The Impact of Aging, Calorie Restriction and Dietary Fat on Autophagy Markers and Mitochondrial Ultrastructure and Dynamics in Mouse Skeletal Muscle.

Loss of skeletal muscle mass and function is a hallmark of aging. This phenomenon has been related to a dysregulation of mitochondrial function and proteostasis. Calorie restriction (CR) has been demonstrated to delay aging and preserve function until late in life, particularly in muscle. Recently, we reported the type of dietary fat plays an important role in determining life span extension with 40% CR in male mice. In these conditions, lard fed mice showed an increased longevity compared to mice fed soybean or fish oils. In this article, we analyze the effect of 40% CR on muscle mitochondrial mass, autophagy, and mitochondrial dynamics markers in mice fed these diets. In CR fed animals, lard preserved muscle fibers structure, mitochondrial ultrastructure, and fission/fusion dynamics and autophagy, not only compared to control animals, but also compared with CR mice fed soybean and fish oils as dietary fat. We focus our discussion on dietary fatty acid saturation degree as an essential predictor of life span extension in CR mice.

Cholecalciferol in ethanol-preferring rats muscle fibers increases the number and area of type II fibers.

The chronic use of ethanol causes neuropathy and atrophy of type II fibers and promotes vitamin D decrease. This study evaluated cholecalciferol effects on the deep fibular nerve and extensor digitorum longus (EDL) muscle using an UChB ethanol-preferring rats model. Blood analyses were carried out to measure levels of 25-hydroxycholecalciferol (25(OH)D), calcium (Ca2+), Phosphorus (P), and parathyroid hormone (PTH). It was used EDL muscle to evaluate oxidative stress. The deep fibular nerve and EDL muscle were used for morphologic and morphometric assessment. 25(OH)D plasma levels were higher in the supplemented group and no alterations were observed in other parameters including the oxidative stress evaluation. The G ratio remained constant which indicates nervous conduction normality. Cholecalciferol supplementation promoted an increase in the number and area of type II fibers and a decrease in the area of type I fibers. In the studied model, there was neither alcoholic myopathy nor neuropathy. The EDL muscle glycolytic patterns in the high-drinker UChB rats may be associated with the differential effects of cholecalciferol on metabolism and protein synthesis in skeletal muscle.

Deletion of the microtubule-associated protein 6 (MAP6) results in skeletal muscle dysfunction.

BACKGROUND: The skeletal muscle fiber has a specific and precise intracellular organization which is at the basis of an efficient muscle contraction. Microtubules are long known to play a major role in the function and organization of many cells, but in skeletal muscle, the contribution of the microtubule cytoskeleton to the efficiency of contraction has only recently been studied. The microtubule network is dynamic and is regulated by many microtubule-associated proteins (MAPs). In the present study, the role of the MAP6 protein in skeletal muscle organization and function has been studied using the MAP6 knockout mouse line. METHODS: The presence of MAP6 transcripts and proteins was shown in mouse muscle homogenates and primary culture using RT-PCR and western blot. The in vivo evaluation of muscle force of MAP6 knockout (KO) mice was performed on anesthetized animals using electrostimulation coupled to mechanical measurement and multimodal magnetic resonance. The impact of MAP6 deletion on microtubule organization and intracellular structures was studied using immunofluorescent labeling and electron microscopy, and on calcium release for muscle contraction using Fluo-4 calcium imaging on cultured myotubes. Statistical analysis was performed using Student's t test or the Mann-Whitney test. RESULTS: We demonstrate the presence of MAP6 transcripts and proteins in skeletal muscle. Deletion of MAP6 results in a large number of muscle modifications: muscle weakness associated with slight muscle atrophy, alterations of microtubule network and sarcoplasmic reticulum organization, and reduction in calcium release. CONCLUSION: Altogether, our results demonstrate that MAP6 is involved in skeletal muscle function. Its deletion results in alterations in skeletal muscle contraction which contribute to the global deleterious phenotype of the MAP6 KO mice. As MAP6 KO mouse line is a model for schizophrenia, our work points to a possible muscle weakness associated to some forms of schizophrenia.

