Interactions between dissociated rat sympathetic neurons and skeletal muscle cells developing in cell culture. I. Cholinergic transmission

Sympathetic neurons, dissociated from superior cervical ganglia of newborn rats, and skeletal muscle cells were grown together in mass cultures containing many neurons (ca. 1000–3000) and myotubes, and in microcultures containing only one to three neurons and one or a few myotubes. When these neurons grow under the influence of certain nonneuronal cells many of them acquire cholinergic functions; in the absence of this influence they remain adrenergic. In the present study, the influence of the skeletal muscle cells was so effective that under certain conditions more than 75% of the neurons expressed cholinergic function as judged by their ability to form excitatory cholinergic synapses with myotubes (from rat and chick) and with each other. Stimulation of single neurons often gave rise in the myotubes to simple (direct) postsynaptic potentials (ejp's) and/or complex responses comprising a burst of ejp's that evoked one or more spikes; it appeared that these complex responses involved the activation of interneuronal pathways. In microcultures, a single neuron often made cholinergic synapses with itself (“autapse”) and/or with another neuron as well as with one or more myotubes. The nicotinic blocking agents, tubocurare (dTC), α-bungarotoxin (α-BuTx), and hexamethonium (C₆), attenuated or abolished the ejp's at moderate concentrations; the muscarinic blocker, atropine, was effective only at high concentrations. At several neuron-myotube junctions, the acetylcholine (ACh) receptors had dTC sensitivity similar to adult extrajunctional receptors; however, when different junctions were pooled the average dTC sensitivity was intermediate between that of adult end plate and extrajunctional receptors. The junctional C₆ sensitivity was much higher than expected from the action of the drug at the adult mammalian end plate. As in other studies, chemical transmission from neuron to neuron was also nicotinic cholinergic, but the nicotinic receptors on the myotubes were pharmacologically distinct from those on the neurons.     

The effect on butyryl-CoA dehydrogenase of reagents specific for nucleophilic sulphur.

The technique of erythrocyte-mediated microinjection has been successfully adapted for use with cultured muscle cells. Erythrocytes were fused with primary chick myotube cultures with poly(ethylene glycol), and fluorescent antibodies to haemoglobin demonstrated that this protein was injected into the sarcoplasm of myotubes. The microinjection treatment did not significantly alter protein metabolism in the muscle cells as monitored by rates of synthesis and degradation of muscle proteins. 125I-labelled ribonuclease A and bovine serum albumin were degraded with the expected exponential decay kinetics after microinjection into muscle cells, and the half-life of ribonuclease A (40 h) was approximately twice that of bovine serum albumin (17 h). The degradation of ribonuclease A in the muscle cells was enhanced 1.6-fold in the absence of horse serum and chick-embryo extract, whereas the degradation of bovine serum albumin was not altered during deprivation. These results are characteristic of the breakdown of microinjected ribonuclease A and bovine serum albumin in other cell types. Therefore, our experiments indicate the erythrocyte-mediated microinjection is a valid technique to study protein degradation in primary chick muscle cultures.     

Properties of a murine monocytic tumor cell line J-774 in vitro. I. Morphology and endocytosis.

A murine monocytic tumor cell line J-774 was maintained in culture in the presence or absence of endotoxin, in an attempt to induce differentiation similar to that found in activated peritoneal macrophages. The morphology and Fc and C₃ receptor functions of attachment and ingestion were compared in the treated and untreated cultures. J-774 cells maintained in culture for 72 h seemed to resemble endotoxin-activated macrophages rather than normal peritoneal macrophages. A striking amount of ruffling was observed on the surface of the cells cultured for 3–4 days both in the presence and in the absence of endotoxin. As compared to the peritoneal macrophage where particles attached to the C₃ receptors are not ingested unless the cells are activated, J-744 cells attached and ingested particles via the C₃ receptor even without stimulation. The presence of endotoxin in the culture medium of these cells gave rise to more efficient phagocytosis but did not effect the temperature sensitivity of the phagocytic receptors. Both in treated and untreated cultures attachment to the Fc receptor was less dependent on the temperature than that of the C₃ receptor while ingestion was more sensitive in the Fc receptor as compared with the C₃ receptor.     

