Masataka Kawai

Keio University, Tokyo, Tokyo-to, Japan

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Publications (74)239.75 Total impact

  • Masataka Kawai, Graham Lamb, Stefan Galler
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    ABSTRACT: This Special Issue showcases one view point, one original work, and four review articles covering a range of important aspects of skeletal and cardiac muscle functions in health and disease. These topics include little known history of muscle biology in the nineteenth century, the role of β-tropomyosin in contraction, the role of titin in muscle elasticity and its modulation by calcium, oxidation and other stresses, the determinants of contractile functions in cardiomyocytes, the way in which their defects contribute to heart failure, and how defects in calcium storage and release underlie central core disease (CCD) and exertional heat stroke (EHS). The Special Issue also includes 270 contributed abstracts that were presented during the conference.The first article is by Stefan Galler, the EMC chair for 2014. It provides a very interesting historic viewpoint on muscle biology. Dr. Galler reports that the fine structure of cross-striated muscle and its changes during contraction were so ...
    Journal of Muscle Research and Cell Motility 12/2014; · 1.36 Impact Factor
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    ABSTRACT: Human cardiac actin mutants E99K and A230V were expressed with baculovirus/insect cells and used to reconstitute the thin-filament of bovine cardiac (BVC) muscle fibers, together with tropomyosin (Tm) and troponin (Tn) purified from bovine ventricles. Effects of [Ca(2+)], [ATP], and [phosphate] on tension and its transients were studied at 25°C. In the absence of Tm/Tn, both mutants significantly decreased the tension of actin filament reconstituted fibers (WT: 0.75±0.06 T0, E99K: 0.58±0.04 T0, A230V: 0.58±0.03 T0), where T0 is active tension of native fibers (T0=26.9±1.1kPa, N=41), indicating diminished actin-myosin interactions. However, in the presence of Tm and Tn, WT, E99K, and A230V recovered tension (0.85±0.06 T0, 0.89±0.06 T0, and 0.85±0.05 T0, respectively), demonstrating the compensatory effect of Tm/Tn. Ca(2+) sensitivity (pCa50) increased (5.59±0.02, 5.80±0.03, 5.77±0.03, respectively) and cooperativity (nH) decreased (2.6±0.3, 1.87±0.21, 1.60±0.11, respectively). The kinetic constants of the cross-bridge cycle were deduced using sinusoidal analysis. E99K did not show any significant changes in any of the kinetic constants compared to those of WT. A230V caused a decrease in K1 (ATP association constant), k2 and k-2 (rate constants of the cross-bridge detachment step). The cross-bridge distribution was similar among WT, E99K, and A230V. In conclusion, our experiments demonstrate that the first step of HCM pathogenesis with E99K is increased pCa50 and decreased nH, which result in larger tension during partial activation to cause a diastolic problem. The effect on nH is more severe with A230V. In addition, A230V has a problem of decreased cross-bridge kinetics, which affects the normal functions of the cross-bridge cycle and may contribute to the first step of the HCM pathogenesis. Copyright © 2014 Elsevier Ltd. All rights reserved.
