Effect of electrical stimulation post mortem of bovine muscle on the binding of glycolytic enzymes. Functional and structural implications.
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ABSTRACT:
SUBMITTER: Clarke FM
PROVIDER: S-EPMC1161508 | biostudies-other | 1980 Jan
REPOSITORIES: biostudies-other
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Project description: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 degrees 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.
| S-EPMC2234424 | biostudies-literature
Project description:1. Post-mortem changes in the concentrations of the following compounds in ox sternomandibularis muscles stored in nitrogen at 1 degrees , 5 degrees and 15 degrees are reported: P(i) creatine phosphate, hexose monophosphates, fructose diphosphate, triose phosphates, alpha-glycerophosphate, phosphoglycerates, lactate, ATP, ADP, AMP, NAD(+) and total nucleotides. Some results obtained with muscles stored at 37 degrees are included. 2. At the time the muscles were placed at controlled temperatures (about 1.5hr. post mortem) the phosphorus in the compounds measured accounted for 91+/-6% (s.d.) of the total acid-soluble phosphate. 3. The results indicated that at all temperatures the activities of the phosphorylase and phosphofructokinase steps limited the rate and the extent of post-mortem glycolysis. 4. The large variations in hexose monophosphate concentrations during storage indicated that the ratio of phosphorylase to phosphofructokinase activity varied considerably with time and temperature. 5. Between 3.5 and 7hr. post mortem the rates of glycolysis and of ATP turnover were not slower at 5 degrees that at 15 degrees , and were probably faster at 1 degrees . The significance of this finding is discussed.
| S-EPMC1198283 | biostudies-other
Project description:The reconstituted glycolytic system described previously (Scopes, 1973) was used to simulate post-mortem glycolytic metabolism in muscle. The effects of the following factors have been investigated: ATPase (adenosine triphosphatase) amount, AMP deaminase amount, percentage of the phosphorylase in the a form and the effect of diluting the glycolytic enzyme complex as a whole. It was confirmed that the rate of metabolism was solely dependent on the amount of ATPase present and that various concentrations of the glycolytic enzymes had no effect over a wide range encompassing the variation found in anatomically different muscles. The extent of metabolism, represented by the value of the ;ultimate' pH, depended markedly on the amount of phosphorylase in the a form; as little as 1% of the a form resulted in a considerably lower pH than in its absence. To a lesser extent the amount of AMP deaminase also affected the ultimate pH, but this was probably only significant for comparisons of genetically distinct muscles with widely differing amounts of AMP deaminase. The reconstituted system behaved almost identically with regard to post-mortem glycolytic metabolism compared with intact muscle tissue. It is concluded that the controlling effectors found with the reconstituted system apply to intact muscle also.
| S-EPMC1168213 | biostudies-other
Project description:Development of tissue-engineered hearts for treatment of myocardial infarction or biologic pacemakers has been hindered by the production of mostly arrhythmic or in-synergistic constructs. Electrical stimulation (ES) of these constructs has been shown to produce tissues with greater twitch force and better adrenergic response. To further our understanding of the mechanisms underlying the effect of ES, we fabricated a bioreactor capable of delivering continuous or intermittent waveforms of various types to multiple constructs simultaneously. In this study, we examined the effect of an intermittent biphasic square wave on our artificial heart muscle (AHM) composed of neonatal rat cardiac cells and fibrin gel. Twitch forces, spontaneous contraction rates, biopotentials, gene expression profiles, and histologic observations were examined for the ES protocol over a 12 day culture period. We demonstrate improved consistency between samples for twitch force and contraction rate, and higher normalized twitch force amplitudes for electrically stimulated AHMs. Improvements in electrophysiology within the AHM were noted by higher conduction velocities and lower latency in electrical response for electrically stimulated AHMs. Genes expressing key electrophysiologic and structural markers peaked at days 6 and 8 of culture, only a few days after the initiation of ES. These results may be used for optimization strategies to establish protocols for producing AHMs capable of replacing damaged heart tissue in either a contractile or electrophysiologic capacity. Optimized AHMs can lead to alternative treatments to heart failure and alleviate the limited donor supply crisis.
