Muscle RNA Research Articles

Longitudinal Analysis of Gene Expression in Porcine Skeletal Muscle After Post-Injection Local Injury

The purpose of this study is to describe the time course of gene expression in a skeletal muscle local injury induced by an intramuscular (IM) injection, and to compare the dynamics of gene expression with pathological events. Ten piglets received 4 IM injections of propylene glycol in the longissimus dorsi muscles 6 h, 2, 7, and 21 days before euthanasia, where control and injected muscle sites were sampled for RNA isolation and microscopic examination. The hybridization of nylon cDNA microarrays was carried out with radioactive probes obtained from the muscle RNA. 153 genes were found under- or over-expressed at least once among the investigated time-conditions. The eight most discriminant genes were also identified: Two genes (GTP-binding protein RAD and Ankyrin repeat domain protein) were over-expressed at 6 h and six genes between 2 and 21 days (Osteonectin, Fibronectin, Matrix metalloproteinase-2, Collagen alpha 1(I) chain, Collagen alpha 2(I) chain, and Thymosin beta-4). Necrosis, inflammation and regeneration were observed through both the dynamics of gene expression profiles and through the microscopic examinations. Our data demonstrate that several pathways are involved in post-injection muscle injury, and that necrosis, inflammation and regeneration are not sequential but occur in parallel.

The use of scale increment as a means of indicating fish growth and growth impairment

Growth impairments were studied for three juvenile fish species: the red drum ( Sciaenops ocellatus), the orange-spotted grouper ( Epinephelus coioides) and the marine medaka ( Oryzia melastigma). Fish were subjected to long-term (i) food deprivation, (ii) exposure to benzo[ a]pyrene, (iii) hypoxia, respectively. Fish growth (fork length and body weight) and scale increments (circulus number and radius length) were measured. Results of Pearson correlation analysis showed a significant positive correlation between scale increment and growth for the red drum (fast growing) and the marine medaka (small sized) ( r = 0.741–0.882, p < 0.001). For the slow growing orange-spotted grouper, a significant correlation was found only between body weight and circulus number ( r = 0.406, p < 0.05). Overall, scale circulus number is more consistently linked to fish growth than that of radius length. Importantly, our findings demonstrate that daily circulus deposition on scales of fast-growing red drum is feasible to indicate daily growth of the fish. Moreover, scale increment exhibited the strongest correlation with fish growth as compared to that of serum T 3 level and muscle RNA:DNA ratio. Our data support the use of scale increment as a means of indicating fish growth and growth impairment, particularly for the fast growing fish.

The modulator effect of GH on skeletal muscle lysosomal enzymes is dietary protein dependent

Objective The purpose of this work is to determine whether changes in dietary protein level could alter the modulator effect that GH has on the muscle lysosomal system by influencing the hydrolytic activities of cathepsin D, acid RNase and DNase II and the participation of these enzymes in muscle growth. Design BALB/c female mice were fed a diet containing 20% (HP) or 12% (MP) protein ad libitum and were treated with either saline (s) or rhGH (GH) (74 ng/g) for 29 days. Body weight and feed intake were recorded daily. At 25, 30, 35, 40, 45 and 50 days of age, five mice from each group were slaughtered and nucleic acids and protein concentrations and cathepsin D, acid RNase and DNase II activities in gastrocnemius muscle were analysed. Correlation coefficients were used to analyse the links between the activity of each enzyme with its substrate. Results GH-treatment induced a depletion–recovery response in muscle growth through a compensatory mechanism. Changes in protein content, DNA and RNA concentrations were related to changes in lysosomal enzyme activities. Muscle cathepsin D activity in saline mice fell as the dietary protein concentration increased. GH-treatment reversed this effect by enhancing the proteolytic activity in muscle of well-fed mice and inhibiting it in mice fed a 12% protein diet. This inversion appears to be related to the different mechanism elicited by GH-treatment on skeletal muscle protein growth in each dietary group. An opposite trend was observed in muscle acid nuclease activities. Acid RNase and DNase II increased according to the dietary protein concentration, since a 12% protein diet induced a lower catabolism, especially on muscle DNA of saline mice. In contrast, GH-treatment decreased acid RNase and DNase II activities, but only in mice fed a 20% protein diet, perhaps leading to spare muscle RNA for protein synthesis, as well as to the inhibition of DNA degradation during catch-up growth. A lower dietary protein concentration appeared to reverse the GH protective effect on nucleic acids. Conclusions GH seems to act as a dietary protein-dependent modulator of the skeletal muscle lysosomal enzyme activity. These lysosomal enzymes play a role during muscle growth in GH-treated post-weaning mice by modifying muscle protein and DNA and RNA degradation.

