Philippe Gailly

Involvement of TRPC in the abnormal calcium influx observed in dystrophic (mdx) mouse skeletal muscle fibers.

Duchenne muscular dystrophy results from the lack of dystrophin, a cytoskeletal protein associated with the inner surface membrane, in skeletal muscle. The absence of dystrophin induces an abnormal increase of sarcolemmal calcium influx through cationic channels in adult skeletal muscle fibers from dystrophic (mdx) mice. We observed that the activity of these channels was increased after depletion of the stores of calcium with thapsigargin or caffeine. By analogy with the situation observed in nonexcitable cells, we therefore hypothesized that these store-operated channels could belong to the transient receptor potential channel (TRPC) family. We measured the expression of TRPC isoforms in normal and mdx adult skeletal muscles fibers, and among the seven known isoforms, five were detected (TRPC1, 2, 3, 4, and 6) by RT-PCR. Western blot analysis and immunocytochemistry of normal and mdx muscle fibers demonstrated the localization of TRPC1, 4, and 6 proteins at the plasma membrane. Therefore, an antisense strategy was used to repress these TRPC isoforms. In parallel with the repression of the TRPCs, we observed that the occurrence of calcium leak channels was decreased to one tenth of its control value (patch-clamp technique), showing the involvement of TRPC in the abnormal calcium influx observed in dystrophic fibers.

Targeting the alpha 1 subunit of the sodium pump to combat glioblastoma cells

OBJECTIVEIon transporters play pivotal roles in cancer cell migration in general and in glioblastomas (GBMs) in particular. However, the specific role of Na+/K+-ATPase (the sodium pump) and, in particular, its α1 subunit, has remained unexplored in GBMs. MATERIALS AND METHODSThe expression of Na+/K+-ATPase α1 in GBM clinical samples, normal brain tissue, and a human GBM cell line has been investigated. Using the novel cardenolide UNBS1450 (Unibioscreen, Brussels, Belgium), which is a ligand of the sodium pump, we have characterized the effects of inhibiting Na+/K+-ATPase α1 in human GBM cells with respect to cell proliferation; morphology; impact on intracellular Na+, Ca2+, and adenosine triphosphate; and changes in the actin cytoskeleton. We have investigated the mechanism by which UNBS1450 overcomes the apoptosis resistance of GBMs and determined its anti-tumor effects in comparative studies in vitro in GBM cell viability assays and in vivo using an orthotopic human GBM xenograft model. RESULTSOverall, the α1 subunit of Na+/K+-ATPase is highly expressed in a majority of glioblastomas compared with normal brain tissues, and by binding to this subunit in human U373-MG GBM cells, UNBS1450 impairs cell proliferation and migration via an intracellular adenosine triphosphate decrease-mediated disorganization of the actin cytoskeleton and cytotoxic proautophagic effects. UNBS1450 also significantly increases the in vivo survival of mice orthotopically grafted with U373-MG GBM cells. CONCLUSIONInhibition of the Na+/K+-ATPase α1 subunit in human GBM cells impairs both cell migration and cell proliferation.

Critical evaluation of cytosolic calcium determination in resting muscle fibres from normal and dystrophic (mdx) mice.

The fluorescent probe Fura-2/AM was used to determine cytosolic free calcium concentration in soleus muscle and in isolated flexor digitorum brevis fibres. This required a precise calibration; therefore, each calibration parameter was studied in situ. The influence of the dye concentration on calcium measurements was also examined. This precise calibration technique was used to compare absolute free calcium concentration in resting preparations from dystrophic (mdx) and control (C57) mice. We showed that the behavior of the dye was not similar in C57 and in mdx muscles. For this reason, we did not confirm the previous results that cytosolic free calcium concentration is increased in mdx muscles.

Skeletal muscle hypertrophy and regeneration: interplay between the myogenic regulatory factors (MRFs) and insulin-like growth factors (IGFs) pathways.

Adult skeletal muscle can regenerate in response to muscle damage. This ability is conferred by the presence of myogenic stem cells called satellite cells. In response to stimuli such as injury or exercise, these cells become activated and express myogenic regulatory factors (MRFs), i.e., transcription factors of the myogenic lineage including Myf5, MyoD, myogenin, and Mrf4 to proliferate and differentiate into myofibers. The MRF family of proteins controls the transcription of important muscle-specific proteins such as myosin heavy chain and muscle creatine kinase. Different growth factors are secreted during muscle repair among which insulin-like growth factors (IGFs) are the only ones that promote both muscle cell proliferation and differentiation and that play a key role in muscle regeneration and hypertrophy. Different isoforms of IGFs are expressed during muscle repair: IGF-IEa, IGF-IEb, or IGF-IEc (also known as mechano growth factor, MGF) and IGF-II. MGF is expressed first and is observed in satellite cells and in proliferating myoblasts whereas IGF-Ia and IGF-II expression occurs at the state of muscle fiber formation. Interestingly, several studies report the induction of MRFs in response to IGFs stimulation. Inversely, IGFs expression may also be regulated by MRFs. Various mechanisms are proposed to support these interactions. In this review, we describe the general process of muscle hypertrophy and regeneration and decipher the interactions between the two groups of factors involved in the process.

