Lipid-lowering drugs are widely used for the prevention and cure of cardiovascular diseases (CVD)... more Lipid-lowering drugs are widely used for the prevention and cure of cardiovascular diseases (CVD) [...]
Skeletal muscle tissue has the important function of supporting and defending the organism. It is... more Skeletal muscle tissue has the important function of supporting and defending the organism. It is the largest apparatus in the human body, and its function is important for contraction and movements. In addition, it is involved in the regulation of protein synthesis and degradation. In fact, inhibition of protein synthesis and/or activation of catabolism determines a pathological condition called muscle atrophy. Muscle atrophy is a reduction in muscle mass resulting in a partial or complete loss of function. It has been established that many physiopathological conditions can cause a reduction in muscle mass. Nevertheless, it is not well known the molecular mechanisms and signaling processes causing this dramatic event. There are multiple concomitant processes involved in muscle atrophy. In fact, the gene transcription of some factors, oxidative stress mechanisms, and the alteration of ion transport through specific ion channels may contribute to muscle function impairment. In this r...
Amyotrophic Lateral Sclerosis is a neurodegenerative disease caused by progressive loss of motor ... more Amyotrophic Lateral Sclerosis is a neurodegenerative disease caused by progressive loss of motor neurons, which severely compromises skeletal muscle function. Evidence shows that muscle may act as a molecular powerhouse, whose final signals generate in patients a progressive loss of voluntary muscle function and weakness leading to paralysis. This pathology is the result of a complex cascade of events that involves a crosstalk among motor neurons, glia, and muscles, and evolves through the action of converging toxic mechanisms. In fact, mitochondrial dysfunction, which leads to oxidative stress, is one of the mechanisms causing cell death. It is a common denominator for the two existing forms of the disease: sporadic and familial. Other factors include excitotoxicity, inflammation, and protein aggregation. Currently, there are limited cures. The only approved drug for therapy is riluzole, that modestly prolongs survival, with edaravone now waiting for new clinical trial aimed to cla...
The 14th Meeting of the Interuniversity Institute of Myology (IIM), October 12-15, 2017 - Assisi,... more The 14th Meeting of the Interuniversity Institute of Myology (IIM), October 12-15, 2017 - Assisi, Italy gathered together researchers from Italy, European and North-American countries to discuss recent results on muscle research. The program showcased keynote lectures from world-renowned international speakers presenting advances in muscle physiology, bioengineering, metabolism and therapeutics. Based on selection from submitted abstracts, participants presented their novel, unpublished results in seven oral communication and two poster sessions. Particular emphasis was devoted to young trainees. For example, trainees where directly involved in organizing a scientific session and three round tables tailored to the interests of their peers. The meeting attracted a broad audience from Italy, various European countries and from North America. It offered a unique opportunity to all researchers involved in the field of muscle biology to exchange ideas and foster scientific collaborations...
Statins are the most prescribed and effective drugs to treat cardiovascular diseases (CVD). Never... more 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 s...
The ClC-1 chloride channel 1 is important for muscle function as it stabilizes resting membrane p... more The ClC-1 chloride channel 1 is important for muscle function as it stabilizes resting membrane potential and helps to repolarize the membrane after action potentials. We investigated the contribution of ClC-1 to adaptation of skeletal muscles to needs induced by the different stages of life. We analyzed the ClC-1 gene and protein expression as well as mRNA levels of protein kinase C (PKC) alpha and theta involved in ClC-1 modulation, in soleus (SOL) and extensor digitorum longus (EDL) muscles of rats in all stage of life. The cellular localization of ClC-1 in relation to age was also investigated. Our data show that during muscle development ClC-1 expression differs according to phenotype. In fasttwitch EDL muscles ClC-1 expression increased 10-fold starting at 7 days up to 8 months of life. Conversely, in slow-twitch SOL muscles ClC-1 expression remained constant until 33 days of life and subsequently increased fivefold to reach the adult value. Aging induced a downregulation of gene and protein ClC-1 expression in both muscle types analyzed. The mRNA of PKC-theta revealed the same trend as ClC-1 except in old age, whereas the mRNA of PKC-alpha increased only after 2 months of age. Also, we found that the ClC-1 is localized in both membrane and cytoplasm, in fibers of 12-day-old rats, becoming perfectly localized on the membrane in 2-month-old rats. This study could represent a point of comparison helpful for the identification of accurate pharmacological strategies for all the pathological situations in which ClC-1 protein is altered.
The discovery of pathogenetic mechanisms is essential to identify new therapeutic approaches in A... more 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-...
FASEB journal : official publication of the Federation of American Societies for Experimental Biology, Feb 2, 2017
Muscle fibers lacking dystrophin undergo a long-term alteration of Ca2+ homeostasis, partially ca... more 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 st...