Long-term stability of RNA in post-mortem bovine skeletal muscle, liver and subcutaneous adipose tissues

BackgroundRecovering high quality intact RNA from post-mortem tissue is of major concern for gene expression studies in animals and humans. Since the availability of post-mortem tissue is often associated with substantial delay, it is important that we understand the temporal variation in the stability of total RNA and of individual gene transcripts so as to be able to appropriately interpret the data generated from such studies. Hence, the objective of this experiment was to qualitatively and quantitatively assess the integrity of total and messenger RNA extracted from bovine skeletal muscle, subcutaneous adipose tissue and liver stored at 4°C at a range of time points up to 22 days post-mortem. These conditions were designed to mimic the environment prevailing during the transport of beef from the abattoir to retail outlets.ResultsThe 28S and 18S rRNA molecules of total RNA were intact for up to 24 h post-mortem in liver and adipose tissues and up to 8 days post-mortem in skeletal muscle. The mRNA of housekeeping genes (GAPDH and ACTB) and two diet-related genes (RBP5 and SCD) were detectable up to 22 days post-mortem in skeletal muscle. While the mRNA stability of the two housekeeping genes was different in skeletal muscle and liver, they were similar to each other in adipose tissue. After 22 days post-mortem, the relative abundance of RBP5 gene was increased in skeletal muscle and in adipose tissue and decreased in liver. During this period, the relative abundance of SCD gene also increased in skeletal muscle whereas it decreased in both adipose tissue and liver.ConclusionStability of RNA in three tissues (skeletal muscle, subcutaneous adipose tissue and liver) subjected to long-term post-mortem storage at refrigeration temperature indicated that skeletal muscle can be a suitable tissue for recovering biologically useful RNA for gene expression studies even if the tissue is subjected to post-mortem storage for weeks, whereas adipose tissue and liver should be processed within 24 hours post-mortem.

Estimation of genetic trends from 1977 to 1998 of body composition and physiological state of Large White pigs at birth

Genetic trends for body composition and blood plasma parameters of newborn piglets were estimated through the comparison of two groups of pigs (G77 and G98, respectively) produced by inseminating Large White (LW) sows with semen from LW boars born either in 1977 or in 1998. Random samples of 18 G77 and 19 G98 newborn piglets were used for whole carcass and tissue sampling. Plasma concentrations of glucose, albumin and IGF-1 were determined on 75 G77 and 90 G98 piglets from 18 litters. The G98 piglets had less carcass dry matter, protein and energy (P < 0.01) than their G77 counterparts. When expressed in g/kg birth weight, livers were lighter (P < 0.001) and contained less glycogen (P < 0.01) in G98 piglets, with no difference in the activity of the hepatic glucose-6-phosphatase between G98 and G77 piglets. Concentrations of protein, DNA, RNA in longissimus dorsi muscle were unaffected by selection. Plasma concentrations of glucose (P < 0.05) and IGF-1 (P < 0.01) were lower in G98 than in G77 piglets. On the whole, the results suggest that the improvement in lean growth rate and in sow prolificacy from 1977 to 1998 has resulted in a lower maturity of piglets at birth.

