Cytochrome c transcriptional activation and mRNA stability during contractile activity in skeletal muscle (original) (raw)
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Contractile activity induces adaptations in the expression of genes encoding skeletal muscle mitochon-drial proteins; however, the putative signals responsible for these adaptations remain unknown. We used electrical stimulation (5 Hz, 65 V) of C2C12 muscle cells in culture to define some of the mechanisms involved in contractile activity-induced changes in cytochrome c gene expression. Chronic contractile activity (4 days, 3 h/day) augmented cytochrome c mRNA by 1.6-fold above control cells. This was likely mediated by increases in transcriptional activation, because cells transfected with full-length (؊726 base pairs) or minimal (؊66 base pairs) cytochrome c promoter/chlor-amphenicol acetyltransferase reporter constructs demonstrated contractile activity-induced 1.5-1.7-fold increases in the absence of contractile activity-induced increases in mRNA stability. Transcriptional activation of the ؊726 promoter was abolished when muscle contraction was inhibited at various subcellu-lar locations by pretreatment with either the Na ؉ channel blocker tetrodotoxin, the intracellular Ca 2؉ chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N,N-tet-raacetic acid tetra(acetoxymethyl) ester, or the myosin ATPase inhibitor 2,3-butanedione monoxime. It was further reduced in unstimulated cells when mitochon-drial ATP synthesis was impaired using the uncoupler 2,4-dinitrophenol. Because the contractile activity-induced response was evident within the minimal promoter , electromobility shift assays performed within the first intron (؉75 to ؉104 base pairs) containing Sp1 sites revealed an elevated DNA binding in response to contractile activity. This was paralleled by increases in Sp1 protein levels. Sp1 overexpression studies also led to increases in cytochrome c transactivation and mRNA levels. These data suggest that variations in the rate of mitochondrial ATP synthesis are important in determining cytochrome c gene expression in muscle cells and that this is mediated, in part, by Sp1-induced increases in cytochrome c transcription.
Increased contractile activity decreases RNA-protein interaction in the 3'-UTR of cytochrome c mRNA
1996
Increased contractile activity decreases RNA-protein interaction in the 3'-UTR of cytochrome c mRNA. Am. J. Physiol. 271 (CeZl PhysioZ. 40): Cll57-Cl166, 1996.-This study was designed to gain an insight into mechanisms by which cytochrome c gene expression is enhanced by increased contractile activity in skeletal muscle. When rat tibialis anterior muscles were stimulated (10 Hz, 0.25 ms) for 0,2,6,12, or 24 h or 2,5,9, or 13 days (n = 4 for each time point), cytochrome c protein (enzyme-linked immunosorbent assay) and mRNA (Northern blot analysis) concentrations started to increase by 9 days, and this was associated with concurrent decreases in cytochrome c mRNAprotein interaction (RNA gel mobility shift assay). We found that the decreased RNA-protein interaction in the stimulated muscle extract was restored by ultracentrifugation (150,000 g, 1 h) in the supernatant fraction. The 150,000 g pellet fraction of stimulated muscle was capable of inhibiting the RNA-protein interaction in control tibialis anterior muscles. These results provide evidence of an inhibitory factor that is responsible for decreasing RNA-protein interaction in the 3'-untranslated region of cytochrome c mRNA in continuously stimulated muscle.
Molecular and Cellular Biochemistry, 2013
Sarcoplasmic and t-tubule membrane proteins regulating sarcoplasmic Ca 2? concentration exhibit fibretype-dependent isoform expression, and play central roles in muscle contraction and relaxation. The purpose of this study was to evaluate the effects of in vitro electrical stimulation on the mRNA expression of components involved in Ca 2? regulation in oxidative and glycolytic skeletal muscle. The mRNA level of Ca 2? -ATPase (SERCA1, 2), calsequestrin (CASQ1, 2), ryanodine receptor (RyR1), and dihydropyridine receptor (Cacna1) was assessed in rat extensor digitorum longus (EDL) and soleus (SOL) muscles at 4 h of recovery following in vitro stimulations (either short intensive (SHO) 60 Hz, 5 min, or prolonged moderate (PRO) 20 Hz, 40 min). Stimulation induced acute regulation of the mRNA level of Ca 2? -regulating proteins in a manner that does not follow typical fibre-type-specific transitions. In general, stimulation decreased mRNA content of all proteins studied. Most prominent down-regulation was observed for Cacna1 (26 and 32 % after SHO and PRO, respectively, in SOL; 19 % after SHO in EDL). SERCA1, SERCA2, CASQ1, CASQ2, and RyR1 mRNA content also decreased significantly in both muscles relative to resting control. Of notice is that hexokinase II mRNA content was increased in EDL and unchanged in SOL underlining the specificity of the down-regulation of mRNA of Ca 2? regulatory proteins. The results demonstrate contraction-induced down-regulation of mRNAs for the main components of Ca 2? -regulating system in skeletal muscle. The down-regulation of both isoforms of SERCA and CASQ after a single electrical stimulation session suggests that adaptations to repeated stimulation involve further regulatory mechanisms in addition to acute mRNA responses.
