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.

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.

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.

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.

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.

Denervation-induced changes in myosin heavy chain expression in the rat diaphragm muscle

2003 ◽  
Vol 95 (2) ◽  
pp. 611-619 ◽  
Author(s):  
Paige C. Geiger ◽  
Jeffrey P. Bailey ◽  
Wen-Zhi Zhan ◽  
Carlos B. Mantilla ◽  
Gary C. Sieck
Keyword(s):  
Protein Expression ◽  
Mrna Expression ◽  
Myosin Heavy Chain ◽  
Heavy Chain ◽  
Protein Isoforms ◽  
Diaphragm Muscle ◽  
Northern Analysis ◽  
Mrna Levels ◽  
Rat Diaphragm ◽  
Mrna Isoforms

Unilateral denervation (Dnv) of the rat diaphragm muscle (Diam) markedly alters expression of myosin heavy chain (MHC) isoforms. After 2 wk of Diam Dnv, MHC content per half-sarcomere decreases in fibers expressing MHC2X and MHC2B. We hypothesized that changes in MHC protein expression parallel changes in MHC mRNA expression. Relative MHC isoform mRNA levels were determined by Northern analysis after 1, 3, 7, and 14 days of Dnv of the rat Diam. MHC protein expression was determined by SDS-PAGE. Changes in MHC isoform protein and mRNA expression were not concurrent. Expression of MHCSlow and MHC2X mRNA isoforms decreased dramatically by 3 days of Dnv, whereas that of MHC2A and MHC2B did not change. Expression of all MHC protein isoforms decreased by 3 days of Dnv. We observed a differential effect of rat Diam Dnv on MHC isoform protein and mRNA expression. The time course of the changes in MHC isoform mRNA and protein expression suggests a predominant effect of altered protein turnover rates on MHC protein expression instead of altered transcription after Dnv.

Impact of β-myosin heavy chain isoform expression on cross-bridge cycling kinetics

2005 ◽  
Vol 288 (2) ◽  
pp. H896-H903 ◽  
Author(s):  
Veronica L. M. Rundell ◽  
Vlasios Manaves ◽  
Anne F. Martin ◽  
Pieter P. de Tombe
Keyword(s):  
Myosin Heavy Chain ◽  
Heavy Chain ◽  
Atp Hydrolysis ◽  
High Strain ◽  
Male Rats ◽  
Tension Development ◽  
Myosin Heavy Chain Isoform ◽  
Cross Bridge ◽  
Wide Range ◽  
Tension Cost

Myosin heavy chain (MHC) isoforms α and β have intrinsically different ATP hydrolysis activities (ATPase) and therefore cross-bridge cycling rates in solution. There is considerable evidence of altered MHC expression in rodent cardiac disease models; however, the effect of incremental β-MHC expression over a wide range on the rate of high-strain, isometric cross-bridge cycling is yet to be ascertained. We treated male rats with 6-propyl-2-thiouracil (PTU; 0.8 g/l in drinking water) for short intervals (6, 11, 16, and 21 days) to generate cardiac MHC patterns in transition from predominantly α-MHC to predominantly β-MHC. Steady-state calcium-dependent tension development and tension-dependent ATP consumption (tension cost; proportional to cross-bridge cycling) were measured in chemically permeabilized (skinned) right ventricular muscles at 20°C. To assess dynamic cross-bridge cycling kinetics, the rate of force redevelopment ( ktr) was determined after rapid release-restretch of fully activated muscles. MHC isoform content in each experimental muscle was measured by SDS-PAGE and densitometry. α-MHC content decreased significantly and progressively with length of PTU treatment [68 ± 5%, 58 ± 4%, 37 ± 4%, and 27 ± 6% for 6, 11, 16, and 21 days, respectively; P < 0.001 (ANOVA)]. Tension cost decreased, linearly, with decreased α-MHC content [6.7 ± 0.4, 5.6 ± 0.5, 4.0 ± 0.4, and 3.9 ± 0.3 ATPase/tension for 6, 11, 16, and 21 days, respectively; P < 0.001 (ANOVA)]. Likewise, ktr was significantly and progressively depressed with length of PTU treatment [11.1 ± 0.6, 9.1 ± 0.5, 8.2 ± 0.7, and 6.2 ± 0.3 s−1 for 6, 11, 16, and 21 days, respectively; P < 0.05 (ANOVA)] Thus cross-bridge cycling, under high strain, for α-MHC is three times higher than for β-MHC. Furthermore, under isometric conditions, α-MHC and β-MHC cross bridges hydrolyze ATP independently of one another.

