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The TnI SURE/FIRE chimeric enhancer drives transcription in adult fast muscles. (A) The 130-bp TnI SURE/FIRE chimeric enhancer consists of the pan muscle-specific half of the SURE (red) plus the complementary downstream half of the FIRE, which harbors the two potential NFAT sites S1 and S2 (underlined). The E-box and MEF-2, CACC, and CAGG motifs conserved between SURE and FIRE are shown. (B) Luciferase reporter levels in solei (red) and EDL (blue) muscles of SURE/FIRE-luc transgenic mice harvested at P7, P14, and P28. Measurements represent the mean SEM (n 5 for each bar; * , P 0.05; Kruskal-Wallis test with Dunn's post test). Assays were normalized for protein.
Source publication
Adult skeletal muscles retain an adaptive capacity to switch between slow- and fast-twitch properties that largely depend on motoneuron activity. The NFAT (nuclear factor of activated T cells) family of calcium-dependent transcription factors has been implicated in the up-regulation of genes encoding slow contractile proteins in response to slow-pa...
Contexts in source publication
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... Given the similar organization of the SURE and FIRE, and the bipartite organization of SURE (15), we asked whether the downstream half of FIRE directs reporter expression to fast-twitch muscles. A chimeric SURE/FIRE enhancer was constructed as shown in Fig. 1A. This 130-bp chimeric enhancer was linked to the luciferase reporter and used to generate transgenic mice. As observed with the endogenous TnIf gene (15,20), the luciferase reporter was expressed in both the slow soleus and fast extensor digitorum longus (EDL) muscles during the first postnatal week (P7), and as the muscles matured, ...
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... reporter and used to generate transgenic mice. As observed with the endogenous TnIf gene (15,20), the luciferase reporter was expressed in both the slow soleus and fast extensor digitorum longus (EDL) muscles during the first postnatal week (P7), and as the muscles matured, reporter activity became pro- gressively more restricted to the EDL (Fig. 1B). These results indicated that the sequence between 724 and 776 harbors elements that contribute to its selective expression in fast muscles. Sequence analysis of this region using TransFac, a bioinformatics tool to search for transcription factor binding sites, identified two A/G-rich elements as potential NFAT binding sites that we ...
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... we tested the effects of NFATc1 knockdown in adult TnI SURE/FIRE chimeric transgenic mice (see Fig. 1), where the minimal 130-bp enhancer is integrated into chromatin and more tightly regulated. As shown in Fig. 6C, knockdown of NFATc1 had no detectable effect in the fast-twitch EDL but significantly in- creased luciferase levels in the slow-twitch soleus (19% 4% vs. 3% 0.2%). As expected, luciferase activity in the NFATc1 ...
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... TnIs and TnIf genes represent a good model for studying skeletal muscle fiber-type specificity because these two isoforms are restricted to either slow type I (expressing MHC-I) or fast type II (expressing MHC-IIb, MHC-IIXd/x, MHC-IIa) myofibers, as sum- marized in [supporting information (SI) Fig. S1]. Like other genes encoding contractile proteins, TnI transcription during embryonic/ early fetal development is regulated by the MRF (i.e., MyoD) and MEF-2 families of factors that bind cis elements harbored in the SURE and FIRE enhancers (21,26,27). The differential expres- sion of TnIs and TnIf in diverse fibers is already evident ...
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... harbored in the SURE and FIRE enhancers (21,26,27). The differential expres- sion of TnIs and TnIf in diverse fibers is already evident during late fetal and perinatal development (15,20), but their expression becomes more confined to slow-and fast-twitch myofibers after P7 when motoneurons depolarize muscles with distinct activity pat- terns (Fig. 1B and refs. 7 and 20). We have previously shown that slow and fast activity patterns differentially regulate TnI expression in adult rodent muscles (23)(24)(25). Fast-patterned activity enhances TnIf and reduces TnIs transcription, whereas slow-patterned stim- uli in slow muscles enhance TnIs and repress TnIf transcription (25). These observations raised ...
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... on the complexes that bind DNA. Our work suggests that the opposing effects of patterned stimuli on the transcription of genes encoding distinct contractile protein isoforms and metabolic enzymes may function to increase the degree of myofiber specialization, while maintaining a range of activity-dependent plasticity (see model presented in Fig. ...
