scholarly journals Tissue-specific transcription of the cardiac myosin light-chain 2 gene is regulated by an upstream repressor element.

1991 ◽  
Vol 11 (3) ◽  
pp. 1676-1685 ◽  
Author(s):  
R A Shen ◽  
S K Goswami ◽  
E Mascareno ◽  
A Kumar ◽  
M A Siddiqui
Keyword(s):  
Gene Expression ◽  
Skeletal Muscle ◽  
Cardiac Muscle ◽  
Light Chain ◽  
Myosin Light Chain ◽  
Muscle Cells ◽  
Skeletal Muscle Cells ◽  
Tissue Specific ◽  
Myosin Light Chain 2 ◽  
Functional Components

Physiological expression of the cardiac muscle myosin light-chain 2 (MLC-2) gene in chickens is restricted to cardiac muscle tissue only, at least during the late embryonic to adult stages of development. The mechanism by which cardiac MLC-2 gene expression is repressed in differentiated noncardiac muscle tissues is unknown. Using sequential 5'-deletion mutants of the cardiac MLC-2 promoter introduced into primary skeletal muscle cells in culture, we have demonstrated that a 89-bp region, designated the cardiac-specific sequence (CSS), is essential for repression of cardiac MLC-2 expression in skeletal muscle. Removal of the CSS sequence alone allows transcription in skeletal muscle cells without affecting the transcriptional activity of the promoter in cardiac muscle cells. DNase I footprinting and gel shift assays indicate that protein binding to sequences in the CSS domain occurs readily in nuclear extracts obtained from skeletal muscle but not in extracts isolated under identical conditions from cardiac muscle. Thus, it appears that a negative regulatory mechanism accounts for the lack of expression of the cardiac MLC-2 gene in skeletal muscle and that the CSS element and its binding proteins are important functional components of the regulatory apparatus which ensures the developmental program for cardiac tissue-specific gene expression.

Tissue-specific transcription of the cardiac myosin light-chain 2 gene is regulated by an upstream repressor element

1991 ◽  
Vol 11 (3) ◽  
pp. 1676-1685
Author(s):  
R A Shen ◽  
S K Goswami ◽  
E Mascareno ◽  
A Kumar ◽  
M A Siddiqui
Keyword(s):  
Gene Expression ◽  
Skeletal Muscle ◽  
Cardiac Muscle ◽  
Light Chain ◽  
Myosin Light Chain ◽  
Muscle Cells ◽  
Skeletal Muscle Cells ◽  
Tissue Specific ◽  
Myosin Light Chain 2 ◽  
Functional Components

Physiological expression of the cardiac muscle myosin light-chain 2 (MLC-2) gene in chickens is restricted to cardiac muscle tissue only, at least during the late embryonic to adult stages of development. The mechanism by which cardiac MLC-2 gene expression is repressed in differentiated noncardiac muscle tissues is unknown. Using sequential 5'-deletion mutants of the cardiac MLC-2 promoter introduced into primary skeletal muscle cells in culture, we have demonstrated that a 89-bp region, designated the cardiac-specific sequence (CSS), is essential for repression of cardiac MLC-2 expression in skeletal muscle. Removal of the CSS sequence alone allows transcription in skeletal muscle cells without affecting the transcriptional activity of the promoter in cardiac muscle cells. DNase I footprinting and gel shift assays indicate that protein binding to sequences in the CSS domain occurs readily in nuclear extracts obtained from skeletal muscle but not in extracts isolated under identical conditions from cardiac muscle. Thus, it appears that a negative regulatory mechanism accounts for the lack of expression of the cardiac MLC-2 gene in skeletal muscle and that the CSS element and its binding proteins are important functional components of the regulatory apparatus which ensures the developmental program for cardiac tissue-specific gene expression.

