MyoD mRNA Research Articles

MyoD mRNA expression in skeletal muscle of patients with myotonic dystrophy

To explore the mRNA expression of MyoD gene in the skeletal muscles of myotonic dystrophy (MD) patients. Muscle biopsy specimens were obtained from the biceps muscles of arm of 4 MD patients and 4 healthy controls. Semi-quantitative reverse transcription polymerase chain reaction was performed to evaluate the mRNA expression of MyoD in the specimens. The mRNA expression index of MyoD of the MD patients was (0.267 +/- 0.114), significantly lower than that of the healthy controls [(0.788 +/- 0.136), P = 0.001]. Down-regulated mRNA expression of MyoD gene is involved in the mechanism of DM.

MyoD and Myogenin mRNA Levels after Single Session of Treadmill Exercise in Rat Skeletal Muscle

[Purpose] We examined mRNA levels of MyoD and myogenin in rat skeletal muscle after a single session of treadmill running. [Subjects] A total of 26 male Sprague-Dawley rats aged 4 weeks were used in this study. [Methods] Rats were run on a 16° decline for 30 min. Treadmill speeds were 0, 16, 20, 24 and 28 m/min. At 72 h post-exercise, soleus (SOL) and extensor digitorum longus (EDL) muscles were extracted. The expression of MyoD and myogenin mRNA were analyzed by reverse transcription polymerase chain reaction (RT-PCR) and quantitative RT-PCR. [Results] MyoD and myogenin mRNA expressions were detected in all groups. Significant differences in myogenin levels were apparent in SOL between 16 m/min and 28 m/min. MyoD levels in SOL at 16, 24, 28 m/min were lower, while myogenin levels at 20, 24 and 28 m/min were higher, when compared with the 0 m/min group, although significant differences were not seen. [Conclusion] These results suggest that a single session of exercise has little effect on proliferation of satellite cells, or myotube production.

The transcriptional cascade associated with creatine kinase down-regulation and mitochondrial biogenesis in mice sarcoma

The tissue-specific expressions of creatine kinase (CK) isoforms are regulated by the coordinated action of various transcription factors. The myogenic differentiation factor D (MyoD) family of proteins and the myocyte-specific enhancer binding factor 2 family of transcription factors are important in regulating the muscle-specific expression of cytosolic muscle-type CK (MCK) and mitochondrial CKs. As reported in some related studies, TNF-α mediated degradation of MyoD and myogenin mRNA may lead to severe muscle wasting and cachexia, which is characterized by a low transcript level of MCK and myosin heavy chain proteins. In our previous study, we reported on a complete loss of total CK activity and expression when sarcoma was induced in mouse skeletal muscle (Patra et al. FEBS J. 275 (2008) 3236–3247). This study aimed at investigating the transcriptional cascade of CK down-regulation in carcinogen-induced sarcoma in mouse muscle. Both CK deficiency and enhanced nitric oxide synthase (NOS) were known to augment mitochondrial biogenesis, so we also explored the activation of the transcriptional cascade of mitochondrial biogenesis in this cancer. We observed the activation of the TNF-α-mediated nitric oxide production pathway with NFκB activation and concomitant degradation of MyoD and myogenin mRNA. Exploration of mitochondrial biogenesis revealed high cytochrome c oxidase activity and mitochondrial DNA content in sarcoma. The PGC-related co-activator seems to have a major role in regulating mitochondrial biogenesis by upregulating nuclear respiratory factors and mitochondrial transcription factor A. From the above findings, it can be concluded that severe muscle degeneration leads to CK down-regulation in sarcoma, and that the stimulation of mitochondrial biogenesis indicated a scenario representing both CK deficiency and NOS overexpression on the one hand, and altered bioenergetic profiling on the other.