Effects of nitric oxide on the contraction of skeletal muscle.

Abstract. A review of the literature suggests that the effects of nitric oxide (NO) on skeletal muscles fibers can be classified in two groups. In the first, the effects of NO are direct, due to nitrosation or metal nitrosylation of target proteins: depression of isometric force, shortening velocity of loaded or unloaded contractions, glycolysis and mitochondrial respiration. The effect on calcium release channels varies, being inhibitory at low and stimulatory at high NO concentrations. The general consequence of the direct effects of NO is to ‘brake’ the contraction and its associated metabolism. In the second group, the effects of NO are mediated by cGMP: increase of the shortening velocity of loaded or unloaded contractions, maximal mechanical power, initial rate of force development, frequency of tetanic fusion, glucose uptake, glycolysis and mitochondrial respiration; decreases of half relaxation time of tetanus and twitch, twitch time-to-peak, force maintained during unfused tetanus and of stimulus-associated calcium release. There is negligible effect on maximal force of isometric twitch and tetanus. The general consequence of cGMP-mediated effects of NO is to improve mechanical and metabolic muscle power, similar to a transformation of slow-twitch to fast-twitch muscle, an effect that we may summarize as a ‘slow-to-fast’ shift.

Role of TRPC1 channel in skeletal muscle function

Skeletal muscle contraction is reputed not to depend on extracellular Ca2+. Indeed, stricto sensu, excitation-contraction coupling does not necessitate entry of Ca2+. However, we previously observed that, during sustained activity (repeated contractions), entry of Ca2+ is needed to maintain force production. In the present study, we evaluated the possible involvement of the canonical transient receptor potential (TRPC)1 ion channel in this entry of Ca2+ and investigated its possible role in muscle function. Patch-clamp experiments reveal the presence of a small-conductance channel (13 pS) that is completely lost in adult fibers from TRPC1(-/-) mice. The influx of Ca2+ through TRPC1 channels represents a minor part of the entry of Ca(2+) into muscle fibers at rest, and the activity of the channel is not store dependent. The lack of TRPC1 does not affect intracellular Ca2+ concentration ([Ca2+](i)) transients reached during a single isometric contraction. However, the involvement of TRPC1-related Ca2+ entry is clearly emphasized in muscle fatigue. Indeed, muscles from TRPC1(-/-) mice stimulated repeatedly progressively display lower [Ca2+](i) transients than those observed in TRPC1(+/+) fibers, and they also present an accentuated progressive loss of force. Interestingly, muscles from TRPC1(-/-) mice display a smaller fiber cross-sectional area, generate less force per cross-sectional area, and contain less myofibrillar proteins than their controls. They do not present other signs of myopathy. In agreement with in vitro experiments, TRPC1(-/-) mice present an important decrease of endurance of physical activity. We conclude that TRPC1 ion channels modulate the entry of Ca(2+) during repeated contractions and help muscles to maintain their force during sustained repeated contractions.

Functional role of store-operated and stretch-activated channels in murine adult skeletal muscle fibres.

In skeletal muscle, Ca2+ is implicated in contraction, and in regulation of gene expression. An alteration of [Ca2+]i homeostasis is responsible, at least partially, for the muscle degeneration that occurs after eccentric contractions in Duchenne muscular dystrophy, a disease characterized by the loss of the cytoskeletal protein dystrophin. Using patch clamp in the cell‐attached configuration, we characterized the store‐operated channels (SOCs) and the stretch‐activated channels (SACs) present in isolated mouse skeletal muscle. SOCs were voltage independent, had a unitary conductance between 7 and 8 pS (110 mm Ca2+ in the pipette), and their open probability increased when the sarcoplasmic reticulum was depleted by thapsigargin. These SOCs were identical to those previously described in the pathophysiology of Duchenne muscular dystrophy. Under the same experimental conditions, we detected a channel activity that was increased by applying a negative pressure to the patch electrode. The SACs responsible for this current had the same unitary conductance and current–voltage relationship as those observed for SOCs. SOCs and SACs had a similar sensitivity to pharmacological agents such as Gd3+, SKF‐96365, 2‐aminoethoxydiphenyl borate and GsMTx4 toxin. Moreover, stimulation with IGF‐1 increased the occurrence of the activity of both channel types. Together, these observations suggest that SOCs and SACs might belong to the same population or share common constituents. From a functional point of view, treatment of soleus muscle with SKF‐96365 or GsMTx4 toxin increased its sensitivity to a fatigue protocol, suggesting that the influx of Ca2+ that occurs through these channels during contraction is also involved in force maintaining during repeated stimulations.

