Effect of unilateral denervation on maximum specific force in rat diaphragm muscle fibers

2001 ◽  
Vol 90 (4) ◽  
pp. 1196-1204 ◽  
Author(s):  
Paige C. Geiger ◽  
Mark J. Cody ◽  
Rebecca L. Macken ◽  
Megan E. Bayrd ◽  
Gary C. Sieck
Keyword(s):  
Diaphragm Muscle ◽  
Specific Force ◽  
Rat Diaphragm ◽  
Standard Curve ◽  
Fast Myosin ◽  
Sds Page ◽  
Fast Myosin Heavy Chain ◽  
Cross Bridges ◽  
Triton X ◽  
Permeabilized Fibers

We hypothesize that 1) the effect of denervation (DNV) is more pronounced in fibers expressing fast myosin heavy chain (MHC) isoforms and 2) the effect of DNV on maximum specific force reflects a reduction in MHC content per half sarcomere or the number of cross bridges in parallel. Studies were performed on single Triton X-100-permeabilized fibers activated at a pCa (−log Ca2+ concentration) of 4.0. MHC content per half sarcomere was determined by densitometric analysis of SDS-PAGE gels and comparison to a standard curve of known MHC concentrations. After 2 of wk DNV, the maximum specific force of fibers expressing MHC2X was reduced by ∼40% (MHC2Bexpression was absent), whereas the maximum specific force of fibers expressing MHC2A and MHCslow decreased by only ∼20%. DNV also reduced the MHC content in fibers expressing MHC2X, with no effect on fibers expressing MHC2A and MHCslow. When normalized for MHC content per half sarcomere, force generated by DNV fibers expressing MHC2X and MHC2A was decreased compared with control fibers. These results suggest the force per cross bridge is also affected by DNV.

Effects of hypothyroidism on maximum specific force in rat diaphragm muscle fibers

2002 ◽  
Vol 92 (4) ◽  
pp. 1506-1514 ◽  
Author(s):  
Paige C. Geiger ◽  
Mark J. Cody ◽  
Young Soo Han ◽  
Larry W. Hunter ◽  
Wen-Zhi Zhan ◽  
...  
Keyword(s):  
Microscopic Analysis ◽  
Thick Filament ◽  
Volume Density ◽  
Diaphragm Muscle ◽  
Specific Force ◽  
Rat Diaphragm ◽  
Electron Microscopic ◽  
Standard Curve ◽  
Triton X ◽  
Permeabilized Fibers

We hypothesized that 1) hypothyroidism (Hyp) decreases myosin heavy chain (MHC) content per half-sarcomere in diaphragm muscle (Diam) fibers, 2) Hyp decreases the maximum specific force (Fmax) of Diam fibers because of the reduction in MHC content per half-sarcomere, and 3) Hyp affects MHC content per half-sarcomere and Fmax to a greater extent in fibers expressing MHC type 2X (MHC2X) and/or MHC type 2B (MHC2B). Studies were performed on single Triton X-permeabilized fibers activated at pCa 4.0. MHC content per half-sarcomere was determined by densitometric analysis of SDS-polyacrylamide gels and comparison with a standard curve of known MHC concentrations. After 3 wk of Hyp, MHC content per half-sarcomere was reduced in fibers expressing MHC2X and/or MHC2B. On the basis of electron-microscopic analysis, this reduction in MHC content was also reflected by a decrease in myofibrillar volume density and thick filament density. Hyp decreased Fmax across all MHC isoforms; however, the greatest decrease occurred in fibers expressing fast MHC isoforms (∼40 vs. ∼20% for fibers expressing slow MHC isoforms). When normalized for MHC content per half-sarcomere, force generated by Hyp fibers expressing MHC2A was reduced compared with control fibers, whereas force per half-sarcomere MHC content was higher for fibers expressing MHC2X and/or MHC2B in the Hyp Diam than for controls. These results indicate that the effect of Hyp is more pronounced on fibers expressing MHC2Xand/or MHC2B and that the reduction of Fmaxwith Hyp may be at least partially attributed to a decrease in MHC content per half-sarcomere but not to changes in force per cross bridge.

