Localization of cardiac (alpha)-myosin heavy chain mRNA is regulated by its 3′ untranslated region via mechanical activity and translational block

1997 ◽  
Vol 110 (23) ◽  
pp. 2969-2978 ◽  
Author(s):  
P. Goldspink ◽  
W. Sharp ◽  
B. Russell
Keyword(s):  
Myosin Heavy Chain ◽  
Cardiac Myocytes ◽  
Heavy Chain ◽  
Untranslated Region ◽  
Contractile Activity ◽  
Intracellular Localization ◽  
Mechanical Activity ◽  
Mrna Levels ◽  
Coding Region ◽  
Reporter Protein

We have altered the spontaneous contractile activity of neonatal cardiac myocytes in culture to investigate the re-lationship between mechanical forces, myofibril assembly, and the localization and translation of (alpha)-myosin heavy chain mRNA. Immunofluorescence and in situ hybridization techniques revealed that contracting myocytes display well aligned myofibrils and a diffuse distribution of (alpha)-myosin heavy chain mRNA. Inhibition of contractile activity with the calcium channel blocker verapamil (10 microM) resulted in myofibril disassembly and a perinuclear mRNA distribution within six hours. There was a significant decrease (P<0. 05) of mRNA levels, 5 to 15 micron away from the nucleus following 6 hours of verapamil treatment compared with control cells. Inhibition of protein synthesis with cycloheximide (10 microM) also resulted in perinuclear mRNA localization despite having little effect on contractile activity or myofibril assembly. To determine if the 3′ untranslated region of (alpha)-myosin heavy chain mRNA was sufficient for localizing the entire message, a chimeric construct composed of beta-galactosidase coding region followed by (alpha)-myosin heavy chain 3′ untranslated region sequences was made as a reporter plasmid and transfected into cultured myocytes. A perinuclear accumulation of ss-galactosidase was exhibited in many of the contractile arrested cells (48.3+/−2.4%, n=7). In contrast, significantly fewer (P<0.05) contracting control (29.1+/−3.3%, n=7) and strongly contracting, isoproterenol-treated cells (27.2+/−6.1%, n=3) exhibited a perinuclear localization of protein. The distribution of the reporter protein was not affected by the contractile state in cells transfected with a constitutively translated 3′UTR. We propose that mechanical activity of neonatal cardiac myocytes regulates the intracellular localization of alpha-myosin heavy chain mRNA via the 3′ untranslated region mediated by an initial block in translation.

Mechanical activity in heart regulates translation of α-myosin heavy chain mRNA but not its localization

1999 ◽  
Vol 276 (6) ◽  
pp. H2013-H2019 ◽  
Author(s):  
Gordana Nikcevic ◽  
Maria C. Heidkamp ◽  
Merja Perhonen ◽  
Brenda Russell
Keyword(s):  
Myosin Heavy Chain ◽  
Heavy Chain ◽  
Mrna Translation ◽  
Neonatal Rat ◽  
Mechanical Activity ◽  
Mrna Levels ◽  
Rat Cardiomyocytes ◽  
Significant Difference ◽  
Polysomal Fraction ◽  
Mechanical Transduction

Mechanical inactivity depresses protein expression in cardiac muscle tissue and results in atrophy. We explore the mechanical transduction mechanism in spontaneously beating neonatal rat cardiomyocytes expressing the α-myosin heavy chain (α-MyHC) isoform by interfering with cross-bridge function [2,3-butanedione monoxime (BDM), 7.5 mM] without affecting cell calcium. The polysome content and α-MyHC mRNA levels in fractions from a sucrose gradient were analyzed. BDM treatment blocked translation at initiation (162 ± 12% in the nonpolysomal RNA fraction and 43 ± 6% in the polysomal fraction, relative to control as 100%; P < 0.05). There was an increase in α-MyHC mRNA from the nonpolysomal fraction (120.5 ± 7.7%; P < 0.05 compared with control) with no significant change in the heavy polysomes. In situ hybridization of α-MyHC mRNA was used to estimate message abundance as a function of the distance from the nucleus. The mRNA was dispersed through the cytoplasm in spontaneously beating cells as well as in BDM-treated cells (no significant difference). We conclude that direct inhibition of contractile machinery, but not calcium, regulates initiation of α-MyHC mRNA translation. However, calcium, not pure mechanical signals, appears to be important for message localization.