Myotube elasticity of an amyotrophic lateral sclerosis mouse model.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that affects the motor system leading to generalized paralysis and death of patients. The understanding of early pathogenic mechanisms will help to define early diagnostics criteria that will eventually provide basis for efficient therapeutics. Early symptoms of ALS usually include muscle weakness or stiffness. Therefore, mechanical response of differentiated myotubes from primary cultures of mice, expressing the ALS-causing SOD1 G93A mutation, was examined by atomic force microscopy. Simultaneous acquisition of topography and cell elasticity of ALS myotubes was performed by force mapping method, compared with healthy myotubes and supplemented with immunofluorescence and qRT-PCR studies. Wild type myotubes reveal a significant difference in elasticity between a narrow and a wide population, consistent with maturation occurring with higher actin expression relative to myosin together with larger myotube width. However, this is not true for SOD1 G93A expressing myotubes, where a significant shift of thin population towards higher elastic modulus values was observed. We provide evidence that SOD1 mutant induces structural changes that occurs very early in muscle development and well before symptomatic stage of the disease. These findings could significantly contribute to the understanding of the role of skeletal muscle in ALS pathogenesis.

Primary skeletal muscle cells cultured on gelatin bead microcarriers develop structural and biochemical features characteristic of adult skeletal muscle.

A primary skeletal muscle cell culture, in which myoblasts derived from newborn rabbit hindlimb muscles grow on gelatin bead microcarriers in suspension and differentiate into myotubes, has been established previously. In the course of differentiation and beginning spontaneous contractions, these multinucleated myotubes do not detach from their support. Here, we describe the development of the primary myotubes with respect to their ultrastructural differentiation. Scanning electron microscopy reveals that myotubes not only grow around the surface of one carrier bead but also attach themselves to neighboring carriers, forming bridges between carriers. Transmission electron microscopy demonstrates highly ordered myofibrils, T-tubules, and sarcoplasmic reticulum. The functionality of the contractile apparatus is evidenced by contractile activity that occurs spontaneously or can be elicited by electrostimulation. Creatine kinase activity increases steadily until day 20 of culture. Regarding the expression of isoforms of myosin heavy chains (MHC), we could demonstrate that from day 16 on, no non-adult MHC isoform mRNAs are present. Instead, on day 28 the myotubes express predominantly adult fast MHCIId/x mRNA and protein. This MHC pattern resembles that of fast muscles of adult rabbits. In contrast, primary myotubes grown on matrigel-covered culture dishes express substantial amounts of non-adult MHC protein even on day 21. To conclude, primary myotubes grown on microcarriers in their later stages exhibit many features of adult skeletal muscle and characteristics of fast type II fibers. Thus, the culture represents an excellent model of adult fast skeletal muscle, for example, when investigating molecular mechanisms of fast-to-slow fiber-type transformation.

Lifelong physical exercise delays age-associated skeletal muscle decline.

Aging is usually accompanied by a significant reduction in muscle mass and force. To determine the relative contribution of inactivity and aging per se to this decay, we compared muscle function and structure in (a) male participants belonging to a group of well-trained seniors (average of 70 years) who exercised regularly in their previous 30 years and (b) age-matched healthy sedentary seniors with (c) active young men (average of 27 years). The results collected show that relative to their sedentary cohorts, muscle from senior sportsmen have: (a) greater maximal isometric force and function, (b) better preserved fiber morphology and ultrastructure of intracellular organelles involved in Ca(2+) handling and ATP production, (c) preserved muscle fibers size resulting from fiber rescue by reinnervation, and (d) lowered expression of genes related to autophagy and reactive oxygen species detoxification. All together, our results indicate that: (a) skeletal muscle of senior sportsmen is actually more similar to that of adults than to that of age-matched sedentaries and (b) signaling pathways controlling muscle mass and metabolism are differently modulated in senior sportsmen to guarantee maintenance of skeletal muscle structure, function, bioenergetic characteristics, and phenotype. Thus, regular physical activity is a good strategy to attenuate age-related general decay of muscle structure and function (ClinicalTrials.gov: NCT01679977).

Antioxidant SkQ1 delays sarcopenia-associated damage of mitochondrial ultrastructure.

A comparative electron-microscopic study of ultrastructure of mitochondria in skeletal muscles of the 3- and 24-month-old Wistar and OXYS rats revealed age-dependent changes in both general organization of the mitochondrial reticulum and ultrastructure of mitochondria. The most pronounced ultrastructure changes were detected in the OXYS rats suffering from permanent oxidative stress. In the OXYS rats, significant changes in mitochondrial ultrastructure were detected already at the age of 3 months. Among them, there were the appearance of megamitochondria and reduction of cristae resulting in formation of cristae-free regions inside mitochondria. In the 24-month-old OXYS rats, mitochondrial reticulum was completely destroyed. In the isotropic region of muscle fiber, only small solitary mitochondria were present. There appeared regions of lysed myofibrils as well as vast regions filled with autophagosomes. A mitochondrial antioxidant SkQ1 (given to rats with food daily in the dose of 250 nmol/kg of body weight for 5 months beginning from the age of 19 months) prevented development of age-dependent destructive changes in both the Wistar and OXYS rats.

