Theaflavin Promotes Myogenic Differentiation by Regulating the Cell Cycle and Surface Mechanical Properties of C2C12 Cells.

Aging and muscle diseases often lead to a decline in the differentiation capacity of myoblasts, which in turn results in the deterioration of skeletal muscle (SkM) function and impairment of regeneration ability after injury. Theaflavins, the "gold molecules" found in black tea, have been reported to possess various biological activities and have a positive effect on maintaining human health. In this study, we found that among the four theaflavins (theaflavin (TF1), theaflavin-3-gallate (TF2A), theaflavin-3'-gallate (TF2B), and theaflavin-3,3'-digallate (TF3) monomers), TF1 (20 µM) significantly promoted the fusion index of myoblasts, number of mature myotubes, and degree of myotube development. By combining transcriptomics, bioinformatics, and molecular biology experiments, we showed that TF1 may promote myoblast differentiation by (1) regulating the withdrawal of myoblasts from the cell cycle, inducing the release of myogenic factors (MyoD, MyoG, and MyHC) and accelerating myogenic differentiation and (2) regulating the adhesion force of myoblasts and mechanical properties of mature myotubes and promoting the migration, fusion, and development of myoblasts. In conclusion, our study outcomes show that TF1 can promote myoblast differentiation and regulate myotube mechanical properties. It is a potential dietary supplement for the elderly. Our findings provide a new scientific basis for the relationship between tea drinking and aging.

Flexible Multielectrode Array for Skeletal Muscle Conditioning, Acetylcholine Receptor Stabilization and Epimysial Recording After Critical Peripheral Nerve Injury.

Complete re-innervation after a traumatic injury severing a muscle's peripheral nerve may take years. During this time, the denervated muscle atrophies and loses acetylcholine receptors, a vital component of the neuromuscular junction, limiting functional recovery. One common clinical treatment for atrophy is electrical stimulation; however, epimysial electrodes currently used are bulky and often fail due to an excessive inflammatory response. Additionally, there remains a need for a device providing in vivo monitoring of neuromuscular regeneration and the maintenance of acetylcholine receptors. Here, an implantable, flexible microelectrode array (MEA) was developed that provides surface neuromuscular stimulation and recording during long-term denervation. Methods: The MEA uses a flexible polyimide elastomer and an array of gold-based microelectrodes featuring Peano curve motifs, which together maintain electrode flexibility. The devices were implanted along the denervated gastrocnemius muscles of 5 rats. These rats underwent therapeutic stimulation using the MEA daily beginning on post-operative day 2. Another 5 rats underwent tibial nerve resection without implantation of MEA. Tissues were harvested on post-operative day 14 and evaluated for quantification of acetylcholine receptors and muscle fiber area using immunofluorescence and histological staining. Results: The Young's modulus was 1.67 GPa, which is comparable to native tendon and muscle. The devices successfully recorded electromyogram data when implanted in rats. When compared to untreated denervated muscles, MEA therapy attenuated atrophy by maintaining larger muscle fiber cross-sectional areas (p < 0.05). Furthermore, the acetylcholine receptor areas were markedly larger with MEA treatment (p < 0.05). Conclusions: This proof-of-concept work successfully demonstrates the ability to combine conformability, tensile strength-enhancing metal micropatterning, electrical stimulation and recording into a functional implant for both epimysial stimulation and recording.

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.

Characterization of fish myofibrillar protein film incorporated with catechin-Kradon extract.

This study investigates the properties of films made from fish myofibrillar protein (FMP) incorporated with combinations of catechin-Kradon extract (C/K) (20%, w/w) at different ratios. The experimental films were compared to a polyvinyl chloride (PVC). The thickness and the transparency of the films were in the range of 0.014-0.015mm and 3.65-3.77, respectively. Significant decreases were observed in elongation at break (35-122%), water vapor permeability (0.54-1.26×10-10gm-1s-1Pa-1) properties, and a gradual decrease in L* value was pronounced when the proportions of catechins were increased (P<0.05). FMP films incorporated with C/K demonstrated to have very good barrier properties to UV light and also enhanced the thermal stability of the developed FMP films. All film samples had a smooth surface; however coarser surfaces were observed when the amounts of catechins were increased. Furthermore, the developed FMP films showed high antioxidant capability. However, no significant antimicrobial activity was observed in this film. It is determined that FMP films enriched with C/K can be used for food packaging, with particular potential for active packaging. However, as compared with the PVC, the FMP films need further development, especially in terms of their mechanical and water barrier properties.