Contractile arrest accelerates myosin heavy chain degradation in neonatal rat heart cells

1992 ◽  
Vol 263 (3) ◽  
pp. C642-C652 ◽  
Author(s):  
A. M. Samarel ◽  
M. L. Spragia ◽  
V. Maloney ◽  
S. A. Kamal ◽  
G. L. Engelmann
Keyword(s):  
Myosin Heavy Chain ◽  
Heavy Chain ◽  
Contractile Activity ◽  
Neonatal Rat ◽  
Mechanical Activity ◽  
Mechanical Forces ◽  
Channel Blockade ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Rate Limiting

Mechanical forces influence the growth and metabolism of a variety of cells, including cultured neonatal rat ventricular myocytes. To determine whether mechanical activity affected the synthesis and turnover of myosin heavy chain (MHC) in these striated muscle cells, MHC fractional degradative rates were measured in spontaneously beating cells and in arrested myocytes in which contractile activity was prevented by L-channel blockade (with verapamil, nifedipine, nisoldipine, and diltiazem) or K+ depolarization. MHC degradative rates were measured as the difference between rates of MHC synthesis and accumulation and in pulse-chase biosynthetic labeling experiments. Both methods indicated that contractile arrest markedly increased MHC degradation. Contractile arrest produced by L-channel blockade accelerated MHC degradation to a greater extent than K+ depolarization. The signal transduction pathway linking contractile activity to alterations in MHC degradation did not involve protein kinase C (PKC), because MHC degradation was unaffected by activating PKC in arrested cells or inhibiting PKC in spontaneously beating cells. Chloroquine and E-64 did not suppress the accelerated MHC degradation, suggesting that the rate-limiting step in MHC turnover occurred before degradative processing by cellular proteinases. Using a computer simulation, we hypothesize that the rate-limiting step in MHC turnover preceded (or was coincident with) MHC release from thick filaments. Thus mechanical forces may influence MHC half-life by regulating the rate of myosin disassembly.

Regulation of rat ventricular myosin heavy chain expression by serum and contractile activity

1994 ◽  
Vol 267 (2) ◽  
pp. C520-C528 ◽  
Author(s):  
M. Qi ◽  
K. Ojamaa ◽  
E. G. Eleftheriades ◽  
I. Klein ◽  
A. M. Samarel
Keyword(s):  
Myosin Heavy Chain ◽  
Growth Medium ◽  
Heavy Chain ◽  
Ventricular Myocytes ◽  
Contractile Activity ◽  
Cellular Growth ◽  
Neonatal Rat ◽  
Mrna Levels ◽  
Serum Free ◽  
Serum Stimulation

To quantitatively analyze the effects of serum stimulation and contractile activity and their interaction on cellular growth and cardiac myosin heavy chain (MHC) gene expression, spontaneously contracting neonatal rat ventricular myocytes in primary culture were maintained in serum-free growth medium or growth medium supplemented with fetal bovine serum. Contractile activity in paired cultures was inhibited by addition of the calcium channel blocker verapamil (10 microM) to the culture medium. Both serum stimulation and contractile activity produced myocyte hypertrophy as assessed by increases in total protein, total RNA, protein-to-DNA ratios, and total MHC protein content. MHC isoenzyme analysis indicated that both MHC-alpha and MHC-beta proteins accumulated in response to serum stimulation and/or contractile activity. The increases in MHC-beta protein resulting from serum stimulation and contractile activity occurred in parallel with increases in MHC-beta mRNA. In contrast, MHC-alpha mRNA levels were relatively unaffected by serum stimulation but appeared to decrease in response to contractile activity. The protein kinase inhibitor staurosporine (5 nM) reduced MHC-beta expression in serum-free, contracting cultures and also prevented the serum-induced increase in MHC-beta mRNA observed in both contracting and arrested myocytes. Staurosporine also increased MHC-alpha mRNA levels in serum-free, contracting, and verapamil-arrested myocytes. These data suggest that both humoral and mechanical factors regulate MHC isoenzyme expression and cellular growth in neonatal ventricular myocytes.

