scholarly journals Potentiation in mouse lumbrical muscle without myosin light chain phosphorylation: Is resting calcium responsible?

2013 ◽  
Vol 141 (3) ◽  
pp. 297-308 ◽  
Author(s):  
Ian C. Smith ◽  
William Gittings ◽  
Jian Huang ◽  
Elliott M. McMillan ◽  
Joe Quadrilatero ◽  
...  
Keyword(s):  
Light Chain ◽  
Myosin Light Chain ◽  
Small Magnitude ◽  
Fluorescent Indicators ◽  
Twitch Force ◽  
Myosin Heavy Chain Isoform ◽  
Fura 2 ◽  
Lumbrical Muscle ◽  
Isometric Twitch ◽  
Fast Twitch

The increase in isometric twitch force observed in fast-twitch rodent muscles during or after activity, known universally as potentiation, is normally associated with myosin regulatory light chain (RLC) phosphorylation. Interestingly, fast muscles from mice devoid of detectable skeletal myosin light chain kinase (skMLCK) retain a reduced ability to potentiate twitch force, indicating the presence of a secondary origin for this characteristic feature of the fast muscle phenotype. The purpose of this study was to assess changes in intracellular cytosolic free Ca2+ concentration ([Ca2+]i) after a potentiating stimulus in mouse lumbrical muscle (37°C). Lumbricals were loaded with the Ca2+-sensitive fluorescent indicators fura-2 or furaptra to detect changes in resting and peak, respectively, intracellular Ca2+ levels caused by 2.5 s of 20-Hz stimulation. Although this protocol produced an immediate increase in twitch force of 17 ± 3% (all data are n = 10) (P < 0.01), this potentiation dissipated quickly and was absent 30 s afterward. Fura-2 fluorescence signals at rest were increased by 11.1 ± 1.3% (P < 0.01) during potentiation, indicating a significant increase in resting [Ca2+]i. Interestingly, furaptra signals showed no change to either the amplitude or the duration of the intracellular Ca2+ transients (ICTs) that triggered potentiated twitches during this time (P < 0.50). Immunofluorescence work showed that 77% of lumbrical fibers expressed myosin heavy chain isoform IIx and/or IIb, but with low expression of skMLCK and high expression of myosin phosphatase targeting subunit 2. As a result, lumbrical muscles displayed no detectable RLC phosphorylation either at rest or after stimulation. We conclude that stimulation-induced elevations in resting [Ca2+]i, in the absence of change in the ICT, are responsible for a small-magnitude, short-lived potentiation of isometric twitch force. If operative in other fast-twitch muscles, this mechanism may complement the potentiating influence of myosin RLC phosphorylation.

Myosin light chain phosphorylation-dephosphorylation in mammalian skeletal muscle

1982 ◽  
Vol 242 (3) ◽  
pp. C234-C241 ◽  
Author(s):  
D. R. Manning ◽  
J. T. Stull
Keyword(s):  
Skeletal Muscle ◽  
Light Chain ◽  
Extensor Digitorum Longus ◽  
Myosin Light Chain ◽  
Extensor Digitorum Longus Muscle ◽  
Extensor Digitorum ◽  
Mammalian Skeletal Muscle ◽  
Phosphate Content ◽  
Longus Muscle ◽  
Fast Twitch

Phosphorylation of the myosin light chain 2 (LC2) subunit was examined in rat fast-twitch and slow-twitch skeletal muscles in response to repetitive stimulation at 23 and 35 degrees C and on incubation of fast-twitch skeletal muscle with isoproterenol. After a 1-s tetany at 35 degrees C, LC2 phosphate content in extensor digitorum longus muscle increased rapidly and transiently from 0.21 to 0.51 mol phosphate/mol LC2. This pattern of phosphorylation was similar to that observed at 23 degrees C. Increases in LC2 phosphate content were dependent on the frequency and duration of stimulation. In soleus muscle LC2 phosphate content was minimal following a 1-s tetany but increased markedly following more prolonged tetanies. On incubation of extensor digitorum longus muscle with isoproterenol (20 microM), LC2 phosphate content did not change, whereas phosphorylase a levels increased. A positive correlation existed between LC2 phosphate content and potentiation of peak twitch tension in both types of muscles, suggesting a physiological function for LC2 phosphorylation.