Molecular Biological Changes in Reloaded Skeletal Muscles after Rat Hindlimb Suspension

[Purpose] The purpose of this study was to clarify the expression changes of MyoD mRNA under reloading rat skeletal muscles after hindlimb suspension. [Subjects] The subjects were 27 Wistar male rats aged 8 weeks old. [Methods] They were randomly divided into a control group (n=5) and 4 experimental groups (n=5 to 6). The experimental groups were all submitted to 14 days of hindlimb suspension provoking muscle disuse atrophy, and afterwards they were assigned to no-reload hindlimb suspended group (HS) and 2, 7 and 14 days reloading groups (2D, 7D, 14D). [Results] The MyoD mRNA expression level increased in the HS group was significantly higher in the 2D reloading group and decreased to controls levels in the 7D reloading group. [Conclusion] The MyoD mRNA expression results suggest that reloading after hindlimb suspension stimulates satellite cell activation. Furthermore, the peak of MyoD mRNA expression was observed after 2 days of reloading.

MyoD, Myogenin and Myosin Heavy Chain mRNA Expression in Rat Skeletal Muscle after a Single Session of Low-Intensity Treadmill Exercise

[Purpose] We examined the time course of changes in the activation of muscle satellite cells and muscle protein synthesis after a single session of low-intensity treadmill exercise. [Subjects] A total of 25 male Wistar rats aged 11 weeks were used. [Methods] Rats were run on a treadmill with a 16° decline for 30 min at 24 m/min, except for the control group (CON). Blood was collected to measure creatine phosphokinase (CPK) and both soleus muscles were removed to analyze histological and muscle gene expression at 24, 48, 72 and 96 h post-exercise. [Results] CPK levels in exercised rats at 24 and 96 h were significantly higher than in CON, and CPK levels at 96 h were higher than those at 48 h. Infiltration of macrophages and gathering of small myofibers were detected in the exercise group, but there were no significant differences in MyoD, myogenin, myosin heavy chain (MHC) -1 and MHC-2a mRNA levels. [Conclusion] These results suggest that muscle satellite cells and muscle protein synthesis are not significantly activated, despite the occurrence of myotrauma, in the acute phase (24 h to 96 h) after a single session of low-intensity exercise.

Expression of Caspase Family and Muscle- and Apoptosis-Specific Genes during Skeletal Myogenesis in Mouse Embryo

The caspases (Casps) are a family of cysteine proteases that are known to regulate apoptotic signaling. Apoptosis by activation of Casp is strongly associated with embryonal development and regeneration in many organs, therefore indicating that disorders caused by homozygous mutation in Casp genes can result in embryonic lethality. In the present study, the authors investigated the causative relationship between skeletal myogenesis and the activation of Casps by analyzing their dynamics during mouse embryogenesis. Individual myogenetic tissues were obtained from C57BL/6 mouse embryos aged 12.5-17.5 days post-conception (dpc), and the expression of Casps was analyzed by histochemical and molecular biological methods. Immunoreactions for Casp-3, -9 and -12 were detected first in myoblasts, increasing according to embryonal development, as a result of which myoblasts differentiated into myotube cells. On the other hand, the immunoreaction for ssDNA, which is well-known as an apoptosis marker, was little detected during the skeletal myogenesis. Quantification analysis for Casp mRNA expression by RT-PCR as well as by in situ hybridization showed a peak at 15.5 dpc but a decrease at 17.5 dpc. Similar dynamics were detected for Myod1 mRNA, one of the muscle regulatory factors, but not for Fasl, Bax and Rock1, apoptosis-associated factors during skeletal myogenesis. These results suggest that the activation of Casps in skeletal myogenesis is deeply associated with myoblast differentiation, but not directly related to apoptosis.

Co‐expression of IGF‐1 family members with myogenic regulatory factors following acute damaging muscle‐lengthening contractions in humans