Contractile properties of the developing diaphragm correlate with myosin heavy chain phenotype

1994 ◽  
Vol 77 (1) ◽  
pp. 481-487 ◽  
Author(s):  
B. D. Johnson ◽  
L. E. Wilson ◽  
W. Z. Zhan ◽  
J. F. Watchko ◽  
M. J. Daood ◽  
...  
Keyword(s):  
Myosin Heavy Chain ◽  
Heavy Chain ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Diaphragm Muscle ◽  
Shortening Velocity ◽  
Cross Bridge ◽  
Isoform Expression ◽  
The Relationship ◽  
Laser Densitometry

The objective of this study was to determine the relationship between developmental transitions in myosin heavy chain (MHC) composition and changes in maximum unloaded shortening velocity (Vo) and maximum specific force (Po) of the rat diaphragm muscle. The diaphragm was excised at postnatal days 0, 3, 7, 14, 21, and 28 and in adults. MHC isoform expression was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and laser densitometry. In muscle fiber bundles, Vo was determined at 15 degrees C by use of the “slack” test. Isometric Po was determined at 15 and 26 degrees C. Simple and stepwise regressions were used to evaluate the correlations between Vo, Po, and MHC phenotype transitions and the various developmental ages. The progressive increases in Vo and Po with age were found to be inversely correlated to MHC-neonatal isoform expression (r2 = -0.84 and -0.63, respectively) and positively correlated to MHC-2X (r2 = 0.78 and 0.57) and MHC-2B (r2 = 0.51 and 0.40) isoform expression (P < 0.001). Changes in MHC-neonatal isoform expression contributed to most of the developmental variance in Vo and Po, with changes in MHC-2X and MHC-2B expression also contributing significant increments to total variance. The postnatal increase in Vo most likely relates to differences in the actomyosin adenosinetriphosphatase activity between neonatal and adult fast MHC phenotypes. The increase in Po may reflect inherent differences in myofibrillar density, cross-bridge cycling kinetics, and/or the force produced per cross bridge among fibers composed of the different MHC isoforms.

Effects of perinatal undernutrition on elimination of immature myosin isoforms in the rat diaphragm

1991 ◽  
Vol 261 (2) ◽  
pp. L49-L54 ◽  
Author(s):  
B. S. Brozanski ◽  
M. J. Daood ◽  
W. A. LaFramboise ◽  
J. F. Watchko ◽  
T. P. Foley ◽  
...  
Keyword(s):  
Myosin Heavy Chain ◽  
Heavy Chain ◽  
Time Course ◽  
Myosin Light Chain ◽  
Diaphragm Muscle ◽  
Myosin Isoforms ◽  
Rat Diaphragm ◽  
Two Dimensional Gel Electrophoresis ◽  
Significant Difference ◽  
Immunocytochemical Techniques

