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 Effect of creatine on contents of myosin heavy chain and myosin-heavy-chain mRNA in steady-state chicken muscle-cell cultures

1984 ◽  
Vol 218 (3) ◽  
pp. 871-876 ◽  
Author(s):  
R B Young ◽  
R M Denome
Keyword(s):  
Steady State ◽  
Myosin Heavy Chain ◽  
Muscle Cell ◽  
Cell Cultures ◽  
Heavy Chain ◽  
Synthesis Rate ◽  
Adult Skeletal Muscle ◽  
Muscle Cultures ◽  
Chain Synthesis

Embryonic-chick muscle cells reach a steady state with respect to protein metabolism after approx. 1 week in cell culture. To determine if this steady state could be altered by the administration of agents that have been reported to stimulate myosin heavy-chain synthesis, 7-day muscle-cell cultures were treated with 0-1 mM-creatine. Incorporation of [3H]leucine into myosin heavy chain was stimulated by 30-40% at the optimum creatine concentration (0.2 mM), but this stimulation was blocked when actinomycin D (10 micrograms/ml) was also present. However, the quantity of myosin-heavy-chain mRNA as measured by hybridization in vitro was only 15% higher in creatine-treated cultures, and was therefore not entirely responsible for the observed effect. It is important to note that creatine only exerted its action on myosin-heavy-chain synthesis rate in steady-state cultures; creatine was ineffective in altering this rate in rapidly differentiating 3-day muscle cultures. Finally, muscle-cell cultures that had been grown for the entire 7-day culture period in the presence of 0.2 mM-creatine were assayed for quantity of myosin heavy chain. Control and creatine-treated cultures contained 12.7 +/- 1.5 and 20.5 +/- 1.8 micrograms/dish respectively. In conclusion, creatine apparently enhances the quantity of myosin heavy chain in steady-state embryonic muscle-cell cultures, but it probably does not mediate regulation of myosin content in adult skeletal muscle.

Control of AMP deaminase 1 binding to myosin heavy chain

1998 ◽  
Vol 275 (3) ◽  
pp. C870-C881 ◽  
Author(s):  
Ichiro Hisatome ◽  
Takayuki Morisaki ◽  
Hiroshi Kamma ◽  
Takako Sugama ◽  
Hiroko Morisaki ◽  
...  
Keyword(s):  
Myosin Heavy Chain ◽  
Heavy Chain ◽  
Sequence Similarity ◽  
Critical Role ◽  
Energy Charge ◽  
Adenylate Energy Charge ◽  
Amp Deaminase ◽  
Amino Terminal

AMP deaminase (AMPD) plays a central role in preserving the adenylate energy charge in myocytes following exercise and in producing intermediates for the citric acid cycle in muscle. Prior studies have demonstrated that AMPD1 binds to myosin heavy chain (MHC) in vitro; binding to the myofibril varies with the state of muscle contraction in vivo, and binding of AMPD1 to MHC is required for activation of this enzyme in myocytes. The present study has identified three domains in AMPD1 that influence binding of this enzyme to MHC using a cotransfection model that permits assessment of mutations introduced into the AMPD1 peptide. One domain that encompasses residues 178–333 of this 727-amino acid peptide is essential for binding of AMPD1 to MHC. This region of AMPD1 shares sequence similarity with several regions of titin, another MHC binding protein. Two additional domains regulate binding of this peptide to MHC in response to intracellular and extracellular signals. A nucleotide binding site, which is located at residues 660–674, controls binding of AMPD1 to MHC in response to changes in intracellular ATP concentration. Deletion analyses demonstrate that the amino-terminal 65 residues of AMPD1 play a critical role in modulating the sensitivity to ATP-induced inhibition of MHC binding. Alternative splicing of the AMPD1 gene product, which alters the sequence of residues 8–12, produces two AMPD1 isoforms that exhibit different MHC binding properties in the presence of ATP. These findings are discussed in the context of the various roles proposed for AMPD in energy production in the myocyte.

Differential regulation by thyroid hormones of myosin heavy chain α and β mRNAs in the rat ventricular myocardium

1989 ◽  
Vol 122 (1) ◽  
pp. 193-200 ◽  
Author(s):  
N. K. Green ◽  
J. A. Franklyn ◽  
J. A. O. Ahlquist ◽  
M. D. Gammage ◽  
M. C. Sheppard
Keyword(s):  
Myosin Heavy Chain ◽  
Cardiac Myocytes ◽  
Heavy Chain ◽  
Ventricular Myocardium ◽  
Mhc Genes ◽  
Specific Effects ◽  
Dependent Inhibition ◽  
Dose Dependent