The muscular force transmission system: role of the intramuscular connective tissue.

The objective of this review is to analyze in detail the microscopic structure and relations among muscular fibers, endomysium, perimysium, epimysium and deep fasciae. In particular, the multilayer organization and the collagen fiber orientation of these elements are reported. The endomysium, perimysium, epimysium and deep fasciae have not just a role of containment, limiting the expansion of the muscle with the disposition in concentric layers of the collagen tissue, but are fundamental elements for the transmission of muscular force, each one with a specific role. From this review it appears that the muscular fibers should not be studied as isolated elements, but as a complex inseparable from their fibrous components. The force expressed by a muscle depends not only on its anatomical structure, but also the angle at which its fibers are attached to the intramuscular connective tissue and the relation with the epimysium and deep fasciae.

Oxidative stress and successful antioxidant treatment in models of RYR1-related myopathy.

The skeletal muscle ryanodine receptor is an essential component of the excitation-contraction coupling apparatus. Mutations in RYR1 are associated with several congenital myopathies (termed RYR1-related myopathies) that are the most common non-dystrophic muscle diseases of childhood. Currently, no treatments exist for these disorders. Although the primary pathogenic abnormality involves defective excitation-contraction coupling, other abnormalities likely play a role in disease pathogenesis. In an effort to discover novel pathogenic mechanisms, we analysed two complementary models of RYR1-related myopathies, the relatively relaxed zebrafish and cultured myotubes from patients with RYR1-related myopathies. Expression array analysis in the zebrafish disclosed significant abnormalities in pathways associated with cellular stress. Subsequent studies focused on oxidative stress in relatively relaxed zebrafish and RYR1-related myopathy myotubes and demonstrated increased oxidant activity, the presence of oxidative stress markers, excessive production of oxidants by mitochondria and diminished survival under oxidant conditions. Exposure to the antioxidant N-acetylcysteine reduced oxidative stress and improved survival in the RYR1-related myopathies human myotubes ex vivo and led to significant restoration of aspects of muscle function in the relatively relaxed zebrafish, thereby confirming its efficacy in vivo. We conclude that oxidative stress is an important pathophysiological mechanism in RYR1-related myopathies and that N-acetylcysteine is a successful treatment modality ex vivo and in a vertebrate disease model. We propose that N-acetylcysteine represents the first potential therapeutic strategy for these debilitating muscle diseases.

Calsequestrin (CASQ1) rescues function and structure of calcium release units in skeletal muscles of CASQ1-null mice.

Amplitude of Ca(2+) transients, ultrastructure of Ca(2+) release units, and molecular composition of sarcoplasmic reticulum (SR) are altered in fast-twitch skeletal muscles of calsequestrin-1 (CASQ1)-null mice. To determine whether such changes are directly caused by CASQ1 ablation or are instead the result of adaptive mechanisms, here we assessed ability of CASQ1 in rescuing the null phenotype. In vivo reintroduction of CASQ1 was carried out by cDNA electro transfer in flexor digitorum brevis muscle of the mouse. Exogenous CASQ1 was found to be correctly targeted to the junctional SR (jSR), as judged by immunofluorescence and confocal microscopy; terminal cisternae (TC) lumen was filled with electron dense material and its width was significantly increased, as judged by electron microscopy; peak amplitude of Ca(2+) transients was significantly increased compared with null muscle fibers transfected only with green fluorescent protein (control); and finally, transfected fibers were able to sustain cytosolic Ca(2+) concentration during prolonged tetanic stimulation. Only the expression of TC proteins, such as calsequestrin 2, sarcalumenin, and triadin, was not rescued as judged by Western blot. Thus our results support the view that CASQ1 plays a key role in both Ca(2+) homeostasis and TC structure.

Fenofibrate, a PPAR{alpha} agonist, decreases atrogenes and myostatin expression and improves arthritis-induced skeletal muscle atrophy.