Statin therapy may induce skeletal muscle damage ranging from myalgia to severe rhabdomyolysis. O... more Statin therapy may induce skeletal muscle damage ranging from myalgia to severe rhabdomyolysis. Our previous preclinical studies showed that statin treatment in rats involves the reduction of skeletal muscle ClC-1 chloride channel expression and related chloride conductance (gCl). An increase of the activity of protein kinase C theta (PKC theta) isoform, able to inactivate ClC-1, may contribute to destabilize sarcolemma excitability. These effects can be detrimental for muscle function leading to druginduced myopathy. Our goal is to study the causes of statin-induced muscle side effects in patients at the aim to identify biological markers useful to prevent and counteract statin-induced muscle damage. We examined 10 patients, who experienced myalgia and hyper-CK-emia after starting statin therapy compared to 9 non-myopathic subjects not using lipid-lowering drugs. Western Blot (WB) analysis showed a 40% reduction of ClC-1 protein and increased expression of phosphorylated PKC in muscle biopsies of statin-treated patients with respect to untreated subjects, independently from their age and statin type. Real-time PCR analysis showed that despite reduction of the protein, the ClC-1 mRNA was not significantly changed, suggesting posttranscriptional modification. The mRNA expression of a series of genes was also evaluated. MuRF-1 was increased in accord with muscle atrophy, MEF-2, calcineurin (CN) and GLUT-4 transporter were reduced, suggesting altered transcription, alteration of glucose homeostasis and energy deficit. Accordingly, the phosphorylated form of AMPK, measured by WB, was increased, suggesting cytoprotective process activation. In parallel, mRNA expression of Notch-1, involved in muscle cell proliferation, was highly expressed in statin-treated patients, indicating active regeneration. Also, PGC-1-alpha and isocitrate-dehydrogenase increased expression together with increased activity of mitochondrial citrate-synthase, measured by spectrophotometric assay, suggests mitochondrial biogenesis. Thus, the reduction of ClC-1 protein and consequent sarcolemma hyperexcitability together with energy deficiency appear to be among the most important alterations to be associated with statin-related risk of myopathy in
Journal of cachexia, sarcopenia and muscle, Jan 10, 2017
Cachexia is a wasting condition associated with cancer types and, at the same time, is a serious ... more 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. By a multidisciplinary approach ranging from cytofluorometry and electrophysiology to gene expression and histology, we characterized the calcium homeostasis in fast-twitch extensor digitorum longus (EDL) muscle of adult...
Taurine is a natural amino acid present as free form in many mammalian tissues and in particular ... more Taurine is a natural amino acid present as free form in many mammalian tissues and in particular in skeletal muscle. Taurine exerts many physiological functions, including membrane stabilization, osmoregulation and cytoprotective effects, antioxidant and anti-inflammatory actions as well as modulation of intracellular calcium concentration and ion channel function. In addition taurine may control muscle metabolism and gene expression, through yet unclear mechanisms. This review summarizes the effects of taurine on specific muscle targets and pathways as well as its therapeutic potential to restore skeletal muscle function and performance in various pathological conditions. Evidences support the link between alteration of intracellular taurine level in skeletal muscle and different pathophysiological conditions, such as disuse-induced muscle atrophy, muscular dystrophy and/or senescence, reinforcing the interest towards its exogenous supplementation. In addition, taurine treatment can be beneficial to reduce sarcolemmal hyper-excitability in myotonia-related syndromes. Although further studies are necessary to fill the gaps between animals and humans, the benefit of the amino acid appears to be due to its multiple actions on cellular functions while toxicity seems relatively low. Human clinical trials using taurine in various pathologies such as diabetes, cardiovascular and neurological disorders have been performed and may represent a guideline for designing specific studies in patients of neuromuscular diseases.
American Journal of Physiology-Cell Physiology, 2014
Angiotensin II (ANG II) plays a role in muscle wasting and remodeling; however, little evidence s... more Angiotensin II (ANG II) plays a role in muscle wasting and remodeling; however, little evidence shows its direct effects on specific muscle functions. We presently investigated the acute in vitro effects of ANG II on resting ionic conductance and calcium homeostasis of mouse extensor digitorum longus (EDL) muscle fibers, based on previous findings that in vivo inhibition of ANG II counteracts the impairment of macroscopic ClC-1 chloride channel conductance (gCl) in the mdx mouse model of muscular dystrophy. By means of intracellular microelectrode recordings we found that ANG II reduced gCl in the nanomolar range and in a concentration-dependent manner (EC50= 0.06 μM) meanwhile increasing potassium conductance (gK). Both effects were inhibited by the ANG II receptors type 1 (AT1)-receptor antagonist losartan and the protein kinase C inhibitor chelerythrine; no antagonism was observed with the AT2antagonist PD123,319. The scavenger of reactive oxygen species (ROS) N-acetyl cysteine a...