Similar publications
The molecular mechanisms generating muscle diversity during development are unknown. The phenotypic properties of slow- and fast-twitch myofibers are determined by the selective transcription of genes coding for contractile proteins and metabolic enzymes in these muscles, properties that fail to develop in cultured muscle. Using transgenic mice, we...
Citations
... Several of the transcription factors identified in our analysis of regulatory networks are well known regulators of skeletal muscle phenotype and function, such as NFAT, SOX, SIX and MYOD1 [2,3,93]. Our enriched factors identified by isolating the myonuclei prior to epigenetic analysis are largely in agreement with a recent study using an alternative bioinformatic approach to identify and investigate the epigenetic regulatory environment in slow and fast muscles at the single nuclei level [85]. ...
Muscle cells have different phenotypes adapted to different usage, and can be grossly divided into fast/glycolytic and slow/oxidative types. While most muscles contain a mixture of such fiber types, we aimed at providing a genome-wide analysis of the epigenetic landscape by ChIP-Seq in two muscle extremes, the fast/glycolytic extensor digitorum longus (EDL) and slow/oxidative soleus muscles. Muscle is a heterogeneous tissue where up to 60% of the nuclei can be of a different origin. Since cellular homogeneity is critical in epigenome-wide association studies we developed a new method for purifying skeletal muscle nuclei from whole tissue, based on the nuclear envelope protein Pericentriolar material 1 (PCM1) being a specific marker for myonuclei. Using antibody labelling and a magnetic-assisted sorting approach, we were able to sort out myonuclei with 95% purity in muscles from mice, rats and humans. The sorting eliminated influence from the other cell types in the tissue and improved the myo-specific signal. A genome-wide comparison of the epigenetic landscape in EDL and soleus reflected the differences in the functional properties of the two muscles, and revealed distinct regulatory programs involving distal enhancers, including a glycolytic super-enhancer in the EDL. The two muscles were also regulated by different sets of transcription factors; e.g. in soleus, binding sites for MEF2C, NFATC2 and PPARA were enriched, while in EDL MYOD1 and SIX1 binding sites were found to be overrepresented. In addition, more novel transcription factors for muscle regulation such as members of the MAF family, ZFX and ZBTB14 were identified.
... Moreover, these results at the nerve terminal are coincident with the functional coupling between PKA and calcineurin to modulate the fast-to-slow transition in the muscular fibers [93]. In brief, calcineurin dephosphorylates to activate NFAT and allows its translocation to the nucleus where it upregulates slow muscle genes [94] and downregulate fast ones in an activity-dependent manner [95]. Consequently, calcineurin enhances the transcription of slow specific genes [96] and decreased PKA activity facilitates it. ...
Physical exercise improves motor control and related cognitive abilities and reinforces neuroprotective mechanisms in the nervous system. As peripheral nerves interact with skeletal muscles at the neuromuscular junction, modifications of this bidirectional communication by physical activity are positive to preserve this synapse as it increases quantal content and resistance to fatigue, acetylcholine receptors expansion, and myocytes’ fast-to-slow functional transition. Here, we provide the intermediate step between physical activity and functional and morphological changes by analyzing the molecular adaptations in the skeletal muscle of the full BDNF/TrkB downstream signaling pathway, directly involved in acetylcholine release and synapse maintenance. After 45 days of training at different intensities, the BDNF/TrkB molecular phenotype of trained muscles from male B6SJLF1/J mice undergo a fast-to-slow transition without affecting motor neuron size. We provide further knowledge to understand how exercise induces muscle molecular adaptations towards a slower phenotype, resistant to prolonged trains of stimulation or activity that can be useful as therapeutic tools.
... A reduction of slow fibres is also observed in calcineurin knock-out mice [151] and in mice overexpressing the calcineurin inhibitor regulator of calcineurin 1 (RCAN1) [152]. On the other hand, it has been shown that the nuclear factor of activated T cells (NFAT) functions as a repressor of fast properties in slow muscles [153], and is involved in the fast-to-slow phenotype switch induced by aerobic exercise. This effect is due to the NFATs ability to inhibit MyoD action, by binding to its N-terminal transcription activating domain and blocking the recruitment of the histone acetyltransferase p300 [154]. ...