A new serum-responsive, cardiac tissue-specific transcription factor that recognizes the MEF-2 site in the myosin light chain-2 promoter

1993 ◽  
Vol 13 (2) ◽  
pp. 1222-1231
Author(s):  
M D Zhou ◽  
S K Goswami ◽  
M E Martin ◽  
M A Siddiqui
Keyword(s):  
Transcription Factor ◽  
Cardiac Muscle ◽  
Light Chain ◽  
Myosin Light Chain ◽  
Cardiac Tissue ◽  
Muscle Cells ◽  
Cardiac Muscle Cells ◽  
Tissue Specific ◽  
Specific Transcription Factor ◽  
Myosin Light Chain 2

We have identified a serum-responsive, cardiac tissue-specific transcription factor, BBF-1, that recognizes an AT-rich sequence (element B), identical to the myocyte enhancer factor (MEF-2) target site, in the cardiac myosin light chain-2 (MLC-2) promoter. Deletion of the element B sequence alone from the cardiac MLC-2 promoter causes, as does that of the MEF-2 site from other promoters and the enhancer of skeletal muscle genes, a marked reduction of transcription. BBF-1 is distinguishable from cardiac MEF-2 on the basis of immunoprecipitation with an antibody which recognizes MEF-2 but not BBF-1. Unlike MEF-2, BBF-1 is present exclusively in nuclear extracts from cardiac muscle cells cultured in a medium containing a high concentration of serum. Removal of serum from culture medium abolishes BBF-1 activity selectively with a concomitant loss of the positive regulatory effect of element B on MLC-2 gene transcription, indicating that there is a correlation between the BBF-1 binding activity and the tissue-specific role of the element B (MEF-2 site) sequence. The loss of element B-mediated activation of transcription is reversed following the refeeding of cells with serum-containing medium. These data demonstrate that cardiac muscle cells contain two distinct protein factors, MEF-2 and BBF-1, which bind to the same target site but that, unlike MEF-2, BBF-1 is serum inducible and cardiac tissue specific. BBF-1 thus appears to be a crucial member of the MEF-2 family of proteins which will serve as an important tool in understanding the regulatory mechanism(s) underlying cardiogenic differentiation.

scholarly journals A new serum-responsive, cardiac tissue-specific transcription factor that recognizes the MEF-2 site in the myosin light chain-2 promoter.

1993 ◽  
Vol 13 (2) ◽  
pp. 1222-1231 ◽  
Author(s):  
M D Zhou ◽  
S K Goswami ◽  
M E Martin ◽  
M A Siddiqui
Keyword(s):  
Transcription Factor ◽  
Cardiac Muscle ◽  
Light Chain ◽  
Myosin Light Chain ◽  
Cardiac Tissue ◽  
Muscle Cells ◽  
Cardiac Muscle Cells ◽  
Tissue Specific ◽  
Specific Transcription Factor ◽  
Myosin Light Chain 2

We have identified a serum-responsive, cardiac tissue-specific transcription factor, BBF-1, that recognizes an AT-rich sequence (element B), identical to the myocyte enhancer factor (MEF-2) target site, in the cardiac myosin light chain-2 (MLC-2) promoter. Deletion of the element B sequence alone from the cardiac MLC-2 promoter causes, as does that of the MEF-2 site from other promoters and the enhancer of skeletal muscle genes, a marked reduction of transcription. BBF-1 is distinguishable from cardiac MEF-2 on the basis of immunoprecipitation with an antibody which recognizes MEF-2 but not BBF-1. Unlike MEF-2, BBF-1 is present exclusively in nuclear extracts from cardiac muscle cells cultured in a medium containing a high concentration of serum. Removal of serum from culture medium abolishes BBF-1 activity selectively with a concomitant loss of the positive regulatory effect of element B on MLC-2 gene transcription, indicating that there is a correlation between the BBF-1 binding activity and the tissue-specific role of the element B (MEF-2 site) sequence. The loss of element B-mediated activation of transcription is reversed following the refeeding of cells with serum-containing medium. These data demonstrate that cardiac muscle cells contain two distinct protein factors, MEF-2 and BBF-1, which bind to the same target site but that, unlike MEF-2, BBF-1 is serum inducible and cardiac tissue specific. BBF-1 thus appears to be a crucial member of the MEF-2 family of proteins which will serve as an important tool in understanding the regulatory mechanism(s) underlying cardiogenic differentiation.