Muscle disuse produced by hindlimb unloading (HU) induces severe atrophy and slow-to-fast fibre t... more Muscle disuse produced by hindlimb unloading (HU) induces severe atrophy and slow-to-fast fibre type transition of the slow-twitch soleus muscle (Sol). After 2 weeks HU, the resting ClC-1 chloride conductance (g Cl) of sarcolemma, which controls muscle excitability, increases in Sol toward a value typical of the fast-twitch EDL muscle. After 3 days of HU, the g Cl increases as well before initiation of fibre type transition. Since ClC-1 channels are acutely silenced by PKC-dependent phosphorylation, we studied the modulation of g Cl by PKC and serine-threonine phosphatase in Sol during HU, using a number of pharmacological tools. We show that a fraction of ClC-1 channels of control Sol are maintained in an inactive state by PKC basal activity, which contributes to the lower g Cl in control Sol compared to EDL. After 14 days of HU, PKC/phosphatase manipulation produces effects on Sol g Cl that corroborate the partial slow-to-fast transition. After 3 days of HU, the early increase of g Cl in Sol is entirely attributable to a reduction of PKC activity and/or activation of phosphatase, maintaining ClC-1 channels in a fully active state. Accordingly, we found that HU reduces expression of PKCα, ε, and θ isoenzymes in Sol and EDL muscles and reduces total PKC activity. Moreover, we show that the rheobase current is increased in Sol muscle fibres as soon as after 3 days of HU, most probably in relation to the increased g Cl. In conclusion, Sol muscle disuse is characterized by a rapid reduction of PKC activity, which reduces muscle excitability and is likely to contribute to disuse-induced muscle impairment.
Using fura-2 and the manganese quenching technique, we show here that sarcolemmal permeability to... more 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. Key words: resting cytosolic calcium • sarcolemmal permeability to cations • slow-twitch and fast-twitch skeletal muscles • hindlimb unloading-induced slow-to-fast transition • fura-2 espite their high degree of differentiation, adult muscles are still able to adapt in response to altered physiological request, for instance, when an organism is exposed to new environmental conditions. This is the case of the antigravity muscles when exposed to hypogravity during space flight or in the ground-based model of simulated microgravity, the rat hindlimb unloading (HU) (1-3). During both space flight and HU, the soleus muscle undergoes atrophy and slow-to-fast phenotype transition. Studies have provided strong evidence that HU-induced muscle wasting is mainly related to an increased protein breakdown via activation of a calcium-independent and ATP-ubiquitin-dependent proteolysis pathway (2-5). However, HU-induced slow-to-fast transition is characterized by changes in the expression of numerous specific genes. For instance, the fast isoforms of estenso myosin heavy chain (MHC) and sarcoplasmic reticulum Ca-ATPase (SERCA) are up-regulated in response to HU, inducing a D shortening of relaxation and contraction times (6, 7). Also, electrophysiological and metabolic properties of soleus muscle are modified according to slow-to-fast transition, with, for example, an altered expression of sodium, chloride, and aquaporin-4 channels (8-10); a reduction of oxidative enzymes; and an increase of glycolytic enzyme activity (11). Despite the numerous studies on the HU-induced slow-to-fast transition of soleus muscle, the mechanisms that trigger gene expression changes during this process remain unclear (3). Calcium ion exerts a pivotal role in regulating muscle physiology (12). For instance, at shortterm, it triggers contraction, while at longer term, it is involved in modulation of gene expression. Interestingly, alteration of calcium homeostasis can induce muscle fiber phenotype changes (13-16). One of the better examples of this process was provided by Kubis et al. (13). It is known that intracellular cytosolic free calcium ([Ca 2+ ] i) at rest is muscle-type specific, being higher in muscle fibers from slow-twitch muscles (14, 17, 18). Kubis and collaborators have shown that in vitro application of a calcium ionophore to rabbit fast skeletal muscle cells in culture induces resting [Ca 2+ ] i increase and consequent fast-to-slow transformation, which are both reversible (13, 15). In this example, both a down-regulation of the type II fast MHC and an increased expression of the type I slow MHC isoforms were observed, suggesting that resting [Ca 2+ ] i is involved in both slow-and fast-twitch phenotype determinism and maintenance.