    Journal of Molecular and Cellular Cardiology 11/2014; 79C:123-132. · 5.15 Impact Factor
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    ABSTRACT: Recombinant WT human cardiac actin (WT actin) was expressed using the baculovirus/insect cell expression system, purified, and used to reconstitute the thin-filament of bovine cardiac muscle fibers, together with bovine cardiac tropomyosin (Tm) and troponin (Tn). Effects of [Ca(2+)], [ATP], [phosphate] and [ADP] on tension and tension transients were studied at 25°C by using sinusoidal analysis, and the results were compared with those of native fibers and fibers reconstituted with purified bovine cardiac actin (BVC actin). In actin filament reconstituted fibers (without Tm/Tn), those reconstituted with WT actin showed exactly the same active tension as those reconstituted with purified BVC actin (WT: 0.75±0.06T0, N=11; BVC: 0.73±0.07T0, N=12, where T0 is tension of original fibers before extraction). After Tm/Tn reconstitution, fibers reconstituted with WT actin generated 0.85±0.06T0 (N=11) compared to 0.98±0.04T0 (N=12) recovered by those reconstituted with BVC actin. In the presence of Tm/Tn, WT actin reconstituted fibers showed exactly the same Ca(2+) sensitivity as those of the native fibers and BVC actin reconstituted fibers (pCa50: native fibers: 5.69±0.01, N=10; WT: 5.69±0.02, N=11; BVC: 5.68±0.02, N=12). Sinusoidal analysis showed that the cross-bridge kinetics were the same among native fibers, BVC actin reconstituted fibers, and WT actin reconstituted fibers, followed by reconstitution of Tm/Tn. These results demonstrate that baculovirus/insect cell expressed actin has no significant differences from tissue purified actin and can be used for thin-filament reconstitution assays. One hypertrophic cardiomyopathy (HCM) causing actin mutant A331P actin was also expressed and studied similarly, and the results were compared to those of the WT actin. In the reconstituted fibers, A331P significantly decreased the tension both in the absence of Tm/Tn (0.55±0.03T0, N=13) and in their presence (0.65±0.02T0, N=13) compared to those of the WT (0.75±0.06T0 and 0.85±0.06T0, respectively, N=11). A331P also showed decreased pCa50 (5.57±0.03, N=13) compared to that of WT (5.69±0.02, N=11). The cross-bridge kinetics and its distribution were similar between WT and A331P actin reconstituted fibers, indicating that force/cross-bridge was decreased by A331P. In conclusion, A331P causes a weakened cross-bridge force, which leads to a decreased active tension, reduces left-ventricular ejection fraction, and eventually results in the HCM phenotype.
    Journal of Molecular and Cellular Cardiology 04/2014; · 5.15 Impact Factor
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    ABSTRACT: Recombinant WT human cardiac actin (WT actin) was expressed using the baculovirus/insect cell expression system, purified, and used to reconstitute the thin-filament of bovine cardiac muscle fibers, together with bovine cardiac tropomyosin (Tm) and troponin (Tn). Effects of [Ca2 +], [ATP], [phosphate] and [ADP] on tension and tension transients were studied at 25 °C by using sinusoidal analysis, and the results were compared with those of native fibers and fibers reconstituted with purified bovine cardiac actin (BVC actin). In actin filament reconstituted fibers (without Tm/Tn), those reconstituted with WT actin showed exactly the same active tension as those reconstituted with purified BVC actin (WT: 0.75 ± 0.06 T0, N = 11; BVC: 0.73 ± 0.07 T0, N = 12, where T0 is tension of original fibers before extraction). After Tm/Tn reconstitution, fibers reconstituted with WT actin generated 0.85 ± 0.06 T0 (N = 11) compared to 0.98 ± 0.04 T0 (N = 12) recovered by those reconstituted with BVC actin. In the presence of Tm/Tn, WT actin reconstituted fibers showed exactly the same Ca2 + sensitivity as those of the native fibers and BVC actin reconstituted fibers (pCa50: native fibers: 5.69 ± 0.01, N = 10; WT: 5.69 ± 0.02, N = 11; BVC: 5.68 ± 0.02, N = 12). Sinusoidal analysis showed that the cross-bridge kinetics were the same among native fibers, BVC actin reconstituted fibers, and WT actin reconstituted fibers, followed by reconstitution of Tm/Tn. These results demonstrate that baculovirus/insect cell expressed actin has no significant differences from tissue purified actin and can be used for thin-filament reconstitution assays. One hypertrophic cardiomyopathy (HCM) causing actin mutant A331P actin was also expressed and studied similarly, and the results were compared to those of the WT actin. In the reconstituted fibers, A331P significantly decreased the tension both in the absence of Tm/Tn (0.55 ± 0.03 T0, N = 13) and in their presence (0.65 ± 0.02 T0, N = 13) compared to those of the WT (0.75 ± 0.06 T0 and 0.85 ± 0.06 T0, respectively, N = 11). A331P also showed decreased pCa50 (5.57 ± 0.03, N = 13) compared to that of WT (5.69 ± 0.02, N = 11). The cross-bridge kinetics and its distribution were similar between WT and A331P actin reconstituted fibers, indicating that force/cross-bridge was decreased by A331P. In conclusion, A331P causes a weakened cross-bridge force, which leads to a decreased active tension, reduces left-ventricular ejection fraction, and eventually results in the HCM phenotype.