Maximum specific force depends on myosin heavy chain content in rat diaphragm muscle fibers

2000 ◽  
Vol 89 (2) ◽  
pp. 695-703 ◽  
Author(s):  
Paige C. Geiger ◽  
Mark J. Cody ◽  
Rebecca L. Macken ◽  
Gary C. Sieck
Keyword(s):  
Myosin Heavy Chain ◽  
Heavy Chain ◽  
Fiber Type ◽  
Diaphragm Muscle ◽  
Specific Force ◽  
Rat Diaphragm ◽  
Cross Sectional ◽  
High Calcium ◽  
Cross Bridges ◽  
Triton X

In the present study, myosin heavy chain (MHC) content per half sarcomere, an estimate of the number of cross bridges available for force generation, was determined in rat diaphragm muscle (Diam) fibers expressing different MHC isoforms. We hypothesize that fiber-type differences in maximum specific force [force per cross-sectional area (CSA)] reflect the number of cross bridges present per CSA. Studies were performed on single, Triton X-100-permeabilized rat Diam fibers. Maximum specific force was determined by activation of single Diamfibers in the presence of a high-calcium solution (pCa, −log Ca2+ concentration of 4.0). SDS-PAGE and Western blot analyses were used to determine MHC isoform composition and MHC content per half sarcomere. Differences in maximum specific force across fast MHC isoforms were eliminated when controlled for half-sarcomere MHC content. However, the force produced by slow fibers remained below that of fast fibers when normalized for the number of cross bridges available. On the basis of these results, the lower force produced by slow fibers may be due to less force per cross bridge compared with fast fibers.

ATP consumption rate per cross bridge depends on myosin heavy chain isoform

2003 ◽  
Vol 94 (6) ◽  
pp. 2188-2196 ◽  
Author(s):  
Young-Soo Han ◽  
Paige C. Geiger ◽  
Mark J. Cody ◽  
Rebecca L. Macken ◽  
Gary C. Sieck
Keyword(s):  
Myosin Heavy Chain ◽  
Heavy Chain ◽  
Consumption Rate ◽  
Diaphragm Muscle ◽  
Rat Diaphragm ◽  
Myosin Heavy Chain Isoform ◽  
Standard Curve ◽  
Cross Bridge ◽  
Sds Page ◽  
Triton X

In the present study, we tested the hypothesis that intrinsic differences in ATP consumption rate per cross bridge exist across rat diaphragm muscle (Diam) fibers expressing different myosin heavy chain (MHC) isoforms. During maximum Ca2+ activation (pCa 4.0) of single, Triton X-permeabilized Diam fibers, isometric ATP consumption rate was determined by using an NADH-linked fluorometric technique. The MHC concentration in single Diam fibers was determined by densitometric analysis of SDS-PAGE gels and comparison to a standard curve of known MHC concentrations. Isometric ATP consumption rate varied across Diam fibers expressing different MHC isoforms, being highest in fibers expressing MHC2X (1.14 ± 0.08 nmol · mm−3 · s−1) and/or MHC2B (1.33 ± 0.08 nmol · mm−3 · s−1), followed by fibers expressing MHC2A (0.77 ± 0.11 nmol · mm−3 · s−1) and MHCSlow (0.46 ± 0.03 nmol · mm−3 · s−1). These differences in ATP consumption rate also persisted when it was normalized for MHC concentration in single Diam fibers. Normalized ATP consumption rate for MHC concentration varied across Diam fibers expressing different MHC isoforms, being highest in fibers expressing MHC2X (2.02 ± 0.19 s−1) and/or MHC2B (2.64 ± 0.15 s−1), followed by fibers expressing MHC2A(1.57 ± 0.16 s−1) and MHCSlow (0.77 ± 0.05 s−1). On the basis of these results, we conclude that there are intrinsic differences in ATP consumption rate per cross bridge in Diam fibers expressing MHC isoforms.

scholarly journals Selected Contribution: Mechanisms underlying increased force generation by rat diaphragm muscle fibers during development

2001 ◽  
Vol 90 (1) ◽  
pp. 380-388 ◽  
Author(s):  
Paige C. Geiger ◽  
Mark J. Cody ◽  
Rebecca L. Macken ◽  
Megan E. Bayrd ◽  
Yun-Hua Fang ◽  
...  
Keyword(s):  
Postnatal Development ◽  
Developmental Change ◽  
Diaphragm Muscle ◽  
Rat Diaphragm ◽  
Cross Sectional ◽  
Early Postnatal Development ◽  
Standard Curve ◽  
Cross Bridge ◽  
Sds Page ◽  
Cross Bridges

It has been found that maximum specific force (Fmax; force per cross-sectional area) of rat diaphragm muscle doubles from birth to 84 days (adult). We hypothesize that this developmental change in Fmax reflects an increase in myosin heavy chain (MHC) content per half-sarcomere (an estimate of the number of cross bridges in parallel) and/or a greater force per cross bridge in fibers expressing fast MHC isoforms compared with slow and neonatal MHC isoforms (MHCslow and MHCneo, respectively). Single Triton 100-X-permeabilized fibers were activated at a pCa of 4.0. MHC isoform expression was determined by SDS-PAGE. MHC content per half-sarcomere was determined by densitometric analysis and comparison to a standard curve of known MHC concentrations. MHC content per half-sarcomere progressively increased during early postnatal development. When normalized for MHC content per half-sarcomere, fibers expressing MHCslow and coexpressing MHCneoproduced less force than fibers expressing fast MHC isoforms. We conclude that lower force per cross bridge in fibers expressing MHCslow and MHCneo contributes to the lower Fmax seen in early postnatal development.

