Time course of in vitro development of engineered skeletal muscle: contractility and myosin heavy chain content

2006 ◽  
Vol 39 ◽  
pp. S227
Author(s):  
R.G. Dennis ◽  
P.E. Kosnik
Keyword(s):  
Skeletal Muscle ◽  
Myosin Heavy Chain ◽  
Heavy Chain ◽  
Time Course ◽  
Muscle Contractility ◽  
In Vitro Development ◽  
Vitro Development

Differential patterns of transcript accumulation during human myogenesis

1987 ◽  
Vol 7 (11) ◽  
pp. 4100-4114
Author(s):  
P Gunning ◽  
E Hardeman ◽  
R Wade ◽  
P Ponte ◽  
W Bains ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Carbonic Anhydrase ◽  
Myosin Heavy Chain ◽  
Heavy Chain ◽  
Time Course ◽  
Human Skeletal Muscle ◽  
Transcript Accumulation ◽  
Mrna Accumulation ◽  
Adult Human ◽  
Alpha Actin

We evaluated the extent to which muscle-specific genes display identical patterns of mRNA accumulation during human myogenesis. Cloned satellite cells isolated from adult human skeletal muscle were expanded in culture, and RNA was isolated from low- and high-confluence cells and from fusing cultures over a 15-day time course. The accumulation of over 20 different transcripts was compared in these samples with that in fetal and adult human skeletal muscle. The expression of carbonic anhydrase 3, myoglobin, HSP83, and mRNAs encoding eight unknown proteins were examined in human myogenic cultures. In general, the expression of most of the mRNAs was induced after fusion to form myotubes. However, several exceptions, including carbonic anhydrase and myoglobin, showed no detectable expression in early myotubes. Comparison of all transcripts demonstrated little, if any, identity of mRNA accumulation patterns. Similar variability was also seen for mRNAs which were also expressed in nonmuscle cells. Accumulation of mRNAs encoding alpha-skeletal, alpha-cardiac, beta- and gamma-actin, total myosin heavy chain, and alpha- and beta-tubulin also displayed discordant regulation, which has important implications for sarcomere assembly. Cardiac actin was the only muscle-specific transcript that was detected in low-confluency cells and was the major alpha-actin mRNA at all times in fusing cultures. Skeletal actin was transiently induced in fusing cultures and then reduced by an order of magnitude. Total myosin heavy-chain mRNA accumulation lagged behind that of alpha-actin. Whereas beta- and gamma-actin displayed a sharp decrease after initiation of fusion and thereafter did not change, alpha- and beta-tubulin were transiently induced to a high level during the time course in culture. We conclude that each gene may have its own unique determinants of transcript accumulation and that the phenotype of a muscle may not be determined so much by which genes are active or silent but rather by the extent to which their transcript levels are modulated. Finally, we observed that patterns of transcript accumulation established within the myotube cultures were consistent with the hypothesis that myoblasts isolated from adult tissue recapitulate a myogenic developmental program. However, we also detected a transient appearance of adult skeletal muscle-specific transcripts in high-confluence myoblast cultures. This indicates that the initial differentiation of these myoblasts may reflect a more complex process than simple recapitulation of development.

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

Cells ◽  
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.

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

2001 ◽  
Vol 12 (5) ◽  
pp. 1499-1508 ◽  
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.

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

1987 ◽  
Vol 35 (7) ◽  
pp. 717-722 ◽  
Author(s):  
M M Robinson
Keyword(s):  
Skeletal Muscle ◽  
Creatine Kinase ◽  
Myosin Heavy Chain ◽  
Heavy Chain ◽  
Fixation Procedure ◽  
Creatine Kinase B ◽  
Immunofluorescent Analysis ◽  
Embryonic Muscle ◽  
Creatine Kinase Isozymes

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.

scholarly journals Differential patterns of transcript accumulation during human myogenesis.

1987 ◽  
Vol 7 (11) ◽  
pp. 4100-4114 ◽  
Author(s):  
P Gunning ◽  
E Hardeman ◽  
R Wade ◽  
P Ponte ◽  
W Bains ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Carbonic Anhydrase ◽  
Myosin Heavy Chain ◽  
Heavy Chain ◽  
Time Course ◽  
Human Skeletal Muscle ◽  
Transcript Accumulation ◽  
Mrna Accumulation ◽  
Adult Human ◽  
Alpha Actin

We evaluated the extent to which muscle-specific genes display identical patterns of mRNA accumulation during human myogenesis. Cloned satellite cells isolated from adult human skeletal muscle were expanded in culture, and RNA was isolated from low- and high-confluence cells and from fusing cultures over a 15-day time course. The accumulation of over 20 different transcripts was compared in these samples with that in fetal and adult human skeletal muscle. The expression of carbonic anhydrase 3, myoglobin, HSP83, and mRNAs encoding eight unknown proteins were examined in human myogenic cultures. In general, the expression of most of the mRNAs was induced after fusion to form myotubes. However, several exceptions, including carbonic anhydrase and myoglobin, showed no detectable expression in early myotubes. Comparison of all transcripts demonstrated little, if any, identity of mRNA accumulation patterns. Similar variability was also seen for mRNAs which were also expressed in nonmuscle cells. Accumulation of mRNAs encoding alpha-skeletal, alpha-cardiac, beta- and gamma-actin, total myosin heavy chain, and alpha- and beta-tubulin also displayed discordant regulation, which has important implications for sarcomere assembly. Cardiac actin was the only muscle-specific transcript that was detected in low-confluency cells and was the major alpha-actin mRNA at all times in fusing cultures. Skeletal actin was transiently induced in fusing cultures and then reduced by an order of magnitude. Total myosin heavy-chain mRNA accumulation lagged behind that of alpha-actin. Whereas beta- and gamma-actin displayed a sharp decrease after initiation of fusion and thereafter did not change, alpha- and beta-tubulin were transiently induced to a high level during the time course in culture. We conclude that each gene may have its own unique determinants of transcript accumulation and that the phenotype of a muscle may not be determined so much by which genes are active or silent but rather by the extent to which their transcript levels are modulated. Finally, we observed that patterns of transcript accumulation established within the myotube cultures were consistent with the hypothesis that myoblasts isolated from adult tissue recapitulate a myogenic developmental program. However, we also detected a transient appearance of adult skeletal muscle-specific transcripts in high-confluence myoblast cultures. This indicates that the initial differentiation of these myoblasts may reflect a more complex process than simple recapitulation of development.

Sign in / Sign up

Export Citation Format

Share Document