Slow-twitch muscles, devoted to postural maintenance, experience atrophy and weakness during musc... more Slow-twitch muscles, devoted to postural maintenance, experience atrophy and weakness during muscle disuse due to bedrest, aging or spaceflight. These conditions impair motion activities and can have survival implications. Human and animal studies demonstrate the anabolic role of IGF-1 on skeletal muscle suggesting its interest as a muscle disuse countermeasure. Thus, we tested the role of IGF-1 overexpression on skeletal muscle alteration due to hindlimb unloading (HU) by using MLC/mIgf-1 transgenic mice expressing IGF-1 under the transcriptional control of MLC promoter, selectively activated in skeletal muscle. HU produced atrophy in soleus muscle, in terms of muscle weight and fiber cross-sectional area (CSA) reduction, and up-regulation of atrophy gene MuRF1. In parallel, the disuse-induced slow-to-fast fiber transition was confirmed by an increase of the fast-type of the Myosin Heavy Chain (MHC), a decrease of PGC-1a expression and an increase of histone deacetylase-5 (HDAC5). Consistently, functional parameters such as the resting chloride conductance (gCl) together with ClC-1 chloride channel expression were increased and the contractile parameters were modified in soleus muscle of HU mice. Surprisingly, IGF-1 overexpression in HU mice was unable to counteract the loss of muscle weight and the decrease of fiber CSA. However, the expression of MuRF1 was recovered, suggesting early effects on muscle atrophy. Although the expression of PGC-1a and MHC were not improved in IGF-1-HU mice, the expression of HDAC5 was recovered. Importantly, the HU-induced increase of gCl was fully contrasted in IGF-1 transgenic mice, as well as the changes in contractile parameters. These results indicate that, even if local expression does not seem to attenuate HU-induced atrophy and slow-to-fast phenotype transition, it exerts early molecular effects on gene expression which can counteract the HU-induced modification of electrical and contractile properties. MuRF1 and HDAC5 can be attractive therapeutic targets for pharmacological countermeasures and then deserve further investigations.
Journal of Pharmacology and Experimental Therapeutics, 2007
The mechanism by which the 3-hydroxy-3-methyl-glutaryl-CoA reductase inhibitors (statins) induce ... more The mechanism by which the 3-hydroxy-3-methyl-glutaryl-CoA reductase inhibitors (statins) induce skeletal muscle injury is still under debate. By using fura-2 cytofluorimetry on intact extensor digitorum longus muscle fibers, here we provided the first evidence that 2 months in vivo chronic treatment of rats with fluvastatin (5 and 20 mg kg Ϫ1) and atorvastatin (5 and 10 mg kg Ϫ1) caused an alteration of calcium homeostasis. All treated animals showed a significant increase of resting cytosolic calcium [Ca 2ϩ ] i , up to 60% with the higher fluvastatin dose and up to 20% with the other treatments. The [Ca 2ϩ ] i rise induced by statin administration was not due to an increase of sarcolemmal permeability to calcium. Furthermore, the treatments reduced This study was supported by Italian "Ministero dell'Istruzione, dell'Università e della Ricerca" (FIRB RBAU015E9T) (to D.C.C.). Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
A pivotal role has been ascribed to oxidative stress in determining the imbalance between protein... more A pivotal role has been ascribed to oxidative stress in determining the imbalance between protein synthesis and degradation leading to muscle atrophy in many pathological conditions and in disuse. However, a large variability in disuse-induced alteration of redox homeostasis through muscles, models and species emerges from the literature. Whereas the causal role of oxidative stress appears well established in the mechanical ventilation model, findings are less compelling in the hindlimb unloaded mice and very limited in humans. The mere coexistence of muscle atrophy, indirect indexes of increased reactive oxygen species (ROS) production and impairment of antioxidant defence systems, in fact, does not unequivocally support a causal role of oxidative stress in the phenomenon. We hypothesise that in some muscles, models and species only, due to a large redox imbalance, the leading phenomena are activation of proteolysis and massive oxidation of proteins, which would become more suscept...
The preparation of a new series of 2-aryloxy-3-phenyl-propanoic acids, resulting from the introdu... more The preparation of a new series of 2-aryloxy-3-phenyl-propanoic acids, resulting from the introduction of a linker into the diphenyl system of the previously reported PPARalpha/gamma dual agonist 1, allowed the identification of new ligands with improved potency on PPARalpha and unchanged activity on PPARgamma. For the most interesting stereoisomers S-2 and S-4, X-ray studies in PPARgamma and docking experiments in PPARalpha provided a molecular explanation for their different behavior as full and partial agonists of PPARalpha and PPARgamma, respectively. Due to the adverse effects provoked by hypolipidemic drugs on skeletal muscle function, we also investigated the blocking activity of S-2 and S-4 on skeletal muscle membrane chloride channel conductance and found that these ligands have a pharmacological profile more beneficial compared to fibrates currently used in therapy.
In order to clarify the mechanism underlying the reduction of resting membrane chloride conductan... more In order to clarify the mechanism underlying the reduction of resting membrane chloride conductance (g gl) during aging, the levels of mRNA encoding the principal skeletal muscle chloride channel, ClC-1, were measured. Total RNA samples isolated from tibialis anterior muscles of aged (24^29 months old) and adult (3^4 months old) rats were examined for ClC-1 expression using Northern blot analysis, and macroscopic g gl was recorded from extensor digitorum longus muscle fibers from each adult and aged rat in vitro using a two intracellular microelectrode technique. Although interindividual variability was observed, aged rats exhibited a parallel reduction of both g gl and ClC-1 mRNA expression as compared to adult rats. A linear correlation exists between individual values of ClC-1 mRNA and g gl. These results provide evidence that ClC-1 is the main determinant of sarcolemmal g gl and demonstrate that the decrease of g gl observed during aging is associated with a down-regulation of ClC-1 expression in muscle.