Dietary Sodium Butyrate Supplementation Promotes Oxidative Fiber Formation in Mice.

Sodium butyrate (SB), a sodium salt of butyric acid, has been shown to improve the animal production performance. The aim of this work was to test the effect of feeding mice with diets containing different dose of SB (1, 3, and 5%) on oxidative fiber formation. Dietary SB supplementation had no effect on body weights and food intakes. Dietary SB supplementation upregulated the expressions of oxidative fiber-related protein including MyHC I, MyHC IIa, myoglobin, and troponin-I-slow. Dietary SB supplementation also upregulated the expressions of phospho-FoxO1 and MEF2C protein, but did not affect total FoxO1 protein expression. Taken together, these results indicate that dietary SB supplementation promotes oxidative fiber formation in mice, which might be through inactivation of FoxO1 and upregulation of MEF2C expression.

Intrinsic transient tracheal occlusion training and myogenic remodeling of rodent parasternal intercostal fibers.

It is recognized that diaphragm muscle plasticity occurs with mechanical overloads, yet less is known about synergistic parasternal intercostal muscle fiber remodeling. We conducted overload training with intrinsic transient tracheal occlusion (ITTO) exercises in conscious animals. We hypothesized that ITTO would yield significant fiber hypertrophy and myogenic activation that would parallel diaphragm fiber remodeling. Sprague-Dawley rats underwent placement of a tracheal cuff and were randomly assigned to receive daily 10 min sessions of conscious ITTO or observation (sham) over 2 wk. After training, fiber morphology, myosin heavy chain (MHC) isoform composition, cross-sectional area, proportion of Pax7-positive nuclei, and presence of embryonic MHC (eMHC) were quantified. Type IIx/b fibers were 20% larger after ITTO training than with sham training (ITTO: 4,431 +/­ 676 µm2, sham: 3,689 +/­ 400 µm2, p < 0.05), and type I fibers were more prevalent after ITTO (p < 0.01). Expression of Pax7 was increased in ITTO parasternals and diaphragm (p < 0.05). In contrast, the proportion of eMHC-positive fibers was increased only in ITTO parasternals (1.2% [3.4%­0.6%], sham: 0% [0.6%­0%], p < 0.05). Although diaphragm and parasternal type II fibers hypertrophy to a similar degree, myogenic remodeling appears to differ between the two muscles.

Dietary fat influences the expression of contractile and metabolic genes in rat skeletal muscle.

Dietary fat plays a major role in obesity, lipid metabolism, and cardiovascular diseases. To determine whether the intake of different types of dietary fats affect the muscle fiber types that govern the metabolic and contractile properties of the skeletal muscle, we fed male Wistar rats with a 15% fat diet derived from different fat sources. Diets composed of soybean oil (n-6 polyunsaturated fatty acids (PUFA)-rich), fish oil (n-3 PUFA-rich), or lard (low in PUFAs) were administered to the rats for 4 weeks. Myosin heavy chain (MyHC) isoforms were used as biomarkers to delineate the skeletal muscle fiber types. Compared with soybean oil intake, fish oil intake showed significantly lower levels of the fast-type MyHC2B and higher levels of the intermediate-type MyHC2X composition in the extensor digitorum longus (EDL) muscle, which is a fast-type dominant muscle. Concomitantly, MyHC2X mRNA levels in fish oil-fed rats were significantly higher than those observed in the soybean oil-fed rats. The MyHC isoform composition in the lard-fed rats was an intermediate between that of the fish oil and soybean oil-fed rats. Mitochondrial uncoupling protein 3, pyruvate dehydrogenase kinase 4, and porin mRNA showed significantly upregulated levels in the EDL of fish oil-fed rats compared to those observed in soybean oil-fed and lard-fed rats, implying an activation of oxidative metabolism. In contrast, no changes in the composition of MyHC isoforms was observed in the soleus muscle, which is a slow-type dominant muscle. Fatty acid composition in the serum and the muscle was significantly influenced by the type of dietary fat consumed. In conclusion, dietary fat affects the expression of genes related to the contractile and metabolic properties in the fast-type dominant skeletal muscle, where the activation of oxidative metabolism is more pronounced after fish oil intake than that after soybean oil intake.

