT. In support of the therapeutic interest of taurine in dystrophic conditions, we found that it was as effective as ␣-methylprednisolone,

Taurine and Skeletal Muscle Disorders the classical steroid drug used to treat Duchenne patients. Interestingly, taurine treatment also counteracted the exercise-induced decrease in chloride conductance, another specific index of muscle degeneration in dystrophic mice (51). The molecular mechanisms by which taurine exerts its positive effects in dystrophic muscle are not totally clear. Our preliminary fura-2 calcium imaging experiments on intact EDL muscle fibers from exercised mdx mice suggest that taurine is able to decrease the cytosolic calcium level and to potentiate the caffeine-induced calcium release. These observations are in line with the proposed effect of taurine on SR (39). Also, verification is needed of the ability of taurine to act on the voltage-insensitive calcium channels involved in the enhanced sarcolemmal permeability to calcium and/or to counteract the activation of calcium-dependent enzymes involved in both inflammatory reactions and muscle fiber necrosis (1,50,52,53). Furthermore, the degenerative loss of taurine from dystrophic muscle can be worsened by exercise, further supporting the beneficial effect of taurine supplementation in this pathology, characterized by contraction-induced injury (48,54–56). Most importantly, no significant signs of toxicity were observed upon chronic in vivo treatment of mdx mice with taurine, supporting the possibility of its clinical trial in Duchenne patients.

ACKNOWLEDGMENTS Supported by Telethon-Italy (projects 1150 and 1208). The authors wish to thank Professor Alberto Giotti and Professor Ryan Huxtable for continuous support to their work in the taurine field.

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