Transferrin receptor numbers and transferrin and iron uptake in cultured chick muscle cells at different stages of development

The mechanism of iron uptake and the changes which occur during cellular development of muscle cells were investigated using primary cultures of chick embryo breast muscle. Replicating presumptive myoblasts were examined in exponential growth and after growth had plateaued. These were compared to the terminally differentiated cell type, the myotube. All cells, regardless of the state of growth or differentiation, had specific receptors for transferrin. Presumptive myoblasts in exponential growth had more transferrin receptors (3.78 +/- 0.24 X 10(10) receptors/micrograms DNA) than when division had ceased (1.70 +/- 0.14 X 10(10) receptors/micrograms DNA), while myotubes had 3.80 +/- 0.26 X 10(10) receptors/micrograms DNA. Iron uptake occurred by receptor-mediated endocytosis of transferrin. While iron was accumulated by the cells, apotransferrin was released in an undegraded form. There was a close correlation between the molar rates of endocytosis of transferrin and iron. Maximum rates of iron uptake were significantly higher in myotubes than in presumptive myoblasts in either exponential growth or after growth had plateaued. There were two rates of exocytosis of transferrin, implying the existence of two intracellular pathways for transferrin. These experiments demonstrate that iron uptake by muscle cells in culture occurs by receptor-mediated endocytosis of transferrin and that transferrin receptor numbers and the kinetics of transferrin and iron uptake vary with development of the cells.     

Turnover of Adrenergic Receptors Under Normal and Desensitized Conditions

Abstract

Alpha₁ and beta adrenergic receptor metabolism was investigated by studying receptor reappearance after an irreversible blockade. Phenoxybenzamine was used to irreversibly block alpha₁ adrenergic receptors both in vitro in the BC₃H₁ cell line and in vivo in rat submaxillary glands. In these two systems, the alpha₁ adrenergic receptor reappearance followed a monoexponential kinetic allowing to determine the half-life of the receptor (23h in vitro, 33h in vivo) as well as the rate of receptor synthesis and degradation. the receptor reappearance was due to receptor synthesis since it was blocked by cycloheximide. The irreversible blockade of beta adrenergic receptors was done with an alkylating beta adrenergic antagonist that we recently developped : Br-pindolol (1). This ligand has high efficiency and blocked at 10^?7 M 80-90% of the beta adrenergic receptors present in C₆ glioma cells in culture. After this irreversible blockade, receptors reappeared only during cell division. At confluency, when cells did not significantly divide, receptor synthesis could hardly be detectable. Therefore, at confluency, the metabolic stability of the beta adrenergic receptor is considerable, compared to that of the alpha₁ adrenergic receptor. This stability was confirmed by the observation that after an almost complete “down-regulation” of the beta adrenergic receptor, receptor repopulation of the C₆ glioma cells was total and occured in the presence of cycloheximide.     

Quantification of hormone-induced atrophy of large myotubes from C2C12 and L6 cells: atrophy-inducible and atrophy-resistant C2C12 myotubes

Myofiber atrophy is the final outcome of muscle wasting induced by catabolic factors such as glucocorticoids and thyroid hormones. We set up an in vitro system to define the catabolic reaction based on myotube atrophy. Both mouse C₂C₁₂ and rat L6 cells were used. C₂C₁₂ myotube formation was improved by replacing horse serum with the serum substitute Ultroser G. A new method was developed to quantify size changes of large (0.5–1 mm) myotubes only, excluding remaining myoblasts and small myotubes. Dexamethasone reduced myotube size by 30% in L6 but not in C₂C₁₂ myotubes. Expression of the glucocorticoid receptor was twofold higher in L6 myotubes than in C₂C₁₂ myotubes. In both cell lines, 3,3',5-triiodo-L-thyronine (T₃) did not induce a significant size reduction. Expression of the major T₃ receptor (T₃R1) was higher in L6 myotubes. We investigated whether the changes in myotube size are related to changes in atrogin-1 expression, as this enzyme is thought to be a key factor in the initiation of muscle atrophy. Dexamethasone induced a twofold increase of atrogin-1 mRNA; again, only L6 myotubes were susceptible. Interestingly, atrogin-1 expression in Ultroser G-fused C₂C₁₂ myotubes was lower than that in horse serum-fused myotubes. Furthermore, dexamethasone treatment increased atrogin-1 expression only in horse serum-fused myotubes but not in Ultroser G-fused myotubes. Ultroser G-induced fusion may result in atrophy-resistant C₂C₁₂ myotubes. Therefore, C₂C₁₂ myotubes offer an ideal system in which to study skeletal muscle atrophy because, depending on differentiation conditions, C₂C₁₂ cells produce atrophy-inducible and atrophy-resistant myotubes. glucocorticoids; nuclear receptors; atrogin     