Physical activity (PA) has been central in the life of our species for most of its history, and thus shaped our physiology during evolution. However, only recently the health consequences of a sedentary lifestyle, and of highly energetic diets, are becoming clear. It has been also acknowledged that lifestyle and diet can induce epigenetic modifications which modify chromatin structure and gene expression, thus causing even heritable metabolic outcomes. Many studies have shown that PA can reverse at least some of the unwanted effects of sedentary lifestyle, and can also contribute in delaying brain aging and degenerative pathologies such as Alzheimer's Disease, diabetes, and multiple sclerosis. Most importantly, PA improves cognitive processes and memory, has analgesic and antidepressant effects, and even induces a sense of wellbeing, giving strength to the ancient principle of "menssanain corporesano" (i.e., a sound mind in a sound body). In this review we will discuss the potential mechanisms underlying the effects of PA on brain health, focusing on hormones, neurotrophins, and neurotransmitters, the release of which is modulated by PA, as well as on the intra- and extra-cellular pathways that regulate the expression of some of the genes involved.
... More specifically, upregulation of calcineurin promotes type I fibers gene products, whereas the inhibition of calcineurin promotes type II fibers (17). This signaling pathway is related with calcium concentration, which can be affected by exercise (24). ...
Metaxas, T, Mandroukas, A, Michailidis, Y, Koutlianos, N, Christoulas, K, and Ekblom, B. Correlation of fiber-type composition and sprint performance in youth soccer players. J Strength Cond Res XX(X): 000-000, 2019-The aim of this study was to examine the correlation between muscle fiber type and sprint performance in elite young soccer players of different age groups of the same team. Twenty-eight young players participated in this study (group U15, n = 8; group U13, n = 9; and group U11, n = 11). Anthropometric assessments, acceleration (10 m), and Bangsbo modified sprint test (30 m) were performed. Muscle biopsies were obtained from the vastus lateralis, and after that, fiber-type composition was determined by immunohistochemistry. No significant correlations were found between the sprint test and muscle fiber distribution for the groups U13 and U11 (p > 0.05). Also, no correlations were found between cross-sectional areas in the types of fibers with the sprint test in all groups (p > 0.05). A positive correlation was found between type I fibers and the performance in the acceleration test (10 m) (r = 0.77, p < 0.05) was found only in group U15 and a negative correlation between type IIA fibers and the performance in the acceleration test (10 m) (r = -0.89, p < 0.05). The correlations were observed only in group U15, which may indicate that the duration and the intensity of the soccer systematic training can affect the plasticity of the muscle fibers. Specific soccer training in youth is one of the factors that can affect fiber-type plasticity. The specific training programs and status of U15 are more intensive, and the exercises are oriented more to improve physical fitness.
... In peripheral tissues, NFATs play key roles in phenotype switching. Activation/anergy of T lymphocytes (Hogan, 2017), myotube formation and fiber-type commitment (Horsley and Pavlath, 2002;McCullagh et al., 2004;Rana et al., 2008), cardiomyocyte hypertrophy (Molkentin, 2004), vascular smooth muscle cell migration and proliferation (Liu et al., 2004;Karpurapu et al., 2010;Kundumani-Sridharan et al., 2013), and bone and joint remodeling (Sitara and Aliprantis, 2010) all depend critically on the NFAT pathway. Though not as extensively investigated in the CNS, several studies suggest that NFATs play a key role in the activation of astrocytes and microglia, as well (Nagamoto-Combs and Combs, 2010;Furman and Norris, 2014). ...
Mounting evidence supports a fundamental role for Ca2+ dysregulation in astrocyte activation. Though the activated astrocyte phenotype is complex, cell-type targeting approaches have revealed a number of detrimental roles of activated astrocytes involving neuroinflammation, release of synaptotoxic factors and loss of glutamate regulation. Work from our lab and others has suggested that the Ca2+/calmodulin dependent protein phosphatase, calcineurin (CN), provides a critical link between Ca2+ dysregulation and the activated astrocyte phenotype. A proteolyzed, hyperactivated form of CN appears at high levels in activated astrocytes in both human tissue and rodent tissue around regions of amyloid and vascular pathology. Similar upregulation of the CN-dependent transcription factor nuclear factor of activated T cells (NFAT4) also appears in activated astrocytes in mouse models of Alzheimer’s disease (ADs) and traumatic brain injury (TBI). Major consequences of hyperactivated CN/NFAT4 signaling in astrocytes are neuroinflammation, synapse dysfunction and glutamate dysregulation/excitotoxicity, which will be covered in this review article.