Force-calcium relationship depends on myosin heavy chain and troponin isoforms in rat diaphragm muscle fibers

1999 ◽  
Vol 87 (5) ◽  
pp. 1894-1900 ◽  
Author(s):  
Paige C. Geiger ◽  
Mark J. Cody ◽  
Gary C. Sieck
Keyword(s):  
Myosin Heavy Chain ◽  
Heavy Chain ◽  
Fiber Type ◽  
Muscle Fibers ◽  
Diaphragm Muscle ◽  
Rat Diaphragm ◽  
Single Fibers ◽  
Maximal Force ◽  
Sds Page ◽  
Triton X

The present study examined Ca2+ sensitivity of diaphragm muscle (Diam) fibers expressing different myosin heavy chain (MHC) isoforms. We hypothesized that Diam fibers expressing the MHCslow isoform have greater Ca2+ sensitivity than fibers expressing fast MHC isoforms and that this fiber-type difference in Ca2+ sensitivity reflects the isoform composition of the troponin (Tn) complex (TnC, TnT, and TnI). Studies were performed in single Triton-X-permeabilized Diam fibers. The Ca2+ concentration at which 50% maximal force was generated (pCa50) was determined for each fiber. SDS-PAGE and Western analyses were used to determine the MHC and Tn isoform composition of single fibers. The pCa50 for Diam fibers expressing MHCslow was significantly greater than that of fibers expressing fast MHC isoforms, and this greater Ca2+ sensitivity was associated with expression of slow isoforms of the Tn complex. However, some Diam fibers expressing MHCslow contained the fast TnC isoform. These results suggest that the combination of TnT, TnI, and TnC isoforms may determine Ca2+ sensitivity in Diam fibers.

Denervation alters myosin heavy chain expression and contractility of developing rat diaphragm muscle

2000 ◽  
Vol 89 (3) ◽  
pp. 1106-1113 ◽  
Author(s):  
Gary C. Sieck ◽  
Wen-Zhi Zhan
Keyword(s):  
Myosin Heavy Chain ◽  
Heavy Chain ◽  
Contractile Properties ◽  
Diaphragm Muscle ◽  
Specific Force ◽  
Rat Diaphragm ◽  
Shortening Velocity ◽  
Relative Expression ◽  
Isoform Expression ◽  
Developing Rat

We hypothesized that unilateral denervation (DNV) of the rat diaphragm muscle (Diam) in neonates at postnatal day 7 (D-7) alters normal transitions of myosin heavy chain (MHC) isoform expression and thereby affects postnatal changes in maximum specific force (Po) and maximum unloaded shortening velocity ( V o). The relative expression of different MHC isoforms was analyzed electrophoretically. With DNV at D-7, expression of MHCneo in the Diam persisted, and emergence of MHC2X and MHC2B was delayed. By D-21 and D-28, relative expression of MHC2A and MHC2Bwas reduced in DNV compared with control (CTL) animals. Expression of MHCneo also reappeared in adult Diam by 2–3 wk after DNV, and relative expression of MHC2B was reduced. At each age, Po was reduced and V o was slowed by DNV, compared with CTL. In CTL Diam, postnatal changes in Po and V o were associated with an increase in fast MHC isoform expression. In DNV Diam, no such association existed. We conclude that, in the Diam, DNV induces alterations in both MHC isoform expression and contractile properties, which are not necessarily causally linked.

scholarly journals Impact of Extraction Method on the Detection of Quality Biomarkers in Normal vs. DFD Meat

Foods ◽  
2021 ◽  
Vol 10 (5) ◽  
pp. 1097
Author(s):  
Laura González-Blanco ◽  
Yolanda Diñeiro ◽  
Andrea Díaz-Luis ◽  
Ana Coto-Montes ◽  
Mamen Oliván ◽  
...  
Keyword(s):  
Extraction Method ◽  
Protein Pattern ◽  
Stress Protein ◽  
Electrophoretic Pattern ◽  
Extraction Methods ◽  
Sds Page ◽  
Weight Protein ◽  
Triton X ◽  
Protein Extractability ◽  
Selection Of

