Magali Louis

Beneficial effects of creatine supplementation in dystrophic patients

The effect of creatine (Cr) supplementation on muscle function and body composition of 12 boys with Duchenne muscular dystrophy and three with Becker dystrophy was evaluated by a randomized double‐blind cross‐over study (3 g Cr or maltodextrin daily for 3 months, with wash‐out period of 2 months). After placebo, no change was observed in maximal voluntary contraction (MVC) and resistance to fatigue, whereas total joint stiffness (TJS) was increased by ∼25% (P < 0.05). The patients receiving Cr did not show any change in TJS, improved MVC by 15% (P = 0.02), and almost doubled their resistance to fatigue (P < 0.001). In patients still independent of a wheelchair (n = 5), bone mineral density increased by 3% (P < 0.05), and urinary excretion of collagen type I cross‐linking N‐telopeptide declined to about one third (P < 0.001) after Cr. No adverse effect was observed. Thus, Cr may provide some symptomatic benefit in these patients. Muscle Nerve 27: 604–610, 2003

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.

Creatine increases IGF-I and myogenic regulatory factor mRNA in C2C12 cells

Addition of creatine to the differentiation medium of C2C12 cells leads to hypertrophy of the myotubes. To investigate the implication of insulin‐like growth factor I (IGF‐I) and myogenic regulatory factors (MRFs) in this hypertrophy, their mRNA levels were assessed during the first 72 h of differentiation. Creatine significantly increased the IGF‐I mRNA level over the whole investigated period of time, whereas the MRF mRNA levels were only augmented at precise moments, suggesting a general activation mechanism for IGF‐I and a specifically regulated mechanism for MRF transcription. Our results suggest therefore that creatine‐induced hypertrophy of C2C12 cells is at least partially mediated by overexpression of IGF‐I and MRFs.

Tumor-Associated Antigen Preferentially Expressed Antigen of Melanoma (PRAME) Induces Caspase-Independent Cell Death In vitro and Reduces Tumorigenicity In vivo

Preferentially expressed antigen of melanoma (PRAME) is expressed in a wide variety of tumors, but in contrast with most other tumor associated antigens, it is also expressed in leukemias. The physiologic role of PRAME remains elusive. Interestingly, PRAME expression is correlated with a favorable prognosis in childhood acute leukemias. Moreover, a high expression of PRAME seems to be predominantly found in acute leukemias carrying a favorable prognosis. On these clinical observations, we assumed that PRAME could be involved in the regulation of cell death or cell cycle. In this study, we show that transient overexpression of PRAME induces a caspase-independent cell death in cultured cell lines (CHO-K1 and HeLa). Cells stably transfected with PRAME also exhibit a decreased proliferation rate due, at least partially, to an elevated basal rate of cell death. Immunocytochemistry of a FLAG-tagged PRAME, in vivo imaging of an enhanced green fluorescent protein-tagged PRAME, and Western blotting after cell fractionation reveal a nuclear localization of the protein. Using a microarray-based approach, we show that KG-1 leukemic cells stably transfected with PRAME present a significant decrease of expression of the heat-shock protein Hsp27, the cyclin-dependent kinase inhibitor p21, and the calcium-binding protein S100A4. The expression of these three proteins is known to inhibit apoptosis and has been associated with an unfavorable prognosis in a series of cancers. Finally, repression of PRAME expression by a short interfering RNA strategy increases tumorigenicity of K562 leukemic cells in nude mice. We suggest that all these observations might explain the favorable prognosis of the leukemias expressing high levels of PRAME.

Effect of creatine supplementation on skeletal muscle of mdx mice

Dystrophic mice (mdx) and their controls (C57/Bl10) were fed for 1 month with a diet with or without creatine (Cr) enrichment. Cr supplementation reduced mass (by 19%, P < 0.01) and mean fiber surface (by 25%, P < 0.05) of fast‐twitch mdx muscles. In both strains, tetanic tension increased slightly (9.2%) without reaching statistical significance (P = 0.08), and relaxation time increased by 16% (P < 0.001). However, Cr had no protective effect on the other hallmarks of dystrophy such as susceptibility to eccentric contractions; large numbers of centrally nucleated fibers in tibialis anterior; and elevated total calcium content, which increased by 85% (P = 0.008) in gastrocnemius mdx muscles. In conclusion, Cr may be a positive intervention for improving function of dystrophic muscle. Muscle Nerve 29: 687–692, 2004

Tumor associated antigen PRAME is a marker of favorable prognosis in childhood acute myeloid leukemia patients and modifies the expression of S100A4, Hsp 27, p21, IL-8 and IGFBP-2 in vitro and in vivo.

Preferentially expressed antigen of melanoma (PRAME) is expressed in a wide variety of tumors, but in contrast with most other tumor associated antigens, it is also expressed in leukemias. In a previous study, we showed that overexpression of PRAME induced apoptosis, inhibited cell proliferation and reduced tumorigenicity of leukemic cells in vivo. We also demonstrated that PRAME overexpression induced the repression of three genes (Hsp27, S100A4 and p21) associated with an unfavorable prognosis in leukemia. Here, we further investigated the mechanisms of PRAME-induced tumor suppression in vitro and in vivo. We performed a gene profiling study by analysing PRAME shRNA-silenced leukemic cells on high-density micro-arrays (Affymetrix) and found that PRAME altered the expression of two additional genes potentially involved in cancerogenesis and cancer progression: IL-8 and IGFBP-2. In a series of 28 acute myeloid leukemia pediatric patients, we observed that PRAME expression was associated with an increased leukemia-free survival. Importantly, the correlation between PRAME expression in leukemic cell lines and the decreased expression of Hsp27, S100A4, p21, IL-8 and the increased expression of IGFBP-2 was also observed in vivo, in leukemic patients. Our results suggest that the favorable prognosis of PRAME could be mediated, at least in part, by the modified expression of those genes.

