Intracellular Calcium and Myosin Isoform Transitions

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Adult fast muscle fibers express distinct myosin heavy chains (MyHC) in differing proportions, but the mechanisms underlying their differential expression remain undefined. We used a variety of in vitro and in vivo approaches to explore the contribution of transcriptional regulation to adult fast MyHC expression. Here we show that 800 -1000 bp of a sequence upstream of the three mouse adult fast MyHC genes (Ia, IIb, and IId/x) are sufficient to drive muscle-specific and fiber-specific expression in vivo. We show that the upstream promoter region of the gene most abundantly expressed in mouse skeletal muscles, IIb MyHC, retains binding activity and transcriptional activation for three positive transcription factors, the serum response factor, Oct-1, and myocyte enhancer factor-2, whereas the other two genes (IIa and IId/x) have nucleotide substitutions in these sites that reduce binding and transcriptional activation. Finally, we demonstrate that regions upstream of 300 bp modulate the effects of these elements. Together, these data demonstrate that the quantitative differences in MyHC expression in mouse skeletal muscle have evolved at least in part through the elimination of positive-acting transcription factor binding sites.

Section: Methodsmentioning

confidence: 99%

Myocyte Enhancer Factor-2 and Serum Response Factor Binding Elements Regulate Fast Myosin Heavy Chain Transcription in Vivo

Allen¹,

Weber²,

Sycuro³

et al. 2005

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“…C, 2.0-kb SOCS-3 promoter activity increases during myoblast differentiation. C2C12 myoblasts were transiently transfected with the 2.0-kb mouse SOCS-3 promoter, and the cells were either maintained in the myoblast state or induced to differentiate for either 24, 48, or 72 h. At the defined time point, the luciferase activity was subsequently quantified as previously described (21). The data are expressed as the fold increases in promoter activity over the promoterless control plasmid (pGL3).…”

Section: Statisticsmentioning

confidence: 99%

“…The 2.0-kb SOCS-3 promoter or the empty vector (pGL3-luciferase) were co-transfected with either a vector expressing a dmSTAT3 or the corresponding empty vector to the dmSTAT3 into proliferating C2C12 myoblasts. After completion of the transfection, the myoblasts were induced to differentiate in differentiation medium with or without IGF-I (15 ng/ml) for 48 h. The luciferase activity was subsequently quantified and normalized as previously described (21). * indicates a value significantly different the control group and the dominant negative STAT3 IGF-I group (p Ͻ 0.05).…”

Section: Fig 4 Stat3 Phosphorylation Increases During Myoblast Diffmentioning

confidence: 99%

See 1 more Smart Citation

SOCS-3 Induces Myoblast Differentiation

Spangenburg

2005

Myoblast differentiation is characterized by a sequence of events that includes an increase in insulinlike growth factor (IGF)-I and contractile gene expression. The increase in IGF-I expression activates cell signaling mechanisms that participate in the differentiation process. One potential contributor is the SOCS-3 (suppressor of cytokine signaling-3) gene, which regulates signaling mechanisms and may be sensitive to changes in IGF-I concentrations. For the first time, the role of SOCS-3 is investigated in myoblast differentiation. SOCS-3 mRNA levels and SOCS-3 transcriptional activity increase during myoblast differentiation. SOCS-3 gene expression is induced, at least in part, by activation of the IGF-I receptor during myoblast differentiation. Overexpression of SOCS-3 cDNA significantly increased transcriptional activation of the 2.0-kb skeletal ␣-actin promoter in differentiating C2C12 myoblasts. In addition, overexpression of SOCS-3 specifically increased serum response factor-driven transcriptional activity but had no effect on nuclear-factor of activated T cell-driven transcriptional activity. SOCS-3 overexpression induced skeletal ␣-actin transcription in a myoblast cell line that cannot respond to endogenous IGF-I, indicating that SOCS-3 can contribute to the myoblast differentiation process in the absence of IGF-I. These data suggest that IGF-I induces myoblast differentiation, in part, by increasing SOCS-3 expression.

