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We previously reported in HEK 293T cells that silencing the mitochondrial peptidyl prolyl isomerase cyclophilin-D (Cyp-D) reduces V o 2 . We now report that in vivo Cyp-D ablation using constitutive Cyp-D knockout (KO) mice also reduces V o 2 both at rest (∼15%) and during treadmill exercise (∼12%). Yet, despite V o 2 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/β-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.

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-β (TGF-β) signaling, and Krüppel-like factor 10 (KLF10) is an immediate early gene that is transcriptionally activated in response to TGF-β signaling. It encodes a transcriptional regulator that mediates the effects of TGF-β 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-β signaling in chronically damaged regenerating muscle., (Copyright © 2018 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

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., (Copyright © 2017 Elsevier B.V. All rights reserved.)

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 C 2 C 12 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., (Copyright © 2017 Elsevier B.V. All rights reserved.)

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/EBPβ 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/EBPβ 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/EBPβ 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/EBPβ, 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., (Copyright © 2016 Elsevier B.V. All rights reserved.)

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., (Copyright © 2015 Elsevier Inc. All rights reserved.)

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., (Copyright © 2013 Wiley Periodicals, Inc.)

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.

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.

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.

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.

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.

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., (Copyright (c) 2010 Elsevier Inc. All rights reserved.)

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 (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.

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.

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.

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.

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.

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.

Skeletal muscle fiber type is regulated by innervation-induced cell signaling including calcium release mechanisms that lead to transcriptional activation of fiber type-specific genes. Avian fast pectoralis major (PM) and slow medial adductor (MA) muscles differentially control expression of the slow myosin heavy chain 2 (slow MyHC2) gene. We report here that slow MyHC2 gene expression in fast PM muscle fibers is repressed by endogenous activity of the ryanodine receptor 1 (RyR1). Inhibition of RyR1 with ryanodine led to expression of the slow MyHC2 gene in innervated PM muscle fibers in vitro. Administration of ryanodine to innervated PM muscle fibers also decreased protein kinase C (PKC) activity, the reduction of which is necessary for slow MyHC2 gene expression in both PM and MA muscle fibers. Furthermore, RyR1 inhibition increased slow MyHC2 promoter activity in innervated PM muscle fibers and enhanced transcriptional activities of nuclear factor of activated T cells (NFAT) and myocyte enhancer factor 2 (MEF2), as well as their interactions with their respective binding sites of the slow MyHC2 promoter. These results indicate that RyR1 activity in innervated fast PM muscle fibers contributes to the cell type-specific repression of slow muscle specific genes.

Skeletal muscle fiber type is regulated, in part, by innervation leading to transcriptional regulation of fiber type-specific genes. Here, we report the initial characterization of the transcriptional regulation of the slow myosin heavy chain 2 (MyHC2) promoter in innervated and noninnervated slow medial adductor (MA) and fast pectoralis major (PM) muscle fibers in cell culture. The proximal 1358 bp of slow MyHC2 upstream DNA contains a functional E-box and binding sites for myocyte enhancer factor 2 (MEF2) and nuclear factor of activated T cells (NFAT). Mutagenesis studies indicated that both MEF2 and NFAT binding sites are required for innervation-induced slow MyHC2 promoter activity in MA muscle fibers. However, MEF2 transcription factor activity was unaffected by innervation and did not demonstrate fiber type-specific interactions with the slow MyHC2 MEF2 binding site. NFAT transcription factor activity did increase in innervated MA muscle fibers and not in PM muscle fibers, indicating innervation and muscle fiber type-specific regulation. However, transfection of constitutively active NFAT indicated that NFAT is insufficient to induce slow MyHC2 gene expression in either fast PM or slow MA muscle fibers without innervation. These results indicate the requirement for MEF2 and NFAT in innervation-induced slow MyHC2 gene expression and suggest that additional innervation-dependent and fiber type-specific control of slow MyHC2 gene expression resides in MA and PM muscle fibers, respectively., (2004 Wiley-Liss, Inc.)

