Improved exercise capacity in cyclophilin-D knockout mice associated with enhanced oxygen utilization efficiency and augmented glucose uptake via AMPK-TBC1D1 signaling nexus.

We previously reported in HEK 293T cells that silencing the mitochondrial peptidyl prolyl isomerase cyclophilin-D (Cyp-D) reduces Vo2. We now report that in vivo Cyp-D ablation using constitutive Cyp-D knockout (KO) mice also reduces Vo2 both at rest (∼15%) and during treadmill exercise (∼12%). Yet, despite Vo2 reduction, these Cyp-D KO mice ran longer (1071 ± 77 vs. 785 ± 79 m; P = 0.002), for longer time (43 ± 3 vs. 34 ± 3 min; P = 0.004), and at higher speed (34 ± 1 vs. 29 ± 1 m/s; P ≤ 0.001), resulting in increased work (87 ± 6 vs. 58 ± 6 J; P ≤ 0.001). There were parallel reductions in carbon dioxide production, but of lesser magnitude, yielding a 2.3% increase in the respiratory exchange ratio consistent with increased glucose utilization as respiratory substrate. In addition, primary skeletal muscle cells of Cyp-D KO mice subjected to electrical stimulation exhibited higher glucose uptake (4.4 ± 0.55 vs. 2.6 ± 0.04 pmol/mg/min; P ≤ 0.001) with enhanced AMPK activation (0.58 ± 0.06 vs. 0.38 ± 0.03 pAMPK/ß-tubulin ratio; P ≤ 0.01) and TBC1 (Tre-2/USP6, BUB2, Cdc16) domain family, member 1 (TBC1D1) inactivation. Likewise, pharmacological activation of AMPK also increased glucose uptake (3.2 ± 0.3 vs. 2.3 ± 0.2 pmol/mg/min; P ≤ 0.001). Moreover, lactate and ATP levels were increased in these cells. Taken together, Cyp-D ablation triggered an adaptive response resulting in increased exercise capacity despite less oxygen utilization associated with increased glucose uptake and utilization involving AMPK-TBC1D1 signaling nexus.-Radhakrishnan, J., Baetiong, A., Kaufman, H., Huynh, M., Leschinsky, A., Fresquez, A., White, C., DiMario, J. X., Gazmuri, R. J. Improved exercise capacity in cyclophilin-D knockout mice associated with enhanced oxygen utilization efficiency and augmented glucose uptake via AMPK-TBC1D1 signaling nexus.

KLF10 Gene Expression Modulates Fibrosis in Dystrophic Skeletal Muscle.

Dystrophic skeletal muscle is characterized by fibrotic accumulation of extracellular matrix components that compromise muscle structure, function, and capacity for regeneration. Tissue fibrosis is often initiated and sustained through transforming growth factor-ß (TGF-ß) signaling, and Krüppel-like factor 10 (KLF10) is an immediate early gene that is transcriptionally activated in response to TGF-ß signaling. It encodes a transcriptional regulator that mediates the effects of TGF-ß signaling in a variety of cell types. This report presents results of investigation of the effects of loss of KLF10 gene expression in wild-type and dystrophic (mdx) skeletal muscle. On the basis of RT-PCR, Western blot, and histological analyses of mouse tibialis anterior and diaphragm muscles, collagen type I (Col1a1) and fibronectin gene expression and protein deposition were increased in KLF10-/- mice, contributing to increased fibrosis. KLF10-/- mice displayed increased expression of genes encoding SMAD2, SMAD3, and SMAD7, particularly in diaphragm muscle. SMAD4 gene expression was unchanged. Expression of the extracellular matrix remodeling genes, MMP2 and TIMP1, was also increased in KLF10-deficient mouse muscle. Histological analyses and assays of hydroxyproline content indicated that the loss of KLF10 increased fibrosis. Dystrophic KLF10-null mice also had reduced grip strength. The effects of loss of KLF10 gene expression were most pronounced in dystrophic diaphragm muscle, suggesting that KLF10 moderates the fibrotic effects of TGF-ß signaling in chronically damaged regenerating muscle.

EMX2 activates slow myosin heavy chain 2 gene expression in embryonic muscle fibers.