    Journal of Molecular and Cellular Cardiology 01/2014; · 5.15 Impact Factor
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    Li Wang, Masataka Kawai
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    ABSTRACT: A slackening to zero tension by large length release (~20 %) and a restretch of active muscle fibres cause a fall and a redevelopment in tension. According to the model of Brenner (Proc Natl Acad Sci USA 85(9):3265-3269, 1988), the rate constant of tension redevelopment (k TR) is the sum of attachment and detachment rate constants, hence is limited by the fast reaction. Here we propose a model in which, after restretch, cross-bridges cycle many times by stretching series elastic elements, hence k TR is limited by a slow reaction. To set up this model, we made an assumption that the stepping rate (v) decreases linearly with tension (F), which is consistent with the Fenn effect. The distance traveled by a cross-bridge stretches series elastic elements with stiffness σ. With these assumptions, we set up a first order differential equation, which results in an exponential time course with the rate constant k TR = ση 0 ν 0(1 - λ)/F 1, where λ = ν 1/ν 0, η = step size, the subscript 0 indicates unloaded condition, and the subscript 1 indicate isometric condition. We demonstrate that the ATP hydrolysis rate (=[myosin head]/ν 0) is proportionate to k TR as the ambient temperature is changed, and that the published data fit to this relationship well if λ = 0.28. We conclude that k TR is limited by the cross-bridge turnover rate; hence it represents the rate constant of the slowest reaction of the cross-bridge cycle, i.e. the ADP isomerization step before ADP is released.
    Journal of Muscle Research and Cell Motility 10/2013; · 1.36 Impact Factor
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    ABSTRACT: Aging is commonly defined as the accumulation of diverse deleterious changes in cells and tissues with advancing age. To investigate whether aging changes are involved in the lacrimal glands of chronic graft-versus-host disease (cGVHD) model mice, we obtained the specimens from cGVHD model mice, untreated aged and young mice, and examined by histopathology, and immunoblotting. Oxidative stress markers, 8-OHdG, 4-HNE, and hexonoyl lesion (HEL), and other aging markers, p16 and p38, were used to assess the samples. The infiltrating mononuclear cells and endothelia of capillaries in the cGVHD and aged mice expressed the oxidative stress markers and other aging markers, but not in the young mice. Histological changes and the expression of aging markers in the samples from cGVHD mice exhibited similar features to those in aging mice. These results suggest that changes that typically appear with advanced age occur earlier in the lives of mice with lacrimal gland cGVHD.
    Scientific Reports 08/2013; 3:2455. · 5.08 Impact Factor
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    ABSTRACT: Mechanical properties of skinned papillary muscle fibers from transgenic mice expressing familial hypertrophic cardiomyopathy associated mutations D166V and R58Q in myosin regulatory light chain were investigated. Elementary steps and the apparent rate constants of the cross-bridge cycle were characterized from the tension transients induced by sinusoidal length changes during maximal Ca(2+) activation, together with ATP, ADP, and Pi studies. The tension-pCa relation was also tested in two sets of solutions with differing Pi and ionic strength. Our results showed that in both mutants, the fast apparent rate constant 2πc and the rate constants of the cross-bridge detachment step (k2) were smaller than those of wild type (WT), demonstrating the slower cross-bridge kinetics. D166V showed significantly smaller ATP (K1) and ADP (K0) association constants than WT, displaying weaker ATP binding and easier ADP release, whereas those of R58Q were not significantly different from WT. In tension-pCa study, both D166V and R58Q mutations exhibited increased Ca(2+) sensitivity and less cooperativity. We conclude that, while the two FHC mutations have similar clinical manifestations and prognosis, some of the mechanical parameters of cross-bridges (K0, K1) are differently modified, whereas some others (Ca(2+)-sensitivity, cooperativity, k2) are similarly modified by these two FHC associated mutations.