Arthritis is a chronic inflammatory illness that induces cachexia, which has a direct impact on morbidity and mortality. Fenofibrate, a selective PPARα activator prescribed to treat human dyslipidemia, has been reported to decrease inflammation in rheumatoid arthritis patients. The aim of this study was to elucidate whether fenofibrate is able to ameliorate skeletal muscle wasting in adjuvant-induced arthritis, an experimental model of rheumatoid arthritis. On day 4 after adjuvant injection, control and arthritic rats were treated with 300 mg/kg fenofibrate until day 15, when all rats were euthanized. Fenofibrate decreased external signs of arthritis and liver TNFα and blocked arthritis-induced decreased in PPARα expression in the gastrocnemius muscle. Arthritis decreased gastrocnemius weight, which results from a decrease in cross-section area and myofiber size, whereas fenofibrate administration to arthritic rats attenuated the decrease in both gastrocnemius weight and fast myofiber size. Fenofibrate treatment prevented arthritis-induced increase in atrogin-1 and MuRF1 expression in the gastrocnemius. Neither arthritis nor fenofibrate administration modify Akt-FoxO3 signaling. Myostatin expression was not modified by arthritis, but fenofibrate decreased myostatin expression in the gastrocnemius of arthritic rats. Arthritis increased muscle expression of MyoD, PCNA, and myogenin in the rats treated with vehicle but not in those treated with fenofibrate. The results indicate that, in experimental arthritis, fenofibrate decreases skeletal muscle atrophy through inhibition of the ubiquitin-proteasome system and myostatin.

Functional electrical stimulation of denervated muscles: an experimental evaluation.

For many years there has been a dearth of effective treatment options for the severe wasting and secondary consequences of motor nerve injury. In recent years, however, an intensive regime of electrical stimulation has been shown to have considerable therapeutic benefits. This article reviews the results of an extensive study designed to address the clinically relevant issues in an appropriate animal model. The study reveals both the benefits and the limitations of the technique, but strongly endorses the therapeutic advantages of introducing a program of stimulation during the initial, nondegenerative phase of the muscle response to nerve or root injury.

Acupuncture muscle channel in the subcutaneous layer of rat skin.

Using a mixed-dye injection technique, we found a novel kind of muscle fiber with a lumen, established its precise location in the subcutaneous muscle layer along the acupuncture muscle of the bladder line, and determined its detailed ultrastructure. The channels with flowing liquid were a novel kind of muscle fibers with lumens and they were located in the subcutaneous muscle layer of rat. Their detection was realized by using chrome-hematoxylin and a mixture of fluorescent nanoparticles and commercial Pelikan ink. These acupuncture muscle channels were hidden among the neighboring skin skeletal muscle fibers and were barely distinguishable from them with light microscopes. Only with a transmission electron microscope were their characteristic features shown to be different from normal skin skeletal muscle. These features included undifferentiated muscle fibers that resembled immature myofibrils without Z-lines and reassembled telophase nuclei.

Combined enzymatic and antioxidative treatment reduces ischemia-reperfusion injury in rabbit skeletal muscle.

BACKGROUND: Ischemia/reperfusion (I/R) injury is characterized by the production of oxygen-free radicals leading to disturbances in vasomotility (microvascular constriction) and microvascular permeability (interstitial edema formation). The objective was to evaluate the effect of the combined antioxidative and enzymatic preparation Phlogenzym on I/R injury of skeletal muscle. MATERIALS AND METHODS: A rabbit hindlimb model of I/R (2.5/2 h) was used (IR group). Phlogenzym, containing rutin, trypsin, and bromelain, was applied enterally (60 mg/kg body weight) as a bolus 30 min prior to ischemia (Ph group). Sham-operated animals served as controls (CO group). Plasma malondialdehyde, potassium, and microvascular perfusion (monitored by laser flowmetry) were assessed. Histomorphometry and electron microscopy were performed from major adductor muscles. RESULTS: Two hours after reperfusion, potassium levels were significantly elevated in IR compared to Ph group (6.7 +/- 1.2 versus 4.9 +/- 0.9 mmol/l, P < 0.006). Enhanced lipid peroxidation, apparent by increased plasma malondialdehyde levels, was ameliorated in the Ph group (1.0 +/- 0.1 versus 0.7 +/- 0.1 nmol/ml, P < 0.0001). No-reflow (reduction of blood flow by 62% in IR group) was not observed in the Ph group (P < 0.004). Phlogenzym treatment prevented microvascular constriction (17.6 +/- 2.3 versus 12.6 +/- 1.1 microm(2), P < 0.0001) and mollified interstitial edema (21.5 +/- 2.0 versus 26.0 +/- 3.7%, P < 0.017), resulting in mild ultrastructural alterations in contrast to pronounced sarcolemmal and mitochondrial damage in untreated rabbits. CONCLUSIONS: Phlogenzym had a protective effect on skeletal muscle during I/R injury expressed by prevention of no-reflow and preservation of muscle tissue.