Avian myogenesis is partly characterized by commitment of distinct myoblast cell lineages to the formation of specific muscle fiber types. Previous studies have identified the transcription factor EMX2 as a regulator of slow myosin heavy chain 2 (MyHC2) gene expression in fast/slow primary muscle fibers. We report here the interaction of EMX2 with the slow MyHC2 transcriptional regulatory region in fast/slow embryonic muscle fibers. Promoter activity and electromobility shift assays localized the site of interaction of EMX2 with the slow MyHC2 gene within a defined binding site located between 3336 and 3326bp from the 3' end of the cloned slow MyHC2 DNA containing the transcriptional regulatory region. Using clonally-derived myoblasts stably committed to the formation of fast/slow muscle fibers, we also report the effect of altered EMX2 gene expression on genome-wide gene expression within these myoblasts. Increased EMX2 gene expression in fast/slow myoblasts caused altered gene expression of 1185 genes, indicating that EMX2 plays a central role in the gene expression profile of embryonic myoblasts.

Sp3 controls fibroblast growth factor receptor 4 gene activity during myogenic differentiation.

Fibroblast growth factor/fibroblast growth factor receptor (FGF/FGFR) signaling is a critical component in the regulation of myoblast proliferation and differentiation. The transient FGFR4 gene expression during the transition from proliferating myoblasts to differentiated myotubes indicates that FGFR4 regulates this critical phase of myogenesis. The Specificity Protein (SP) family of transcription factors controls FGFR family member gene activity. We sought to determine if members of the Sp family regulate mouse FGFR4 gene activity during myogenic differentiation. RT-PCR and western blot analysis of FGFR4 mRNA and protein revealed transient expression over 72h, with peak expression between 24 and 36h after addition of differentiation medium to C2C12 myogenic cultures. Sp3 also displayed a transient expression pattern with peak expression occurring after 6h of differentiation. We cloned a 1527bp fragment of the mouse FGFR4 promoter into a luciferase reporter. This FGFR4 promoter contains eight putative Sp binding sites and directed luciferase gene activity comparable to native FGFR4 expression. Overexpression of Sp1 and Sp3 showed that Sp1 repressed FGFR4 gene activity, and Sp3 activated FGFR4 gene activity during myogenic differentiation. Mutational analyses of multiple Sp binding sites within the FGFR4 promoter revealed that three of these sites were transcriptionally active. Electromobility shift assays and chromatin immunoprecipitation of the area containing the activator sites showed that Sp3 bound to this promoter location.

C/EBPα represses slow myosin heavy chain 2 gene expression in developing avian myotubes.

BACKGROUND: The CCAAT/enhancer binding proteins (C/EBP) comprise a family of transcription factors that regulate many cellular processes. Little is known of their function during embryonic and fetal myogenesis. Slow myosin heavy chain 2 (MyHC2) is a marker of the slow avian skeletal muscle fiber type, and slow MyHC2 gene regulation involves molecular pathways that lead to muscle fiber type diversification. METHODS: The biological effects of C/EBPα and C/EBPß expression were analyzed by use of a general C/EBP activity reporter and by slow MyHC2 promoter-reporter constructs transfected into specific myogenic cell lineages. The effects of C/EBPα and C/EBPß expression were also analyzed by immunocytochemical detection of slow MyHC2. C/EBPα interaction with the slow MyHC2 promoter was assessed by electromobility shift assays. RESULTS: C/EBPα and C/EBPß are present in embryonic fast and fast/slow avian myogenic lineages. Overexpression of C/EBPα cDNA repressed slow MyHC2 promoter activity in embryonic myotubes and in both electrically stimulated fetal myotubes. Deletion analysis of the slow MyHC2 promoter-luciferase reporter demonstrated that the transcriptional repression mediated by C/EBPα occurs within the first 222bp upstream from exon 1 of the slow MyHC2 gene. Electromobility shift assays determined that C/EBPα can bind to a non-canonical C/EBP site within the slow MyHC2 gene, and mutation of this site reduced transcriptional repression of the slow MyHC2 gene. CONCLUSION: C/EBPα, but not C/EBPß, represses slow MyHC2 promoter activity via a non-canonical C/EBP binding element. GENERAL SIGNIFICANCE: Members of the C/EBP family of transcription factors differentially regulate genes indicative of distinct muscle fiber types.