... In contrast to the activation of NFATc3 by Gα13 in fibroblasts (24), adenoviral infection of myotubes with a constitutively active Gα13 (Q229L mutant, hereafter referred to as Gα13QL) robustly inhibited the basal luciferase activity as well as that enhanced by ectopically overexpressed NFATc1 ( Figure 4D). Among NFAT proteins, NFATc1 is the predominant isoform expressed in adult skeletal muscle (22,25). So, it is conceivable that alterations in NFATc1 mainly contribute to overall NFAT activity downstream of Gα13. ...
Skeletal muscle is a key organ in energy homeostasis owing to its high requirement for nutrients. Heterotrimeric G proteins converge signals from cell-surface receptors to potentiate or blunt responses against environmental changes. Here, we show that muscle-specific ablation of Gα13 in mice promotes reprogramming of myofibers to the oxidative type, with resultant increases in mitochondrial biogenesis and cellular respiration. Mechanistically, Gα13 and its downstream effector RhoA suppressed nuclear factor of activated T cells 1 (NFATc1), a chief regulator of myofiber conversion, by increasing Rho-associated kinase 2-mediated (Rock2-mediated) phosphorylation at Ser243. Ser243 phosphorylation of NFATc1 was reduced after exercise, but was higher in obese animals. Consequently, Gα13 ablation in muscles enhanced whole-body energy metabolism and increased insulin sensitivity, thus affording protection from diet-induced obesity and hepatic steatosis. Our results define Gα13 as a switch regulator of myofiber reprogramming, implying that modulations of Gα13 and its downstream effectors in skeletal muscle are a potential therapeutic approach to treating metabolic diseases.
... Our study showed that heat increased the phosphorylation of CREB; on the other hand, heat did not change the phosphorylation of C/EBP (data not shown). Calcineurin is a potent effector of NFAT and acts as a repressor of certain genes (14,49,57). The present study revealed that a calcineurin inhibitor, FK506, increased the mRNA expression level of IL-6. ...
Interluekin-6 (IL-6) is released from skeletal muscle cells and induced by exercise, heat, catecholamine, glucose, lipopolysaccharide, reactive oxygen species, and inflammation. However, the mechanism, which induces release of IL-6 from skeletal muscle cells, remains unknown. Thermo-sensitive transient receptor potential (TRP) proteins such as TRPV1-4 play vital roles in cellular functions. In this study we hypothesized that TRPV1 senses heat, transmits a signal into the nucleus, and produces IL-6. The purpose of the present study is to investigate the underlying mechanisms, where skeletal muscle cells sense heat and respond to it. When mouse myoblast cells were exposed to temperatures ranging from 37℃ to 42℃ for two hours, the mRNA expression of IL-6 increased in a temperature-dependent manner. Heat also increased IL-6 secretion in myoblast cells. A fura-2 fluorescence dual-wavelength excitation method showed that heat increased the intracellular calcium flux temperature-dependently. TRPV1 agonists, capsaicin and NADA, increased the intracellular calcium flux and the IL-6 mRNA expression, while TRPV1 antagonists, AMG9810 and SB366791, and the siRNA mediated knockdown of TRPV1 decreased them. TRPV2, 3, and 4 agonists did not change the intracellular calcium flux. Western blotting with inhibitors demonstrated that heat increased the phosphorylation levels of TRPV1 followed by PKC and CREB. PKC inhibitors, Gö6983 and staurosporine, CREB inhibitors, curcumin and naphthol AS-E, and the knockdown of CREB suppressed the heat induced increases of IL-6. These results indicate that heat increases IL-6 in skeletal muscle cells through the TRPV1, PKC, and CREB signal transduction pathway.
... Several observations have suggested that transcription factor families including members of the MyoD, Mef2, and NFAT families, which are essential in myogenesis, also function as mediators through which neural activity regulates the types of transcripts and proteins expressed in a muscle fiber (4,11,12,15,16,22,34,52,70,71). This speculation is supported by studies showing that changes in electrical activity in skeletal muscles of adult animals are coupled to alterations in the levels of these transcriptional regulators, which in turn lead to modifications in the transcription of contraction-associated genes (1,16,47,54). However, the role that Mef2, NFAT, and MyoD families play in regulating muscle properties other than changes in contractile protein isoforms has not been fully determined. ...