Phosphorylation of myosin in permeabilized mammalian cardiac and skeletal muscle cells

1986 ◽  
Vol 250 (4) ◽  
pp. C657-C660 ◽  
Author(s):  
H. L. Sweeney ◽  
J. T. Stull
Keyword(s):  
Skeletal Muscle ◽  
Cardiac Muscle ◽  
Light Chain ◽  
Myosin Light Chain Kinase ◽  
Skeletal Muscles ◽  
Myosin Light Chain ◽  
Muscle Cells ◽  
Ventricular Muscle ◽  
Myosin Phosphorylation ◽  
Maximal Activation

The effect of myosin phosphorylation on tension production at less than 50% maximal activation by Ca2+ was examined in rabbit psoas and ventricular muscle. For psoas fibers, tension was determined at pCa 6.0, 5.8, 5.6, 5.5, and 5.4. Myosin light chain kinase (0.15 microM) and calmodulin (2 microM) were added, and the fibers were incubated at pCa 5.4, which resulted in an increase in light chain phosphorylation (P-light chain) from 5-10 to 60-75%. After 5 min, the sequence of pCa activations was repeated. An identical protocol was followed for cardiac muscle, except the activation solutions were pCa 6.2, 6.0, 5.9, 5.8, and 5.6. Phosphorylation of P-light chain increased tension in both permeabilized cardiac and skeletal muscle fibers. The effect manifested itself as a leftward shift in the pCa-tension relationship at levels below 50% maximal activation, with a decrease in the slope of the pCa-tension relationship. These results indicate that P-light chain phosphorylation affects actin-myosin interactions in cardiac and skeletal muscles at submaximal levels of Ca2+ activation

scholarly journals Impact of Adenosine A2 Receptor Ligands on BCL2 Expression in Skeletal Muscle Cells

2021 ◽  
Vol 11 (5) ◽  
pp. 2272
Author(s):  
Mansour Haddad
Keyword(s):  
Gene Expression ◽  
Skeletal Muscle ◽  
Adenosine Receptors ◽  
Muscle Cells ◽  
Skeletal Muscle Cells ◽  
Qrt Pcr ◽  
A2a Receptor ◽  
Adenosine A2a ◽  
Bcl2 Expression ◽  
Bcl2 Gene

Background: Adenosine plays the role of regulating cell differentiation, proliferation, and apoptosis in various kinds of cells through the B-cell lymphoma 2 (BCL2) pathway. Objectives: Since anti-apoptotic (BCL2) expression plays a role in controlling apoptosis in some cell lines, this study was designed to investigate whether adenosine analogue, NECA (non-selective adenosine receptors agonist), selective adenosine A2B receptor antagonist, PSB 603, and a selective adenosine A2A receptor agonist, CG21680, affect BCL2-gene expression in the skeletal muscle cells of rats. The purpose of this investigation was to test the hypothesis that CG21680 treatment would significantly intensify BCL2 gene expression in rat skeletal muscle. Methods: Flasks measuring 25 cm2 were employed in culturing the rat L6 skeletal muscle cells. After treating these differential cells, the relative mRNA expression level for the BCL2 gene, at varying conditions of treatment, was measured using quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Results: From the qRT-PCR analysis results, it was concluded that BCL2 expression was markedly amplified after selective adenosine A2A receptor agonist, CGS21680 (p < 0.01) treatment. More prospective validation for the adenosine receptors’ contribution in modulating apoptosis by NECA was delivered by the outcomes from the combined pre-treatment of the cells with NECA and PSB 603. These outcomes show that when starved skeletal muscle cells are treated with a combination of NECA and 100 nM PSB 603, there was a substantial decrease in comparison to either treatment used on its own. Conclusions: This study’s results showed, for the first time, an increase in BCL2 gene expression within skeletal muscle after CGS21680 treatment. Hence, the prospective escalation in BCL2 protein expression might have a protective role to play against apoptosis and avert damage to the skeletal muscle.