    Journal of Molecular and Cellular Cardiology 05/2013; · 5.15 Impact Factor
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    Fan Bai, Li Wang, Masataka Kawai
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    ABSTRACT: Tropomyosin (Tm) is the key regulatory component of the thin-filament and plays a central role in the cardiac muscle's cooperative activation mechanism. Many mutations of cardiac Tm are related to hypertrophic cardiomyopathy (HCM), dilated cardiomyopathy (DCM), and left ventricular noncompaction (LVNC). Using the thin-filament extraction/reconstitution technique, we are able to incorporate various Tm mutants and protein isoforms into a muscle fiber environment to study their roles in Ca(2+) regulation, cross-bridge kinetics, and force generation. The thin-filament reconstitution technique poses several advantages compared to other in vitro and in vivo methods: (1) Tm mutants and isoforms are placed into the real muscle fiber environment to exhibit their effect on a level much higher than simple protein complexes; (2) only the primary and immediate effects of Tm mutants are studied in the thin-filament reconstituted myocardium; (3) lethal mutants of Tm can be studied without causing a problem; and (4) inexpensive. In transgenic models, various secondary effects (myocyte disarray, ECM fibrosis, altered protein phosphorylation levels, etc.) also affect the performance of the myocardium, making it very difficult to isolate the primary effect of the mutation. Our studies on Tm have demonstrated that: (1) Tm positively enhances the hydrophobic interaction between actin and myosin in the "closed state", which in turn enhances the isometric tension; (2) Tm's seven periodical repeats carry distinct functions, with the 3rd period being essential for the tension enhancement; (3) Tm mutants lead to HCM by impairing the relaxation on one hand, and lead to DCM by over inhibition of the AM interaction on the other hand. Ca(2+) sensitivity is affected by inorganic phosphate, ionic strength, and phosphorylation of constituent proteins; hence it may not be the primary cause of the pathogenesis. Here, we review our current knowledge regarding Tm's effect on the actomyosin interaction and the early molecular pathogenesis of Tm mutation related to HCM, DCM, and LVNC.
    Journal of Muscle Research and Cell Motility 05/2013; · 1.36 Impact Factor
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    ABSTRACT: Three troponin T (TnT) mutants that cause hypertrophic, restrictive, and dilated cardiomyopathy (I79N, ΔE96, and ΔK210, respectively), were examined using the thin-filament extraction/reconstitution technique. Effects of Ca(2+), ATP, phosphate, and ADP concentrations on force and its transients were studied at 25°C. Maximal Ca(2+) tension (THC) and Ca(2+)-activatable tension (Tact), respectively, were similar among I79N, ΔE96, and WT, whereas ΔK210 led to a significantly lower THC (∼20% less) and Tact (∼25% less) than did WT. In pCa solution containing 8 mM Pi and ionic strength adjusted to 200 mM, the Ca(2+) sensitivity (pCa50) of I79N (5.63 ± 0.02) and ΔE96 (5.60 ± 0.03) was significantly greater than that of WT (5.45 ± 0.04), but the pCa50 of ΔK210 (5.54 ± 0.04) remained similar to that of WT. Five equilibrium constants were deduced using sinusoidal analysis. All three mutants showed significantly lower K0 (ADP association constant) and larger K4 (equilibrium constant of force generation step) relative to the corresponding values for WT. I79N and ΔK210 were associated with a K2 (equilibrium constant of cross-bridge detachment step) significantly lower than that of ΔE96 and WT. These results demonstrated that at pCa 4.66, the force/cross-bridge is ∼18% less in I79N and ∼41% less in ΔK210 than that in WT. These results indicate that the molecular pathogenesis of the cardiac TnT mutation-related cardiomyopathies is different for each mutation.
    Biophysical Journal 05/2013; 104(9):1979-88. · 3.67 Impact Factor
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    ABSTRACT: Cross-bridge kinetics were studied at 20 °C in cardiac muscle strips from transgenic (Tg) mice expressing N-terminal 43 amino acid truncation mutation (Δ43) of myosin essential light chain (ELC), and the results were compared to those from Tg-wild type (WT) mice. Sinusoidal length changes were applied to activated skinned papillary muscle strips to induce tension transients, from which two exponential processes were deduced to characterize the cross-bridge kinetics. Their two rate constants were studied as functions of ATP, phosphate (Pi), ADP, and Ca(2+) concentrations to characterize elementary steps of the cross-bridge cycle consisting of six states. Our results demonstrate for the first time that the cross-bridge kinetics of Δ43 are accelerated owing to an acceleration of the rate constant k (2) of the cross-bridge detachment step, and that the number of strongly attached cross-bridges are decreased because of a reduction of the equilibrium constant K (4) of the force generation step. The isometric tension and stiffness of Δ43 are diminished compared to WT, but the force per cross-bridge is not changed. Stiffness measurement during rigor induction demonstrates a reduction in the stiffness in Δ43, indicating that the N-terminal extension of ELC forms an extra linkage between the myosin cross-bridge and actin. The tension-pCa study demonstrates that there is no Ca(2+) sensitivity change with Δ43, but the cooperativity is diminished. These results demonstrate the importance of the N-terminal extension of ELC in maintaining the myosin motor function during force generation and optimal cardiac performance.