Lipid-lowering drugs are widely used for the prevention and cure of cardiovascular diseases (CVD)... more Lipid-lowering drugs are widely used for the prevention and cure of cardiovascular diseases (CVD) [...]
Skeletal muscle tissue has the important function of supporting and defending the organism. It is... more Skeletal muscle tissue has the important function of supporting and defending the organism. It is the largest apparatus in the human body, and its function is important for contraction and movements. In addition, it is involved in the regulation of protein synthesis and degradation. In fact, inhibition of protein synthesis and/or activation of catabolism determines a pathological condition called muscle atrophy. Muscle atrophy is a reduction in muscle mass resulting in a partial or complete loss of function. It has been established that many physiopathological conditions can cause a reduction in muscle mass. Nevertheless, it is not well known the molecular mechanisms and signaling processes causing this dramatic event. There are multiple concomitant processes involved in muscle atrophy. In fact, the gene transcription of some factors, oxidative stress mechanisms, and the alteration of ion transport through specific ion channels may contribute to muscle function impairment. In this r...
Amyotrophic Lateral Sclerosis is a neurodegenerative disease caused by progressive loss of motor ... more Amyotrophic Lateral Sclerosis is a neurodegenerative disease caused by progressive loss of motor neurons, which severely compromises skeletal muscle function. Evidence shows that muscle may act as a molecular powerhouse, whose final signals generate in patients a progressive loss of voluntary muscle function and weakness leading to paralysis. This pathology is the result of a complex cascade of events that involves a crosstalk among motor neurons, glia, and muscles, and evolves through the action of converging toxic mechanisms. In fact, mitochondrial dysfunction, which leads to oxidative stress, is one of the mechanisms causing cell death. It is a common denominator for the two existing forms of the disease: sporadic and familial. Other factors include excitotoxicity, inflammation, and protein aggregation. Currently, there are limited cures. The only approved drug for therapy is riluzole, that modestly prolongs survival, with edaravone now waiting for new clinical trial aimed to cla...
The 14th Meeting of the Interuniversity Institute of Myology (IIM), October 12-15, 2017 - Assisi,... more The 14th Meeting of the Interuniversity Institute of Myology (IIM), October 12-15, 2017 - Assisi, Italy gathered together researchers from Italy, European and North-American countries to discuss recent results on muscle research. The program showcased keynote lectures from world-renowned international speakers presenting advances in muscle physiology, bioengineering, metabolism and therapeutics. Based on selection from submitted abstracts, participants presented their novel, unpublished results in seven oral communication and two poster sessions. Particular emphasis was devoted to young trainees. For example, trainees where directly involved in organizing a scientific session and three round tables tailored to the interests of their peers. The meeting attracted a broad audience from Italy, various European countries and from North America. It offered a unique opportunity to all researchers involved in the field of muscle biology to exchange ideas and foster scientific collaborations...
Statins are the most prescribed and effective drugs to treat cardiovascular diseases (CVD). Never... more 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 s...
The ClC-1 chloride channel 1 is important for muscle function as it stabilizes resting membrane p... more The ClC-1 chloride channel 1 is important for muscle function as it stabilizes resting membrane potential and helps to repolarize the membrane after action potentials. We investigated the contribution of ClC-1 to adaptation of skeletal muscles to needs induced by the different stages of life. We analyzed the ClC-1 gene and protein expression as well as mRNA levels of protein kinase C (PKC) alpha and theta involved in ClC-1 modulation, in soleus (SOL) and extensor digitorum longus (EDL) muscles of rats in all stage of life. The cellular localization of ClC-1 in relation to age was also investigated. Our data show that during muscle development ClC-1 expression differs according to phenotype. In fasttwitch EDL muscles ClC-1 expression increased 10-fold starting at 7 days up to 8 months of life. Conversely, in slow-twitch SOL muscles ClC-1 expression remained constant until 33 days of life and subsequently increased fivefold to reach the adult value. Aging induced a downregulation of gene and protein ClC-1 expression in both muscle types analyzed. The mRNA of PKC-theta revealed the same trend as ClC-1 except in old age, whereas the mRNA of PKC-alpha increased only after 2 months of age. Also, we found that the ClC-1 is localized in both membrane and cytoplasm, in fibers of 12-day-old rats, becoming perfectly localized on the membrane in 2-month-old rats. This study could represent a point of comparison helpful for the identification of accurate pharmacological strategies for all the pathological situations in which ClC-1 protein is altered.
The discovery of pathogenetic mechanisms is essential to identify new therapeutic approaches in A... more 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-...
FASEB journal : official publication of the Federation of American Societies for Experimental Biology, Feb 2, 2017
Muscle fibers lacking dystrophin undergo a long-term alteration of Ca2+ homeostasis, partially ca... more 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 st...