Vastus medialis: anatomical and functional considerations and implications based upon human and cadaveric studies.

OBJECTIVE: To provide an electrophysiological and functional description of the vastus medialis (VM) and contrast it to an anatomical description. METHODS: Motor points of all superficial portions of the quadriceps were identified on the dominant side of 8 human subjects and electrically stimulated to achieve a light contraction to trace and measure the orientation of the fibers. Electromyography of the VM was then recorded over 2 motor points during isometric and isokinetic maximum knee extensions. An independent laboratory dissected 39 cadaveric specimens focusing on fiber orientations and distal insertions of the VM. RESULTS: Results revealed 5 motor points for the quadriceps: 1 point for the vastus lateralis, 1 point for the rectus femoris (RF), and 3 points for the VM. The 3 VM motor points suggest 3 separate groups of fibers: proximal (pf), median (mf), distal (df). Fiber orientations ranged from 45 degrees for VMpfs to 55 degrees for VMdfs. Motor point stimulation and anatomical dissection clearly showed that the VMpfs and VMmfs were inserted on a tendon common to the RF, whereas VMdfs were attached directly to the medial aspect of the patella. Furthermore, the VMpfs were more active (P < .05) than VMdfs during maximum knee extensions. CONCLUSION: The anatomy, motor points, and electromyography clearly support an important distinction between the VMpfs and VMdfs. The role of the VMpfs would be one of assisting the RF in knee extension, whereas the VMdfs would track the patella medially without participating in knee extension. Because of these anatomical and functional differences, the VMpfs and VMdfs should be addressed very differently during quadriceps rehabilitation in patellofemoral dysfunctions.

Electrical stimulation attenuates denervation and age-related atrophy in extensor digitorum longus muscles of old rats.

Skeletal muscles of old rats and elderly humans lose muscle mass and maximum force. Denervation is a major cause of age-related muscle atrophy and weakness, because denervated fibers do not contract, and undergo atrophy. At any age, surgical denervation causes even more dramatic muscle atrophy and loss in force than aging does. Electrical stimulation that generates tetanic contractions of denervated muscles reduces the denervation-induced declines. We investigated whether a stimulation protocol that maintains mass and force of denervated extensor digitorum longus muscles of adult rats would also maintain these properties in denervated muscles of old rats during a 2-month period of age-induced declines in these properties. Contractile activity generated by the electrical stimulation eliminated age-related losses in muscle mass and reduced the deficit in force by 50%. These data provide support for the hypothesis that during aging, lack of contractile activity in fibers contributes to muscle atrophy and weakness.

The effect of resistance training combined with timed ingestion of protein on muscle fiber size and muscle strength.

Acute muscle protein metabolism is modulated not only by resistance exercise but also by amino acids. However, less is known about the long-term hypertrophic effect of protein supplementation in combination with resistance training. The present study was designed to compare the effect of 14 weeks of resistance training combined with timed ingestion of isoenergetic protein vs carbohydrate supplementation on muscle fiber hypertrophy and mechanical muscle performance. Supplementation was administered before and immediately after each training bout and, in addition, in the morning on nontraining days. Muscle biopsy specimens were obtained from the vastus lateralis muscle and analyzed for muscle fiber cross-sectional area. Squat jump and countermovement jump were performed on a force platform to determine vertical jump height. Peak torque during slow (30 degrees s-1) and fast (240 degrees s-1) concentric and eccentric contractions of the knee extensor muscle was measured in an isokinetic dynamometer. After 14 weeks of resistance training, the protein group showed hypertrophy of type I (18% +/- 5%; P < .01) and type II (26% +/- 5%; P < .01) muscle fibers, whereas no change above baseline occurred in the carbohydrate group. Squat jump height increased only in the protein group, whereas countermovement jump height and peak torque during slow isokinetic muscle contraction increased similarly in both groups. In conclusion, a minor advantage of protein supplementation over carbohydrate supplementation during resistance training on mechanical muscle function was found. However, the present results may have relevance for individuals who are particularly interested in gaining muscle size.

Long-term denervation in humans causes degeneration of both contractile and excitation-contraction coupling apparatus, which is reversible by functional electrical stimulation (FES): a role for myofiber regeneration?