Myosin heavy chain gene expression in neonatal rat heart cells: effects of [Ca2+]i and contractile activity

1997 ◽  
Vol 273 (2) ◽  
pp. C394-C403 ◽  
Author(s):  
M. Qi ◽  
J. L. Puglisi ◽  
K. L. Byron ◽  
K. Ojamaa ◽  
I. Klein ◽  
...  
Keyword(s):  
Gene Expression ◽  
Myosin Heavy Chain ◽  
Heavy Chain ◽  
Promoter Activity ◽  
Contractile Activity ◽  
Neonatal Rat ◽  
Luciferase Expression ◽  
Heart Cells ◽  
Mrna Levels ◽  
Mechanical Signals

To determine if mechanical signals or alterations in intracellular Ca2+ concentration ([Ca2+]i) affect myosin heavy chain (MHC) gene expression in spontaneously beating, neonatal rat ventricular myocytes, contractile activity was inhibited with verapamil, KCl, or 2,3-butanedione monoxime (BDM), and their acute and chronic effects on myocyte shortening, [Ca2+]i, and MHC gene expression were examined. Despite their differing effects on [Ca2+]i, verapamil, KCl, and BDM all inhibited contractile activity and markedly downregulated beta-MHC mRNA levels to 24 +/- 5, 21 +/- 7, and 6 +/- 2% of contracting cells, respectively. In contrast, these inhibitors of contraction upregulated alpha-MHC mRNA levels to 163 +/- 19, 156 +/- 7, and 198 +/- 20% of contracting cells, respectively. Transient transfection with a rat beta-MHC promoter-luciferase expression plasmid demonstrated that all inhibitors of contraction significantly decreased beta-MHC promoter activity. Paradoxically, contractile arrest also inhibited alpha-MHC promoter activity, suggesting that increased alpha-MHC mRNA levels resulted from posttranscriptional mechanisms. Actinomycin D mRNA stability assays indicated that alpha-MHC mRNA half-life was prolonged in noncontracting cells (33 h) compared with contracting myocytes (14 h). Contraction-dependent alterations in MHC gene expression were not dependent on release of angiotensin II or other growth factors into the culture medium. Thus intrinsic mechanical signals rather than alterations in [Ca2+]i regulate alpha-MHC and beta-MHC gene expression by both transcriptional and posttranscriptional mechanisms.

Tenotomy decreases reporter protein synthesis via the 3′-untranslated region of the β-myosin heavy chain mRNA

2000 ◽  
Vol 279 (1) ◽  
pp. C257-C265 ◽  
Author(s):  
William W. Ashley ◽  
Brenda Russell
Keyword(s):  
Protein Expression ◽  
Myosin Heavy Chain ◽  
Heavy Chain ◽  
Protein Interactions ◽  
Untranslated Region ◽  
Critical Role ◽  
Mobility Shift ◽  
Reporter Protein ◽  
Adult Rat ◽  
Protein Factor

We tested the hypothesis that the β-myosin heavy chain (β-MHC) 3′-untranslated region (UTR) mediates decreased protein expression after tenotomy of the rat soleus. We also tested the hypothesis that decreased protein expression is the result of RNA-protein interactions within the 3′-UTR. β-MHC was chosen for study because of its critical role in the function of postural muscles such as soleus. Adult rat soleus muscles were directly injected with luciferase (LUC) reporter constructs containing either the β-MHC or SV40 3′-UTR. After 48 h of tenotomy, there was no significant effect on LUC expression in the SV40 3′-UTR group. In the β-MHC 3′-UTR group, LUC expression was 37.3 ± 4% ( n = 5, P = 0.03) of that in sham controls. Gel mobility shift assays showed that a protein factor specifically interacts with the β-MHC 3′-UTR and that tenotomy significantly increases the level of this interaction (25 ± 7%, n = 5, P = 0.02). Thus the β-MHC 3′-UTR is directly involved in decreased protein expression that is probably due to increased RNA-protein binding within the UTR.

scholarly journals Tissue-Restricted Expression of the Cardiac α-Myosin Heavy Chain Gene Is Controlled by a Downstream Repressor Element Containing a Palindrome of Two Ets-Binding Sites

1998 ◽  
Vol 18 (12) ◽  
pp. 7243-7258 ◽  
Author(s):  
Madhu Gupta ◽  
Radovan Zak ◽  
Towia A. Libermann ◽  
Mahesh P. Gupta
Keyword(s):  
Gene Regulation ◽  
Myosin Heavy Chain ◽  
Cardiac Myocytes ◽  
Heavy Chain ◽  
Binding Sites ◽  
Cardiac Myocyte ◽  
Specific Expression ◽  
Tissue Specific Expression ◽  
Nonmuscle Cells ◽  
Muscle Gene