Muscle regeneration following injury is dependent on the ability of muscle satellite cells to activate, proliferate and fuse with damaged fibres. This process is controlled by the myogenic regulatory factors (MRF). Little is known about the temporal relation of the MRF with the expression of known myogenic growth factors (i.e. IGF-1) in humans following muscle damage. Eight subjects (20.6 +/- 2.1 years; 81.4 +/- 9.8 kg) performed 300 lengthening contractions (180 deg s(-1)) of their knee extensors in one leg on a dynamometer. Blood and muscle samples were collected before and at 4 (T4), 24 (T24), 72 (T72) and 120 h (T120) post-exercise. Mechano growth factor (MGF), IGF-1Ea and IGF-1Eb mRNA were quantified. Serum IGF-1 did not change over the post-exercise time course. IGF-1Ea and IGF-1Eb mRNA increased approximately 4- to 6-fold by T72 (P < 0.01) and MGF mRNA expression peaked at T24 (P = 0.005). MyoD mRNA expression increased approximately 2-fold at T4 (P < 0.05). Myf5 expression peaked at T24 (P < 0.05), while MRF4 and myogenin mRNA expression peaked at T72 (P < 0.05). Myf5 expression strongly correlated with the increase in MGF mRNA (r(2) = 0.83; P = 0.03), while MRF4 was correlated with both IGF-1Ea and -Eb (r(2) = 0.90; r(2) = 0.81, respectively; P < 0.05). Immunofluorescence analysis showed IGF-1 protein expression localized to satellite cells at T24, and to satellite cells and the myofibre at T72 and T120; IGF-1 was not detected at T0 or T4. These results suggest that the temporal response of MGF is probably related to the activation/proliferation phase of the myogenic programme as marked by an increase in both Myf5 and MyoD, while IGF-1Ea and -Eb may be temporally related to differentiation as marked by an increase in MRF4 and myogenin expression following acute muscle damage.

Myostatin preferentially down-regulates the expression of fast 2x myosin heavy chain in cattle

Myostatin is involved in an inhibitor of muscular growth and differentiation. Myoblasts derived from double-muscled Japanese shorthorn cattle (DM myoblasts) with absence of functional myostatin had higher abilities to proliferate and differentiate than myoblasts derived from normal-muscled cattle (NM myoblasts). In DM myoblasts, mRNA expressions of fetal myosin heavy chain (MyHC) in growth medium and of fast 2a and 2x MyHC in fusion medium were significantly greater than that in NM myoblasts. No significant difference existed in expressions of embryonic and slow MyHC mRNA between DM and NM myoblasts. The expression of MyoD mRNA was suppressed in myoblasts by administration of myostatin. Two cloned DM myoblast strains (DMc) were established. Addition of myostatin for DMc resulted in less myotube formation and suppression of mRNA expression of fast 2x MyHC. These findings suggest that the endogenous myostatin preferentially down-regulates the expression of the fast 2x MyHC and participates in differentiation of myofiber types during early bovine myogenesis.

Expression of growth-related genes in young and older human skeletal muscle following an acute stimulation of protein synthesis

Muscle growth is associated with an activation of the mTOR signaling pathway and satellite cell regulators. The purpose of this study was to determine whether 17 selected genes associated with mTOR/muscle protein synthesis and the satellite cells/myogenic program are differentially expressed in young and older human skeletal muscle at rest and in response to a potent anabolic stimulus [resistance exercise + essential amino acid ingestion (RE+EAA)]. Twelve male subjects (6 young, 6 old) completed a bout of heavy resistance exercise. Muscle biopsies were obtained before and at 3 and 6 h post RE+EAA. Subjects ingested leucine-enriched essential amino acids at 1 h postexercise. mRNA expression was determined using qRT-PCR. At rest, hVps34 mRNA was elevated in the older subjects (P < 0.05) while there was a tendency for levels of myoD, myogenin, and TSC2 mRNA to be higher than young. The anabolic stimulus (RE+EAA) altered mRNAs associated with mTOR regulation. Notably, REDD2 decreased in both age groups (P < 0.05) but the expression of Rheb mRNA increased only in the young. Finally, cMyc mRNA was elevated (P < 0.05) in both young and old at 6 h post RE+EAA. Furthermore, RE+EAA also increased expression of several mRNAs associated with satellite function in the young (P < 0.05), while expression of these mRNAs did not change in the old. We conclude that several anabolic genes in muscle are more responsive in young men post RE+EAA. Our data provide new insights into the regulation of genes important for transcription and translation in young and old human skeletal muscle post RE+EAA.