Bovine fast-twitch myosin light chain 1: cloning and mRNA amount in muscle of cattle treated with clenbuterol

1995 ◽  
Vol 268 (5) ◽  
pp. E858-E865 ◽  
Author(s):  
S. B. Smith ◽  
S. K. Davis ◽  
J. J. Wilson ◽  
R. T. Stone ◽  
F. Y. Wu ◽  
...  
Keyword(s):  
Light Chain ◽  
Cdna Clone ◽  
Myosin Light Chain ◽  
Day Treatment ◽  
Succinic Dehydrogenase ◽  
Longissimus Dorsi ◽  
Beta Adrenergic ◽  
Longissimus Dorsi Muscle ◽  
Dorsi Muscle ◽  
Fast Twitch

The cDNA clone encoding the fast-twitch isoform of myosin light chain 1 (MLC-1f) was isolated from bovine longissimus dorsi muscle and sequenced in M13 and pUC8. An 0.8-kb subclone, produced by digestion of the cDNA with EcoRI, contained the portion of the molecule common to MLC-1f and MLC-3f. The cDNA in pUC8 contained an additional 81 bp upstream of the EcoR I digestion site, which was unique to MLC-1f. The cDNA clone was used to measure MLC-1f mRNA in longissimus dorsi muscle of cattle chronically administered the beta-adrenergic agonist clenbuterol. Treatment with clenbuterol for 50 days increased succinic dehydrogenase negative (type IIB) and positive (types I and IIA) myofiber cross-sectional areas by 25%. After the 50-day treatment period, the amount of MLC-1f mRNA was 90% greater in longissimus dorsi muscle of treated animals than in the initial group. This effect was lost when clenbuterol treatment was withdrawn for a 78-day period, during which time muscle growth in the treated animals stopped completely. We conclude that we have cloned the bovine cDNA for MLC-1f, which has provided additional evidence that beta-adrenergic agonists increase myofibrillar gene expression.

Expression of MRF4, a myogenic helix-loop-helix protein, produces multiple changes in the myogenic program of BC3H-1 cells

1992 ◽  
Vol 12 (6) ◽  
pp. 2484-2492
Author(s):  
N E Block ◽  
J B Miller
Keyword(s):  
Myosin Heavy Chain ◽  
Heavy Chain ◽  
Light Chain ◽  
Myosin Light Chain ◽  
Myogenic Regulatory Factors ◽  
Regulatory Factors ◽  
Myosin Heavy Chain Isoform ◽  
Helix Loop Helix ◽  
Fast Myosin ◽  
Myogenic Program

Expression of MRF4, a myogenic regulatory factor of the basic helix-loop-helix type, produced multiple changes in the myogenic program of the BC3H-1 cell line. BC3H-1 cells that stably expressed exogenous MRF4 were prepared and termed BR cell lines. Upon differentiation, the BR cells were found to have three muscle-specific properties (endogenous MyoD expression, myoblast fusion, and fast myosin light-chain 1 expression) that the parent BC3H-1 cells did not have. Of the four known myogenic regulatory factors (MyoD, myogenin, Myf-5, and MRF4), only MRF4 was capable of activating expression of the endogenous BC3H-1 myoD gene. In addition, the pattern of Myf-5 expression in BR cells was the opposite of that in BC3H-1 cells. Myf-5 expression was low in BR myoblasts and showed a small increase upon myotube formation, whereas Myf-5 expression was high in BC3H-1 myoblasts and decreased upon differentiation. Though the MRF4-transfected BR cells fused to form large myotubes and expressed fast myosin light-chain 1, the pattern of myosin heavy-chain isoform expression was the same in the BR and the nonfusing parent BC3H-1 cells, suggesting that factors in addition to the MyoD family members regulate myosin heavy-chain isoform expression patterns in BC3H-1 cells. In contrast to the changes produced by MRF4 expression, overexpression of Myf-5 did not alter BC3H-1 myogenesis. The results suggest that differential expression of the myogenic regulatory factors of the MyoD family may be one mechanism for generating cells with diverse myogenic phenotypes.