Micropatterning contractile C2C12 myotubes embedded in a fibrin gel

Contractile C₂C₁₂ myotube line patterns embedded in a fibrin gel have been developed to afford a physiologically relevant and stable bioassay system. The C₂C₁₂ myotube/fibrin gel system was prepared by transferring a myotube monolayer from a glass substrate to a fibrin gel while retaining the original line patterns of myotubes. To endow the myotubes with contractile activity, a series of electrical pulses was applied through a pair of carbon electrodes placed at either side of a fibrin gel separately. The frequency and magnitude of myotube contraction were functions of the pulse frequency and duration, respectively. We found that the myotubes supported by an elastic fibrin gel maintained their line patterns and contractile activities for a longer period of time (1 week) than myotubes adhered on a conventional culture dish. Biotechnol. Bioeng. 2010;105: 1161–1167. © 2009 Wiley Periodicals, Inc.     

Pharmacological Blockade of Cannabinoid CB1 Receptors in Diet-Induced Obesity Regulates Mitochondrial Dihydrolipoamide Dehydrogenase in Muscle

Cannabinoid CB₁ receptors peripherally modulate energy metabolism. Here, we investigated the role of CB₁ receptors in the expression of glucose/pyruvate/tricarboxylic acid (TCA) metabolism in rat abdominal muscle. Dihydrolipoamide dehydrogenase (DLD), a flavoprotein component (E3) of α-ketoacid dehydrogenase complexes with diaphorase activity in mitochondria, was specifically analyzed. After assessing the effectiveness of the CB₁ receptor antagonist AM251 (3 mg kg^-1, 14 days) on food intake and body weight, we could identified seven key enzymes from either glycolytic pathway or TCA cycle—regulated by both diet and CB₁ receptor activity—through comprehensive proteomic approaches involving two-dimensional electrophoresis and MALDI-TOF/LC-ESI trap mass spectrometry. These enzymes were glucose 6-phosphate isomerase (GPI), triosephosphate isomerase (TPI), enolase (Eno3), lactate dehydrogenase (LDHa), glyoxalase-1 (Glo1) and the mitochondrial DLD, whose expressions were modified by AM251 in hypercaloric diet-induced obesity. Specifically, AM251 blocked high-carbohydrate diet (HCD)-induced expression of GPI, TPI, Eno3 and LDHa, suggesting a down-regulation of glucose/pyruvate/lactate pathways under glucose availability. AM251 reversed the HCD-inhibited expression of Glo1 and DLD in the muscle, and the DLD and CB₁ receptor expression in the mitochondrial fraction. Interestingly, we identified the presence of CB₁ receptors at the membrane of striate muscle mitochondria. DLD over-expression was confirmed in muscle of CB ₁ ^-/- mice. AM251 increased the pyruvate dehydrogenase and glutathione reductase activity in C₂C₁₂ myotubes, and the diaphorase/oxidative activity in the mitochondria fraction. These results indicated an up-regulation of methylglyoxal and TCA cycle activity. Findings suggest that CB₁ receptors in muscle modulate glucose/pyruvate/lactate pathways and mitochondrial oxidative activity by targeting DLD.     

Acetycholine receptor production and incorporation into membranes of developing muscle fibers

Using electrophysiological and quantitative autoradiographic techniques, we studied the kinetics of acetylcholine (ACh) receptor production and incorporation into membranes of muscle fibers developing in culture. These studies were performed by utilizing ¹²⁵I-labeled α-Bungarotoxin (α-BGT) which binds irreversibly to ACh receptors. α-BGT binding to ACh-sensitive muscle cells in culture correlates well with the level of ACh sensitivity. α-BGT binds to myotubes with two different apparent rates. The slow component of binding is due to the incorporation of new receptors into the membrane at a rate of 90 receptors/μm² per hour. However, the ACh receptor density increases at a rate of only 35 receptors/μm² per hour as the result of a concurrent increase in cell surface area. The α-BGT-receptor complexes turn over slowly and the rate of receptor incorporation is not affected by the presence of α-BGT. Inhibition of protein synthesis with cycloheximide depresses receptor incorporation, the percent inhibition increasing with time in cycloheximide. Overnight treatment in actinomycin D has no effect, but inhibition of ATP synthesis with dinitrophenol and iodoacetate or incubation in the cold inhibits the appearance of new ACh receptors.     