“…of Physiology, Han- (MKK6EE) reportedly promotes expression of fast but not slow MyHC protein while enhancing differentiation of C2C12 muscle cells (16). However, in C2C12 myotubes fast MyHCIIa promoter activity was found to be unaffected by p38 inhibition (17). The p38 MAPK pathway promotes skeletal muscle differentiation at least in part via muscle regulatory factors, recruitment of chromatin remodelling enzymes, and the transcription factor myocyte enhancer factor-2C (MEF-2C) (9,18).…”

mentioning

confidence: 99%

The p38α/β Mitogen-activated Protein Kinases Mediate Recruitment of CREB-binding Protein to Preserve Fast Myosin Heavy Chain IId/x Gene Activity in Myotubes

Meißner¹,

Chang

Kubis³

et al. 2007

In skeletal muscle, the transformation of fast into slow fiber type is accompanied by shifts in fiber type-specific gene expression that includes down-regulation of the adult fast fiber myosin heavy chain IId/x (MyHCIId/x) gene. Here, we report that the mitogen-activated protein kinases (MAPKs) p38␣/␤ regulate MyHCIId/x gene expression. Electrical stimulation of rabbit skeletal muscle cells with a slow fiber type activity pattern and treatment of C2C12 myotubes with Ca 2؉ -ionophore inhibited p38␣/␤ MAPKs and reduced fast fiber type MyHC protein expression and promoter activity. Pharmacological inhibition of p38␣/␤ also down-regulated MyHCII gene expression. In controls, binding of the myocyte enhancer factor-2 (MEF-2) isoforms C and D as a heterodimer to a proximal consensus site within the MyHCIId/x promoter and recruitment of a transcriptional coactivator, the CREB-binding protein CBP, were observed. Overexpression of wild type MEF-2C but not of a MEF-2C mutant that cannot be phosphorylated by p38 induced promoter activity. Mutation of the MEF-2-binding site decreased the inducing effect of overexpressed CBP. Inhibition of p38␣/␤ MAPKs abolished CBP binding, whereas enforced induction of p38 by activated MAPK kinase 6 (MKK6EE) enhanced binding of CBP and increased promoter activity. Furthermore, knockdown of endogenous CBP by RNA interference eliminated promoter activation by MEF-2C or MKK6EE. In electrical stimulated and Ca 2؉ -ionophore-treated myotubes, CBP was absent in complex formation at that site. Taken together, the data indicate that p38␣/␤ MAPKs-mediated coactivator recruitment at a proximal MEF-2 site is important for MyHCIId/x gene regulation in skeletal muscle.Skeletal muscle fibers have been classified into fiber types based on their contraction speed, force development, fatigability, and metabolic functions (1). The characteristic fiber types are fast twitch glycolytic (type IID/X and IIB), fast twitch oxidative/glycolytic (type IIA), and slow twitch oxidative (type I/␤). Fast fibers express the IID/X, IIB, or IIA isoform of the myosin heavy chain (MyHC), 3 whereas slow fibers predominantly express the type I/␤ isoform. The functional, biochemical, and morphological differences between fiber types are a consequence of different fiber-specific gene expression patterns. Fibers are characterized by a remarkable plasticity and can be modulated by chronic low frequency electrical stimulation depending on the imposed activity pattern (2). Physiologically, this transformation process in fiber type occurs in response to altered demands, such as increases in muscle activity (3). Fiber type shifts are also observed during aging and disease. The importance of changes in intracellular Ca 2ϩ as a trigger for fiber type transformation has clearly been demonstrated by a Ca 2ϩ -ionophore-induced fast-to-slow transformation in primary skeletal muscle cells (4, 5). The involvement of calcineurin, a Ca 2ϩ -calmodulin-regulated serine/threonine phosphatase, in transducing the Ca 2ϩ -signal into altered gene expre...