Fixation and immunofluorescent analysis of creatine kinase isozymes in embryonic skeletal muscle.

Pectoral muscles from chicken embryos of various ages were examined with immunofluorescent and radiolabeled probes for the presence of brain-type creatine kinase (B-CK), muscle-specific creatine kinase (M-CK), muscle-specific myosin heavy chain (MHC), and cycling cells. The diffusible creatine kinase isozymes were not detectable by indirect immunofluorescence after standard histological fixation of embryonic muscle. However, a fixation procedure was devised that permitted immunodetection of the creatine kinase isozymes (particularly B-CK) in embryonic tissue from all stages of development studied. B-CK, M-CK, and MHC were all detected in post-mitotic muscle cells, but only B-CK was detected in cycling cells. Correlations between these findings and in vitro observations of a deterministic muscle lineage are discussed.

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ProNGF/p75NTR Axis Drives Fiber Type Specification by Inducing the Fast-Glycolytic Phenotype in Mouse Skeletal Muscle Cells

Cells ◽

10.3390/cells9102232 ◽

2020 ◽

Vol 9 (10) ◽

pp. 2232

Author(s):

Valentina Pallottini ◽

Mayra Colardo ◽

Claudia Tonini ◽

Noemi Martella ◽

Georgios Strimpakos ◽

...

Keyword(s):

Skeletal Muscle ◽

Myosin Heavy Chain ◽

Heavy Chain ◽

Myogenic Differentiation ◽

Fiber Type ◽

Pcr Analysis ◽

Mdx Mouse ◽

Mdx Mice ◽

Muscle Physiology

Despite its undisputable role in the homeostatic regulation of the nervous system, the nerve growth factor (NGF) also governs the relevant cellular processes in other tissues and organs. In this study, we aimed at assessing the expression and the putative involvement of NGF signaling in skeletal muscle physiology. To reach this objective, we employed satellite cell-derived myoblasts as an in vitro culture model. In vivo experiments were performed on Tibialis anterior from wild-type mice and an mdx mouse model of Duchenne muscular dystrophy. Targets of interest were mainly assessed by means of morphological, Western blot and qRT-PCR analysis. The results show that proNGF is involved in myogenic differentiation. Importantly, the proNGF/p75NTR pathway orchestrates a slow-to-fast fiber type transition by counteracting the expression of slow myosin heavy chain and that of oxidative markers. Concurrently, proNGF/p75NTR activation facilitates the induction of fast myosin heavy chain and of fast/glycolytic markers. Furthermore, we also provided evidence that the oxidative metabolism is impaired in mdx mice, and that these alterations are paralleled by a prominent buildup of proNGF and p75NTR. These findings underline that the proNGF/p75NTR pathway may play a crucial role in fiber type determination and suggest its prospective modulation as an innovative therapeutic approach to counteract muscle disorders.

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Regulation of Myosin Heavy Chain Expression during Rat Skeletal Muscle Development In Vitro

Molecular Biology of the Cell ◽

10.1091/mbc.12.5.1499 ◽

2001 ◽

Vol 12 (5) ◽

pp. 1499-1508 ◽

Cited By ~ 39

Author(s):

Carol E. Torgan ◽

Mathew P. Daniels

Keyword(s):

Skeletal Muscle ◽

T Cells ◽

Cyclosporin A ◽

Myosin Heavy Chain ◽

Heavy Chain ◽

Fiber Formation ◽

Immunofluorescent Staining ◽

Nuclear Factor ◽

Activated T Cells

Signals that determine fast- and slow-twitch phenotypes of skeletal muscle fibers are thought to stem from depolarization, with concomitant contraction and activation of calcium-dependent pathways. We examined the roles of contraction and activation of calcineurin (CN) in regulation of slow and fast myosin heavy chain (MHC) protein expression during muscle fiber formation in vitro. Myotubes formed from embryonic day 21 rat myoblasts contracted spontaneously, and ∼10% expressed slow MHC after 12 d in culture, as seen by immunofluorescent staining. Transfection with a constitutively active form of calcineurin (CN*) increased slow MHC by 2.5-fold as determined by Western blot. This effect was attenuated 35% by treatment with tetrodotoxin and 90% by administration of the selective inhibitor of CN, cyclosporin A. Conversely, cyclosporin A alone increased fast MHC by twofold. Cotransfection with VIVIT, a peptide that selectively inhibits calcineurin-induced activation of the nuclear factor of activated T-cells, blocked the effect of CN* on slow MHC by 70% but had no effect on fast MHC. The results suggest that contractile activity-dependent expression of slow MHC is mediated largely through the CN–nuclear factor of activated T-cells pathway, whereas suppression of fast MHC expression may be independent of nuclear factor of activated T-cells.

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SPECIFICITY OF CREATINE IN THE CONTROL OF MUSCLE PROTEIN SYNTHESIS

The Journal of Cell Biology ◽

10.1083/jcb.62.1.145 ◽

1974 ◽

Vol 62 (1) ◽

pp. 145-151 ◽

Cited By ~ 55

Author(s):

Joanne S. Ingwall ◽

Cynthia D. Weiner ◽

Manuel F. Morales ◽

Elaine Davis ◽

Frank E. Stockdale

Keyword(s):

Skeletal Muscle ◽

Amino Acids ◽

Protein Synthesis ◽

Myosin Heavy Chain ◽

Heavy Chain ◽

Muscle Protein ◽

Muscle Protein Synthesis ◽

Muscle Creatine ◽

Embryonic Muscle ◽

Chain Synthesis

This study provides additional evidence that creatine, an end product of contraction unique to muscle, is involved in the control of muscle protein synthesis. Creatine is shown to stimulate selectively the rate of synthesis of two major contractile proteins, actin and myosin heavy chain, in cultures of differentiating skeletal muscle. Creatine affects only the rate of synthesis and not the rate of degradation. Several creatine analogs are as effective as creatine in stimulating muscle protein synthesis, creatinine and amino acids such as arginine and glycine are not. Creatine stimulates myosin heavy chain synthesis twofold in cultures of embryonic muscle grown in either normal or dialyzed media.

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