A Simple Method for Rapid Separation of Endothelial and Smooth Muscle mRNA Reveals Na+/K+-ATPase Alpha-Subunit Distribution in Rat Arteries

Background/Aims: The endothelium has been recognized as a key component in the regulation of blood vessels. We designed a simple procedure to separate endothelial and smooth muscle RNA from rat aorta and mesenteric artery and used this method to establish the distribution of Na⁺/K⁺-ATPase α-subunit isoforms (NaKα1, NaKα2 and NaKα3) within the arterial wall. Methods: Rat aorta was perfused with Tripure, a reagent for RNA isolation, yielding 3 successive RNA fractions (E1–E3) and the remaining tissular RNA (Ao[E–]). A similar procedure was applied to the mesenteric artery. Gene expression was studied by semiquantitative reverse-transcription polymerase chain reaction. Results: Compared to unperfused aorta (Ao[E+]), typical endothelial mRNAs were enriched 3- to 5-fold in E1–E3 but almost absent in Ao[E–], whereas smooth muscle mRNAs were low in E1–E3 but similarly expressed in Ao[E–] and Ao[E+]. NaKα1 was uniformly expressed in all fractions, NaKα2 closely followed the expression pattern of smooth muscle markers and NaKα3 expression was weak and attributable to blood contamination. Comparable results were obtained with the mesenteric artery. Conclusion: We conclude that, in aorta and mesenteric artery, Tripure perfusion allows for a rapid and reliable separation of endothelial mRNA from smooth muscle mRNA, and that endothelium only expresses NaKα1, whereas smooth muscle expresses NaKα1 and NaKα2, but not NaKα3.

Effect of Gender and Aerobic Training Status on Plasma and Skeletal Muscle Fibrinolytic Potential

Fibrinolysis is the capacity to lyse fibrin clots through the activity of the plasminogen system. Plasminogen is primarily activated into active plasmin by the enzyme tissue plasminogen activator (tPA), while this process is primarily inhibited by plasminogen activator inhibitor-1 (PAI-1). Endothelial cells are the primary site of tPA and PAI-1 synthesis and release. However, tPA and PAI-1 are expressed in skeletal muscle tissue, and PAI-1 is also expressed in adipose tissue. Both of these tissues are influenced by gender and aerobic exercise training. PURPOSE: To cross-sectionally investigate the effect of gender and aerobic exercise status on plasma and skeletal muscle expression of tPA and PAI-1. METHODS: Skeletal muscle and plasma tPA and PAI-1 gene expression were measured in four groups of young subjects; trained males (TM, n = 7, mean age 21.6 ± 0.4 y), untrained males (UM, n = 6, mean age 21.5 ± 0.4 y), trained females (TF, n = 8, mean age 24.4 ± 2.1 y), and untrained females (UF, n = 6, mean age 22.8 ± 1.2 y). To measure tPA and PAI-1 activity and antigen, 5 ml of venous blood were collected into an acidified citrate solution after a 12h fast. Women were tested 4 d post-menses. Samples were spun to obtain platelet-poor plasma and stored at −80C until assayed. All blood samples were obtained prior to the muscle biopsy procedure. Muscle samples were obtained from the vastus lateralis muscle via a percutaneous muscle biopsy following a 12-hour fast. Total RNA was extracted from the muscle samples, and subjected to reverse transcription. Skeletal muscle tPA and PAI-1 gene expression was then assessed by the polymerase chain reaction using gene specific primers. RESULTS: There was no affect of gender or aerobic training status on plasma tPA activity or antigen, muscle tPA RNA expression, or muscle PAI-1 RNA expression. There was a significant effect of training status on plasma PAI-1 activity, such that the trained groups had significantly lower PAI-1 activity compared to the untrained groups (6.0 ± 1.5 vs. 13.0 ± 2.1 IU/ml, p<0.05). Additionally, there was a significant inverse relationship between plasma PAI-1 activity and VO2max (r = −0.386, p < 0.05). CONCLUSIONS: Gender does not have an influence on plasma tPA activity or antigen, muscle RNA expression of tPA or PAI-1. Aerobic exercise does have a positive affect on fibrinolytic capacity, as evidenced by the lower PAI-1 activity in the trained subjects. This effect appears to be mediated through increased fitness level, as indicated by the negative correlation between VO2max and PAI-1 activity.