Statin therapy may induce skeletal muscle damage ranging from myalgia to severe rhabdomyolysis. O... more Statin therapy may induce skeletal muscle damage ranging from myalgia to severe rhabdomyolysis. Our previous preclinical studies showed that statin treatment in rats involves the reduction of skeletal muscle ClC-1 chloride channel expression and related chloride conductance (gCl). An increase of the activity of protein kinase C theta (PKC theta) isoform, able to inactivate ClC-1, may contribute to destabilize sarcolemma excitability. These effects can be detrimental for muscle function leading to druginduced myopathy. Our goal is to study the causes of statin-induced muscle side effects in patients at the aim to identify biological markers useful to prevent and counteract statin-induced muscle damage. We examined 10 patients, who experienced myalgia and hyper-CK-emia after starting statin therapy compared to 9 non-myopathic subjects not using lipid-lowering drugs. Western Blot (WB) analysis showed a 40% reduction of ClC-1 protein and increased expression of phosphorylated PKC in muscle biopsies of statin-treated patients with respect to untreated subjects, independently from their age and statin type. Real-time PCR analysis showed that despite reduction of the protein, the ClC-1 mRNA was not significantly changed, suggesting posttranscriptional modification. The mRNA expression of a series of genes was also evaluated. MuRF-1 was increased in accord with muscle atrophy, MEF-2, calcineurin (CN) and GLUT-4 transporter were reduced, suggesting altered transcription, alteration of glucose homeostasis and energy deficit. Accordingly, the phosphorylated form of AMPK, measured by WB, was increased, suggesting cytoprotective process activation. In parallel, mRNA expression of Notch-1, involved in muscle cell proliferation, was highly expressed in statin-treated patients, indicating active regeneration. Also, PGC-1-alpha and isocitrate-dehydrogenase increased expression together with increased activity of mitochondrial citrate-synthase, measured by spectrophotometric assay, suggests mitochondrial biogenesis. Thus, the reduction of ClC-1 protein and consequent sarcolemma hyperexcitability together with energy deficiency appear to be among the most important alterations to be associated with statin-related risk of myopathy in
Journal of cachexia, sarcopenia and muscle, Jan 10, 2017
Cachexia is a wasting condition associated with cancer types and, at the same time, is a serious ... more 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. By a multidisciplinary approach ranging from cytofluorometry and electrophysiology to gene expression and histology, we characterized the calcium homeostasis in fast-twitch extensor digitorum longus (EDL) muscle of adult...
Taurine is a natural amino acid present as free form in many mammalian tissues and in particular ... more Taurine is a natural amino acid present as free form in many mammalian tissues and in particular in skeletal muscle. Taurine exerts many physiological functions, including membrane stabilization, osmoregulation and cytoprotective effects, antioxidant and anti-inflammatory actions as well as modulation of intracellular calcium concentration and ion channel function. In addition taurine may control muscle metabolism and gene expression, through yet unclear mechanisms. This review summarizes the effects of taurine on specific muscle targets and pathways as well as its therapeutic potential to restore skeletal muscle function and performance in various pathological conditions. Evidences support the link between alteration of intracellular taurine level in skeletal muscle and different pathophysiological conditions, such as disuse-induced muscle atrophy, muscular dystrophy and/or senescence, reinforcing the interest towards its exogenous supplementation. In addition, taurine treatment can be beneficial to reduce sarcolemmal hyper-excitability in myotonia-related syndromes. Although further studies are necessary to fill the gaps between animals and humans, the benefit of the amino acid appears to be due to its multiple actions on cellular functions while toxicity seems relatively low. Human clinical trials using taurine in various pathologies such as diabetes, cardiovascular and neurological disorders have been performed and may represent a guideline for designing specific studies in patients of neuromuscular diseases.
American Journal of Physiology-Cell Physiology, 2014
Angiotensin II (ANG II) plays a role in muscle wasting and remodeling; however, little evidence s... more Angiotensin II (ANG II) plays a role in muscle wasting and remodeling; however, little evidence shows its direct effects on specific muscle functions. We presently investigated the acute in vitro effects of ANG II on resting ionic conductance and calcium homeostasis of mouse extensor digitorum longus (EDL) muscle fibers, based on previous findings that in vivo inhibition of ANG II counteracts the impairment of macroscopic ClC-1 chloride channel conductance (gCl) in the mdx mouse model of muscular dystrophy. By means of intracellular microelectrode recordings we found that ANG II reduced gCl in the nanomolar range and in a concentration-dependent manner (EC50= 0.06 μM) meanwhile increasing potassium conductance (gK). Both effects were inhibited by the ANG II receptors type 1 (AT1)-receptor antagonist losartan and the protein kinase C inhibitor chelerythrine; no antagonism was observed with the AT2antagonist PD123,319. The scavenger of reactive oxygen species (ROS) N-acetyl cysteine a...