Catabolism of terbuthylazine by mixed bacterial culture originating from s-triazine-contaminated soil

The s-triazine herbicide terbuthylazine (TERB) has been used as the main substitute of atrazine in many EU countries for more than 10 years. However, the ecological consequences of this substitution are still not fully understood. Since the fate of triazine herbicides is primarily dependent on microbial degradation, in this paper, we investigated the ability of a mixed bacterial culture, M3-T, originating from s-triazine-contaminated soil, to degrade TERB in liquid culture and soil microcosms. The M3-T culture grown in mineral medium with TERB as the N source and citrate as the C source degraded 50 mg L^?1 of TERB within 3 days of incubation. The culture was capable of degrading TERB as the sole C and N source, though at slower degradation kinetics. A thorough LC-MS analysis of the biodegradation media showed the formation of hydroxyterbuthylazine (TERB-OH) and N-t-butylammelide (TBA) as major metabolites, and desethylterbuthylazine (DET), hydroxydesethylterbuthylazine (DET-OH) and cyanuric acid (CA) as minor metabolites in the TERB degradation pathway. TBA was identified as a bottleneck in the catabolic pathway leading to its transient accumulation in culture media. The supplementation of glucose as the exogenous C source had no effect on TBA degradation, whereas citrate inhibited its disappearance. The addition of M3-T to sterile soil artificially contaminated with TERB at 3 mg kg^?1 of soil resulted in an accelerated TERB degradation with t _1/2 value being about 40 times shorter than that achieved by the native microbial community. Catabolic versatility of M3-T culture makes it a promising seed culture for accelerating biotransformation processes in s-triazine-contaminated environment.     

Biodegradation of phenanthrene and analysis of degrading cultures in the presence of a model organo-mineral matrix and of a simulated NAPL phase

Two mixed bacterial cultures (C_B-BT and C_I-AT) degraded phenanthrene when it was: (i) in the presence of either hexadecane as a non aqueous phase liquid or a montmorillonite–Al(OH)x-humic acid complex as a model organo-mineral matrix; (ii) sorbed to the complex, either alone or in the presence of hexadecane. The cultures had different kinetic behaviours towards phenanthrene with or without hexadecane. The degradation of Phe alone as well as that of Phe in hexadecane ended in 8 and 15 days with C_B-BT and C_I-AT cultures, respectively. Hexadecane increased Phe bioavailability for C_I-AT bacteria which degraded Phe according to first-order kinetics. The same effect was observed for C_B-BT bacteria, but with an initial 2 days lag phase and in accordance with zero-order kinetics. The presence of hexadecane did not affect the degradation of phenanthrene sorbed and aged on the complex by C_I-AT culture. This capability was exhibited also after experimental aging of 30 days. The dynamics of the bacterial community composition was investigated through PCR-DGGE (denaturing gradient gel electrophoresis) of 16S rRNA gene fragments. Individual bands changed their intensity during the incubation time, implying that particular microbe’s relative abundance changed according to the culture conditions. Isolation of phenanthrene and/or hexadecane degraders was in accord with cultivation-independent data. Growth-dependent changes in the cell surface hydrophobicity of the two cultures and of the isolates suggested that modulation of cell surface hydrophobicity probably played an important role for an efficient phenanthrene assimilation/uptake.     

Cell death-resistance of differentiated myotubes is associated with enhanced anti-apoptotic mechanisms compared to myoblasts