Mechanism of fenvalerate-withdrawal-dependent recovery in metabolism of a catfish, Clarias batrachus

The effects of protein synthesis inhibitors on fenvalerate-withdrawal-dependent recovery in citrate synthase (CS), glucose 6-phosphate dehydrogenase (G6-PDH), lactate dehydrogenase (LDH), RNA, and protein of brain, liver, and skeletal muscle of Clarias batrachus were studied to explore possible mode of action of a pyrethroid insecticide at metabolic level. The administration of actinomycin D or cycloheximide inhibited partially or completely the withdrawal-dependent increase in enzyme activities, RNA, and protein contents, substantiating de novo synthesis of these macromolecules during recovery. The inhibition was more pronounced in response to cycloheximide reflecting recovery in translation process. This also suggests the possibility of fenvalerate-induced impairment of metabolism through inhibition of enzyme synthesis.

Effects of intra-duodenal provison of limiting amino acids on serum concentrations of immunoglobulins and tissue concentrations of DNA and RNA in growing goats fed a maize stover-based diet

The study was conducted to investigate the effects of intra-duodenal infusion of methionine (Met), lysine (Lys) and leucine (Leu) on dry matter intake (DMI), serum concentrations of total protein (TP), IgA, IgG, IgM and PUN, as well as tissue DNA and RNA concentrations in growing goats. In Experiment 1, 3 Liuyang Black growing wether goats (10.0 ± 0.1 kg) fitted with ruminal, proximal duodenal and terminal ileal fistulae were used to determine the optimal amounts of the limiting amino acids infused into the lumen of the duodenum on the basis of the intestinal digestibility of amino acids and the amino acid pattern in muscle protein. This experiment established the infusion amounts of Met, Lys and Leu to be 0.77 g/day, 0.91 g/day and 0.58 g/day, respectively. In Experiment 2, 12 Liuyang black growing wether goats (10.0 ± 0.2 kg), cannulated in the duodenum, were randomly divided into four groups according to body weights. The four treatment groups were the 21% removal of Met, Lys or Leu from the mixed Met, Lys and Leu solution, and a control group (full amounts of Met, Lys and Leu). Blood samples were taken from the jugular vein on day 8 before slaughter to measure changes in serum total protein (TP), IgA, IgG, IgM and plasma urea nitrogen (PUN), as well as DNA and RNA in skeletal muscle and jejunal mucosa. Serum TP and IgA were unaffected ( P > 0.05) by the 21% removal of Met, Lys or Leu, compared with the control group. Serum IgG and IgM concentrations were lower ( P < 0.05) in response to the 21% removal of Met or Lys, but PUN concentration were higher ( P < 0.05) in response to the 21% Met removal, in comparison with the control group. Concentrations of DNA and RNA in both skeletal muscle and jejunal mucosa decreased in response to the 21% removal of Met and Lys, compared with the control group. Collectively, results indicate that the extent, to which the immune response and tissue protein-synthetic capacity are affected by the intestinal removal of essential amino acids, appear to be related to the order of their limitation in the lumen of the duodenum.