Effect of ractopamine-HCl supplementation for 28 days on carcass characteristics, muscle fiber morphometrics, and whole muscle yields of six distinct muscles of the loin and round.

This study evaluated the effects of ractopamine-HCl (RAC) supplementation on carcass characteristics, muscle fiber morphometrics, and tenderness. Thirty-four steers (2 groups, 4 replicates) were fed RAC or carrier for 28 days prior to harvest. Seventy-two hours postmortem, the Longissimus lumborum (LL), Gracilis (GRA), Vastus lateralis (VL), Rectus femoris (RF), Semimembranosus (SM), and Adductor (ADD) were dissected from each carcass. Commodity weight, denuded weight, and muscle dimensions were collected. RAC supplementation tended to affect dressing percentage (P=0.15) and muscle firmness (P<0.15), and significantly affected lean maturity (P<0.05) and marbling score (P<0.05). With the exception of the LL and GRA (P<0.05), RAC had no effect on muscle dimensions. RAC did not influence the tenderness of vacuum-packaged, aged steaks as measured by Warner-Bratzler shear force. Muscle fiber size within the six muscles was unchanged (P>0.05) by RAC. Thus, RAC improves carcass parameters without a negative impact on tenderness.

Vitamin D and its role in skeletal muscle.

PURPOSE OF REVIEW: Vitamin D is best known for its role in regulating calcium homeostasis and in strengthening bone. However, it has become increasingly clear that it also has important beneficial effects beyond the skeleton, including muscle. This review summarizes current knowledge about the role of vitamin D in skeletal muscle tissue and physical performance. RECENT FINDINGS: Molecular mechanisms of vitamin D action in muscle tissue include genomic and nongenomic effects via a receptor present in muscle cells. Knockout mouse models of the vitamin D receptor provide insight into understanding the direct effects of vitamin D on muscle tissue. Vitamin D status is positively associated with physical performance and inversely associated with risk of falling. Vitamin D supplementation has been shown to improve tests of muscle performance, reduce falls, and possibly impact on muscle fiber composition and morphology in vitamin D deficient older adults. SUMMARY: Further studies are needed to fully characterize the underlying mechanisms of vitamin D action in human muscle tissue, to understand how these actions translate into changes in muscle cell morphology and improvements in physical performance, and to define the 25-hydroxyvitamin D level at which to achieve these beneficial effects in muscle.

The effect of 4 weeks beta-alanine supplementation and isokinetic training on carnosine concentrations in type I and II human skeletal muscle fibres.

Seven male students were supplemented with beta-alanine (beta-ALG) for 4 weeks (6.4 g day(-1)) and seven with a matching placebo (PLG). Subjects undertook 4 weeks of isokinetic training with the right leg (T) whilst the left leg was untrained (UT), serving as a control. Each training session consisted of 10 x 10 maximal 90 degrees extension and flexion contractions at 180 degrees /s using a Kin-Com isokinetic dynamometer, with 1 min rest between bouts. Muscle biopsies were taken from the vastus lateralis immediately before and at the end of the supplementation period. Following freeze drying muscle fibres were dissected and characterised by their MHC profile, as type I, IIa, IIx, or as hybrids of these. Carnosine was measured by HPLC. There was a significant increase in carnosine in both T and UT legs of the beta-ALG (9.63 +/- 3.92 mmol kg(-1) dry muscle and 6.55 +/- 2.36 mmol kg(-1) dry muscle respectively). There was a significant increase in the carnosine content of all fibre phentotypes, with no significant difference between types. There were no significant differences in the changes in muscle or in fibres between the T and UT legs. In contrast there was no significant change in the carnosine content in either the T or UT legs with placebo. The results indicate that 4 weeks training has no effect on the muscle carnosine content. Whilst an increase was seen with beta-alanine supplementation, this was not further influenced by training. These findings suggest that beta-alanine availability is the main factor regulating muscle carnosine synthesis.