| S-EPMC5469367 | biostudies-literature
Project description:Stimulation of the mouse hindlimb via the sciatic nerve was used to induce contractions for 4 hours to investigate acute muscle gene activation in a model of muscle phenotype conversion. Initial force production (1.6 + 0.1 g/g body weight) declined 45% within 10 min and was maintained for the remainder of the experiment. Force returned to initial levels upon completion of the study. An immediate-early growth response was present in the EDL (FOS, JUN, ATF3, MAFK) with a similar but attenuated pattern in the soleus. Transcript profiles showed decreased fast fiber specific mRNA (myosin heavy chains 2A, 2B; troponins T3, I; -tropomyosin, m-creatine kinase) and increased slow transcripts (myosin heavy chain slow/1, troponin C, tropomyosin 3) in the EDL. Histological analysis of the EDL revealed glycogen depletion without inflammatory cell infiltration or myofiber damage in stimulated vs. control muscles. Several fiber type specific transcription factors (EYA1, TEAD1, NFATc1 and c4, PPARG, PPARGC1 and β, BHLHB2) increased in the EDL along with transcription factors characteristic of embryogenesis (KLF4, SOX17, TCF15, PKNOX1, ELAV). No established in vivo satellite cell markers or the genes activated during our parallel studies of satellite cell proliferation in vitro (CYCLINS A2, B2, C, E1, MyoD) increased in the stimulated muscles. These data indicated that onset of fast to slow phenotype conversion occurred in the EDL within 4 hours of stimulation without satellite cell recruitment or muscle injury but was driven by phenotype specific transcription factors from resident fiber myonuclei including activation of nascent developmental transcriptional programs. Adult male Swiss Webster mice (30-35 g) were anesthetized, a bipolar electrode was implanted adjacent to the sciatic nerve and the hindlimb immobilized. The voltage-force relation was determined to establish supramaximal stimulation conditions and the length-tension relation was determined to set the resting length for maximum twitch tension. Contractions were induced by sciatic nerve stimulation (0.5 msec duration, 2-5 volts). The muscles were allowed to rest 15 minutes for full metabolic recovery at physiologic temperatures. Supramaximal stimulation was applied at a rate of 10 Hz for 4 hours. At the end of each experiment the soleus muscles were carefully dissected and flash frozen in liquid nitrogen for analysis of mRNA expression via microarray analysis. The contralateral, unstimulated Soleus provided a genetically matched, paired control for each specimen.
2010-06-01 | E-GEOD-14391 | biostudies-arrayexpress
Project description:Stimulation of the mouse hindlimb via the sciatic nerve was used to induce contractions for 4 hours to investigate acute muscle gene activation in a model of muscle phenotype conversion. Initial force production (1.6 + 0.1 g/g body weight) declined 45% within 10 min and was maintained for the remainder of the experiment. Force returned to initial levels upon completion of the study. An immediate-early growth response was present in the EDL (FOS, JUN, ATF3, MAFK) with a similar but attenuated pattern in the soleus. Transcript profiles showed decreased fast fiber specific mRNA (myosin heavy chains 2A, 2B; troponins T3, I; alpha-tropomyosin, m-creatine kinase) and increased slow transcripts (myosin heavy chain slow/1beta, troponin C, tropomyosin 3gamma) in the EDL. Histological analysis of the EDL revealed glycogen depletion without inflammatory cell infiltration or myofiber damage in stimulated vs. control muscles. Several fiber type specific transcription factors (EYA1, TEAD1, NFATc1 and c4, PPARG, PPARGC1alpha and beta, BHLHB2) increased in the EDL along with transcription factors characteristic of embryogenesis (KLF4, SOX17, TCF15, PKNOX1, ELAV). No established in vivo satellite cell markers or the genes activated during our parallel studies of satellite cell proliferation in vitro (CYCLINS A2, B2, C, E1, MyoD) increased in the stimulated muscles. These data indicated that onset of fast to slow phenotype conversion occurred in the EDL within 4 hours of stimulation without satellite cell recruitment or muscle injury but was driven by phenotype specific transcription factors from resident fiber myonuclei including activation of nascent developmental transcriptional programs.
2010-06-01 | GSE14391 | GEO
Project description:Adult male Swiss Webster mice (30-35 g) were anesthetized, a bipolar electrode was implanted adjacent to the sciatic nerve and the hindlimb immobilized. The voltage-force relation was determined to establish supramaximal stimulation conditions and the length-tension relation was determined to set the resting length for maximum twitch tension. Contractions were induced by sciatic nerve stimulation (0.5 msec duration, 2-5 volts). The muscles were allowed to rest 15 minutes for full metabolic recovery at physiologic temperatures. Supramaximal stimulation was applied at a rate of 10 Hz for 4 hours. At the end of each experiment the extensor digitorum longus (EDL) muscles were carefully dissected and flash frozen in liquid nitrogen for analysis of mRNA expression via microarray analysis. The contralateral, unstimulated EDL provided a genetically matched, paired control for each specimen.