The objective of this work was to demonstrate how the extraction method affects the reliability of biomarker detection and how this detection depends on the biomarker location within the cell compartment. Different extraction methods were used to study the sarcoplasmic and myofibrillar fractions of the Longissimus thoracis et lumborum muscle of young bulls of the Asturiana de los Valles breed in two quality grades, standard (Control) or dark, firm, and dry (DFD) meat. Protein extractability and the expression of some of the main meat quality biomarkers—oxidative status (lipoperoxidation (LPO) and catalase activity (CAT)), proteome (SDS-PAGE electrophoretic pattern), and cell stress protein (Hsp70)—were analyzed. In the sarcoplasmic fraction, buffers containing Triton X-100 showed significantly higher protein extractability, LPO, and higher intensity of high-molecular-weight protein bands, whereas the TES buffer was more sensitive to distinguishing differences in the protein pattern between the Control and DFD meat. In the myofibrillar fraction, samples extracted with the lysis buffer showed significantly higher protein extractability, whereas samples extracted with the non-denaturing buffer showed higher results for LPO, CAT, and Hsp70, and higher-intensity bands in the electrophoretic pattern. These findings highlight the need for the careful selection of the extraction method used to analyze the different biomarkers considering their cellular location to adapt the extractive process.

A metalloproteinase extracellularly released byCrithidia deanei

2003 ◽  
Vol 49 (10) ◽  
pp. 625-632 ◽  
Author(s):  
Claudia Masini d'Avila-Levy ◽  
Rodrigo F Souza ◽  
Rosana C Gomes ◽  
Alane B Vermelho ◽  
Marta H Branquinha
Keyword(s):  
Molecular Mass ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Residual Activity ◽  
Major Protein ◽  
Motile Cells ◽  
Sds Page ◽  
Triton X

Actively motile cells from a cured strain of Crithidia deanei released proteins in phosphate buffer (pH 7.4). The molecular mass of the released polypeptides, which included some proteinases, ranged from 19 to 116 kDa. One of the major protein bands was purified to homogeneity by a combination of anion-exchange and gel filtration chromatographs. The apparent molecular mass of this protein was estimated to be 62 kDa by sodium dodecyl sulfate – polyacrylamide gel electrophoresis (SDS–PAGE). The incorporation of gelatin into SDS–PAGE showed that the purified protein presented proteolytic activity in a position corresponding to a molecular mass of 60 kDa. The enzyme was optimally active at 37 °C and pH 6.0 and showed 25% of residual activity at 28 °C for 30 min. The proteinase was inhibited by 1,10-phenanthroline and EDTA, showing that it belonged to the metalloproteinase class. A polyclonal antibody to the leishmanial gp63 reacted strongly with the released C. deanei protease. After Triton X-114 extraction, an enzyme similar to the purified metalloproteinase was detected in aqueous and detergent-rich phases. The detection of an extracellular metalloproteinase produced by C. deanei and some other Crithidia species suggests a potential role of this released enzyme in substrate degradation that may be relevant to the survival of trypanosomatids in the host.Key words: endosymbiont, trypanosomatid, extracellular, proteinase.

scholarly journals Corticosteroid effects on isotonic contractile properties of rat diaphragm muscle

1997 ◽  
Vol 83 (4) ◽  
pp. 1062-1067 ◽  
Author(s):  
Roland H. H. Van Balkom ◽  
Wen-Zhi Zhan ◽  
Y. S. Prakash ◽  
P. N. Richard Dekhuijzen ◽  
Gary C. Sieck
Keyword(s):  
Isometric Force ◽  
Contractile Properties ◽  
Diaphragm Muscle ◽  
Peak Power Output ◽  
Rat Diaphragm ◽  
Fiber Morphology ◽  
Shortening Velocity ◽  
Maximum Isometric Force ◽  
Induced Changes ◽  
Isotonic Contractions

Van Balkom, Roland H. H., Wen-Zhi Zhan, Y. S. Prakash, P. N. Richard Dekhuijzen, and Gary C. Sieck. Corticosteroid effects on isotonic contractile properties of rat diaphragm muscle. J. Appl. Physiol. 83(4): 1062–1067, 1997.—The effects of corticosteroids (CS) on diaphragm muscle (Diam) fiber morphology and contractile properties were evaluated in three groups of rats: controls (Ctl), surgical sham and weight-matched controls (Sham), and CS-treated (6 mg ⋅ kg−1 ⋅ day−1prednisolone at 2.5 ml/h for 3 wk). In the CS-treated Diam, there was a selective atrophy of type IIx and IIb fibers, compared with a generalized atrophy of all fibers in the Sham group. Maximum isometric force was reduced by 20% in the CS group compared with both Ctl and Sham. Maximum shortening velocity in the CS Diamwas slowed by ∼20% compared with Ctl and Sham. Peak power output of the CS Diam was only 60% of Ctl and 70% of Sham. Endurance to repeated isotonic contractions improved in the CS-treated Diam compared with Ctl. We conclude that the atrophy of type IIx and IIb fibers in the Diam can only partially account for the CS-induced changes in isotonic contractile properties. Other factors such as reduced myofibrillar density or altered cross-bridge cycling kinetics are also likely to contribute to the effects of CS treatment.

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