Gene expression in skeletal muscle fibers is regulated by innervation and intrinsic fiber properties. To determine the mechanism of repression of slow MyHC2 expression in innervated fast pectoralis major (PM) fibers, we investigated the function of the muscarinic acetylcholine receptor (mAchR) and G(alpha)q. Both mAchR and G(alpha)q are abundant in medial adductor (MA) and PM fibers, and mAchR and G(alpha)q interact in these fibers. Whereas innervation of PM fibers was insufficient to induce slow MyHC2 expression, inhibition of mAchR activity with atropine in innervated PM fibers induced slow MyHC2 expression. Increased G(alpha)q activity repressed slow MyHC2 expression to nondetectable levels in innervated MA fibers. Reduced mAchR activity decreased PKC activity in PM fibers, and increased G(alpha)q activity increased PKC activity in PM and MA fibers. Decreased PKC activity in atropine-treated innervated PM fibers correlated with slow MyHC2 expression. These data suggest that slow MyHC2 repression in innervated fast PM fibers is mediated by cell signaling involving mAchRs, G(alpha)q, and PKC.

Growth factor receptors mediate cell signaling events that regulate a diverse array of cellular activities including cell proliferation, homeostasis, and differentiation of both normal and cancer cells. Studies of the mechanisms governing transcription of growth factor receptor genes have revealed common structural features of their promoters. These common features include GC rich promoter regions and multiple Sp factor binding sites based upon which most of these promoters are transactivated. Mechanisms of growth factor receptor promoter activation via these common structural features will be reviewed, with particular attention to control of FGFR1 promoter activity in skeletal muscle cells. Of equal importance in cellular function is the repression of growth factor receptor signaling and gene expression. Mechanisms that repress growth factor receptor promoter activity operate via direct repression at transcriptional activator binding sites and via protein-protein interactions that abrogate activator function. Mechanisms of growth factor receptor transcriptional repression will be considered in the context of known tumor suppressors, transcription activator availability, as well as in light of emerging potential Sp1-like transcriptional repressors.

Expression of the fibroblast growth factor receptor 1 (FGFR1) gene in skeletal muscle is positively regulated in proliferating myoblasts and declines during differentiation. We have characterized the cis-regulatory elements in the proximal region of the FGFR1 promoter which render positive transcriptional activity. Multiple elements between -69 and -14 activate the FGFR1 promoter. Myoblast transfections revealed that potential Sp transcription factor binding sites are required for promoter activity. Electromobility shift assays indicated that myoblast nuclear proteins specifically bind to these cis-elements and that differentiated myotube nuclear extracts do not form these same complexes. In addition, Southwestern blot analysis detected binding of the most proximal Sp motif to a Sp1-like protein present in myoblast nuclear extracts but not in myotubes. In corroboration, Sp1 and Sp3 proteins were detected only in myoblasts and not in differentiated myotubes. Finally, transfection of Drosophila SL2 cells showed that Sp1 is a positive regulator of FGFR1 promoter activity and that Sp3 is a coactivator via the proximal Sp binding sites. These studies demonstrate that the FGFR1 promoter is activated by Sp transcription factors in proliferating myoblasts and demonstrate at least part of the mechanism by which FGFR1 gene expression is down-regulated in differentiated muscle fibers.

Skeletal myoblast cell proliferation and subsequent differentiation are dependent on developmentally regulated expression of the fibroblast growth factor receptor 1 (FGFR1) gene. We have previously reported the isolation and initial characterization of the chicken FGFR1 gene (cek1) promoter. Both distal and proximal regions of the promoter were identified as necessary for developmentally regulated transcriptional activity in proliferating myoblasts, including its down-regulation in differentiated muscle fibers in vitro. Here we report detailed characterization of the molecular mechanism regulating FGFR1 promoter activity via the distal promoter region in proliferating myoblasts. This region was identified as a 242 base pair segment located greater than 1 kilobase upstream from the start of transcription that conferred increased transcriptional activity to a minimal thymidine kinase promoter. This segment contains two Sp1 binding sites. Site directed mutagenesis and transfection studies indicated that both Sp1 sites are functional and both are required for FGFR1 promoter activity. Furthermore, Sp1 binding to the two sites was synergistic enhancing FGFR1 promoter activity. The specificity of Sp1 binding to the two distal promoter cis-elements was verified by electromobility shift and transfection assays employing an Sp1 expression construct. Differences in myoblast versus fibroblast-specific protein-DNA complex formation at these sites correlated with high promoter activity in myoblasts and significantly reduced promoter activity in fibroblasts. These studies for the first time establish a molecular mechanism regulating FGFR1 gene expression during myoblast proliferation.