2010-06-01 | GSE10454 | GEO
Project description:About three decades ago, researchers suggested that metabolic enzymes participate in cellular processes that are unrelated to their catalytic activity, and the term "moonlighting functions" was proposed. Recently developed advanced technologies in the field of RNA interactome capture now unveil the unexpected RNA binding activity of many metabolic enzymes, as exemplified here for the enzymes of glycolysis. Although for most of these proteins a precise binding mechanism, binding conditions, and physiological relevance of the binding events still await in-depth clarification, several well explored examples demonstrate that metabolic enzymes hold crucial functions in post-transcriptional regulation of protein synthesis. This widely conserved RNA-binding function of glycolytic enzymes plays major roles in controlling cell activities. The best explored examples are glyceraldehyde 3-phosphate dehydrogenase, enolase, phosphoglycerate kinase, and pyruvate kinase. This review summarizes current knowledge about the RNA-binding activity of the ten core enzymes of glycolysis in plant, yeast, and animal cells, its regulation and physiological relevance. Apparently, a tight bidirectional regulation connects core metabolism and RNA biology, forcing us to rethink long established functional singularities.
| S-EPMC9745834 | biostudies-literature
Project description:Electrical stimulation affects the deposition of extracellular matrices and cellular differentiation. Type I collagen is one of the most abundant extracellular matrix proteins; however, not much is known about the effects of electrical stimulation on collagen type I deposition in C2C12 cells. Thus, we studied the effects of electrical voltage and stimulation frequency in 3D cultured C2C12 muscle cells in terms of metabolic activity, type I collagen deposition and cell morphology. Electrically excitable C2C12 muscle cells were seeded in collagen scaffolds and stimulated with rectangular signals of voltage (2, 5, 7 V) and frequency (1, 2 Hz), using parallel carbon electrodes spaced 1 cm apart. Metabolic activity was quantified by the glucose:lactate concentration ratio in the medium. Apoptotic activity was assessed by TUNEL staining and changes in collagen deposition were identified by immunohistology. The ultrastructure of the tissue was examined by TEM. Glucose and lactate analysis indicated that all groups had similar metabolic activity. TUNEL stain showed no significant difference in apoptotic damage induced by electrical stimulation compared to the control. Samples stimulated at 2 Hz exhibited reduced collagen deposition compared to the control and 1 Hz stimulated samples. Muscle-protein marker desmin was highly expressed in constructs stimulated with 1 Hz/5 V sample. TEM revealed that the stimulated samples developed highly organized sarcomeres, which coincided with improved contractile properties in the 1 Hz/5 V- and 2 Hz/5 V-stimulated groups. Our data implicate that a specific electrical frequency may modulate type I collagen accumulation and a specific voltage may affect the differentiation of muscle sarcomeres in excitable cells.
| S-EPMC2782921 | biostudies-literature
Project description:BackgroundTranscutaneous neuromuscular electrical stimulation (NMES) can be applied as a complementary intervention to regular exercise training programs. A distinction can be made between high-frequency (HF) NMES and low-frequency (LF) NMES. In order to increase understanding of the mechanisms of functional improvements following NMES, the purpose of this study was to systematically review changes in enzyme activity, muscle fiber type composition and muscle fiber size in human lower-limb skeletal muscles following only NMES.MethodsTrials were collected up to march 2012 and were identified by searching the Medline/PubMed, EMBASE, Cochrane Central Register of Controlled Trials, CINAHL and The Physical Therapy Evidence Database (PEDro) databases and reference lists. 18 trials were reviewed in detail: 8 trials studied changes in enzyme activities, 7 trials studied changes in muscle fiber type composition and 14 trials studied changes in muscle fiber size following NMES.ResultsThe methodological quality generally was poor, and the heterogeneity in study design, study population, NMES features and outcome parameters prohibited the use of meta-analysis. Most of the LF-NMES studies reported significant increases in oxidative enzyme activity, while the results concerning changes in muscle fiber composition and muscle size were conflicting. HF-NMES significantly increased muscle size in 50% of the studies.ConclusionNMES seems to be a training modality resulting in changes in oxidative enzyme activity, skeletal muscle fiber type and skeletal muscle fiber size. However, considering the small sample sizes, the variance in study populations, the non-randomized controlled study designs, the variance in primary outcomes, and the large heterogeneity in NMES protocols, it is difficult to draw definitive conclusions about the effects of stimulation frequencies on muscular changes.
| S-EPMC3760845 | biostudies-literature