The effect of perinatal undernutrition on the postnatal elimination of immature myosin isoforms in rat diaphragm muscle was examined using electrophoretic and immunocytochemical techniques. Electrophoresis of native myosin showed that neonatal bands were present in diaphragm muscles of both control and undernourished rats on day 4. By day 21, the neonatal bands were diminished in the control diaphragm compared with the diaphragm of the undernourished rats. Neonatal bands persisted on postnatal day 30 in the diaphragm of the undernourished rats but not in the diaphragm of control rats. No significant difference in the time course of elimination of embryonic myosin light chain (LCemb) was observed between the diaphragm muscles of control and undernourished rats with two-dimensional gel electrophoresis. Immunocytochemical analysis demonstrated embryonic myosin heavy chain (MHCemb) in all myofibers of the diaphragm muscle of both groups at day 4, but this isoform was not detected in either group by day 14. Reactivity with anti-neonatal myosin heavy chain (MHCneo) indicated that rate of elimination of the MHCneo was delayed in the undernourished state as compared with the normal rats (P less than 0.001). Serum triiodothyronine levels were measured at 14, 21, and 30 days and were significantly lower in the undernourished rats compared with age-matched controls. These data demonstrate that the normal postnatal decrease in MHCneo, but not MHCemb or LCemb, is affected by the nutritional state of the animal. We speculate that these alterations in myosin isoform transitions are induced by hypothyroidism associated with undernutrition.

scholarly journals Regulatory light chain phosphorylation augments length-dependent contraction in PTU-treated rats

2018 ◽  
Vol 151 (1) ◽  
pp. 66-76 ◽  
Author(s):  
Jason J. Breithaupt ◽  
Hannah C. Pulcastro ◽  
Peter O. Awinda ◽  
David C. DeWitt ◽  
Bertrand C.W. Tanner
Keyword(s):  
Myosin Heavy Chain ◽  
Heavy Chain ◽  
Light Chain ◽  
Myosin Heavy Chain Isoforms ◽  
Regulatory Light Chain ◽  
Cardiac Myosin ◽  
Myosin Heavy Chain Isoform ◽  
Cross Bridge ◽  
Detachment Rate ◽  
Length Dependent Activation

Force production by actin–myosin cross-bridges in cardiac muscle is regulated by thin-filament proteins and sarcomere length (SL) throughout the heartbeat. Prior work has shown that myosin regulatory light chain (RLC), which binds to the neck of myosin heavy chain, increases cardiac contractility when phosphorylated. We recently showed that cross-bridge kinetics slow with increasing SLs, and that RLC phosphorylation amplifies this effect, using skinned rat myocardial strips predominantly composed of the faster α-cardiac myosin heavy chain isoform. In the present study, to assess how RLC phosphorylation influences length-dependent myosin function as myosin motor speed varies, we used a propylthiouracil (PTU) diet to induce &gt;95% expression of the slower β-myosin heavy chain isoform in rat cardiac ventricles. We measured the effect of RLC phosphorylation on Ca2+-activated isometric contraction and myosin cross-bridge kinetics (via stochastic length perturbation analysis) in skinned rat papillary muscle strips at 1.9- and 2.2-µm SL. Maximum tension and Ca2+ sensitivity increased with SL, and RLC phosphorylation augmented this response at 2.2-µm SL. Subtle increases in viscoelastic myocardial stiffness occurred with RLC phosphorylation at 2.2-µm SL, but not at 1.9-µm SL, thereby suggesting that RLC phosphorylation increases β-myosin heavy chain binding or stiffness at longer SLs. The cross-bridge detachment rate slowed as SL increased, providing a potential mechanism for prolonged cross-bridge attachment to augment length-dependent activation of contraction at longer SLs. Length-dependent slowing of β-myosin heavy chain detachment rate was not affected by RLC phosphorylation. Together with our previous studies, these data suggest that both α- and β-myosin heavy chain isoforms show a length-dependent activation response and prolonged myosin attachment as SL increases in rat myocardial strips, and that RLC phosphorylation augments length-dependent activation at longer SLs. In comparing cardiac isoforms, however, we found that β-myosin heavy chain consistently showed greater length-dependent sensitivity than α-myosin heavy chain. Our work suggests that RLC phosphorylation is a vital contributor to the regulation of myocardial contractility in both cardiac myosin heavy chain isoforms.

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