Creatine feeding does not enhance intramyocellular glycogen concentration during carbohydrate loading: an in vivo study by 31P- and 13C-MRS

The main aim of this study was to examine the hypothesis that creatine (Cr) feedingenhances myocellular glycogen storage in humans undergoing carbohydrateloading. Twenty trained male subjects were randomly assigned to have their dietssupplemented daily with 252 g of glucose polymer (GP) and either 21 g of Cr (CRGP,n=10) or placebo (PL-GP, n=10) for 5 days. Changes in resting myocellularglycogen and phosphocreatine (PCr) were determined with Magnetic ResonanceSpectroscopy (13C- and 31P-MRS, respectively). After CR-GP, the levels of intramyocellularglycogen increased from 147 ± 13 (standard error) mmol·(kg wet weight-1)to 172 ± 13 mmol·(kg wet weight)-1, while it increased from 134 ± 17 mmol·(kg wetweight)- to 182 ± 17 mmol·(kg wet weight)-1 after PL-GP; the increments in intramyocellularglycogen concentrations were not statistically different. The increment inthe PCr/ATP ratio after CR-GP (+ 0.20 ± 0.12) was significantly different comparedto PL-GP (- 0.34 ± 0.16) (p<0.05). The present results do not support the hypothesisthat Cr loading increases muscle glycogen storage (AU)

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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.

Creatine feeding does not enhance intramyocellular glycogen concentration during carbohydrate loading: an in vivo study by 31P- and 13C-MRS.

The main aim of this study was to examine the hypothesis that creatine (Cr) feeding enhances myocellular glycogen storage in humans undergoing carbohydrate loading. Twenty trained male subjects were randomly assigned to have their diets supplemented daily with 252 g of glucose polymer (GP) and either 21 g of Cr (CR-GP, n = 10) or placebo (PL-GP, n = 10) for 5 days. Changes in resting myocellular glycogen and phosphocreatine (PCr) were determined with Magnetic Resonance Spectroscopy (13C- and 31P-MRS, respectively). After CR-GP, the levels of intramyocellular glycogen increased from 147 +/- 13 (standard error) mmol x (kg wet weight(-1)) to 172 +/- 13 m mol x (kg wet weight)(-1), while it increased from 134 +/- 17 mmol x (kg wet weight)(-) to 182 +/- 17 mmol x (kg wet weight)(-1) after PL-GP; the increments in intramyocellular glycogen concentrations were not statistically different. The increment in the PCr/ATP ratio after CR-GP (+ 0.20 +/- 0.12) was significantly different compared to PL-GP (- 0.34 +/- 0.16) (p < 0.05). The present results do not support the hypothesis that Cr loading increases muscle glycogen storage.

Aerobic training, but not creatine supplementation, alters the gluteus medius muscle.

The aim of the present study was to investigate the effect of oral supplementation of creatine on the muscular responses to aerobic training. Twelve purebred Arabian horses were submitted to aerobic training for 90 d, with and without creatine supplementation, and evaluated with respect to BW and BCS and to the area and frequency of the different types of muscle fibers in the gluteus medius. Supplementation consisted of the daily administration of 75 g of creatine monohydrate mixed into the ration for the 90 d of training. Physical conditioning was conducted on a high-performance treadmill, and training intensity was stipulated by calculating the velocity at which blood lactate reaches 4 mmol/L, determined monthly for each animal. The individual intensity of physical force at 80% of aerobic threshold was established. Morphometry of gluteus medius muscle fibers was performed on frozen sections processed for histochemical analysis of myosin adenosine triphosphatase and immunohistochemistry of slow-contracting myosin. The results demonstrated that the animals maintained a moderate BCS without alteration of BW during the course of training, providing evidence of equilibrium between food intake and caloric expenditure during the study period. The present study demonstrated that aerobic training for 90 d caused hypertrophy of fiber types I (P = 0.04), IIA (P = 0.04), and IIX (P = 0.01), as well as an increase in the relative area occupied by type I fibers (P = 0.02) at the expense of type IIX fibers (P = 0.03), resulting in modifications of the contractile and metabolic characteristics of the gluteus medius muscle. It was not possible to show any beneficial effect from creatine on the skeletal muscle characteristics examined.