Muscle fiber type specific activation of the slow myosin heavy chain 2 promoter by a non-canonical E-box.

Different mechanisms control skeletal muscle fiber type gene expression at specific times in vertebrate development. Embryonic myogenesis leading to formation of primary muscle fibers in avian species is largely directed by myoblast cell commitment to the formation of diverse fiber types. In contrast, development of different secondary fiber types during fetal myogenesis is partly determined by neural influences. In both primary and secondary chicken muscle fibers, differential expression of the slow myosin heavy chain 2 (MyHC2) gene distinguishes fast from fast/slow muscle fibers. This study focused on the transcriptional regulation of the slow MyHC2 gene in primary myotubes formed from distinct fast/slow and fast myogenic cell lineages. Promoter deletion analyses identified a discrete 86 bp promoter segment that conferred fiber type, lineage-specific gene expression in fast/slow versus fast myoblast derived primary myotubes. Sequence analysis and promoter activity assays determined that this segment contains two functional cis-regulatory elements. One element is a non-canonical E-box, and electromobility shift assays demonstrated that both cis-elements interacted with the E-protein, E47. The results indicate that primary muscle fiber type specific expression of the slow MyHC2 gene is controlled by a novel mechanism involving a transcriptional complex that includes E47 at a non-canonical E-box.

Genome-wide expression analysis and EMX2 gene expression in embryonic myoblasts committed to diverse skeletal muscle fiber type fates.

BACKGROUND: Primary skeletal muscle fibers form during embryonic development and are characterized as fast or slow fibers based on contractile protein gene expression. Different avian primary muscle fiber types arise from myoblast lineages committed to formation of diverse fiber types. To understand the basis of embryonic muscle fiber type diversity and the distinct myoblast lineages that generate this diversity, gene expression analyses were conducted on differentiated muscle fiber types and their respective myoblast precursor lineages. RESULTS: Embryonic fast muscle fibers preferentially expressed 718 genes, and embryonic fast/slow muscle fibers differentially expressed 799 genes. Fast and fast/slow myoblast lineages displayed appreciable diversity in their gene expression profiles, indicating diversity of precursor myoblasts. Several genes, including the transcriptional regulator EMX2, were differentially expressed in both fast/slow myoblasts and muscle fibers vs. fast myoblasts and muscle fibers. EMX2 was localized to nuclei of fast/slow myoblasts and muscle fibers and was not detected in fast lineage cells. Furthermore, EMX2 overexpression and knockdown studies indicated that EMX2 is a positive transcriptional regulator of the slow myosin heavy chain 2 (MyHC2) gene promoter activity in fast/slow muscle fibers. CONCLUSIONS: These results indicate the presence of distinct molecular signatures that characterize diverse embryonic myoblast lineages before differentiation.

Repression of myoblast proliferation and fibroblast growth factor receptor 1 promoter activity by KLF10 protein.

BACKGROUND: FGFR1 gene expression regulates myoblast proliferation and differentiation, and its expression is controlled by Krüppel-like transcription factors. RESULTS: KLF10 interacts with the FGFR1 promoter, repressing its activity and cell proliferation. CONCLUSION: KLF10 represses FGFR1 promoter activity and thereby myoblast proliferation. SIGNIFICANCE: A model of transcriptional control of chicken FGFR1 gene regulation during myogenesis is presented. Skeletal muscle development is controlled by regulation of myoblast proliferation and differentiation into muscle fibers. Growth factors such as fibroblast growth factors (FGFs) and their receptors (FGFRs) regulate cell proliferation and differentiation in numerous tissues, including skeletal muscle. Transcriptional regulation of FGFR1 gene expression is developmentally regulated by the Sp1 transcription factor, a member of the Krüppel-like factor (KLF) family of transcriptional regulators. Here, we show that another KLF transcription factor, KLF10, also regulates myoblast proliferation and FGFR1 promoter activity. Expression of KLF10 reduced myoblast proliferation by 86%. KLF10 expression also significantly reduced FGFR1 promoter activity in myoblasts and Sp1-mediated FGFR1 promoter activity in Drosophila SL2 cells. Southwestern blot, electromobility shift, and chromatin immunoprecipitation assays demonstrated that KLF10 bound to the proximal Sp factor binding site of the FGFR1 promoter and reduced Sp1 complex formation with the FGFR1 promoter at that site. These results indicate that KLF10 is an effective repressor of myoblast proliferation and represses FGFR1 promoter activity in these cells via an Sp1 binding site.

