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• Articles by T. Kraft
• Articles by B. Brenner

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• Temperature-Dependence of Isometric Tension and Cross-Bridge...

  Temperature-Dependence of Isometric Tension and Cross-Bridge Kinetics of Cardiac Muscle Fibers Reconstituted with a Tropomyosin Internal Deletion Mutant Biophysical Journal, Volume 91, Issue 11, 1 December 2006, Pages 4230-4240 Xiaoying Lu, Larry S. Tobacman and Masataka Kawai Abstract The effect of temperature on isometric tension and cross-bridge kinetics was studied with a tropomyosin (Tm) internal deletion mutant AS-Δ23Tm (Ala-Ser-Tm Δ(47–123)) in bovine cardiac muscle fibers by using the thin filament extraction and reconstitution technique. The results are compared with those from actin reconstituted alone, cardiac muscle-derived control acetyl-Tm, and recombinant control AS-Tm. In all four reconstituted muscle groups, isometric tension and stiffness increased linearly with temperature in the range 5–40°C for fibers activated in the presence of saturating ATP and Ca. The slopes of the temperature-tension plots of the two controls were very similar, whereas the slope derived from fibers with actin alone had ∼40% the control value, and the slope from mutant Tm had ∼36% the control value. Sinusoidal analysis was performed to study the temperature dependence of cross-bridge kinetics. All three exponential processes A, B, and C were identified in the high temperature range (30–40°C); only processes B and C were identified in the mid-temperature range (15–25°C), and only process C was identified in the low temperature range (5–10°C). At a given temperature, similar apparent rate constants (2a, 2b, 2c) were observed in all four muscle groups, whereas their magnitudes were markedly less in the order of AS-Δ23Tm<Actin<AS-Tm ≈ Acetyl-Tm groups. Our observations are consistent with the hypothesis that Tm enhances hydrophobic and stereospecific interactions (positive allosteric effect) between actin and myosin, but Δ23Tm decreases these interactions (negative allosteric effect). Our observations further indicate that tension/cross-bridge is increased by Tm, but is diminished by Δ23Tm. We conclude that Tm affects the conformation of actin so as to increase the area of hydrophobic interaction between actin and myosin molecules. Abstract | Full Text | PDF (194 kb)

• Structural Features of Cross-Bridges in Isometrically Contra...

  Structural Features of Cross-Bridges in Isometrically Contracting Skeletal Muscle Biophysical Journal, Volume 82, Issue 5, 1 May 2002, Pages 2536-2547 Theresia Kraft, Thomas Mattei, Ante Radocaj, Birgit Piep, Christoph Nocula, Markus Furch and Bernhard Brenner Abstract Two-dimensional x-ray diffraction was used to investigate structural features of cross-bridges that generate force in isometrically contracting skeletal muscle. Diffraction patterns were recorded from arrays of single, chemically skinned rabbit psoas muscle fibers during isometric force generation, under relaxation, and in rigor. In isometric contraction, a rather prominent intensification of the actin layer lines at 5.9 and 5.1nm and of the first actin layer line at 37nm was found compared with those under relaxing conditions. Surprisingly, during isometric contraction, the intensity profile of the 5.9-nm actin layer line was shifted toward the meridian, but the resulting intensity profile was different from that observed in rigor. We particularly addressed the question whether the differences seen between rigor and active contraction might be due to a rigor-like configuration of both myosin heads in the absence of nucleotide (rigor), whereas during active contraction only one head of each myosin molecule is in a rigor-like configuration and the second head is weakly bound. To investigate this question, we created different mixtures of weak binding myosin heads and rigor-like actomyosin complexes by titrating MgATPS at saturating [Ca] into arrays of single muscle fibers. The resulting diffraction patterns were different in several respects from patterns recorded under isometric contraction, particularly in the intensity distribution along the 5.9-nm actin layer line. This result indicates that cross-bridges present during isometric force generation are not simply a mixture of weakly bound and single-headed rigor-like complexes but are rather distinctly different from the rigor-like cross-bridge. Experiments with myosin-S1 and truncated S1 (motor domain) support the idea that for a force generating cross-bridge, disorder due to elastic distortion might involve a larger part of the myosin head than for a nucleotide free, rigor cross-bridge. Abstract | Full Text | PDF (387 kb)

• Tension Recovery in Permeabilized Rat Soleus Muscle Fibers a...

  Tension Recovery in Permeabilized Rat Soleus Muscle Fibers after Rapid Shortening and Restretch Biophysical Journal, Volume 90, Issue 4, 15 February 2006, Pages 1288-1294 Kenneth S. Campbell Abstract Permeabilized rat soleus muscle fibers were subjected to rapid shortening/restretch protocols (20% muscle length, 20ms duration) in solutions with pCa values ranging from 6.5 to 4.5. Force redeveloped after each restretch but temporarily exceeded the steady-state isometric tension reaching a maximum value ∼2.5s after relengthening. The relative size of the overshoot was <5% in pCa 6.5 and pCa 4.5 solutions but equaled 17%±4% at pCa 6.0 (approximately half-maximal Ca activation). Muscle stiffness was estimated during pCa 6.0 activations by imposing length steps at different time intervals after repeated shortening/restretch perturbations. Relative stiffness and relative tension were correlated (<0.001) during recovery, suggesting that tension overshoots reflect a temporary increase in the number of attached cross-bridges. Rates of tension recovery () correlated (<0.001) with the relative residual force prevailing immediately after restretch. Force also recovered to the isometric value more quickly at 5.7≤pCa≤5.9 than at pCa 4.5 (ANOVA, <0.05). These results show that measurements underestimate the rate of isometric force development during submaximal Ca activations and suggest that the rate of tension recovery is limited primarily by the availability of actin binding sites. Abstract | Full Text | PDF (219 kb)

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Abstract

The thiadiazinon derivative EMD 57033 has been found previously in cardiac muscle to increase isometric force generation without a proportional increase in fiber ATPase, thus causing a reduction in tension cost. To analyze the mechanism by which EMD 57033 affects the contractile system, we studied its effects on isometric force, isometric fiber ATPase, the rate constant of force redevelopment (k(redev)), active fiber stiffness, and its effect on Fo, which is the force contribution of a cross-bridge in the force-generating states. We used chemically skinned fibers of the rabbit psoas muscle. It was found that with 50 microM EMD 57033, isometric force increases by more than 50%, whereas Kredev, active stiffness, and isometric fiber ATPase increase by at most 10%. The results show that EMD 57033 causes no changes in cross-bridge turnover kinetics and no changes in active fiber stiffness that would result in a large enough increase in occupancy of the force-generating states to account for the increase in active force. However, plots of force versus length change recorded during stretches and releases (T plots) indicate that in the presence of EMD 57033 the y(o) value (x axis intercept) for the cross-bridges becomes more negative while its absolute value increases. This might suggest a larger cross-bridge strain as the basis for increased active force. Analysis of T plots with and without EMD 57033 shows that the increase in cross-bridge strain is not due to a redistribution of cross-bridges among different force-generating states favoring states of larger strain. Instead, it reflects an increased cross-bridge strain in the main force-generating state. The direct effect of EMD 57033 on the force contribution of cross-bridges in the force-generating states represents an alternative mechanism for a positive inotropic intervention.