Influences on the loin and cellular characteristics of the m. longissimus lumborum of Australian Poll Dorset-sired lambs

Offspring of 4 Poll Dorset rams differing in eye muscle depth estimated breeding values (EBVs) were studied to determine sire, sex, and nutritional influences on cellular characteristics in the longissimus lumborum muscle. At 12 weeks of age, 62 lambs were individually fed a concentrate diet with or without protected nutrients ad libitum for 120 days while 39 lambs were grazed on improved pasture. Sire influenced the percentages of type 2A and 2B/2X myofibres, but not myofibre number or size. Progeny of the highest eye muscle depth EBV ram had less type 2A and more 2B/2X myofibres than the lowest ranking sire. At equivalent carcass weight, amount of RNA and protein in the longissimus muscle was influenced by sire, consistent with differences in eye muscle depth EBVs. Sex had little effect on muscle cellular characteristics, whereas lambs fed pasture had less type 1 myofibres than those fed concentrates and had less muscle RNA and a higher ratio of protein to RNA. The findings demonstrate differences in m. longissimus lumborum cellular characteristics in offspring of sires differing in muscle EBVs. The extent to which these differences relate to the Carwell muscle hypertrophy gene remains to be determined.

Gene expression in human skeletal muscle: alternative normalization method and effect of repeated biopsies

The reverse transcriptase-polymerase chain reaction (RT-PCR) method has lately become widely used to determine transcription and mRNA content in rodent and human muscle samples. However, the common use of endogenous controls for correcting for variance in cDNA between samples is not optimal. Specifically, we investigated (1) a new normalization method based on determining the cDNA content by the flourophores PicoGreen and OliGreen, (2) effect of repeated muscle biopsies on mRNA gene expression, and (3) the spatial heterogeneity in mRNA expression across the muscle. Standard curves using oligo standards revealed a high degree of sensitivity and linearity (2.5-45 ng; R2>0.99) with OliGreen reagent, as was the case for OliGreen analyses with standard curves constructed from serial dilutions of representative RT samples (R2 >0.99 for a ten times dilution range of a representative reversed transcribed (RT) sample). Likewise, PicoGreen reagent detected the RNA:DNA hybrid content in RT samples with great sensitivity. Standard curves constructed from both double-stranded lambda DNA (1-10 ng) and from serial dilutions of representative RT samples consistently resulted in linearity with R2 >0.99. The present determination of cDNA content in reversed transcribed human skeletal muscle RNA samples by both PicoGreen and OliGreen analyses suggests that these fluorophores provide a potential alternative normalization procedure for human gene expression studies. In addition, the present study shows that multiple muscle biopsies obtained from the same muscle do not influence the mRNA response induced by an acute exercise bout for any of the genes examined.

α 2-Macroglobulin in the marine fish Sparus aurata

The α 2-macroglobulin proteinase inhibitors (α 2Ms) are a family of plasma proteins with the unique ability to inhibit a broad spectrum of proteinases, but are also known as binding proteins for many growth factors and cytokines, including growth hormone and members of the transforming growth factor-β superfamily. A partial cDNA (475 amino acids) encoding the C-terminus of α 2M was cloned from the liver of the marine teleostean fish Sparus aurata. The deduced amino acid sequence of the cloned fragment showed 58–60% similarity to carp α 2Ms. Northern blot analysis of hepatic α 2M revealed a transcript of about 5 kb. A transcript of a similar size was detected in 1-day larvae. Steady state levels of α 2M in larvae increased gradually on subsequent days post-hatching. α 2M expression in embryos was determined by RT-PCR and started in embryos aged 8 h post-fertilization, but not earlier. RT-PCR of muscle RNA detected α 2M also in fish muscle, albeit with a lower expression than in the liver. Immunoreactive-α 2M was found in yolk syncytial layer of 3-day larvae and in livers from larvae and adults. Immunoreactive-α 2M was also identified in soluble total proteins from young larvae with a pattern resembling that of plasma. These data demonstrate that the α 2M gene is expressed early in fish development. Moreover, in addition to its major expression in liver, α 2M is expressed also in fish muscle.