    Journal of Muscle Research and Cell Motility 02/2013; · 1.36 Impact Factor
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    ABSTRACT: Cross-bridge kinetics were studied at 20 °C in cardiac muscle strips from transgenic (Tg) mice expressing N-terminal 43 amino acid truncation mutation (Δ43) of myosin essential light chain (ELC), and the results were compared to those from Tg-wild type (WT) mice. Sinusoidal length changes were applied to activated skinned papillary muscle strips to induce tension transients, from which two exponential processes were deduced to characterize the cross-bridge kinetics. Their two rate constants were studied as functions of ATP, phosphate (Pi), ADP, and Ca(2+) concentrations to characterize elementary steps of the cross-bridge cycle consisting of six states. Our results demonstrate for the first time that the cross-bridge kinetics of Δ43 are accelerated owing to an acceleration of the rate constant k (2) of the cross-bridge detachment step, and that the number of strongly attached cross-bridges are decreased because of a reduction of the equilibrium constant K (4) of the force generation step. The isometric tension and stiffness of Δ43 are diminished compared to WT, but the force per cross-bridge is not changed. Stiffness measurement during rigor induction demonstrates a reduction in the stiffness in Δ43, indicating that the N-terminal extension of ELC forms an extra linkage between the myosin cross-bridge and actin. The tension-pCa study demonstrates that there is no Ca(2+) sensitivity change with Δ43, but the cooperativity is diminished. These results demonstrate the importance of the N-terminal extension of ELC in maintaining the myosin motor function during force generation and optimal cardiac performance.
    Journal of Muscle Research and Cell Motility 02/2013; · 1.36 Impact Factor
  • Masataka Kawai, Li Wang
    Biophysical Journal 01/2013; 104(2):308-. · 3.67 Impact Factor
  • Fan Bai, Hannah Caster, John Dawson, Masataka Kawai
    Biophysical Journal 01/2013; 104(2):155-. · 3.67 Impact Factor
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    ABSTRACT: Single myofibrils 50–60 μm length and 2–3 μm diameter were isolated from rabbit psoas muscle fibres, and cross-bridge kinetics were studied by small perturbations of the length (∼0.2%) over a range of 15 frequencies (1–250 Hz). The experiments were performed at 15◦C in the presence of 0.05–10 mM MgATP, 8mM phosphate (Pi), 200 mM ionic strength with KAc (acetate), pCa 4.35–4.65, and pH 7.0. Two exponential processes, B and C, were resolved in tension transients. Their apparent rate constants (2πb and 2πc) increased as the [MgATP] was raised from 0.05 mM to 1mM, and then reached saturation at [MgATP] ≥ 1. Given that these rate constants were similar (c/b ∼1.7) at [Pi] ≥ 4 mM, they were combined to achieve an accurate estimate of the kinetic constants: their sum and product were analysed as functions of [MgATP]. These analyses yielded K1 =2.91 ± 0.31 mM −1, k2 =288 ± 36 s−1, and k−2 =10 ± 21 s−1 (±95% confidence limit, n =13 preparations), based on the cross-bridge model: AM+ATP ↔ (step 1) AM.ATP ↔ (step 2) A+M.ATP, where K1 is the ATP association constant (step 1), k2 is the rate constant of the cross-bridge detachment (step 2), and k−2 is the rate constant of its reversal step. These kinetic constants are respectively comparable to those observed in single fibres from rabbit psoas (K1 =2.35 ± 0.31 mM −1, k2 =243 ± 22 s−1, and k−2 =6 ± 14 s−1; n =8 preparations) when analysed by the same methods and under the same experimental conditions. These values are respectively not significantly different from those obtained in myofibrils, indicating that the same kinetic constants can be deduced from myofibril and muscle fibre studies, in terms of ATP binding and cross-bridge detachments steps. The fact that K1 in myofibrils is 1.2 times that in fibres (P≈0.05) may be explained by a small concentration gradient of ATP, ADP and/or Pi in single fibres.