Skeletal muscle is distinguished from other tissues on the basis of its shape, biochemistry, and physiological function. Based on mammalian studies, fiber size, fiber types, and gene expression profiles are regulated, in part, by the electrical activity exerted by the nervous system. To address whether similar adaptations to changes in electrical activity in skeletal muscle occur in teleosts, we studied these phenotypic properties of ventral muscle in the electric fish Sternopygus macrurus following two and five days of electrical inactivation by spinal transection. Our data show that morphological and biochemical properties of skeletal muscle remained largely unchanged after these treatments. Specifically, the distribution of type I and type II muscle fibers, and the cross-sectional areas of these fiber types observed in control fish remained unaltered after each spinal transection survival period. This response to electrical inactivation was generally reflected at the transcript level using real-time PCR and RNA-seq data by showing little effect on the transcript levels of genes associated with muscle fiber type differentiation and plasticity, the sarcomere complex, and pathways implicated in the regulation of muscle fiber size. Data from this first study characterizing the neural influence on muscle mass and sarcomere gene expression in a teleost is discussed in the context of comparative studies in mammalian model systems and vertebrate species from different lineages.
... Indeed, selective up regulation of calcineurin promotes type I fibers gene products while inhibition of calcineurin promotes type II fibers-specific gene activity [29]. This fiber type switching is controlled via calcineurin mediated activation of nuclear factor of activated T-cells (NFAT), which are a family of transcription factors involved in nerve activity sensing and calcium regulation [30]. A number of other transcription factors, co-activators and co-repressors have also been associated with fiber type switching and have been comprehensively reviewed elsewhere [31]. ...
The plasticity of skeletal muscle can be traced down to extensive metabolic, structural and molecular remodeling at the single fiber level. Skeletal muscle is comprised of different fiber types that are the basis of muscle plasticity in response to various functional demands. Resistance and endurance exercises are two external stimuli that differ in their duration and intensity of contraction and elicit markedly different responses in muscles adaptation. Further, eccentric contractions that are associated with exercise-induced injuries, elicit varied muscle adaptation and regenerative responses. Most adaptive changes are fiber type-specific and are highly influenced by diverse structural, metabolic and functional characteristics of individual fiber types. Regulation of signaling pathways by reactive oxygen species (ROS) and oxidative stress also plays an important role in muscle fiber adaptation during exercise. This review focuses on cellular and molecular responses that regulate the adaptation of skeletal muscle to exercise and exercise-related injuries.
... Unbiased enrichment analyses of SOL-specific and activitydependent circadian genes indicate the Ca 2þ -dependent calcineurin-NFAT pathway is one important mediator of activity-dependent circadian gene expression in slow muscles. Upon dephosphorylation by the phosphatase calcineurin, NFAT proteins translocate to the nucleus and activate or repress target genes [40,41]. We report here that NFAT responds to circadian activity rhythms, as shown by increased nuclear import of NFATc1 and NFATc3 during the active/dark phase, followed by increased transcriptional activity of an NFAT-dependent reporter. ...
Objective
Physical activity and circadian rhythms are well-established determinants of human health and disease, but the relationship between muscle activity and the circadian regulation of muscle genes is a relatively new area of research. It is unknown whether muscle activity and muscle clock rhythms are coupled together, nor whether activity rhythms can drive circadian gene expression in skeletal muscle.
Methods
We compared the circadian transcriptomes of two mouse hindlimb muscles with vastly different circadian activity patterns, the continuously active slow soleus and the sporadically active fast tibialis anterior, in the presence or absence of a functional skeletal muscle clock (skeletal muscle-specific Bmal1 KO). In addition, we compared the effect of denervation on muscle circadian gene expression.
Results
We found that different skeletal muscles exhibit major differences in their circadian transcriptomes, yet core clock gene oscillations were essentially identical in fast and slow muscles. Furthermore, denervation caused relatively minor changes in circadian expression of most core clock genes, yet major differences in expression level, phase and amplitude of many muscle circadian genes.
Conclusions
We report that activity controls the oscillation of around 15% of skeletal muscle circadian genes independently of the core muscle clock, and we have identified the Ca2+-dependent calcineurin-NFAT pathway as an important mediator of activity-dependent circadian gene expression, showing that circadian locomotor activity rhythms drive circadian rhythms of NFAT nuclear translocation and target gene expression.