Rapid combined genotyping of factor V, prothrombin and methylenetetrahydrofolate reductase single nucleotide polymorphisms using minor groove binding DNA oligonucleotides (MGB probes) and real-time polymerase chain reaction

Abstract Risk factors for cardiovascular diseases and venous thromboembolism involve both acquired and hereditary conditions. Among the latter, mutations in genes coding for coagulation factors (factor V Leiden [Arg506Gly], G20210A in the 3′-untranslated region of factor II) and variant C677T of the methylenetetrahydrofolate reductase (MTHFR) are often involved and co-inherited. These three factors were genotyped simultaneously in the same 96-well plate, using a real-time polymerase chain reaction (PCR) Taqman® assay and minor groove binding DNA oligonucleotides (MGB probes). While primers and MGB probes matched their corresponding single nucleotide polymorphism (SNP), the real-time MGB program was identical for each target gene. Homozygous wild-type (WT; −/−), heterozygous (+/−) or homozygous (+/+) variants (n=362) were selected for factor V (n=115, with −/−, 40; +/−, 40; +/+, 35), factor II (n=122, with −/−, 60; +/−, 60; +/+, 2), and MTHFR (n=120, with −/−, 40; +/−, 40; +/+, 40), according to the results of conventional PCR-restriction fragment length polymorphism (PCR-RFLP), but the allelic discrimination was performed blind. Results of the real-time MGB and PCR-RFLP assays were identical. This new assay was easy and fast with high throughput, without risk of molecular carryover, and cost-effective for laboratories utilizing the Taqman or related fluorescence reading methods. These advantages make it particularly suitable for large-scale combined genotyping of several polymorphisms in the routine setting.

Effects of Creatine Supplementation in Males and Females

This study compared the effects of creatine supplementation between genders in 40 subjects: 20 females (F) and 20 males (M). Each gender group was randomly divided into 2 groups: creatine (Cr, n = 10) or placebo (Pl, n = 10). The first 5 days, the participants received 21g.d-1 Cr or Pl and then 3g.d-1 until completion of the supplementation (28 days). To avoid the influence of hormonal cycle in females, the experiment started during the first 5 days of the menstrual cycle (β-oestradiol < 100 pg.ml-1). 24-hour urines and a blood sample were collected to monitor liver and kidney functions. The maximal power output (MPO) was determined during single-knee extension exercises, based on incremental loads and displacement velocities. After a 15-min recovery, the subjects were submitted to a fatigue test. They were instructed to lift up once every 5-s, a load corresponding to MPO. Gender and treatment differences were assessed by a repeated measures ANOVA design with 2 grouping factors (F-M and Cr-Pl). ASAT, ALAT, LDH, γ-GT, alkalin phosphatases as well as creatinine, urea and albumin clearances did not change and did never reach pathological values. Body mass (BM) did not change either in Pl groups, or in the Cr-F group (60.9 ± 3.8 kg vs 61.4 ± 3.9 kg), but it increased (p < 0.001) in the Cr-M group (73.0 ± 6.5 kg vs 74.2 ± 6.9 kg). Nevertheless, no statistical difference was found between genders in the change of BM (time*treatment*gender interaction). Similar significant increases were found in MPO for Cr-F and Cr-M groups (7% and 8%, P = 0.008). The performance during the fatigue test, evaluated by the total external work developed until exhaustion, was largely improved in both Cr groups (89% and 47% in F and M, respectively), with a significant time*treatment*gender interaction (P = 0.024). In conclusion, we did not observed any trouble of the liver and kidney functions during this experimentation. No difference between genders was observed in the change of BM, MPO, but the females improved their performance during the fatigue test more than the males.

Effect of Creatine and Guanidino-Propionic Acid on Myotube Growth

Since the paper of Roger Harris and his collaborators (Clin. Sci. 83: 367-374,1992), creatine has been largely used as an ergogenic supplement, in the hope of improving muscle performance. Recently, positive effects have been observed in patients suffering from neuro-muscular disorders like myopathies, Huntington or Parkinson diseases. A secondary effect, commonly reported, of creatine supplementation is an increase in lean body mass attributed to water retention, muscle hypertrophy or both. In this study, we applied creatine or guanidino-propionic acid (GPA), a creatine analogue, to myotube cultures to test the hypothesis of a possible role of creatine in the mechanism of hypertrophy. C2C12 cells were seeded at 5 × 104 cell/cm2 in DMEM medium (Gibco, Basel, Switzerland) containing 10% of foetal calf serum. When cell confluence reached 70%, the proliferation medium was replaced by a differentiation medium containing 1% horse serum. 20 mM creatine or GPA was added to the medium. Fusion of myobasts into myotubes occurred after 2 days in culture and was completed within 24 hours. Six cultures were prepared in each condition, including a control condition. The myotube content of each culture was counted on the basis of 2 randomly chosen fields. The size of the myotubes was estimated by measuring their diameter. No difference in the number of myotubes in cultures was found. Their diameter was however increased by 40% in the presence of creatine in the medium (P < 0.001) and decreased by 25% in the presence of GPA (P = 0.008). In conclusion, creatine added to the differenciation medium promotes myotube growth while GPA depresses it. As, GPA replaces creatine in the cell and reduces phosphoryl-creatine content, myotube growth could be controlled by the energy status of the cell.