    The Journal of Physiology 05/2012; 590(Pt 14):3361-73. · 4.38 Impact Factor
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    Fan Bai, Heather L Groth, Masataka Kawai
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    ABSTRACT: Two DCM mutants (E40K and E54K) of tropomyosin (Tm) were examined using the thin-filament extraction/reconstitu-tion technique. The effects of the Ca(2+), ATP, phos-phate (Pi), and ADP concentrations on isometric tension and its transients were studied at 25°C, and the results were com-pared to those for the WT protein. Our results indicate that both E40K and E54K have a significantly lower T(HC) (high Ca(2+) ten-sion at pCa 4.66) (E40K: 1.21±0.06 T(a), ±SEM, N = 34; E54K: 1.24±0.07 T(a), N = 28), a significantly lower T(LC) (low- Ca(2+) tension at pCa 7.0) (E40K: 0.07±0.02 T(a), N = 34; E54K: 0.06±0.02 T(a), N = 28), and a significantly lower T(act) (Ca(2+) activatable tension) (T(act) = T(HC)-T(LC,) E40K: 1.15±0.08 T(a), N = 34; E54K: 1.18±0.06 T(a), N = 28) than WT (T(HC) = 1.53±0.07 T(a), T(LC) = 0.12±0.01 T(a), T(act) = 1.40±0.07 T(a), N = 25). All tensions were normalized to T(a) ( = 13.9±0.8 kPa, N = 57), the ten-sion of actin-filament reconstituted cardiac fibers (myocardium) under the standard activating conditions. The Ca(2+) sensitivity (pCa(50)) of E40K (5.23±0.02, N = 34) and E54K (5.24±0.03, N = 28) was similar to that of the WT protein (5.26±0.03, N = 25). The cooper-a-tivity increased significantly in E54K (3.73±0.25, N = 28) compared to WT (2.80±0.17, N = 25). Seven kinetic constants were deduced using sinusoidal analysis at pCa 4.66. These results enabled us to calculate the cross-bridge distribution in the strongly attached states, and thereby deduce the force/cross-bridge. The results indicate that the force/cross-bridge is ∼15% less in E54K than WT, but remains similar to that of the WT protein in the case of E40K. We conclude that over-inhibition of the actomyosin interaction by E40K and E54K Tm mutants leads to a decreased force-generating ability at systole, which is the main mechanism underlying the early pathogenesis of DCM.
    PLoS ONE 01/2012; 7(10):e47471. · 3.53 Impact Factor
  • Biophysical Journal 01/2012; 102(3):156-. · 3.67 Impact Factor
  • Biophysical Journal 01/2012; 102(3):614-. · 3.67 Impact Factor
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    ABSTRACT: To establish α-tropomyosin (Tm)'s structure-function relationships in cooperative regulation of muscle contraction, thin filaments were reconstituted with a variety of Tm mutants (Δ2Tm, Δ3Tm, Δ6Tm, P2sTm, P3sTm, P2P3sTm, P1P5Tm, and wtTm), and force and sliding velocity of the thin filament were studied using an in vitro motility assay. In the case of deletion mutants, Δ indicates which of the quasi-equivalent repeats in Tm was deleted. In the case of period (P) mutants, an Ala cluster was introduced into the indicated period to strengthen the Tm-actin interaction. In P1P5Tm, the N-terminal half of period 5 was substituted with that of period 1 to test the quasi-equivalence of these two Tm periods. The reconstitution included bovine cardiac troponin. Deletion studies revealed that period 3 is important for the positive cooperative effect of Tm on actin filament regulation and that period 2 also contributes to this effect at low ionic strength, but to a lesser degree. Furthermore, Tm with one extra Ala cluster at period 2 (P2s) or period 3 (P3s) did not increase force or velocity, whereas Tm with two extra Ala clusters (P2P3s) increased both force and velocity, demonstrating interaction between these periods. Most mutants did not move in the absence of Ca(2+). Notable exceptions were Δ6Tm and P1P5Tm, which moved near at the full velocity, but with reduced force, which indicate impaired relaxation. These results are consistent with the mechanism that the Tm-actin interaction cooperatively affects actin to result in generation of greater force and velocity.