Interaction of α-catulin with dystrobrevin contributes to integrity of dystrophin complex in muscle.

The dystrophin complex is a multimolecular membrane-associated protein complex whose defects underlie many forms of muscular dystrophy. The dystrophin complex is postulated to function as a structural element that stabilizes the cell membrane by linking the contractile apparatus to the extracellular matrix. A better understanding of how this complex is organized and localized will improve our knowledge of the pathogenic mechanisms of diseases that involve the dystrophin complex. In a Caenorhabditis elegans genetic study, we demonstrate that CTN-1/α-catulin, a cytoskeletal protein, physically interacts with DYB-1/α-dystrobrevin (a component of the dystrophin complex) and that this interaction is critical for the localization of the dystrophin complex near dense bodies, structures analogous to mammalian costameres. We further show that in mouse α-catulin is localized at the sarcolemma and neuromuscular junctions and interacts with α-dystrobrevin and that the level of α-catulin is reduced in α-dystrobrevin-deficient mouse muscle. Intriguingly, in the skeletal muscle of mdx mice lacking dystrophin, we discover that the expression of α-catulin is increased, suggesting a compensatory role of α-catulin in dystrophic muscle. Together, our study demonstrates that the interaction between α-catulin and α-dystrobrevin is evolutionarily conserved in C. elegans and mammalian muscles and strongly suggests that this interaction contributes to the integrity of the dystrophin complex.

Direct electrical stimulation of myogenic cultures for analysis of muscle fiber type control.

Secondary skeletal muscle fiber phenotype is dependent upon depolarization from motor neuron innervation. To study the effects of depolarization on muscle fiber type development, several in vivo and in vitro model systems exist. We have developed a relatively simple-to-use in vitro model system in which differentiated muscle cells are directly electrically stimulated at precise frequencies. This allows for single cell analysis as well as biochemical and molecular analyses of the mechanisms that control skeletal muscle phenotype.

Lineage-based primary muscle fiber type diversification independent of MEF2 and NFAT in chick embryos.

Differences in primary avian skeletal muscle fiber types are based on myoblast cell lineages and independent of innervation. To understand the basis for this mode of myogenesis, embryonic myoblasts specifically committed to the formation of either fast or fast/slow muscle fiber types were isolated, characterized, and examined for their capacities to transcriptionally regulate the slow myosin heavy chain 2 (MyHC2) gene. Myogenic basic helix-loop-helix protein binding sites within the slow MyHC2 promoter were mutated and did not direct fast versus fast/slow muscle fiber type development. Using promoter analyses coupled with overexpression studies and transcriptional sensors, the roles of Nuclear Factor of Activated T cells (NFATc1), and MEF2A in regulation of the slow MyHC2 gene were determined. MEF2A activated the slow MyHC2 promoter in both fast and fast/slow primary muscle fibers. In contrast, NFATc1 repressed promoter activity. These results do not support the roles of MEF2 and NFAT as direct regulators of primary muscle fiber type differences. Rather, the results reflect intrinsic differences in the modes of regulation of the slow MyHC2 gene in primary muscle fiber types.

Bimodal, reciprocal regulation of fibroblast growth factor receptor 1 promoter activity by BTEB1/KLF9 during myogenesis.

Expression of the gene encoding fibroblast growth factor receptor 1 (FGFR1) and subsequent FGFR1-mediated cell signaling controls numerous developmental and disease-related processes. The transcriptional regulation of the FGFR1 gene is central to these developmental events and serves as a molecular model for understanding transcriptional control of growth factor receptor genes. The FGFR1 promoter is activated in proliferating myoblasts via several Sp1-like binding elements. These elements display varying levels of activation potential, suggesting that unique protein-DNA complexes coordinate FGFR1 gene expression via each of these sites. The Krüppel-like factor, BTEB1/KLF9, was expressed in both proliferating myoblasts and differentiated myotubes in vitro. The BTEB1 protein was nuclear-localized in both cell types. BTEB1 activated the FGFR1 promoter via interaction with the Sp1-like binding site located at -59 bp within the FGFR1 promoter. FGFR1 gene expression is down-regulated during myogenic differentiation, and FGFR1 promoter activity is correspondingly reduced. This reduction in FGFR1 promoter activity was attributable to BTEB1 interaction with the same Sp1-like binding site located at -59 bp in the FGFR1 promoter. Therefore, BTEB1 is capable of functioning as a transcriptional activator and repressor of the same promoter via the same DNA-binding element and demonstrates a novel, bimodal role of BTEB1 during myogenesis.