Growth hormone receptor gene expression in the skeletal muscle of normal and double-muscled bovines during foetal development

The expression of the growth hormone receptor (GHR) gene was investigated in semitendinosus muscle during bovine foetal development in both normal and double-muscled Charolais foetuses which differ with respect to muscle development. Northern-blot analysis of foetal muscle RNA preparations with a GHR cDNA probe identified the 4.5 kb GHR mRNA as early as 130 days post-conception. In double-muscled animals, the expression of GHR mRNA increased from 130 to 210 days of gestation while it stayed stable in normal ones. It was significantly higher (P < 0.05) in double-muscled foetuses compared to normal ones from the second third of gestation. Northern-blot analysis of foetal muscle RNA preparations from both genotypes with a beta-actin cDNA probe, revealed lower beta-actin gene expression in double-muscled foetuses than in normal ones, suggesting a delay in the differentiation of muscle cells. In situ hybridisation revealed the localisation of specific GHR mRNA in muscle cells at all gestation stages analysed (130, 170, 210 days post-conception) but not in connective tissue surrounding the muscle cells. At the adult stage, the hybridisation signal was also very high and observed in muscle cells only. These results show the ontogeny of GHR mRNA in bovine muscle and demonstrate a difference between normal and double-muscled animals.

Increased expression of MafBx ubiquitin ligase messenger RNA in skeletal muscle after induction of heart failure: influence of exercise training

Increased expression of MafBx ubiquitin ligase messenger RNA in skeletal muscle after induction of heart failure: influence of exercise training

768. Two Distinct Mechanisms, Silencing of RNA Expression and Loss of Vector DNA, Are Responsible for the Loss of Transgene Expression Following Delivery of Foreign Genes to Skeletal Muscle

Plasmid based delivery of foreign transgenes often results in transient expression in vivo. To investigate the cause of this transient expression we used quantitative PCR to measure the copy number of transgene RNA and vector DNA in the skeletal muscle of C57BL6 mice after electroporation of various plasmids. Gene transfer of a plasmid encoding the mouse interferon beta (mIFN-|[beta]|) gene driven by the CMV promoter (pCMV-mIFN) resulted in mIFN RNA levels between 1 and 5 |[times]| 106 copies per microgram (|[mu]|g) of total RNA that were stable for the first 10 days then dropped by only 10-fold by day 44. Plasmid vector DNA levels were also stable for the first 10 days then dropped 7.5-fold between day 10 and day 44. The ratio of mIFN RNA copies per vector DNA copy reached a peak of 1.8 at day 10 and declined slightly to 0.9 at day 44. These results show that following delivery of a non-foreign transgene, a slow decline in RNA levels occurred that was due to a gradual loss of plasmid DNA. Next, we compared plasmids encoding either the mIFN-|[beta]| or human interferon beta (hIFN-|[beta]|) genes driven by identical CMV promoters (pCMV-hIFN and pCMV-mIFN). At 50 days after electroporation of mouse skeletal muscle, the mean copy number of transgene RNA in the muscles that received the mouse or human IFN-|[beta]| plasmids was 1.1 |[times]| 105 and 7.4 |[times]| 103 copies per |[mu]|g of total RNA, respectively. In contrast, the mean plasmid DNA level was slightly higher in the muscles that received pCMV-hIFN. The mean IFN-|[beta]| RNA to vector DNA ratio was 1.3 in the muscles that received pCMV-mIFN, but only 0.04 (31-fold lower, p<0.001) in the muscles that received pCMV-hIFN. This demonstrates that the lower level of transgene RNA expression achieved with the foreign human IFN-|[beta]| gene as compared to the native mouse IFN-|[beta]| gene was due to reduced gene expression, not to reduced persistence of the vector DNA. When a mixture of pCMV-mIFN and a plasmid encoding human secreted alkaline phosphotase driven by the CMV promoter (pCMV-hSEAP) in a 14:1 ratio was delivered to mouse muscle, mIFN RNA levels fell 350-fold between day 1 and day 10 (p<0.01). The level of pCMV-mIFN plasmid DNA in the same muscles fell 100-fold by day 10 (p<0.01) and the ratio of mIFN RNA copies per copy of pCMV-mIFN DNA dropped slightly from 2.8 at day 1 to 1.1 at day 10, suggesting that there was only a small decrease in transcription. This data demonstrates that following co-delivery of pCMV-hSEAP and pCMV-mIFN, rapid clearance of the CMV-mIFN plasmid DNA from the muscle was primarily responsible for the large drop in mIFN RNA expression.