Human soleus and vastus lateralis muscle protein metabolism with an amino acid infusion.

The calf muscles, compared with the thigh, are less responsive to resistance exercise in ambulatory and bed-rested individuals, apparently due to muscle-specific differences in protein metabolism. We chose to evaluate the efficacy of using amino acids to elevate protein synthesis in the soleus, because amino acids have been shown to have a potent anabolic effect in the vastus lateralis. Mixed muscle protein synthesis in the soleus and vastus lateralis was measured before and after infusion of mixed amino acids in 10 individuals (28 +/- 1 yr). Phosphorylation of ribosomal protein p70 S6 kinase (p70S6K; Thr389) and eukaryotic initiation factor 4E-binding protein-1 (4E-BP1; Thr37/46) was also evaluated at rest and after 3 h of amino acid infusion. Basal protein synthesis was similar (P = 0.126), and amino acids stimulated protein synthesis to a similar extent (P = 0.004) in the vastus lateralis (0.043 +/- 0.011%/h) and soleus (0.032 +/- 0.017%/h). Phosphorylation of p70S6K (P = 0.443) and 4E-BP1 (P = 0.192) was not increased in either muscle; however, the soleus contained more total (P = 0.002) and phosphorylated (P = 0.013) 4E-BP1 than the vastus lateralis. These data support the need for further study of amino acid supplementation as a means to compensate for the reduced effectiveness of calf resistance exercise in ambulatory individuals and those exposed to extended periods of unloading. The greater 4E-BP1 in the soleus suggests that there is a muscle-specific distribution of general translational initiation machinery in human skeletal muscle.

NMR structure of a bifunctional rhodamine labeled N-domain of troponin C complexed with the regulatory "switch" peptide from troponin I: implications for in situ fluorescence studies in muscle fibers.

The structure of the calcium-saturated regulatory domain of skeletal troponin C (sNTnC) complexed with the switch peptide comprising residues 115-131 of troponin I (TnI), and with a bifunctional rhodamine fluorescent label attached to residues 56 (E56C) and 63 (E63C) on the C helix of sNTnC, has been determined using nuclear magnetic resonance (NMR) spectroscopy. The structure shows that the integrity of the C helix is not altered by the E(56,63)C mutations or by the presence of the bifunctional rhodamine and that the label does not interact with the hydrophobic cleft of sNTnC. Moreover, the overall fold of the protein and the position of the TnI peptide are similar to those observed previously with related cardiac NTnC complexes with residues 147-163 of cardiac TnI [Li et al. (1999) Biochemistry 38, 8289-8298] and including the drug bepridil [Wang et al. (2002) J. Biol. Chem. 277, 31124-31133]. The degree of opening of the structure is reduced as compared to that of calcium-saturated sNTnC in the absence of the switch peptide [Gagné et al. (1995) Nat. Struct. Biol. 2, 784-789]. The switch peptide is bound in a shallow and complementary hydrophobic surface cleft largely defined by helices A and B and also has key ionic interactions with sNTnC. These results show that bifunctional rhodamine probes can be attached to surface helices via suitable pairs of solvent-accessible residues that have been mutated to cysteines, without altering the conformation of the labeled domain. A set of such probes can be used to determine the orientation and motion of the target domain in the cellular environment [Corrie et al. (1999) Nature 400, 425-430; Ferguson et al. (2003) Mol. Cell 11(4), in press].

Increased expression of the nicotinic acetylcholine receptor in stimulated muscle.