Excitation-transcription coupling in skeletal muscle: the molecular pathways of exercise
Biological Reviews, 2010
Muscle fibres have different properties with respect to force, contraction speed, endurance, oxidative/glycolytic capacity etc. Although adult muscle fibres are normally post-mitotic with little turnover of cells, the physiological properties of the pre-existing fibres can be changed in the adult animal upon changes in usage such as after exercise. The signal to change is mainly conveyed by alterations in the patterns of nerve-evoked electrical activity, and is to a large extent due to switches in the expression of genes. Thus, an excitation-transcription coupling must exist. It is suggested that changes in nerve-evoked muscle activity lead to a variety of activity correlates such as increases in free intracellular Ca 2+ levels caused by influx across the cell membrane and/or release from the sarcoplasmatic reticulum, concentrations of metabolites such as lipids and ADP, hypoxia and mechanical stress. Such correlates are detected by sensors such as protein kinase C (PKC), calmodulin, AMP-activated kinase (AMPK), peroxisome proliferator-activated receptor δ (PPARδ), and oxygen dependent prolyl hydroxylases that trigger intracellular signaling cascades. These complex cascades involve several transcription factors such as nuclear factor of activated T-cells (NFAT), myocyte enhancer factor 2 (MEF2), myogenic differentiation factor (myoD), myogenin, PPARδ, and sine oculis homeobox 1/eyes absent 1 (Six1/Eya1). These factors might act indirectly by inducing gene products that act back on the cascade, or as ultimate transcription factors binding to and transactivating/repressing genes for the fast and slow isoforms of various contractile proteins and of metabolic enzymes. The determination of size and force is even more complex as this involves not only intracellular signaling within the muscle fibres, but also muscle stem cells called satellite cells. Intercellular signaling substances such as myostatin and insulin-like growth factor 1 (IGF-1) seem to act in a paracrine fashion. Induction of hypertrophy is accompanied by the satellite cells fusing to myofibres and thereby increasing the capacity for protein synthesis. These extra nuclei seem to remain part of the fibre even during subsequent atrophy as a form of muscle memory facilitating retraining. In addition to changes in myonuclear number during hypertrophy, changes in muscle fibre size seem to be caused by alterations in transcription, translation (per nucleus) and protein degradation.
Effects of Endurance Exercise on Cytochrome C Turnover in Skeletal Muscle
Annals of the New York Academy of Sciences, 1977
The adaptive responses of skeletal muscle to repeated daily exercise is dependent upon the type of exercise employed. For example, the responses of muscle to distance running and to weight lifting are different. After repeated bouts of distance running, there is an adaptive increase in the concentration of mitochondria within skeletal muscle, but no significant change in muscular mass.' In contrast, skeletal muscles adapt to repeated bouts of weight lifting by an increase in mass with little change in the concentration of mitochondria.' Thus, adaptations in skeletal muscles are specific to the type of exercise employed during training; and any discussion of the effects of distance running on the turnover of mitochondria1 proteins within skeletal muscle must be restricted to those studies that have employed distance running.
Journal of Physiology, 2010
Exercise training induces mitochondrial biogenesis, but the time course of molecular sequelae that accompany repetitive training stimuli remains to be determined in human skeletal muscle. Therefore, throughout a seven-session, high-intensity interval training period that increaseḋ V O 2 max (12%), we examined the time course of responses of (a) mitochondrial biogenesis and fusion and fission proteins, and (b) selected transcriptional and mitochondrial mRNAs and proteins in human muscle. Muscle biopsies were obtained 4 and 24 h after the 1st, 3rd, 5th and 7th training session. PGC-1α mRNA was increased >10-fold 4 h after the 1st session and returned to control within 24 h. This 'saw-tooth' pattern continued until the 7th bout, with smaller increases after each bout. In contrast, PGC-1α protein was increased 24 h after the 1st bout (23%) and plateaued at +30-40% between the 3rd and 7th bout. Increases in PGC-1β mRNA and protein were more delayed and smaller, and did not persist. Distinct patterns of increases were observed in peroxisome proliferator-activated receptor (PPAR) α and γ protein (1 session), PPAR β/δ mRNA and protein (5 sessions) and nuclear respiratory factor-2 protein (3 sessions) while no changes occurred in mitochondrial transcription factor A protein. Citrate synthase (CS) and β-HAD mRNA were rapidly increased (1 session), followed 2 sessions later (session 3) by increases in CS and β-HAD activities, and mitochondrial DNA. Changes in COX-IV mRNA (session 3) and protein (session 5) were more delayed. Training also increased mitochondrial fission proteins (fission protein-1, >2-fold; dynamin-related protein-1, 47%) and the fusion protein mitofusin-1 (35%) but not mitofusin-2. This study has provided the following novel information: (a) the training-induced increases in transcriptional and mitochondrial proteins appear to result from the cumulative effects of transient bursts in their mRNAs, (b) training-induced mitochondrial biogenesis appears to involve remodelling in addition to increased mitochondrial content, and (c) the 'transcriptional capacity' of human muscle is extremely sensitive, being activated by one training bout.
Changes in transcriptional activity of chronically stimulated fast twitch muscle
FEBS Letters, 1983
from rabbit soleus, normal and 28-day, indirectly stimulated tibialis anterior muscles were translated in an in vitro system. Analysis for translation products by 2-dimensional electrophoresis showed fast myosin light chains in tibialis anterior, and slow myosin light chains in soteus muscle. The stoichiometry of the in vitro translated light chains varies from that seen in normal fast and slow twitch muscles. The stimulated muscle contained mRNA coding, both for fast and slow myosin light chains, although the pattern of slow myosin light chains appears not to be complete at this point of time of the transformation process. Fast and slow twitch muscle Chronic stimulation Myosin Iight chain mRNA In vitro tru~slation