Impaired neuromuscular transmission and skeletal muscle fiber necrosis in mice lacking Na/Ca exchanger 3

We produced and analyzed mice deficient for Na/Ca exchanger 3 (NCX3), a protein that mediates cellular Ca(2+) efflux (forward mode) or Ca(2+) influx (reverse mode) and thus controls intracellular Ca(2+) concentration. NCX3-deficient mice (Ncx3(-/-)) present a skeletal muscle fiber necrosis and a defective neuromuscular transmission, reflecting the absence of NCX3 in the sarcolemma of the muscle fibers and at the neuromuscular junction. The defective neuromuscular transmission is characterized by the presence of electromyographic abnormalities, including low compound muscle action potential amplitude, a decremental response at low-frequency nerve stimulation, an incremental response, and a prominent postexercise facilitation at high-frequency nerve stimulation, as well as neuromuscular blocks. The analysis of quantal transmitter release in Ncx3(-/-) neuromuscular junctions revealed an important facilitation superimposed on the depression of synaptic responses and an elevated delayed release during high-frequency nerve stimulation. It is suggested that Ca(2+) entering nerve terminals is cleared relatively slowly in the absence of NCX3, thereby enhancing residual Ca(2+) and evoked and delayed quantal transmitter release during repetitive nerve stimulation. Our findings indicate that NCX3 plays an important role in vivo in the control of Ca(2+) concentrations in the skeletal muscle fibers and at the neuromuscular junction.

Regulation of store-operated calcium entries and mitochondrial uptake by minidystrophin expression in cultured myotubes.

Defective expression of dystrophin in muscle cells is the primary feature of Duchenne muscular dystrophy (DMD), which is accompanied by fiber necrosis and intracellular calcium mishandling. These features led to the hypothesis that dystrophin could control calcium movements. Calcium mishandling in human DMD myotubes is dependent on contraction and/or calcium release activity, suggesting the involvement of channels being activated during these processes. Forced expression of minidystrophin at the plasma membrane of dystrophin‐deficient Sol8 myotubes reactivates appropriate sarcolemmal expression of dystrophin‐associated proteins and results in normal calcium homeostasis. In active dystrophic myotubes, store‐operated calcium channels could be responsible for a sustained calcium influx in muscle cells. We show here that depletion of calcium stores (sarcoplasmic reticulum) by repetitive activation of calcium release and blockade of SERCA leads to a calcium influx. In myotubes expressing recombinant minidystrophin, these store‐dependent influxes were reduced to a level similar to that observed in myotubes expressing native dystrophin. High store‐dependent calcium influxes in dystrophindeficient myotubes were associated with sustained cytosolic calcium transients and high intramitochondrial entries, while lower store‐dependent calcium influx in myotubes expressing minidystrophin resulted in shorter calcium transients and reduced calcium uptake into mitochondria. We propose that minidystrophin negatively regulates sarcolemmal store‐dependent calcium channels, which reduces store‐dependent calcium influx, as well as its mitochondrial uptake. Forced expression of minidystrophin in dystrophic cells might restore the regulation of sarcolemmal store‐dependent channels, which could protect against calcium mishandling.

Ophiobolin A induces paraptosis-like cell death in human glioblastoma cells by decreasing BKCa channel activity

Glioblastoma multiforme (GBM) is the most lethal and common malignant human brain tumor. The intrinsic resistance of highly invasive GBM cells to radiation- and chemotherapy-induced apoptosis accounts for the generally dismal treatment outcomes. This study investigated ophiobolin A (OP-A), a fungal metabolite from Bipolaris species, for its promising anticancer activity against human GBM cells exhibiting varying degrees of resistance to proapoptotic stimuli. We found that OP-A induced marked changes in the dynamic organization of the F-actin cytoskeleton, and inhibited the proliferation and migration of GBM cells, likely by inhibiting big conductance Ca2+-activated K+ channel (BKCa) channel activity. Moreover, our results indicated that OP-A induced paraptosis-like cell death in GBM cells, which correlated with the vacuolization, possibly brought about by the swelling and fusion of mitochondria and/or the endoplasmic reticulum (ER). In addition, the OP-A-induced cell death did not involve the activation of caspases. We also showed that the expression of BKCa channels colocalized with these two organelles (mitochondria and ER) was affected in this programmed cell death pathway. Thus, this study reveals a novel mechanism of action associated with the anticancer effects of OP-A, which involves the induction of paraptosis through the disruption of internal potassium ion homeostasis. Our findings offer a promising therapeutic strategy to overcome the intrinsic resistance of GBM cells to proapoptotic stimuli.