Expression of mRNA for specific fibroblast growth factors associates with that of the myogenic markers MyoD and proliferating cell nuclear antigen in regenerating and overloaded rat plantaris muscle

To examine the relations between specific fibroblast growth factors (FGFs) and satellite cell activation during muscle regeneration and hypertrophy in vivo, we measured mRNA expression of FGFs and myogenic markers in rat plantaris muscle after bupivacaine administration and synergist ablation. mRNA levels for MyoD, myogenin, proliferating cell nuclear antigen (PCNA), p21, M-cadherin, Pax7, FGF-1, FGF-2, FGF-3, FGF-4, FGF-5, FGF-6, FGF-7, FGF-8 and hepatocyte growth factor (HGF) were measured continually for up to 72 h after bupivacaine administration and synergist ablation. FGF-5, FGF-7 and HGF proteins were immunostained at 72 h after bupivacaine administration. MyoD and PCNA mRNAs started increasing 24 h after bupivacaine administration. Myogenin, p21, M-cadherin and Pax7 mRNAs started to increase after 48 and 72 h. After synergist ablation, MyoD, PCNA, M-cadherin and Pax7 mRNAs had increased at 24 and 48 h, and myogenin and p21 mRNAs at 12 and 24 h. FGF-1, FGF-7 and HGF mRNAs after the treatments started to increase at the same time as MyoD and PCNA mRNAs. FGF-5 was expressed at the same time as MyoD and PCNA mRNAs after bupivacaine administration but did not after the ablation. FGF-2, FGF-3, FGF-4, FGF-6 and FGF-8 mRNAs were not associated with the expression of the myogenic markers. FGF-7 and HGF proteins were expressed in immature muscle fibre nuclei and the extracellular matrix, but FGF-5 protein was preferentially expressed in extracellular matrix. These results indicate that FGF-1, FGF-7 and HGF are associated with specific myogenic marker expression during muscle regeneration and hypertrophy.

Effects of alternagin-C from Bothrops alternatus on gene expression and activity of metalloproteinases in regenerating skeletal muscle

This study evaluated the effects of alternagin-C (ALT-C) on mRNA levels of VEGF, MyoD and matrix metalloproteinase 2 (MMP-2) and on activity of MMPs in injured tibialis anterior (TA) muscle induced by cryolesioning in rats. Thirty-six Wistar rats (3 months old, 258.9 ± 27 g) were divided into five groups: (1) control group; (2) injured TA and analyzed 3 days later; (3) injured TA treated with ALT-C and analyzed 3 days later; (4) injured TA and analyzed 7 days later and (5) injured TA treated with ALT-C and analyzed 7 days later. The injured muscle received 25 μl of ALT-C (50 nM). The injured and uninjured muscle areas were quantified by light microscopy. The MMP activity was evaluated through zymography, and mRNA of MyoD, VEGF and MMP-2 was assessed by quantitative polymerase chain reaction. ALT-C neither reduced the muscle injury area nor altered the pattern of MyoD and VEGF expression in injured muscles. However, ALT-C reduces both MMP-2 mRNA and gelatinolytic activity in injured muscles. The study indicates that ALT-C, at the tested concentrations, did not improve muscle regeneration process in rats. The effect on MMP-2 mRNA and gelatinolytic activity suggests that ALT-C changes the overall balance of ECM protein turnover during muscle regeneration.

PAX3/7 expression coincides with myod during chronic skeletal muscle overload

Paired box (Pax) proteins 3 and 7 are key determinants for embryonic skeletal muscle development by initiating myogenic regulatory factor (MRF) gene expression. We show that Pax3 and 7 participate in adult skeletal muscle plasticity during the initial responses to chronic overload (< or =7 days) and appear to coordinate MyoD expression, a member of the MRF family of genes. Pax3 and 7 mRNA were higher than control within 12 h after initiation of overload, preceded the increase in MyoD mRNA on day 1, and peaked on day 2. On days 3 and 7, Pax7 mRNA remained higher than control, suggesting that satellite cell self-renewal was occurring. Pax3 and 7 and MyoD protein levels were higher than control on days 2 and 3. These data indicate that Pax3 and 7 coordinate the recapitulation of developmental-like regulatory mechanisms in response to growth-inducing stimuli in adult skeletal muscle, presumably through activation of satellite cells.