Muscle disuse produced by hindlimb unloading (HU) induces severe atrophy and slow-to-fast fibre t... more Muscle disuse produced by hindlimb unloading (HU) induces severe atrophy and slow-to-fast fibre type transition of the slow-twitch soleus muscle (Sol). After 2 weeks HU, the resting ClC-1 chloride conductance (g Cl) of sarcolemma, which controls muscle excitability, increases in Sol toward a value typical of the fast-twitch EDL muscle. After 3 days of HU, the g Cl increases as well before initiation of fibre type transition. Since ClC-1 channels are acutely silenced by PKC-dependent phosphorylation, we studied the modulation of g Cl by PKC and serine-threonine phosphatase in Sol during HU, using a number of pharmacological tools. We show that a fraction of ClC-1 channels of control Sol are maintained in an inactive state by PKC basal activity, which contributes to the lower g Cl in control Sol compared to EDL. After 14 days of HU, PKC/phosphatase manipulation produces effects on Sol g Cl that corroborate the partial slow-to-fast transition. After 3 days of HU, the early increase of g Cl in Sol is entirely attributable to a reduction of PKC activity and/or activation of phosphatase, maintaining ClC-1 channels in a fully active state. Accordingly, we found that HU reduces expression of PKCα, ε, and θ isoenzymes in Sol and EDL muscles and reduces total PKC activity. Moreover, we show that the rheobase current is increased in Sol muscle fibres as soon as after 3 days of HU, most probably in relation to the increased g Cl. In conclusion, Sol muscle disuse is characterized by a rapid reduction of PKC activity, which reduces muscle excitability and is likely to contribute to disuse-induced muscle impairment.
Using fura-2 and the manganese quenching technique, we show here that sarcolemmal permeability to... more 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. Key words: resting cytosolic calcium • sarcolemmal permeability to cations • slow-twitch and fast-twitch skeletal muscles • hindlimb unloading-induced slow-to-fast transition • fura-2 espite their high degree of differentiation, adult muscles are still able to adapt in response to altered physiological request, for instance, when an organism is exposed to new environmental conditions. This is the case of the antigravity muscles when exposed to hypogravity during space flight or in the ground-based model of simulated microgravity, the rat hindlimb unloading (HU) (1-3). During both space flight and HU, the soleus muscle undergoes atrophy and slow-to-fast phenotype transition. Studies have provided strong evidence that HU-induced muscle wasting is mainly related to an increased protein breakdown via activation of a calcium-independent and ATP-ubiquitin-dependent proteolysis pathway (2-5). However, HU-induced slow-to-fast transition is characterized by changes in the expression of numerous specific genes. For instance, the fast isoforms of estenso myosin heavy chain (MHC) and sarcoplasmic reticulum Ca-ATPase (SERCA) are up-regulated in response to HU, inducing a D shortening of relaxation and contraction times (6, 7). Also, electrophysiological and metabolic properties of soleus muscle are modified according to slow-to-fast transition, with, for example, an altered expression of sodium, chloride, and aquaporin-4 channels (8-10); a reduction of oxidative enzymes; and an increase of glycolytic enzyme activity (11). Despite the numerous studies on the HU-induced slow-to-fast transition of soleus muscle, the mechanisms that trigger gene expression changes during this process remain unclear (3). Calcium ion exerts a pivotal role in regulating muscle physiology (12). For instance, at shortterm, it triggers contraction, while at longer term, it is involved in modulation of gene expression. Interestingly, alteration of calcium homeostasis can induce muscle fiber phenotype changes (13-16). One of the better examples of this process was provided by Kubis et al. (13). It is known that intracellular cytosolic free calcium ([Ca 2+ ] i) at rest is muscle-type specific, being higher in muscle fibers from slow-twitch muscles (14, 17, 18). Kubis and collaborators have shown that in vitro application of a calcium ionophore to rabbit fast skeletal muscle cells in culture induces resting [Ca 2+ ] i increase and consequent fast-to-slow transformation, which are both reversible (13, 15). In this example, both a down-regulation of the type II fast MHC and an increased expression of the type I slow MHC isoforms were observed, suggesting that resting [Ca 2+ ] i is involved in both slow-and fast-twitch phenotype determinism and maintenance.