ABSTRACT The expression of the α-myosin heavy chain (MHC) gene is restricted primarily to cardiac myocytes. To date, several positive regulatory elements and their binding factors involved in α-MHC gene regulation have been identified; however, the mechanism restricting the expression of this gene to cardiac myocytes has yet to be elucidated. In this study, we have identified by using sequential deletion mutants of the rat cardiac α-MHC gene a 30-bp purine-rich negative regulatory (PNR) element located in the first intronic region that appeared to be essential for the tissue-specific expression of the α-MHC gene. Removal of this element alone elevated (20- to 30-fold) the expression of the α-MHC gene in cardiac myocyte cultures and in heart muscle directly injected with plasmid DNA. Surprisingly, this deletion also allowed a significant expression of the α-MHC gene in HeLa and other nonmuscle cells, where it is normally inactive. The PNR element required upstream sequences of the α-MHC gene for negative gene regulation. By DNase I footprint analysis of the PNR element, a palindrome of two high-affinity Ets-binding sites (CTTCCCTGGAAG) was identified. Furthermore, by analyses of site-specific base-pair mutation, mobility gel shift competition, and UV cross-linking, two different Ets-like proteins from cardiac and HeLa cell nuclear extracts were found to bind to the PNR motif. Moreover, the activity of the PNR-binding factor was found to be increased two- to threefold in adult rat hearts subjected to pressure overload hypertrophy, where the α-MHC gene is usually suppressed. These data demonstrate that the PNR element plays a dual role, both downregulating the expression of the α-MHC gene in cardiac myocytes and silencing the muscle gene activity in nonmuscle cells. Similar palindromic Ets-binding motifs are found conserved in the α-MHC genes from different species and in other cardiac myocyte-restricted genes. These results are the first to reveal a role of the Ets class of proteins in controlling the tissue-specific expression of a cardiac muscle gene.

scholarly journals Contractile activity is required for the expression of neonatal myosin heavy chain in embryonic chick pectoral muscle cultures.

1986 ◽  
Vol 103 (6) ◽  
pp. 2153-2161 ◽  
Author(s):  
L C Cerny ◽  
E Bandman
Keyword(s):  
Monoclonal Antibody ◽  
Myosin Heavy Chain ◽  
Heavy Chain ◽  
Contractile Activity ◽  
Pectoral Muscle ◽  
Chicken Muscle ◽  
High K ◽  
Muscle Cultures ◽  
Spontaneous Contractions

The expression of neonatal myosin heavy chain (MHC) was examined in developing embryonic chicken muscle cultures using a monoclonal antibody (2E9) that has been shown to be specific for that isoform (Bandman, E., 1985, Science (Wash. DC), 227: 780-782). After 1 wk in vitro some myotubes could be stained with the antibody, and the number of cells that reacted with 2E9 increased with time in culture. All myotubes always stained with a second monoclonal antibody that reacted with all MHC isoforms (AG19) or with a third monoclonal antibody that reacted with the embryonic but not the neonatal MHC (EB165). Quantitation by ELISA of an extract from 2-wk cultures demonstrated that the neonatal MHC represented between 10 and 15% of the total myosin. The appearance of the neonatal isoform was inhibited by switching young cultures to medium with a higher [K+] which has been shown to block spontaneous contractions of myotubes in culture. Furthermore, if mature cultures that reacted with the neonatal antibody were placed into high [K+] medium, neonatal MHC disappeared from virtually all myotubes within 3 d. The effect of high [K+] medium was reversible. When cultures maintained in high [K+] medium for 2 wk were placed in standard medium, which permitted the resumption of contractile activity, within 24 h cells began to react with the neonatal specific antibody, and by 72 h many myotubes were strongly positive. Since similar results were also obtained by inhibiting spontaneous contractions with tetrodotoxin, we suggest that the development of contractile activity is not only associated with the maturation of myotubes in culture, but may also be the signal that induces the expression of the neonatal MHC.

scholarly journals GATA-5 Is Involved in Leukemia Inhibitory Factor-responsive Transcription of the β-Myosin Heavy Chain Gene in Cardiac Myocytes

1999 ◽  
Vol 274 (18) ◽  
pp. 12811-12818 ◽  
Author(s):  
Tatsuya Morimoto ◽  
Koji Hasegawa ◽  
Satoshi Kaburagi ◽  
Tsuyoshi Kakita ◽  
Hiroshi Masutani ◽  
...  
Keyword(s):  
Myosin Heavy Chain ◽  
Cardiac Myocytes ◽  
Heavy Chain ◽  
Leukemia Inhibitory Factor ◽  
Inhibitory Factor ◽  
Chain Gene ◽  
Heavy Chain Gene ◽  
Myosin Heavy Chain Gene
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