The Effects Of Loading And Stretch On Transcriptional And Translational Markers

INTRODUCTION To date, literally thousands of studies have examined issues related to muscle atrophy and hypertrophy as reflected by changes in radial size. In contrast, less than 100 studies have examined issues related to the underlying biology of longitudinal growth. Given this consideration, the objective of this study was to contrast changes in transcriptional and translational capacity following: 1) recovery of cross-sectional area via reloading of atrophied muscles; and 2) longitudinal growth as induced by chronic stretch via distraction. METHODS Female Sprague-Dawley rats were randomly assigned to the following groups: 1) control (CON); 2) reloading (RELOAD); and 3) distracted (DIS). Animals assigned to the RELOAD group underwent 4 wks of hindlimb suspension prior to the reloading phase. They were then allowed to undergo normal cage activity for either 4 or 8 days, at which time they were sacrificed. Animals in the DIS group were sacrificed 4 and 8 days following the onset of stretch. The soleus muscle was stretched at a rate of 0.5 mm/day. RESULTS As compared to the CON group, there was a large increase in the concentration of total RNA of the soleus muscle at both the 4 and 8-day time-points for the RELOAD group. Similarly, there was a large increase in the concentration of oligo dT. In contrast, stretch actually produced a small but significant reduction in total RNA, whereas the concentration of oligo dT was unaffected. Reloading also produced large increases in the concentrations of both actin and MHC mRNA levels, whereas just the opposite was observed in the stretched muscles. DISCUSSION Reloading of skeletal muscle produced significant increases in four key markers of growth: i) muscle weight; ii) total RNA pool; iii) the total amount and concentration of mRNA as reflected by oligo dT; and iv) a large increase in the amounts and concentrations of actin and myosin mRNA. Stretch, in contrast, failed to elicit an anabolic profile as defined by our markers. This may be due, in large part, to the joint immobilization that accompanies the stretch model used in this study.

The Effects Of Loading And Stretch On Transcriptional And Translational Markers

INTRODUCTION To date, literally thousands of studies have examined issues related to muscle atrophy and hypertrophy as reflected by changes in radial size. In contrast, less than 100 studies have examined issues related to the underlying biology of longitudinal growth. Given this consideration, the objective of this study was to contrast changes in transcriptional and translational capacity following: 1) recovery of cross-sectional area via reloading of atrophied muscles; and 2) longitudinal growth as induced by chronic stretch via distraction. METHODS Female Sprague-Dawley rats were randomly assigned to the following groups: 1) control (CON); 2) reloading (RELOAD); and 3) distracted (DIS). Animals assigned to the RELOAD group underwent 4 wks of hindlimb suspension prior to the reloading phase. They were then allowed to undergo normal cage activity for either 4 or 8 days, at which time they were sacrificed. Animals in the DIS group were sacrificed 4 and 8 days following the onset of stretch. The soleus muscle was stretched at a rate of 0.5 mm/day. RESULTS As compared to the CON group, there was a large increase in the concentration of total RNA of the soleus muscle at both the 4 and 8-day time-points for the RELOAD group. Similarly, there was a large increase in the concentration of oligo dT. In contrast, stretch actually produced a small but significant reduction in total RNA, whereas the concentration of oligo dT was unaffected. Reloading also produced large increases in the concentrations of both actin and MHC mRNA levels, whereas just the opposite was observed in the stretched muscles. DISCUSSION Reloading of skeletal muscle produced significant increases in four key markers of growth: i) muscle weight; ii) total RNA pool; iii) the total amount and concentration of mRNA as reflected by oligo dT; and iv) a large increase in the amounts and concentrations of actin and myosin mRNA. Stretch, in contrast, failed to elicit an anabolic profile as defined by our markers. This may be due, in large part, to the joint immobilization that accompanies the stretch model used in this study.