Interleukin-6 Deficiency Attenuates the Recovery of Gastrocnemius Muscle Mass from Disuse-Induced Atrophy

Interleukin-6 (IL-6), an inflammatory cytokine, expression is induced during recovery from disuse-induced atrophy in skeletal muscle. However, the functional significance of increased IL-6 expression in response to reload has not been determined. PURPOSE: The purpose of this study was to determine the effect of IL-6 loss on reload-induced muscle morphology and gene expression related to growth. METHODS: Female C57/BL6 (WT, n= 10) and IL-6 knockout (IL-6-/-, n=10) mice (10 wks of age) were randomly assigned to a control, hindlimb suspension, or hindlimb suspension + recovery treatments. To induce disuse-induced atrophy mice underwent 10 days of hindlimb suspension. The recovery groups were allowed normal ambulation following hindlimb suspension for 1, 7, or 14 days. Gene expression was determined using quantitative real-time PCR. RESULTS: 10 days of hindlimb suspension decreased gastrocnemius muscle mass to tibia length ratio 20% in both WT (5.7 ± 0.11 mg/mm vs. 4.6 ± 0.20 mg/mm, p < 0.05) and IL-6-/- mice (5.7 ± 0.20 mg/mm vs. 4.6 ± 0.17 mg/mm, p < 0.05). 7 days of reload returned wild type mice muscle mass to tibia length ratio to control values, whereas IL-6-/- gastrocnemius muscle to tibia was still depressed (p < 0.05). However, 14 days of reload returned gastrocnemius muscle mass of IL-6-/- mice back to control values. 1 day of reload increased IGF-1 gene expression 4-fold (p < 0.05) in WT gastrocnemius muscle. In contrast, IL-6-/- gastrocnemius muscle had a 67% decrease (p < 0.05) in IGF-1 gene expression. 1 day of reload increased myoD mRNA abundance 3-fold (p < 0.05) in WT gastrocnemius muscle. However, 1 day of reload decreased myoD gene expression 85% (p < 0.05) in IL-6-/- gastrocnemius muscle. 1 day of reload increased myogenin gene expression 2-fold (p < 0.05) in WT gastrocnemius muscle whereas; this response was ablated in the IL-6-/- gastrocnemius. CONCLUSIONS: In conclusion, these data point to IL-6 as a critical regulator of the myogenic and anabolic growth programs that are initiated at the onset of recovery from disuse-induced atrophy.

The Influence of Eccentric and Concentric Exercise on Skeletal Muscle Regulators in Young Women

PURPOSE: To evaluate changes in myostatin, follistatin and MyoD mRNA gene expression using concentric exercise (CE) and eccentric exercise (EE) as a stimulus to better understand the mechanisms of muscle hypertrophy in young women. METHODS: Single-leg maximal eccentric or concentric isokinetic exercise was performed using the Cybex NormTM dynamometer (Cybex International Inc. Ronkonkoma, New York) at 60° /s. Ten sets of ten repetitions with 20 second rest between sets were completed by each subject. Three muscle biopsies of the vastus lateralis were obtained from the exercised leg at baseline (1st biopsy), 8 hours after the first exercise bout (2nd biopsy), and 8 hours after the seventh and final exercise bout performed 2 weeks after the first exercise session (3rd biopsy). mRNA gene expression was analyzed using RT-PCR methods. 18s ribosomal RNA was used as the internal standard to determine relative expression of mRNAs for myostatin, follistatin, and MyoD. Paired t-tests were used to compare changes in mRNA gene expression within groups (Eccentric or Concentric). Independent t-tests were performed to test group differences (Eccentric versus Concentric). A probability level of P ≤ 0.05 was used to determine statistical significance with Bonferroni adjustments. RESULTS: There were no significant fold changes in the CE group for myostatin, follistatin, and MyoD mRNA gene expression between the first and second biopsies as well as the first and third biopsies (P>0.17). Similarly, there were no significant changes in the EE group for the same variables (P>0.11). In addition, there were no significant differences between the EE and CE groups (P≥0.05). CONCLUSIONS: Our data suggests that a single bout or multiple bouts (7) of maximal eccentric or concentric exercise does not significantly alter myostatin, follistatin, or MyoD mRNA gene expression in young women. Therefore, the type of exercise stimulus does not appear to differentially alter mRNA gene expression of hypertrophic myogenic regulators.