Muscle fiber type specific induction of slow myosin heavy chain 2 gene expression by electrical stimulation.

Vertebrate skeletal muscle fiber types are defined by a broad array of differentially expressed contractile and metabolic protein genes. The mechanisms that establish and maintain these different fiber types vary throughout development and with changing functional demand. Chicken skeletal muscle fibers can be generally categorized as fast and fast/slow based on expression of the slow myosin heavy chain 2 (MyHC2) gene in fast/slow muscle fibers. To investigate the cellular and molecular mechanisms that control fiber type formation in secondary or fetal muscle fibers, myoblasts from the fast pectoralis major (PM) and fast/slow medial adductor (MA) muscles were isolated, allowed to differentiate in vitro, and electrically stimulated. MA muscle fibers were induced to express the slow MyHC2 gene by electrical stimulation, whereas PM muscle fibers did not express the slow MyHC2 gene under identical stimulation conditions. However, PM muscle fibers did express the slow MyHC2 gene when electrical stimulation was combined with inhibition of inositol triphosphate receptor (IP3R) activity. Electrical stimulation was sufficient to increase nuclear localization of expressed nuclear-factor-of-activated-T-cells (NFAT), NFAT-mediated transcription, and slow MyHC2 promoter activity in MA muscle fibers. In contrast, both electrical stimulation and inhibitors of IP3R activity were required for these effects in PM muscle fibers. Electrical stimulation also increased levels of peroxisome-proliferator-activated receptor-gamma co-activator-1 (PGC-1alpha) protein in PM and MA muscle fibers. These results indicate that MA muscle fibers can be induced by electrical stimulation to express the slow MyHC2 gene and that fast PM muscle fibers are refractory to stimulation-induced slow MyHC2 gene expression due to fast PM muscle fiber specific cellular mechanisms involving IP3R activity.

AP-2 alpha suppresses skeletal myoblast proliferation and represses fibroblast growth factor receptor 1 promoter activity.

Skeletal muscle development is partly characterized by myoblast proliferation and subsequent differentiation into postmitotic muscle fibers. Developmental regulation of expression of the fibroblast growth factor receptor 1 (FGFR1) gene is required for normal myoblast proliferation and muscle formation. As a result, FGFR1 promoter activity is controlled by multiple transcriptional regulatory proteins during both proliferation and differentiation of myogenic cells. The transcription factor AP-2 alpha is present in nuclei of skeletal muscle cells and suppresses myoblast proliferation in vitro. Since FGFR1 gene expression is tightly linked to myoblast proliferation versus differentiation, the FGFR1 promoter was examined for candidate AP-2 alpha binding sites. Mutagenesis studies indicated that a candidate binding site located at -1035 bp functioned as a repressor cis-regulatory element. Furthermore, mutation of this site alleviated AP-2 alpha-mediated repression of FGFR1 promoter activity. Chromatin immunoprecipitation studies demonstrated that AP-2 alpha interacted with the FGFR1 promoter in both proliferating myoblasts and differentiated myotubes. In total, these results indicate that AP-2 alpha is a transcriptional repressor of FGFR1 gene expression during skeletal myogenesis.

Fibroblast growth factor receptor 1 gene expression is required for cardiomyocyte proliferation and is repressed by Sp3.

