scholarly journals Neonatal and adult myosin heavy chain isoforms in a nerve-muscle culture system.

1986 ◽  
Vol 103 (3) ◽  
pp. 995-1005 ◽  
Author(s):  
M S Ecob-Prince ◽  
M Jenkison ◽  
G S Butler-Browne ◽  
R G Whalen
Keyword(s):  
Spinal Cord ◽  
Myosin Heavy Chain ◽  
Heavy Chain ◽  
Culture System ◽  
Contractile Activity ◽  
Muscle Fibers ◽  
Spinal Cord Tissue ◽  
Mouse Muscle ◽  
Embryonic Mouse ◽  
Alpha Bungarotoxin

When adult mouse muscle fibers are co-cultured with embryonic mouse spinal cord, the muscle regenerates to form myotubes that develop cross-striations and contractions. We have investigated the myosin heavy chain (MHC) isoforms present in these cultures using polyclonal antibodies to the neonatal, adult fast, and slow MHC isoforms of rat (all of which were shown to react specifically with the analogous mouse isoforms) in an immunocytochemical assay. The adult fast MHC was absent in newly formed myotubes but was found at later times, although it was absent when the myotubes myotubes were cultured without spinal cord tissue. When nerve-induced muscle contractions were blocked by the continuous presence of alpha-bungarotoxin, there was no decrease in the proportion of fibers that contained adult fast MHC. Neonatal and slow MHC were found at all times in culture, even in the absence of the spinal cord, and so their expression was not thought to be nerve-dependent. Thus, in this culture system, the expression of adult fast MHC required the presence of the spinal cord, but was probably not dependent upon nerve-induced contractile activity in the muscle fibers.

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.

Distribution of myosin heavy chain isoforms in non-weight-bearing rat soleus muscle fibers

1996 ◽  
Vol 81 (6) ◽  
pp. 2540-2546 ◽  
Author(s):  
Robert J. Talmadge ◽  
Roland R. Roy ◽  
V. Reggie Edgerton
Keyword(s):  
Myosin Heavy Chain ◽  
Soleus Muscle ◽  
Heavy Chain ◽  
De Novo ◽  
Weight Bearing ◽  
Muscle Fibers ◽  
Myosin Heavy Chain Isoforms ◽  
Hindlimb Suspension ◽  
Type I ◽  
Rat Soleus Muscle

Talmadge, Robert J., Roland R. Roy, and V. Reggie Edgerton.Distribution of myosin heavy chain isoforms in non-weight-bearing rat soleus muscle fibers. J. Appl. Physiol. 81(6): 2540–2546, 1996.—The effects of 14 days of spaceflight (SF) or hindlimb suspension (HS) (Cosmos 2044) on myosin heavy chain (MHC) isoform content of the rat soleus muscle and single muscle fibers were determined. On the basis of electrophoretic analyses, there was a de novo synthesis of type IIx MHC but no change in either type I or IIa MHC isoform proportions after either SF or HS compared with controls. The percentage of fibers containing only type I MHC decreased by 26 and 23%, and the percentage of fibers with multiple MHCs increased from 6% in controls to 32% in HS and 34% in SF rats. Type IIx MHC was always found in combination with another MHC or combination of MHCs; i.e., no fibers contained type IIx MHC exclusively. These data suggest that the expression of the normal complement of MHC isoforms in the adult rat soleus muscle is dependent, in part, on normal weight bearing and that the absence of weight bearing induces a shift toward type IIx MHC protein expression in the preexisting type I and IIa fibers of the soleus.

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

1996 ◽  
Vol 271 (5) ◽  
pp. C1447-C1456 ◽  
Author(s):  
K. L. Byron ◽  
J. L. Puglisi ◽  
J. R. Holda ◽  
D. Eble ◽  
A. M. Samarel
Keyword(s):  
Myosin Heavy Chain ◽  
Heavy Chain ◽  
Protein Turnover ◽  
Ventricular Myocytes ◽  
Contractile Activity ◽  
Myofibrillar Protein ◽  
Neonatal Rat ◽  
Heart Cells ◽  
Cell Shortening ◽  
Rat Heart Cells

Blockade of L-type Ca2+ channels in spontaneously contracting cultured neonatal rat ventricular myocytes causes contractile arrest, myofibrillar disassembly, and accelerated myofibrillar protein turnover. To determine whether myofibrillar protein turnover. To determine whether myofibrillar atrophy results indirectly from loss of mechanical signals or directly from alterations in intracellular Ca2+ concentration ([Ca2+]i), contractile activity was inhibited with verapamil (10 microM) or 2,3-butanedione monoxime (BDM), and their effects on cell shortening, [Ca2+]i, and myosin heavy chain (MHC) turnover were assessed. Control cells demonstrated spontaneous [Ca2+]i transients (peak amplitude 232 +/- 15 nM, 1-2 Hz) and vigorous contractile activity. Verapamil inhibited shortening by eliminating spontaneous [Ca2+]i transients. Low concentrations of BDM (5.0-7.5 mM) had no effect on basal or peak [Ca2+]i transient amplitude but reduced cell shortening, whereas 10 mM BDM reduced both [Ca2+]i transient amplitude and shortening. Both agents inhibited MHC synthesis, but only verapamil accelerated MHC degradation. Thus MHC half-life does not change in parallel with contractile activity but rather more closely follows changes in [Ca2+]i. [Ca2+]i transients appear critical in maintaining myofibrillar assembly and preventing accelerated MHC proteolysis.

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.

Role of USF1 phosphorylation on cardiac α-myosin heavy chain promoter activity

2002 ◽  
Vol 283 (1) ◽  
pp. H213-H219 ◽  
Author(s):  
Qianxun Xiao ◽  
Agnes Kenessey ◽  
Kaie Ojamaa
Keyword(s):  
Protein Kinase ◽  
Myosin Heavy Chain ◽  
Molecular Mechanism ◽  
Heavy Chain ◽  
Promoter Activity ◽  
Cardiac Myocyte ◽  
Contractile Function ◽  
Contractile Activity ◽  
Cell Mass ◽  
Two Dimensional Gel Electrophoresis

Contractile activity of the cardiac myocyte is required for maintaining cell mass and phenotype, including expression of the cardiac-specific α-myosin heavy chain (α-MHC) gene. An E-box hemodynamic response element (HME) located at position −47 within the α-MHC promoter is both necessary and sufficient to confer contractile responsiveness to the gene and has been shown to bind upstream stimulatory factor-1 (USF1). When studied in spontaneously contracting cardiac myocytes, there is enhanced binding of USF1 to the HME compared with quiescent cells, which correlates with a threefold increase in α-MHC promoter activity. A molecular mechanism by which contractile function modulates α-MHC transcriptional activity may involve signaling via phosphorylation of USF1. The present studies showed that purified rat USF1 was phosphorylated in vitro by protein kinase C (PKC) and cAMP-dependent protein kinase (PKA) but not casein kinase II. Phosphorylated USF1 by either PKC or PKA had increased DNA binding activity to the HME. PKC-mediated phosphorylation also leads to the formation of USF1 multimers as assessed by gel shift assay. Analysis of in vivo phosphorylated nuclear proteins from cultured ventricular myocytes showed that USF1 was phosphorylated, and resolution by two-dimensional gel electrophoresis identified at least two distinct phosphorylated USF1 molecules. These results suggest that endogenous kinases can covalently modify USF1 and provide a potential molecular mechanism by which the contractile stimulus mediates changes in myocyte gene transcription.

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