NF90 Regulates Cell Cycle Exit and Terminal Myogenic Differentiation by Direct Binding to the 3′-Untranslated Region of MyoD and p21WAF1/CIP1 mRNAs

NF90 and splice variant NF110/ILF3/NFAR are double-stranded RNA-binding proteins that regulate gene expression. Mice with targeted disruption of NF90 were engineered. NF90(-/-) mice were born small and weak and succumbed to perinatal death within 12 h because of neuromuscular respiratory failure. Lung inflation and morphology were normal in NF90(-/-) mice. The diaphragm and other skeletal muscles in NF90(-/-) mice demonstrated disorganized arrangement and paucity of myofibers, evidence of myocyte degeneration and increased apoptosis. The expression of myogenic regulators, MyoD, myogenin, and p21WAF1/CIP1, was severely decreased in NF90(-/-) mice. These myogenic transcription factors and cell cycle inhibitors are regulated in part through post-transcriptional mRNA stabilization. Northwestern blotting revealed that NF90 is the principal and specific p21WAF1/CIP1 and MyoD 3'-untranslated region RNA-binding protein in developing skeletal muscles. NF90 regulates transcription factors and a cell cycle inhibitor essential for skeletal muscle differentiation and for survival.

Cellular Fate of Truncated Slow Skeletal Muscle Troponin T Produced by Glu180 Nonsense Mutation in Amish Nemaline Myopathy

A nonsense mutation at codon Glu180 in exon 11 of slow skeletal muscle troponin T (TnT) gene (TNNT1) causes an autosomal-recessive inherited nemaline myopathy. We previously reported the absence of intact or prematurely terminated slow TnT polypeptide in Amish nemaline myopathy (ANM) patient muscle. The present study further investigates the expression and fate of mutant slow TnT in muscle cells. Intact slow TnT mRNA was readily detected in patient muscle, indicating unaffected transcription and RNA splicing. Sequence of the cloned cDNAs revealed the single nucleotide mutation in two alternatively spliced isoforms of slow TnT mRNA. Mutant TNNT1 cDNA is translationally active in Escherichia coli and non-muscle eukaryotic cells, producing the expected truncated slow TnT protein. The mutant mRNA was expressed at significant levels in differentiated C2C12 myotubes, but unlike intact exogenous TnT, truncated slow TnT protein was not detected. Transfective expression in undifferentiated myoblasts produced slow TnT mRNA but not a detectable amount of truncated or intact slow TnT proteins, indicating a muscle cell-specific proteolysis of TnT when it is not integrated into myofilaments. The slow TnT-(1-179) fragment has substantially lower affinity for binding to tropomyosin, in keeping with the loss of one of two tropomyosin-binding sites. Our findings suggest that inefficient incorporation into myofilament is responsible for the instability of mutant slow TnT in ANM muscle. Rapid degradation of the truncated slow TnT protein, rather than instability of the nonsense mRNA, provides the protective mechanism against the potential dominant negative effect of the mutant TnT fragment.

In situ identification of hepatitis C virus RNA in muscle

The authors examined skeletal muscle specimens from four patients with myositis and hepatitis C virus (HCV) infection. PCR analysis identified HCV RNA in muscle homogenates from two patients. In situ hybridization signals for HCV RNA were detected within muscle fibers as well as in infiltrating lymphocytes from the same patients. The results may relate to the pathomechanism of myositis in patients with HCV infection.