Skeletal muscle atrophy is associated with elevated apoptosis while muscle differentiation results in apoptosis resistance, indicating that the role of apoptosis in skeletal muscle is multifaceted. The objective of this study was to investigate mechanisms underlying apoptosis susceptibility in proliferating myoblasts compared to differentiated myotubes and we hypothesized that cell death-resistance in differentiated myotubes is mediated by enhanced anti-apoptotic pathways. C₂C₁₂ myoblasts and myotubes were treated with H₂O₂ or staurosporine (Stsp) to induce cell death. H₂O₂ and Stsp induced DNA fragmentation in more than 50% of myoblasts, but in myotubes less than 10% of nuclei showed apoptotic changes. Mitochondrial membrane potential dissipation was detected with H₂O₂ and Stsp in myoblasts, while this response was greatly diminished in myotubes. Caspase-3 activity was 10-fold higher in myotubes compared to myoblasts, and Stsp caused a significant caspase-3 induction in both. However, exposure to H₂O₂ did not lead to caspase-3 activation in myoblasts, and only to a modest induction in myotubes. A similar response was observed for caspase-2, -8 and -9. Abundance of caspase-inhibitors (apoptosis repressor with caspase recruitment domain (ARC), and heat shock protein (HSP) 70 and -25 was significantly higher in myotubes compared to myoblasts, and in addition ARC was suppressed in response to Stsp in myotubes. Moreover, increased expression of HSPs in myoblasts attenuated cell death in response to H₂O₂ and Stsp. Protein abundance of the pro-apoptotic protein endonuclease G (EndoG) and apoptosis-inducing factor (AIF) was higher in myotubes compared to myoblasts. These results show that resistance to apoptosis in myotubes is increased despite high levels of pro-apoptotic signaling mechanisms, and we suggest that this protective effect is mediated by enhanced anti-caspase mechanisms.     

Ontogeny of insulin binding during chick skeletal myogenesis in vitro.

Our studies show that insulin receptors exist on chicken skeletal muscle cells at all developmental stages in culture. ¹²⁵I-labeled insulin binding at physiological concentrations to mature myotubes demonstrated saturability, binding proportional to cell number, reversibility, and specificity by competition with native hormone which reduced specific binding by 40% with 1 ng/ml and was maximal with 10 μg/ml. Further evidence for specificity was shown by no competition of insulin specific binding with insulin A chain, insulin B chain, growth hormone, and thyrotropin. Two binding sites were detected, with affinity constants of 10¹⁰M^?1 and 2 × 10⁹M^?1. The hormone receptor complex showed rapid dissociation (70% in 30 min) after equilibrium binding. During myogenesis, an increase in insulin receptors occurs from 500 per proliferating myoblast to 3000 per cell equivalent in mature (6 day) myotubes. Since these studies demonstrate that insulin receptors are present and other studies have shown that insulin is present during most of chicken embryogenesis, insulin may regulate muscle development in vivo to a greater degree than previously suspected.     

Differentiation markers of mouse C2C12 and rat L6 myogenic cell lines and the effect of the differentiation medium

Summary The differentiation grade of cells in culture is dependent on the composition of the culture medium. Two commonly used myogenic cell lines, mouse C₂C₁₂ and rat L₆, usually differentiate at a low concentration of horse serum. In this study we compared the effect of horse serum with a medium containing a low percentage of Ultroser G and rat brain extract. The maturation grade was evaluated on the basis of various biochemical, (immuno)histochemical and cell-physiological parameters. Substitution of horse serum by Ultroser G and rat brain extract during the differentiation phase resulted in a higher maturation grade of the myotubes of both cell lines, on the basis of creatine kinase activity and the diameter of the myotubes. In addition, the C₂C₁₂ myotubes display cross-striation, contain a higher percentage of creatine kinase muscle-specific isoenzyme MM, show a ninefold increase in acetylcholine receptor (AChR) clusters, form a continuous basement membrane, and have a lower resting cytosolic Ca²⁺ concentration. L₆ myotubes show a fivefold increase in AChR clusters and a twofold increase in the expression of the mRNA of the ɛ-subunit of AChR. C₂C₁₂ cells show spontaneous contraction and response of cytosolic Ca²⁺ to various stimulants in contrast to L₆ cells which do not. These studies established that the Ultroser G/brain extract medium leads to a higher differentiation grade of both cell lines, but parameters appropriate for use as differentiation markers appear to differ between both cell lines.     