Slow myosin heavy chain 2 (MyHC2) gene expression in fetal avian skeletal muscle fibers is regulated by innervation and protein kinase C (PKC) activity. Fetal chick muscle fibers derived from the slow twitch medial adductor (MA) muscle express slow MyHC2 when innervated in vitro. The same pattern of slow MyHC2 regulation occurs in MA muscle fibers in which PKC activity is inhibited by staurosporine. To further test the function of PKC activity in the regulation of slow MyHC2 expression, wild-type and dominant-negative mutations of PKCalpha and PKCtheta were overexpressed in MA muscle fibers in vitro. Overexpression of wild-type PKCalpha and PKCtheta cDNAs resulted in increased PKC activities in muscle fibers and concomitant repression of slow MyHC2 expression under conditions that normally induced gene expression. Point mutations leading to single amino acid substitutions were generated in the ATP binding domains of PKCalpha and PKCtheta. Overexpression of CMVPKCalphaR368 and CMVPKCthetaR409 resulted in decreased PKC activities in transfected MA muscle fibers. Furthermore, transfection of CMVPKCalphaR368 and CMVPKCthetaR409 mutant constructs into MA muscle fibers did not repress the capacity of these fibers to express slow MyHC2 when cultured in medium containing staurosporine or when innervated. These results indicate that PKC activity represses slow MyHC2 expression and that PKC down-regulation, possibly in response to innervation, is required but not sufficient for slow MyHC2 expression.

Expression of the slow myosin heavy chain (MyHC) 2 gene defines slow versus fast avian skeletal muscle fiber types. Fetal, or secondary, skeletal muscle fibers express slow MyHC isoform genes in developmentally regulated patterns within the embryo, and this patterning is at least partly dependent on innervation in vivo. We have previously shown that slow MyHC 2 gene expression in vitro is regulated by a combination of innervation and cell lineage. This pattern of gene expression was indistinguishable from the pattern observed in vivo in that it was restricted to innervated muscle fibers of slow muscle origin. We show here that slow MyHC 2 gene expression in the slow muscle fiber lineage is regulated by protein kinase C (PKC) activity. Inhibition of PKC activity induced slow MyHC 2 gene expression, and the capacity to express the slow MyHC 2 gene was restricted to muscle fibers of slow muscle (medial adductor) origin. Fast muscle fibers derived from the pectoralis major did not express significant levels of slow MyHC 2 with or without inhibitors of PKC activity. This differential expression pattern coincided with different inherent PKC activities in fast versus slow muscle fiber types. Furthermore, over-expression of an unregulated PKCalpha mutant suppressed slow MyHC 2 gene expression in muscle fibers of the slow lineage. Lastly, denervation of skeletal muscles caused an increase in PKC activity, particularly in the slow medial adductor muscle. This increase in PKC activity was associated with lack of slow MyHC 2 gene expression in vivo. These results provide a mechanistic link between innervation, an intracellular signaling pathway mediated by PKC, and expression of a muscle fiber type-specific contractile protein gene. Dev Dyn 1999;216:177-189., (Copyright 1999 Wiley-Liss, Inc.)

Myogenic cell proliferation and differentiation are regulated by a fibroblast growth factor (FGF) signal transduction cascade mediated by a high-affinity fibroblast growth factor receptor (FGFR). Exogenous FGF added to myogenic cultures has a mitogenic effect promoting myoblast proliferation while repressing differentiation. We have examined the regulation of the FGFR-1 gene (cek-1) in avian myogenic cultures by immunocytochemistry and Northern blot analysis. FGFR-1 protein was readily detected in undifferentiated myoblast cultures and was significantly reduced in differentiated muscle fiber cultures. Similarly, FGFR-1 mRNA was 2.5-fold more abundant in myoblast cultures than in differentiated cultures. To define the molecular mechanism regulating FGFR-1 gene expression in proliferating myoblasts and post-mitotic muscle fibers, we have isolated and partially characterized the avian FGFR-1 gene promoter. Transfection of FGFR-1 promoter-chloramphenicol acetyltransferase gene constructs into myogenic cultures identified two regions regulating expression of this gene in myoblasts. A distal region of 2226 bp conferred a high level of expression in myoblasts. This region functioned in an orientation-dependent manner and interacted with a promoter element(s) in a proximal 1058 bp promoter region to direct transcription. Deletion analysis revealed a 78 bp region that confers a high level of cek1 promoter activity in myoblasts. This DNA segment also contains Spl binding sites and interacts with a component in myoblast nuclear protein extracts. The proximal promoter region alone demonstrated no activity in directing transcription in either myoblasts or muscle fibers. Using the full-length promoter, gene expression was significantly decreased in differentiated muscle fibers relative to undifferentiated myoblasts indicating that the promoter-reporter gene constructs contain elements regulating expression of the endogenous FGFR-1 gene in both myoblasts and muscle fibers.