CMF1–Rb interaction promotes myogenesis in avian skeletal myoblasts

CMF1 protein is expressed in developing striated muscle before the expression of contractile proteins, and depletion of CMF1 in myoblasts results in inability to express muscle-specific proteins. Previous studies of CMF1 identify a functional Rb-binding domain, which is conserved in the murine and human homologues. Here, we show that CMF1 binds Rb family members, while a CMF1 protein with deletion of the Rb-binding domain (Rb-del CMF1) does not. Myogenic cell lines over-expressing Rb-del CMF1 proliferate normally, but exhibit markedly impaired differentiation, including dramatically reduced contractile proteins gene expression and failure to fuse into myotubes. Furthermore, by quantitative real-time polymerase chain reaction, MyoD and Myf5 mRNA levels are comparable to wild-type, while myogenin and contractile protein mRNA levels are significantly attenuated. These data demonstrate that CMF1 regulates myocyte differentiation by interaction with Rb family members to induce expression of myogenic regulatory factors.

Stau1 negatively regulates myogenic differentiation in C2C12 cells

Sequential expression of myogenic regulatory factors (MRFs) such as MyoD and myogenin drives myogenic differentiation. Besides transcriptional activation of MRFs, this process is also coordinated by post-transcriptional regulation; MyoD and myogenin mRNAs are stabilized by RNA-binding protein HuR. Stau1 is known to regulate mRNA stability in a complex with Upf1, which is termed Stau1-mediated mRNA decay (SMD). We describe here that Stau1 is involved in the regulation of myogenesis. We found that knockdown of Stau1 promotes myogenesis including the expression of a muscle-specific marker protein, myoglobin, in C2C12 myoblasts. MyoD induces myogenin expression in response to induction of myogenesis, which is a key step to start myogenesis. The level of MyoD protein was not affected when Stau1 was depleted by siRNA, whereas the levels of myogenin mRNA and protein were increased in Stau1-knockdown cells. Interestingly, myogenin promoter activity was also increased in Stau1-knockdown cells in the absence of induction of myogenesis. Furthermore, Stau1-knockdown cells spontaneously progressed myogenesis including the expression of muscle-specific protein. Although Stau1 is involved in mRNA decay together with Upf1, Upf1-knockdown did not affect progression of myogenesis. Our results suggest that Stau1 negatively regulates myogenesis in C2C12 myoblasts through a mechanism that is different from SMD.

Differences in Muscle Protein Synthesis and Anabolic Signaling in the Postabsorptive State and in Response to Food in 65–80 Year Old Men and Women

Women have less muscle than men but lose it more slowly during aging. To discover potential underlying mechanism(s) for this we evaluated the muscle protein synthesis process in postabsorptive conditions and during feeding in twenty-nine 65–80 year old men (n = 13) and women (n = 16). We discovered that the basal concentration of phosphorylated eEF2Thr56 was ∼40% less (P<0.05) and the basal rate of MPS was ∼30% greater (P = 0.02) in women than in men; the basal concentrations of muscle phosphorylated AktThr308, p70s6kThr389, eIF4ESer209, and eIF4E-BP1Thr37/46 were not different between the sexes. Feeding increased (P<0.05) AktThr308 and p70s6kThr389 phosphorylation to the same extent in men and women but increased (P<0.05) the phosphorylation of eIF4ESer209 and eIF4E-BP1Thr37/46 in men only. Accordingly, feeding increased MPS in men (P<0.01) but not in women. The postabsorptive muscle mRNA concentrations for myoD and myostatin were not different between sexes; feeding doubled myoD mRNA (P<0.05) and halved that of myostatin (P<0.05) in both sexes. Thus, there is sexual dimorphism in MPS and its control in older adults; a greater basal rate of MPS, operating over most of the day may partially explain the slower loss of muscle in older women.