Metabolic properties of human acetylcholine receptors can be characterized on cultured human muscle

Experiments examining acetylcholine receptor (AChR) metabolism in tissue culture have hitherto been limited to animal systems. For many studies, the human AChR on human skeletal muscle provides a more physiologic target. However, previous studies suggested that the levels of AChR produced on cultured human muscle were inadequate for metabolic studies. We demonstrate here that the metabolism of human acetylcholine receptors can be analysed on pure human muscle fibers that develop in tissue culture. Degradation of AChR follows first-order kinetics and is inhibited 85% by leupeptin, demonstrating that proteolysis of human AChR occurs in the lysosome. New AChR continue to appear on the cell surface for 3 h in the presence of cycloheximide, indicating the existence of a pool of intracellular AChR destined for the cell membrane. This pool is equivalent to approximately one-third of the AChR present on the surface of the cell. At any given time, the rate of AChR accumulation on the cell surface can be quantitatively accounted for by the rates of synthesis and degradation. Our results demonstrate that studies on the effects of hormones, neurotoxins or antibodies from patients with autoimmune neuromuscular diseases are now possible with human AChR which develop on intact human muscle myotubes formed in tissue culture.     

Distinct Components of Retrograde CaV1.1-RyR1 Coupling Revealed by a Lethal Mutation in RyR1

The molecular basis for excitation-contraction coupling in skeletal muscle is generally thought to involve conformational coupling between the L-type voltage-gated Ca²⁺ channel (Ca_V1.1) and the type 1 ryanodine receptor (RyR1). This coupling is bidirectional; in addition to the orthograde signal from Ca_V1.1 to RyR1 that triggers Ca²⁺ release from the sarcoplasmic reticulum, retrograde signaling from RyR1 to Ca_V1.1 results in increased amplitude and slowed activation kinetics of macroscopic L-type Ca²⁺ current. Orthograde coupling was previously shown to be ablated by a glycine for glutamate substitution at RyR1 position 4242. In this study, we investigated whether the RyR1-E4242G mutation affects retrograde coupling. L-type current in myotubes homozygous for RyR1-E4242G was substantially reduced in amplitude (∼80%) relative to that observed in myotubes from normal control (wild-type and/or heterozygous) myotubes. Analysis of intramembrane gating charge movements and ionic tail current amplitudes indicated that the reduction in current amplitude during step depolarizations was a consequence of both decreased Ca_V1.1 membrane expression (∼50%) and reduced channel P_o (∼55%). In contrast, activation kinetics of the L-type current in RyR1-E4242G myotubes resembled those of normal myotubes, unlike dyspedic (RyR1 null) myotubes in which the L-type currents have markedly accelerated activation kinetics. Exogenous expression of wild-type RyR1 partially restored L-type current density. From these observations, we conclude that mutating residue E4242 affects RyR1 structures critical for retrograde communication with Ca_V1.1. Moreover, we propose that retrograde coupling has two distinct and separable components that are dependent on different structural elements of RyR1.     

The absence of dysferlin induces the expression of functional connexin-based hemichannels in human myotubes

Background

Mutations in the gene encoding for dysferlin cause recessive autosomal muscular dystrophies called dysferlinopathies. These mutations induce several alterations in skeletal muscles, including, inflammation, increased membrane permeability and cell death. Despite the fact that the etiology of dysferlinopathies is known, the mechanism that explains the aforementioned alterations is still elusive. Therefore, we have now evaluated the potential involvement of connexin based hemichannels in the pathophysiology of dysferlinopathies.

Results

Human deltoid muscle biopsies of 5 Chilean dysferlinopathy patients exhibited the presence of muscular connexins (Cx40.1, Cx43 and Cx45). The presence of these connexins was also observed in human myotubes derived from immortalized myoblasts derived from other patients with mutated forms of dysferlin. In addition to the aforementioned connexins, these myotubes expressed functional connexin based hemichannels, evaluated by ethidium uptake assays, as opposed to myotubes obtained from a normal human muscle cell line, RCMH. This response was reproduced in a knock-down model of dysferlin, by treating RCMH cell line with small hairpin RNA specific for dysferlin (RCMH-sh Dysferlin). Also, the presence of P2X₇ receptor and the transient receptor potential channel, TRPV2, another Ca²⁺ permeable channels, was detected in the myotubes expressing mutated dysferlin, and an elevated resting intracellular Ca²⁺ level was found in the latter myotubes, which was in turn reduced to control levels in the presence of the molecule D4, a selective Cx HCs inhibitor.

Conclusions

The data suggests that dysferlin deficiency, caused by mutation or downregulation of dysferlin, promotes the expression of Cx HCs. Then, the de novo expression Cx HC causes a dysregulation of intracellular free Ca²⁺ levels, which could underlie muscular damage associated to dysferlin mutations. This mechanism could constitute a potential therapeutical target in dysferlinopathies.