    Journal of Molecular Biology 12/2011; 414(5):667-80. · 3.91 Impact Factor
  • Robin Candau, Masataka Kawai
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    ABSTRACT: Our goal is to correlate kinetic constants obtained from fluorescence studies of myofibril suspension with those from mechanical studies of skinned muscle fibers from rabbit psoas. In myofibril studies, the stopped-flow technique with tryptophan fluorescence was used; in muscle fiber studies, tension transients with small amplitude sinusoidal length perturbations were used. All experiments were performed using the equivalent solution conditions (200 mM ionic strength, pH 7.00) at 10°C. The concentration of MgATP was varied to characterize kinetic constants of the ATP binding step 1 (K (1): dissociation constant), the binding induced cross-bridge detachment step 2 (k (2), k (-2): rate constants), and the ATP cleavage step 3 (k (3), k (-3)). In myofibrils we found that K (1) = 0.52 ± 0.08 mM (±95% confidence limits), k (2) = 242 ± 24 s(-1), and k (-2) ≈ 0; in muscle fibers, K (1) = 0.46 ± 0.06 mM, k (2) = 286 ± 32 s(-1), and k (-2) = 57 ± 21 s(-1). From these results, we conclude that myofibrils and muscle fibers exhibit nearly equal ATP binding step, and nearly equal ATP binding induced cross-bridge detachment step. Consequently, there is a good correlation between process C (phase 2 of step analysis) and the cross-bridge detachment step. The reverse detachment step is finite in fibers, but almost absent in myofibrils. We further studied partially cross-linked myofibrils and found little change in steps 2 and 3, indicating that cross-linking does not affect these steps. However, we found that K (1) is 2.5× of native myofibrils, indicating that MgATP binding is weakened by the presence of the extra load. We further studied the phosphate (Pi) effect in myofibrils, and found that Pi is a competitive inhibitor of MgATP, with the inhibitory dissociation constant of ~9 mM. Similar results were also deduced from fiber studies. To characterize the ATP cleavage step in myofibrils, we measured the slow rate constant in fluorescence, and found that k (3) + k (-3) = 16 ± 1 s(-1).
    Journal of Muscle Research and Cell Motility 12/2011; 32(4-5):315-26. · 1.36 Impact Factor
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    ABSTRACT: The myosin essential light chain (ELC) is a structural component of the actomyosin cross-bridge, but its function is poorly understood, especially the role of the cardiac specific N-terminal extension in modulating actomyosin interaction. Here, we generated transgenic (Tg) mice expressing the A57G (alanine to glycine) mutation in the cardiac ELC known to cause familial hypertrophic cardiomyopathy (FHC). The function of the ELC N-terminal extension was investigated with the Tg-Δ43 mouse model, whose myocardium expresses a truncated ELC. Low-angle X-ray diffraction studies on papillary muscle fibers in rigor revealed a decreased interfilament spacing (≈ 1.5 nm) and no alterations in cross-bridge mass distribution in Tg-A57G mice compared to Tg-WT, expressing the full-length nonmutated ELC. The truncation mutation showed a 1.3-fold increase in I(1,1)/I(1,0), indicating a shift of cross-bridge mass from the thick filament backbone toward the thin filaments. Mechanical studies demonstrated increased stiffness in Tg-A57G muscle fibers compared to Tg-WT or Tg-Δ43. The equilibrium constant for the cross-bridge force generation step was smallest in Tg-Δ43. These results support an important role for the N-terminal ELC extension in prepositioning the cross-bridge for optimal force production. Subtle changes in the ELC sequence were sufficient to alter cross-bridge properties and lead to pathological phenotypes.
    The FASEB Journal 09/2011; 25(12):4394-405. · 5.70 Impact Factor

Publication Stats

954 Citations
239.75 Total Impact Points

Institutions

  • 2002–2013
    • Keio University
      • Department of Ophthalmology
      Tokyo, Tokyo-to, Japan
  • 1989–2013
    • University of Iowa
      • Department of Anatomy and Cell Biology
      Iowa City, Iowa, United States
  • 2011
    • French National Institute for Agricultural Research
      Lutetia Parisorum, Île-de-France, France
  • 2000–2011
    • Waseda University
      • Department of Physics
      Tokyo, Tokyo-to, Japan
  • 1985–1988
    • Columbia University
      New York City, New York, United States