ABSTRACT The effect of tri-iodothyronine (T3) treatment on myocardial levels of α and β myosin heavy chain (MHC) mRNAs in the rat was defined in vivo and in vitro. Dose–response experiments were performed in intact hypothyroid and euthyroid rats; in addition, studies in vitro examined the effect of T3 on MHC mRNAs in neonatal cardiac myocytes in primary culture. Specific α and β MHC mRNAs were determined by Northern blot and dot hybridization to oligonucleotide probes complementary to the 3′ untranslated regions of the MHC genes. An increase in myocardial β MHC mRNA was demonstrated in hypothyroidism, accompanied by a reduction in α MHC mRNA. Marked differences in the sensitivity of α and β MHC mRNAs to T3 replacement were found; a dose-dependent increase in α mRNA was evident at 6 h after T3 treatment, in the absence of consistent effects on β mRNA, whereas 72 h after T3 replacement was commenced, stimulatory effects of T3 on α MHC mRNA, evident at all doses, were accompanied by a dose-dependent inhibition of β MHC mRNA. No effect of thyroid status on actin mRNA was found, indicating the specificity of MHC gene regulation. T3 treatment of cardiac myocytes in vitro exerted similar actions on MHC mRNAs to those found in vivo, with a more marked influence on α than β MHC mRNA. These studies of the action of T3 in vivo and in vitro have thus demonstrated specific effects of T3 on pretranslational regulation of the α and β MHC genes, influences which differ not only in terms of stimulation or inhibition, but also in magnitude of effect. Journal of Endocrinology (1989) 122, 193–200

Molecular characterization of a developmentally regulated porcine skeletal myosin heavy chain gene and its 5′ regulatory region

1995 ◽  
Vol 108 (4) ◽  
pp. 1779-1789 ◽  
Author(s):  
K.C. Chang ◽  
K. Fernandes ◽  
M.J. Dauncey
Keyword(s):  
Myosin Heavy Chain ◽  
Heavy Chain ◽  
Transcriptional Start Site ◽  
Regulatory Region ◽  
Chain Gene ◽  
Specific Expression ◽  
Slow Fibre ◽  
Skeletal Myosin

Members of the myosin heavy chain (MyHC) gene family show developmental stage- and spatial-specificity of expression. We report on the characterization and identification of a porcine skeletal fast MyHC gene, including its corresponding 5′ end cDNA and 5′ regulatory region. This MyHC isoform was found exclusively in skeletal muscles from about the last quarter of gestation through to adulthood. Expression of this isoform was higher postnatally and its spatial distribution resembled a rosette cluster; each with a ring of fast fibres surrounding a central slow fibre. This rosette pattern was absent in the adult diaphragm but about 20% of the fibres continued to express this MyHC isoform. Further in vivo expression studies, in a variety of morphologically and functionally diverse muscles, showed that this particular skeletal MyHC isoform was expressed in fast oxidative-glycolytic fibres, suggesting that it was the equivalent of the fast IIA isoform. Two domains in the upstream regulatory region were found to confer differentiation-specific expression on C2 myotubes (−1007 to -828 and -455 to -101), based on in vitro transient expression assays using the chloramphenicol acetyltransferase (CAT) reporter gene. Interestingly, for high levels of CAT expression to occur, a 3′ region, extending from the transcriptional start site to part. of intron 2, must be present in all the DNA constructs used.

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.

Selected Contribution: Improved anoxic tolerance in rat diaphragm following intermittent hypoxia

2001 ◽  
Vol 90 (6) ◽  
pp. 2508-2513 ◽  
Author(s):  
Thomas L. Clanton ◽  
Valerie P. Wright ◽  
Peter J. Reiser ◽  
Paul F. Klawitter ◽  
Nanduri R. Prabhakar
Keyword(s):  
Myosin Heavy Chain ◽  
Heavy Chain ◽  
Intermittent Hypoxia ◽  
Contractile Function ◽  
Myosin Heavy Chain Isoforms ◽  
Adaptive Responses ◽  
Low Frequencies ◽  
Myosin Heavy Chain Isoform ◽  
Obstructive Sleep

Intermittent hypoxia (IH), associated with obstructive sleep apnea, initiates adaptive physiological responses in a variety of organs. Little is known about its influence on diaphragm. IH was simulated by exposing rats to alternating 15-s cycles of 5% O2 and 21% O2 for 5 min, 9 sets/h, 8 h/day, for 10 days. Controls did not experience IH. Diaphragms were excised 20–36 h after IH. Diaphragm bundles were studied in vitro or analyzed for myosin heavy chain isoform composition. No differences in maximum tetanic stress were observed between groups. However, peak twitch stress ( P < 0.005), twitch half-relaxation time ( P < 0.02), and tetanic stress at 20 or 30 Hz ( P < 0.05) were elevated in IH. No differences in expression of myosin heavy chain isoforms or susceptibility to fatigue were seen. Contractile function after 30 min of anoxia (95% N2-5% CO2) was markedly preserved at all stimulation frequencies during IH and at low frequencies after 15 min of reoxygenation. Anoxia-induced increases in passive muscle force were eliminated in the IH animals ( P < 0.01). These results demonstrate that IH induces adaptive responses in the diaphragm that preserve its function in anoxia.

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.

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.

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