Fibroblast growth factor receptor 1 (FGFR1) is the only high-affinity FGFR in the vertebrate myocardium. FGFR1 is a tyrosine kinase receptor and has a non-redundant role in proliferation and differentiation of cardiomyocytes during embryogenesis. Results presented here demonstrate that FGFR1 gene expression declines as neonatal cardiomyocytes develop into adult cardiomyocytes. Furthermore, silencing FGFR1 gene expression reduced neonatal cardiomyocyte proliferation, indicating that FGFR1 gene expression is required for the optimal proliferative capacity of cardiomyocytes. To determine the mechanism that governs FGFR1 gene expression in cardiomyocytes, sequence analysis of the proximal mouse FGFR1 promoter identified a potential binding site for Sp transcription factors. Mutation of this site increased FGFR1 promoter activity compared to the wild-type promoter, indicating the presence of a negative transcriptional regulator of the FGFR1 promoter at this site in cardiomyocytes. Sp3 expression in neonatal cardiomyocytes and Drosophila SL2 cells reduced FGFR1 promoter activity in a dose-dependent manner. Western blots and immunocytochemistry indicated that Sp3 was present in the nuclear and cytoplasmic compartments of neonatal cardiomyocytes. Chromatin-immunoprecipitation studies verified that endogenous Sp3 in cardiomyocytes interacts with the FGFR1 promoter. Transient chromatin-immunoprecipitation studies using wild-type and mutated FGFR1 promoter constructs in SL2 cells identified the specific Sp3 binding site within the FGFR1 promoter. These studies implicate Sp3 as a negative transcriptional regulator of FGFR1 promoter activity in cardiomyocytes and as a suppressor of cardiomyocyte proliferation.

Sp1 is required for transcriptional activation of the fibroblast growth factor receptor 1 gene in neonatal cardiomyocytes.

Fibroblast growth factor receptor 1 (FGFR1) is the predominant FGFR in cardiac tissue and regulates proliferation, differentiation, and maintenance of normal myocardium. During development of cardiac tissue, FGFR1 gene expression regulates cardiomyocyte proliferation. The focus of this study was to determine the molecular mechanism of transcriptional activation of the FGFR1 gene in proliferating neonatal cardiomyocytes. Analysis of DNA sequence of the FGFR1 gene identified three potential Sp factor binding sites located at 49 bp, 68 bp, and 100 bp upstream from the 3' end of the promoter segment. Mutation of each of these sites resulted in a significant decline in FGFR1 promoter activity compared to wild type promoter activity, and combinatorial mutation of all three sites completely abrogated promoter activity to background levels. In addition, overexpression of Sp1 in neonatal cardiomyocytes resulted in a dose-dependent increase in wild type FGFR1 promoter activity. However, Sp1-mediated up-regulation of promoter activity was abrogated when all three Sp interacting sites were mutated. Chromatin immunoprecipitation (ChIP) assays were used to demonstrate direct interactions of Sp1 with the proximal promoter region of the FGFR1 gene in neonatal cardiomyocytes. ChIP assays using Drosophila Schneider Line 2 (SL2) cells transiently transfected with wild type or mutant FGFR1 promoter constructs verified the direct interaction between Sp1 and the three Sp1 interacting sites of the promoter. Western blot analyses indicated that Sp1 was present in cytoplasmic and nuclear extracts of neonatal myocardium. These results indicate that Sp1 is a necessary positive regulator of FGFR1 gene transcription in neonatal cardiomyocytes.

Control of slow myosin heavy chain 2 gene expression by glycogen synthase kinase activity in skeletal muscle fibers.

Skeletal muscle fiber type and expression of slow muscle fiber type specific genes are regulated by fiber type specific cell signaling events initiated by innervation. In avian muscle fibers, expression of the slow myosin heavy chain 2 (MyHC2) gene defines fast versus slow muscle fiber types, and its expression is dependent on the transcription factor, nuclear factor of activated T cells (NFAT). Glycogen synthase kinase 3 (GSK3) phosphorylates NFAT and inhibits its transactivating potential. We report here that expression of the slow MyHC2 gene is dependent on GSK3 activity. Inhibition of GSK3 activity by SB216763 or LiCl induced expression of the slow MyHC2 gene in non-innervated medial adductor (MA) muscle fibers and in innervated fast pectoralis major (PM) muscle fibers. Innervation of MA and PM muscle fibers did not significantly alter GSK3 activity. However, inhibition of GSK3 activity increased NFAT-mediated transcriptional activity, required for full activation of the slow MyHC2 gene, and overexpression of GSK3 reduced NFAT-mediated transcription. Inhibition of GSK3 activity was sufficient to induce slow MyHC2 gene expression in non-innervated MA muscle fibers but not in non-innervated PM muscle fibers, suggesting that fiber type specific mechanisms differentially regulate slow MyHC2 gene expression in innervated muscle fibers.