CARP, a cardiac ankyrin repeat protein, is downstream in the Nkx2-5 homeobox gene pathway

Development ◽  
1997 ◽  
Vol 124 (4) ◽  
pp. 793-804 ◽  
Author(s):  
Y. Zou ◽  
S. Evans ◽  
J. Chen ◽  
H.C. Kuo ◽  
R.P. Harvey ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cardiac Myocytes ◽  
Light Chain ◽  
Myosin Light Chain ◽  
Homeobox Gene ◽  
Ankyrin Repeat ◽  
Repeat Protein ◽  
Ankyrin Repeat Protein ◽  
Myosin Light Chain 2 ◽  
Cardiac Ankyrin Repeat Protein

To identify the molecular pathways that guide cardiac ventricular chamber specification, maturation and morphogenesis, we have sought to characterize factors that regulate the expression of the ventricular myosin light chain-2 gene, one of the earliest markers of ventricular regionalization during mammalian cardiogenesis. Previously, our laboratory identified a 28 bp HF-la/MEF-2 element in the MLC-2v promoter region, which confers cardiac ventricular chamber-specific gene expression during murine cardiogenesis, and showed that the ubiquitous transcription factor YB-1 binds to the HF-la site in conjunction with a co-factor. In a search for interacting co-factors, a nuclear ankyrin-like repeat protein CARP (cardiac ankyrin repeat protein) was isolated from a rat neonatal heart cDNA library by yeast two-hybrid screening, using YB-1 as the bait. Co-immunoprecipitation and GST-CARP pulldown studies reveal that CARP forms a physical complex with YB-1 in cardiac myocytes and immunostaining shows that endogenous CARP is localized in the cardiac myocyte nucleus. Co-transfection assays indicate that CARP can negatively regulate an HF-1-TK minimal promoter in an HF-1 sequence-dependent manner in cardiac myocytes, and CARP displays a transcriptional inhibitory activity when fused to a GAL4 DNA-binding domain in both cardiac and noncardiac cell context. Northern analysis revealed that carp mRNA is highly enriched in the adult heart, with only trace levels in skeletal muscle. During murine embryogenesis, endogenous carp expression was first clearly detected as early as E8.5 specifically in heart and is regulated temporally and spatially in the myocardium. Nkx2-5, the murine homologue of Drosophila gene tinman was previously shown to be required for heart tube looping morphogenesis and ventricular chamber-specific myosin light chain-2 expression during mammalian heart development. In Nkx2-5(−/−)embryos, carp expression was found to be significantly and selectively reduced as assessed by both whole-mount in situ hybridizations and RNase protection assays, suggesting that carp is downstream of the homeobox gene Nkx2-5 in the cardiac regulatory network. Co-transfection assays using a dominant negative mutant Nkx2-5 construct with CARP promoter-luciferase reporter constructs in cardiac myocytes confirms that Nkx2-5 either directly or indirectly regulates carp at the transcriptional level. Finally, a carp promoter-lacZ transgene, which displays cardiac-specific expression in wild-type and Nkx2-5(+/−) background, was also significantly reduced in Nkx2-5(−/−) embryos, indicating that Nkx2-5 either directly or indirectly regulates carp promoter activity during in vivo cardiogenesis as well as in cultured cardiac myocytes. Thus, CARP is a YB-1 associated factor and represents the first identified cardiac-restricted downstream regulatory gene in the homeobox gene Nkx2-5 pathway and may serve as a negative regulator of HF-1-dependent pathways for ventricular muscle gene expression.

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