Over the last 30 years there has been considerable interest in the use of functional electrical stimulation (FES) to restore movement to the limbs of paralyzed patients. Spinal cord injury causes a rapid loss in both muscle mass and contractile force. The atrophy is especially severe when the injury involves lower motoneurons because many months after spinal cord injury, atrophy is complicated by fibrosis and fat substitution. In this study we describe the effects of long-term lower motoneuron denervation of human muscle and present the structural results of muscle trained using FES. By means of an antibody for embryonic myosin, we demonstrate that many regenerative events continue to spontaneously occur in human long-term denervated and degenerated muscle (DDM). In addition, using electron microscopy, we describe i) the overall structure of fibers and myofibrils in long-term denervated and degenerated muscle, including the effects of FES, and ii) the structure and localization of calcium release units, or triads; the structures reputed to activate muscle contraction during excitation-contraction coupling (ECC). Both apparatus undergo disarrangement and re-organization following long-term denervation and FES, respectively. The poor excitability of human long-term DDM fibers, which extends to the first periods of FES training, may be explained in terms of the spatial disorder of the ECC apparatus. Its disorganization and re-organization following long-term denervation and FES, respectively, may play a key role in the parallel disarrangement and re-organization of the myofibrils that characterize denervation and FES training. The present structural studies demonstrate that the protocol used during FES training is effective in reverting long-term denervation atrophy and dystrophy. The mean fiber diameter in FES biopsies is 42.2 +/- 14.8 SD (p < 0.0001 vs DDM 14.9 +/- 6.0 SD); the mean percentile of myofiber area of the biopsy is 94.3 +/- 5.7 SD (p < 0.0001 vs DDM 25.7 +/- 23.7 SD); the mean percentile fat area is 2.1 +/- 2.4 SD (p < 0.001 vs DDM 12.8 +/- 12.1 SD); and the mean percentile connective tissue area is 3.6 +/- 4.6 SD (p < 0.001 vs DDM 61.6 +/- 20.1 SD). In DDM biopsies more than 50% of myofibers have diameter smaller than 10 microm, while the FES-trained subjects have more that 50% of myofibers larger than 30 microm. The recovery of muscle mass seems to be the result of both a size increase of the surviving fibers and the regeneration of new myofibers.

Primary nerve repair by muscle autografts prepared with local anesthetic.

We evaluated the usefulness of muscle autografts obtained immediately after graft preparation with lidocaine injections for primary nerve repair. The right sciatic nerve of adult Wistar rats was sectioned, and muscle grafts obtained 15 min or 24 h after lidocaine injection were used to repair a gap 1.5 cm long. Axon and fiber diameters, as well as myelin thickness, decreased to similar extents for grafts of both time intervals. The G-ratios in the distal stumps of both groups were not different from controls, indicating that regenerated axons had a proper level of myelination. The ultrastructural appearance of the neuromuscular junctions was similar to that of normal samples. These results indicate that there are no restrictions to the use of a muscle graft for primary nerve repair, immediately after lidocaine injection, since the nerve regeneration was comparable to that observed with this type of graft used 24 h after being prepared.

Involvement of micro-calpain (CAPN 1) in muscle cell differentiation.

Several studies have already demonstrated that micro- and milli-calpains (CAPN 1-CAPN 2), calcium-dependent intracellular cysteine-proteases are involved in many biological phenomenon including muscle growth and development. More particularly, recent studies have demonstrated that milli-calpain is implicated in myoblast fusion. Moreover, in primary muscle cells, these proteases do not appear simultaneously throughout muscle cell differentiation. Because micro- and milli-calpains do not have the same intracellular localization, it appears likely that these two calcium-dependent proteases have different biological roles during muscle cell differentiation. The goal of this study is to determine the role of micro-calpain. We therefore, have developed a muscle cell line in which micro-calpain is over-expressed, using the inducible Tet Regulated Expression System. The outcome is observed by following the behavior of different proteins, considered to be potential substrates of the protease. The present study shows important decreases in the expression level of ezrin (68%), vimentin (64%) and caveolin 3 (76%) whereas many other cytoskeletal proteins remain remarkably stable. Concerning the myogenic transcription factors, only the level of myogenin decreased (59%) after the over-expression of micro-calpain. Ultra structural studies have shown that the myofibrils formed near the cell periphery are normally oriented, lying along the longitudinal axis. This regularity is lost progressively towards the cell center where the cytoskeleton presented an increasing disorganization. All these results indicate that micro-calpain is involved in regulation pathway of myogenesis via at least its action on ezrin, vimentin, caveolin 3 and myogenin, a muscle transcription factor.