Skeletal muscle fibers express members of the myosin heavy chain (MyHC) gene family in a fiber-type-specific manner. In avian skeletal muscle it is the expression of the slow MyHC isoforms that most clearly distinguishes slow- from fast-contracting fiber types. Two hypotheses have been proposed to explain fiber-type-specific expression of distinct MyHC genes during development-an intrinsic mechanism based on the formation of different myogenic lineage(s) and an extrinsic, innervation-dependent mechanism. We developed a cell culture model system in which both mechanisms were evaluated during fetal muscle development. Myoblasts isolated from prospective fast (pectoralis major) or slow (medial adductor) fetal chick muscles formed muscle fibers in cell culture, none of which expressed slow MyHC genes. By contrast, when muscle fibers formed from myoblasts derived from the slow muscle were cocultured with neural tube, the muscle fibers expressed a slow MyHC gene, while muscle fibers formed from myoblasts of fast muscle origin continued to express only fast MyHC. Motor endplates formed on the fibers derived from myoblasts of both fast and slow muscle origin in cocultures, and slow MyHC gene expression did not occur when neuromuscular transmission or depolarization was blocked. We have cloned the slow MyHC gene that is expressed in response to innervation and identified it as the slow MyHC 2 gene, the predominant adult slow isoform. cDNAs encoding portions of the three slow myosin heavy chain genes (MyHC1, slow MyHC 2, and slow MyHC 3) were isolated. Only slow MyHC 2 mRNA was demonstrated to be abundant in the cocultures of neural tube and muscle fibers derived from myoblasts of slow muscle origin. Thus, expression of the slow MyHC 2 gene in this in vitro system indicates that formation of slow muscle fiber types is dependent on both myoblast lineage (intrinsic mechanisms) and innervation (extrinsic mechanisms), and suggests neither mechanism alone is sufficient to explain formation of muscle fibers of different types during fetal development.

Myoblasts from embryonic, fetal, and adult quail and chick muscles were transplanted into limb buds of chick embryos to determine if myoblasts can form muscle fibers in heterochronic limbs and to define the conditions that affect the ability of transplanted cells to populate newly developing limb musculature. Myoblasts from each developmental stage were either freshly isolated and transplanted or were cultured prior to transplantation into limb buds of 4- to 5-day (ED4-5) chick embryos. Transplanted myoblasts, regardless of the age of the donor from which they were derived, formed muscle fibers within embryonic limb muscles. Transplanted cloned myoblasts formed muscle fibers, although there was little evidence that the number of transplanted myoblasts significantly increased following transplantation or that they migrated any distance from the site of injection. The fibers that formed from transplanted clonal myoblasts often did not persist in the host limb muscles until ED10. Diminished fiber formation from myoblasts transplanted into host limbs was observed whether myoblasts were cloned or cultured at high density. However, when freshly isolated myoblasts were transplanted, the fibers they formed were numerous, widely dispersed within the limb musculature, and persisted in the muscles until at least ED10. These results indicate that transplanted myoblasts of embryonic, fetal, and adult origin are capable of forming fibers during early limb muscle formation. They also indicate that even in an embryonic chick limb where proliferation of endogenous myoblasts and muscle fiber formation is rapidly progressing, myoblasts that are cultured in vitro do not substantially contribute to long-term muscle fiber formation after they are transplanted into developing limbs. However, when the same myoblasts are freshly isolated and transplanted without prior cell culture, substantial numbers of fibers form and persist after transplantation into developing limbs. Thus, these studies demonstrate that the extent to which transplanted myoblasts fuse to form fibers which persist in host musculature depends upon whether donor myoblasts are freshly isolated or maintained in vitro prior to injection.