Hepatocyte Growth Factor Signaling in Mediating Human Muscle Satellite Cell Activation and Proliferation Following Eccentric Exercise

Eight subjects (20.6 ± 2.1 yr; 81.4 ± 9.8 kg) performed 300 isolateral eccentric contractions (knee extensions at 180 °/s) to evaluate the skeletal muscle satellite cell (SC) response and the potential of hepatocyte growth factor (HGF) signaling in mediating SC activation and proliferation following acute damaging exercise. Muscle biopsies were obtained from the vastus lateralis of the non‐exercised leg prior to exercise (PRE) and the exercised leg at 4 (T4), 24 (T24), 72 (T72), and 120 h (T120) post‐exercise. The number of SC (N‐CAM+) increased (p &lt; 0.001) by T4 and remained elevated at T120 (p = 0.002). Serum HGF increased at T4 (p &lt; 0.05) with a trend for increased muscle HGF and HGF activator (HGFA) protein expression at T24 with active HGF detected in all PRE samples. HGFA inhibitors (HAI‐1 and HAI‐2) protein content increased at T72 and remained elevated at T120 (p &lt; 0.05). Myogenic regulatory factors (MyoD and Myf5) mRNA expression increased at T4 and T24 (p &lt; 0.05) respectively, with increased myogenin mRNA expression at T24 (p &lt; 0.05). Furthermore, plasma interleukin‐6 concentration increased ∼200% (p &lt; 0.05) by T4, illustrating a roll of both local and systemic signaling in the activation and proliferation of SC; with HGF signaling playing an important regulatory role in the SC response to exercise‐induced muscle damage.Supported by NSERC

BMAL1 and CLOCK proteins directly bind to the core enhancer of the Myod1 promoter

The molecular clock regulating circadian rhythms is a network of transcriptional‐translational feedback loops that drive rhythmic expression of genes over a 24‐h period. BMAL1 and CLOCK are two of the main transcription factors that regulate the molecular clock mechanism. Myod1 (myogenic differentiation 1) was identified as having a circadian pattern of expression in adult skeletal muscle. The primary objective of this study was to determine whether CLOCK and BMAL1 bind the Myod1 promoter in a temporal manner. Mice were entrained for 2 weeks in 12 hour light /12 hour dark and then hindlimb muscles were collected every 4 hours for 24 hours. The chromatin immuno‐precipitation (ChIP) assay with nuclear extracts from mouse muscle was performed. We tested binding of BMAL1, CLOCK, and RNA Polymerase to the core enhancer (CE) and distal regulatory region (DRR) of the Myod1 promoter. Results from these studies determined that BMAL1 and CLOCK did bind to the Myod1 promoter but only to the upstream core enhancer CE and not the DRR. Binding of BMAL1 and CLOCK at the CE was also found to oscillate over the course of the circadian cycle. We conclude that Myod1 is a direct clock controlled gene with BMAL1 and CLOCK regulating Myod1 mRNA levels via direct binding to the CE element of the promoter.This research was supported by National Institute of Health grants KAE (AR045617) and JJM (AR053641).

Expression of genes regulating protein synthesis in young and old human muscle following resistance exercise and essential amino acid ingestion

Muscle growth requires an increase in both transcription and translation which results in enhanced muscle protein synthesis. The purpose of this study was to determine whether genes associated with the myogenic program and muscle protein synthesis are differentially expressed in old and young human skeletal muscle after a bout of resistance exercise and essential amino acid (EAA) ingestion. Twelve subjects (6 young, 6 old) completed 8 sets of leg extension exercise (70% 1RM) and muscle biopsies were obtained before and at 1, 3, and 6h post‐exercise. Subjects consumed a 20g leucine‐enriched EAA solution at 1h post‐exercise. RNA was isolated from biopsies and mRNA expression was determined using qRT‐PCR. At baseline, mRNA expression of Myogenin, TSC1/2, and Rheb were greater in old than young (P&lt;0.05). Exercise resulted in increased levels of MyoD mRNA and decreased REDD1 and REDD2 mRNA in both groups (P&lt;0.05). Myogenin mRNA increased at 3 and 6h and IGF‐1 mRNA increased at 6h only in young (P&lt;0.05) suggesting that some genes are more responsive in young muscle following exercise. We conclude that reduced REDD1 and REDD2 mRNA levels are consistent with prolonged elevation of mTOR/S6K1 signaling and protein synthesis following resistance exercise and EAA ingestion in both young and old human muscle. Our data provides new insights into regulation of genes important for transcription and translation in young and old muscle.Funded by NIH grant # P30 AG024832, R01 AR049877, and R01 AR045617