Myosin light chain replacement in the heart

2000 ◽  
Vol 279 (3) ◽  
pp. H1355-H1364 ◽  
Author(s):  
Atsushi Sanbe ◽  
James Gulick ◽  
Eric Hayes ◽  
David Warshaw ◽  
Hanna Osinska ◽  
...  
Keyword(s):  
Myosin Heavy Chain ◽  
Heavy Chain ◽  
Light Chain ◽  
Myosin Light Chain ◽  
Complete Replacement ◽  
Myosin Heavy Chain Isoform ◽  
Hypertrophic Response ◽  
Force Development ◽  
Cross Bridge ◽  
Systolic And Diastolic Function

Myosin-actin cross-bridge kinetics are an important determinant for cardiac systolic and diastolic function. We compared the effects of myosin light chain substitutions on the ability of the fibers to contract in response to calcium and in their ability to produce power. Transgenesis was used to effect essentially complete replacement of the target contractile protein isoform specifically in the heart. Atrial and ventricular fibers derived from the various transgenic (TG) lines were skinned, and the force-velocity relationships, unloaded shortening velocities, and Ca2+-stimulated Mg2+-ATPase activities were determined. Replacement with an ectopic isoform resulted in significant changes in cross-bridge cycling kinetics but without any overt effects on morbidity or mortality. To confirm that this result was not light chain specific, a modified α-myosin heavy chain isoform that resulted in significant changes in force development was also engineered. The animals appeared healthy and have normal lifespans, and the changes in force development did not result in significant remodeling or overt hypertrophy. We conclude that myosin light chains can control aspects of cross-bridge cycling and alter force development. The myosin heavy chain data also show that changes in the kinetics of force development and power output do not necessarily lead to activation of the hypertrophic response or significant cardiac remodeling.

scholarly journals Expression of MRF4, a myogenic helix-loop-helix protein, produces multiple changes in the myogenic program of BC3H-1 cells.

1992 ◽  
Vol 12 (6) ◽  
pp. 2484-2492 ◽  
Author(s):  
N E Block ◽  
J B Miller
Keyword(s):  
Myosin Heavy Chain ◽  
Heavy Chain ◽  
Light Chain ◽  
Myosin Light Chain ◽  
Myogenic Regulatory Factors ◽  
Regulatory Factors ◽  
Myosin Heavy Chain Isoform ◽  
Helix Loop Helix ◽  
Fast Myosin ◽  
Myogenic Program

Expression of MRF4, a myogenic regulatory factor of the basic helix-loop-helix type, produced multiple changes in the myogenic program of the BC3H-1 cell line. BC3H-1 cells that stably expressed exogenous MRF4 were prepared and termed BR cell lines. Upon differentiation, the BR cells were found to have three muscle-specific properties (endogenous MyoD expression, myoblast fusion, and fast myosin light-chain 1 expression) that the parent BC3H-1 cells did not have. Of the four known myogenic regulatory factors (MyoD, myogenin, Myf-5, and MRF4), only MRF4 was capable of activating expression of the endogenous BC3H-1 myoD gene. In addition, the pattern of Myf-5 expression in BR cells was the opposite of that in BC3H-1 cells. Myf-5 expression was low in BR myoblasts and showed a small increase upon myotube formation, whereas Myf-5 expression was high in BC3H-1 myoblasts and decreased upon differentiation. Though the MRF4-transfected BR cells fused to form large myotubes and expressed fast myosin light-chain 1, the pattern of myosin heavy-chain isoform expression was the same in the BR and the nonfusing parent BC3H-1 cells, suggesting that factors in addition to the MyoD family members regulate myosin heavy-chain isoform expression patterns in BC3H-1 cells. In contrast to the changes produced by MRF4 expression, overexpression of Myf-5 did not alter BC3H-1 myogenesis. The results suggest that differential expression of the myogenic regulatory factors of the MyoD family may be one mechanism for generating cells with diverse myogenic phenotypes.

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