Chronic low-frequency stimulation has been used as a model for investigating responses of skeletal muscle fibres to enhanced neuromuscular activity under conditions of maximum activation. Fast-to-slow isoform shifting of markers of the sarcoplasmic reticulum and the contractile apparatus demonstrated successful fibre transitions prior to studying the effect of chronic electro-stimulation on the expression of the nicotinic acetylcholine receptor. Comparative immunoblotting revealed that the alpha- and delta-subunits of the receptor were increased in 10-78 day stimulated specimens, while an associated component of the surface utrophin-glycoprotein complex, beta-dystroglycan, was not drastically changed in stimulated fast skeletal muscle. Previous studies have shown that electro-stimulation induces degeneration of fast glycolytic fibres, trans-differentiation leading to fast-to-slow fibre transitions and activation of muscle precursor cells. In analogy, our results indicate a molecular modification of the central functional unit of the post-synaptic muscle surface within existing neuromuscular junctions and/or during remodelling of nerve-muscle contacts.

Analysis of nucleotide myosin complexes in skeletal muscle fibres by DSC and EPR.

The internal dynamics and thermal unfolding of fibre bundles prepared from rabbit psoas muscle has been studied in the presence of nucleotides by differential scanning calorimetry (DSC) and electron paramagnetic resonance (EPR) spectroscopy. Using ADP, adenosine 5'-triphosphate (ATP), AMP.PNP and inorganic phosphate analogue orthovanadate (V(i)), AlF(4)(-) and BeF(3)(-), three intermediate states of the ATP hydrolysis cycle were simulated in glycerinated muscle fibres. In the main transition of the DSC pattern, three overlapping endotherms were detected in rigor, four in strongly as well as weakly binding state of myosin to actin. Deconvolution procedure showed that the transition temperature of 67.5 degrees C was the same for rigor and strong binding state of myosin. In contrast, nucleotide binding induced shift of the melting temperatures of 52 degrees C and 67.5 degrees C, appeared a new fourth peak at 74 and 77 degrees C and produced changes in the calorimetric enthalpies. The changes of the parameters of the peak functions suggest global rearrangements of the internal structure in myosin heads in the intermediate states. In the presence of ADP or ATP plus phosphate analogue orthovanadate or beryllium fluoride, aluminium fluoride, the conventional EPR spectra of spin-labeled muscle fibres showed large changes in the ordering of the probe molecules, and a new distribution of spin labels appeared. ATP plus orthovanadate induced the orientation disorder of myosin heads; the random population of spin labels gave evidence of large local conformational and motional changes in the internal structure of myosin heads. Saturation transfer EPR measurements reported increased rotational mobility of spin labels in the presence of ATP plus phosphate analogues corresponding to weakly binding state of myosin to actin.

Oral creatine supplementation facilitates the rehabilitation of disuse atrophy and alters the expression of muscle myogenic factors in humans.

1. We investigated the effect of oral creatine supplementation during leg immobilization and rehabilitation on muscle volume and function, and on myogenic transcription factor expression in human subjects. 2. A double-blind trial was performed in young healthy volunteers (n = 22). A cast was used to immobilize the right leg for 2 weeks. Thereafter the subjects participated in a knee-extension rehabilitation programme (3 sessions x week(-1), 10 weeks). Half of the subjects received creatine monohydrate (CR; from 20 g down to 5 g daily), whilst the others ingested placebo (P; maltodextrin). 3. Before and after immobilization, and after 3 and 10 weeks of rehabilitation training, the cross-sectional area (CSA) of the quadriceps muscle was assessed by NMR imaging. In addition, an isokinetic dynamometer was used to measure maximal knee-extension power (Wmax), and needle biopsy samples taken from the vastus lateralis muscle were examined to asses expression of the myogenic transcription factors MyoD, myogenin, Myf5, and MRF4, and muscle fibre diameters. 4. Immobilization decreased quadriceps muscle CSA (approximately 10 %) and Wmax (approximately 25 %) by the same magnitude in both groups. During rehabilitation, CSA and Wmax recovered at a faster rate in CR than in P (P < 0.05 for both parameters). Immobilization changed myogenic factor protein expression in neither P nor CR. However, after rehabilitation myogenin protein expression was increased in P but not in CR (P < 0.05), whilst MRF4 protein expression was increased in CR but not in P (P < 0.05). In addition, the change in MRF4 expression was correlated with the change in mean muscle fibre diameter (r = 0.73, P < 0.05). 5. It is concluded that oral creatine supplementation stimulates muscle hypertrophy during rehabilitative strength training. This effect may be mediated by a creatine-induced change in MRF4 and myogenin expression.