In the limb bud of the 5-day-old avian embryo, when primary muscle fibre formation is beginning and before specific muscles appear, differences in the expression of fast and slow myosin heavy chain genes can be detected among primary fibres of the premuscle masses. Myoblasts that form colonies of fibres of specific types can be isolated from these limb buds. To assess the role of myoblast commitment in specifying fibre types during embryonic development, we cloned myoblasts of specific types from embryonic and adult muscles, transfected them with a reporter gene, and transferred them into developing limb buds. After transfer, cloned myoblasts formed fibres in the limb with the same patterns of myosin heavy chain gene expression as the fibres they formed in cell culture. These results demonstrate that initial skeletal muscle fibre type diversity during avian limb development can originate, in part, from the commitment of distinct myoblast types to the formation of specific fibre types.

At least three slow myosin heavy chain (MHC) isoforms were expressed in skeletal muscles of the developing chicken hindlimb, and differential expression of these slow MHC isoforms produced distinct fiber types from the outset of skeletal muscle myogenesis. Immunohistochemistry with isoform-specific monoclonal antibodies demonstrated differences in MHC content among the fibers of the dorsal and ventral premuscle masses and distinctions among fibers before splitting of the premuscle masses into individual muscles (Hamburger and Hamilton Stage 25). Immunoblot analyses by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of myosin extracted from the hindlimb demonstrated the presence throughout development of different mobility classes of MHCs with epitopes associated with slow MHC isoforms. Immunopeptide mapping showed that one of the MHCs expressed in the embryonic limb was the same slow MHC isoform, slow MHC1 (SMHC1), that is expressed in adult slow muscles. SMHC1 was expressed in the dorsal and ventral premuscle masses, embryonic, fetal, and some neonatal and adult hindlimb muscles. In the embryo and fetus SMHC1 was expressed in future fast, as well as future slow muscles, whereas in the adult only the slow muscles retained expression of SMHC1. Those embryonic muscles destined in the adult to contain slow fibers or mixed fast/slow fibers not only expressed SMHC1, but also an additional slow MHC not previously described, designated as slow MHC3 (SMHC3). Slow MHC3 was shown by immunopeptide mapping to contain a slow MHC epitope (reactive with mAb S58) and to be structurally similar to a MHC expressed in the atria of the adult chicken heart. SMHC3 was designated as a slow MHC isoform because (i) it was expressed only in those muscles destined to be of the slow type in the adult, (ii) it was expressed only in primary fibers of muscles that subsequently are of the slow type, and (iii) it had an epitope demonstrated to be present on other slow, but not fast, isoforms of avian MHC. This study demonstrates that a difference in phenotype between fibers is established very early in the chicken embryo and is based on the fiber type-specific expression of three slow MHC isoforms.

Fiber replacement has been measured in adult mdx mouse limb skeletal muscles. During the first 10 days after birth all fibers appear normal; between Week 3 and 4 there is massive fiber degeneration followed by regeneration in which close to 100% of the fibers are repaired or replaced. New fibers arising in adult mice are characterized by expression of fetal myosin mRNAs in whole muscle extracts, and by staining of individual fibers with an embryonic myosin heavy chain-specific antibody. By 10 weeks of age new fiber replacement rate, indicated by frequency of fibers reacting with antibody, is reduced to about 10%, and by 1 year of age less than 1% of the fibers are being replaced at rates above control. Total fiber number also remains fairly constant. We conclude that the fibers regenerating up to 10 weeks of age become stabilized and do not undergo further rounds of degeneration and regeneration. This is consistent with the observed benign phenotype of adult mdx animals and with the idea that once-regenerated fibers escape the catastrophic dystrophic phenotype by acquiring a function that compensates for their mdx mutation. The mechanism by which regenerated mdx fibers restore adequate function in the absence of dystrophin may, when understood, provide clues to effective nongenetic interventions for muscular dystrophy in humans where regenerated fibers continue to degenerate and where the disease is often fatal.