Slow-twitch muscles, devoted to postural maintenance, experience atrophy and weakness during musc... more Slow-twitch muscles, devoted to postural maintenance, experience atrophy and weakness during muscle disuse due to bedrest, aging or spaceflight. These conditions impair motion activities and can have survival implications. Human and animal studies demonstrate the anabolic role of IGF-1 on skeletal muscle suggesting its interest as a muscle disuse countermeasure. Thus, we tested the role of IGF-1 overexpression on skeletal muscle alteration due to hindlimb unloading (HU) by using MLC/mIgf-1 transgenic mice expressing IGF-1 under the transcriptional control of MLC promoter, selectively activated in skeletal muscle. HU produced atrophy in soleus muscle, in terms of muscle weight and fiber cross-sectional area (CSA) reduction, and up-regulation of atrophy gene MuRF1. In parallel, the disuse-induced slow-to-fast fiber transition was confirmed by an increase of the fast-type of the Myosin Heavy Chain (MHC), a decrease of PGC-1a expression and an increase of histone deacetylase-5 (HDAC5). Consistently, functional parameters such as the resting chloride conductance (gCl) together with ClC-1 chloride channel expression were increased and the contractile parameters were modified in soleus muscle of HU mice. Surprisingly, IGF-1 overexpression in HU mice was unable to counteract the loss of muscle weight and the decrease of fiber CSA. However, the expression of MuRF1 was recovered, suggesting early effects on muscle atrophy. Although the expression of PGC-1a and MHC were not improved in IGF-1-HU mice, the expression of HDAC5 was recovered. Importantly, the HU-induced increase of gCl was fully contrasted in IGF-1 transgenic mice, as well as the changes in contractile parameters. These results indicate that, even if local expression does not seem to attenuate HU-induced atrophy and slow-to-fast phenotype transition, it exerts early molecular effects on gene expression which can counteract the HU-induced modification of electrical and contractile properties. MuRF1 and HDAC5 can be attractive therapeutic targets for pharmacological countermeasures and then deserve further investigations.
Journal of Pharmacology and Experimental Therapeutics, 2007
The mechanism by which the 3-hydroxy-3-methyl-glutaryl-CoA reductase inhibitors (statins) induce ... more The mechanism by which the 3-hydroxy-3-methyl-glutaryl-CoA reductase inhibitors (statins) induce skeletal muscle injury is still under debate. By using fura-2 cytofluorimetry on intact extensor digitorum longus muscle fibers, here we provided the first evidence that 2 months in vivo chronic treatment of rats with fluvastatin (5 and 20 mg kg Ϫ1) and atorvastatin (5 and 10 mg kg Ϫ1) caused an alteration of calcium homeostasis. All treated animals showed a significant increase of resting cytosolic calcium [Ca 2ϩ ] i , up to 60% with the higher fluvastatin dose and up to 20% with the other treatments. The [Ca 2ϩ ] i rise induced by statin administration was not due to an increase of sarcolemmal permeability to calcium. Furthermore, the treatments reduced This study was supported by Italian "Ministero dell'Istruzione, dell'Università e della Ricerca" (FIRB RBAU015E9T) (to D.C.C.). Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
A pivotal role has been ascribed to oxidative stress in determining the imbalance between protein... more A pivotal role has been ascribed to oxidative stress in determining the imbalance between protein synthesis and degradation leading to muscle atrophy in many pathological conditions and in disuse. However, a large variability in disuse-induced alteration of redox homeostasis through muscles, models and species emerges from the literature. Whereas the causal role of oxidative stress appears well established in the mechanical ventilation model, findings are less compelling in the hindlimb unloaded mice and very limited in humans. The mere coexistence of muscle atrophy, indirect indexes of increased reactive oxygen species (ROS) production and impairment of antioxidant defence systems, in fact, does not unequivocally support a causal role of oxidative stress in the phenomenon. We hypothesise that in some muscles, models and species only, due to a large redox imbalance, the leading phenomena are activation of proteolysis and massive oxidation of proteins, which would become more suscept...
The preparation of a new series of 2-aryloxy-3-phenyl-propanoic acids, resulting from the introdu... more The preparation of a new series of 2-aryloxy-3-phenyl-propanoic acids, resulting from the introduction of a linker into the diphenyl system of the previously reported PPARalpha/gamma dual agonist 1, allowed the identification of new ligands with improved potency on PPARalpha and unchanged activity on PPARgamma. For the most interesting stereoisomers S-2 and S-4, X-ray studies in PPARgamma and docking experiments in PPARalpha provided a molecular explanation for their different behavior as full and partial agonists of PPARalpha and PPARgamma, respectively. Due to the adverse effects provoked by hypolipidemic drugs on skeletal muscle function, we also investigated the blocking activity of S-2 and S-4 on skeletal muscle membrane chloride channel conductance and found that these ligands have a pharmacological profile more beneficial compared to fibrates currently used in therapy.
In order to clarify the mechanism underlying the reduction of resting membrane chloride conductan... more In order to clarify the mechanism underlying the reduction of resting membrane chloride conductance (g gl) during aging, the levels of mRNA encoding the principal skeletal muscle chloride channel, ClC-1, were measured. Total RNA samples isolated from tibialis anterior muscles of aged (24^29 months old) and adult (3^4 months old) rats were examined for ClC-1 expression using Northern blot analysis, and macroscopic g gl was recorded from extensor digitorum longus muscle fibers from each adult and aged rat in vitro using a two intracellular microelectrode technique. Although interindividual variability was observed, aged rats exhibited a parallel reduction of both g gl and ClC-1 mRNA expression as compared to adult rats. A linear correlation exists between individual values of ClC-1 mRNA and g gl. These results provide evidence that ClC-1 is the main determinant of sarcolemmal g gl and demonstrate that the decrease of g gl observed during aging is associated with a down-regulation of ClC-1 expression in muscle.
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