Regulation of skeletal muscle fiber type and slow myosin heavy chain 2 gene expression by inositol trisphosphate receptor 1.

Innervation-dependent signaling cascades that control activation of downstream transcription factors regulate expression of skeletal muscle fiber type-specific genes. Many of the innervation-regulated signaling cascades in skeletal muscle are dependent on intracellular calcium and the mechanisms by which calcium is released from the sarcoplasmic reticulum (SR). We report that the inositol trisphosphate receptor 1 (IP3R1), responsible for calcium release from the SR as a slow wave, was more abundant in fast contracting compared to slow contracting avian muscle fibers. Furthermore, inhibition of IP3R1 activity by 2-aminoethoxydiphenylborate (2-APB) and xestospongin D induced a fiber type transition and expression of the slow myosin heavy chain 2 (slow MyHC2) gene in innervated fast muscle fibers. Activation of the slow MyHC2 promoter by IP3R1 inhibition was accompanied by a reduction in protein kinase C activity. In addition, inhibition of IP3R1 activity resulted in a reduction of nuclear factor of activated T cells (NFAT)-dependent transcription and nuclear localization, indicating that IP3R1 activity regulated NFAT transcription factor activity in skeletal muscle fibers. Myocyte enhancer factor 2 (MEF2)-dependent transcriptional activity was increased by innervation, but unaffected by IP3R1 activity. The results indicate that IP3R1 activity regulates muscle fiber type-specific gene expression in innervated muscle fibers.

Dynamic transcriptional regulatory complexes, including E2F4, p107, p130, and Sp1, control fibroblast growth factor receptor 1 gene expression during myogenesis.

Developmentally controlled transcriptional regulation of myogenic cell proliferation and differentiation via expression of the fibroblast growth factor receptor 1 (FGFR1) gene is positively regulated by Sp1 and negatively regulated by E2F4-based transcriptional complexes. We report that p107 and p130 formed transcriptional complexes with E2F4 on the FGFR1 promoter and repressed FGFR1 gene transcription in myogenic cells. However, in Drosophila melanogaster SL2 cells, only p107 was able to repress Sp1-mediated transactivation of the FGFR1 promoter. Gel shift assays using transfected myoblast nuclear extracts showed that ectopic p107 reduced Sp1 occupancy of the proximal Sp binding site of the FGFR1 promoter, and coimmunoprecipitation studies indicated that Sp1 interacts with p107 but not with p130. Gel shift assays also demonstrated that Sp1 interacted with p107 in E2F4-p107 transcriptional complexes in myoblasts. The nature of the repressor transcriptional complex was altered in differentiated muscle fibers by the relative loss of the E2F4-p107-Sp1 transcription complex and replacement by the repressor E2F4-p130 complex. These findings demonstrate that activation and repression of FGFR1 gene transcription is governed by interplay between Sp1, p107, p130, and E2F4 in distinct transcriptional complexes during skeletal muscle development.

Repression of fibroblast growth factor receptor 1 gene expression by E2F4 in skeletal muscle cells.

Fibroblast growth factor receptor 1 (FGFR1) gene expression is positively and negatively regulated during muscle differentiation. We recently reported that FGFR1 gene expression was up-regulated by Sp transcription factors in proliferating myoblasts. However, the mechanism of down-regulation of this gene during differentiation is unknown. We have identified the transcription factor E2F4 as a negative regulator of FGFR1 gene expression. Immunodetection studies revealed that endogenous E2F1 and E2F2 proteins were cytoplasmic in myoblasts and myotubes, whereas E2F4 was abundant in the nuclei of both. Upon overexpression, E2F4 repressed FGFR1 promoter activity in a dose-dependent manner in myoblasts and Drosophila SL2 cells, and mutation of the E2F4 binding site increased FGFR1 promoter activity and reduced E2F4-mediated repression. Gel shift assays detected E2F4 binding to a synthetic FGFR1 E2F4 binding site and chromatin immunoprecipitation assays detected E2F4 binding to the endogenous FGFR1 promoter in proliferating myoblasts and myotubes. The results indicate that FGFR1 promoter activity in skeletal muscle cells is repressed by E2F4.