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RAPID REPORT |
1 Krannert Institute of Cardiology, Indiana University School of Medicine, 1800 N. Capitol Ave, Indianapolis, IN 46202, USA
2 Roudebush VA Medical Center, 1481 W. Tenth St, Indianapolis, IN 46202, USA
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Abstract |
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(Received 10 October 2006;
accepted after revision 8 November 2006;
first published online 9 November 2006)
Corresponding author L. E. Ford: Krannert Institute of Cardiology, Indiana University School of Medicine, 1800 N. Capitol Ave, Indianapolis, IN 46202, USA. Email: lieford{at}iupui.edu
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Introduction |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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The present study was stimulated by the finding that substitution of EGTA for calcium, which caused force to decline to zero over the course of several tetani at 5 min intervals, also caused the birefringence signal to invert before it too declined to zero. This finding suggested that two processes are stimulated at different myofibrillar calcium concentrations during activation. One is myosin light-chain phosphorylation, known to cause folded myosin molecules to unfold and form filaments (Craig et al. 1983) as well as to activate the actomyosin ATPase (Sobieszek, 1977). The second, stimulated at a lower calcium level, was hypothesized to move crossbridges away from thick filaments. To test this hypothesis, birefringence was measured as contraction was inhibited with wortmannin (Nakanishi et al. 1992), which has been shown to inhibit force generation, myosin light-chain phosphorylation, and the increase in number of thick filaments per cell cross-section, without altering the intracellular calcium transient (Qi et al. 2001).
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Methods |
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0.3 mm x
0.8 mm x
5 mm between attachments. Muscles were studied at 37°C in physiological saline containing (mM): NaCl 112.5, NaHCO3 27.5, KCl 4.0, NaH2PO4 1.2, MgSO4 2.0, CaCl2 2, glucose 5, and perfused with 95% O2–5% CO2. Tracheae were stored and muscles dissected in unperfused physiological saline with Hepes buffer substituted for bicarbonate. In some studies, 2 mM EGTA replaced Ca. Wortmannin was obtained in 5 mg quantities from Sigma (St Louis, MO, USA) or Biomol (Plymouth Meeting, PA, USA), dissolved and stored as 10 mM in dimethylsulfoxide for up to 6 weeks at –20°C and added to the physiological saline to make a nominal concentration of 1 or 5 µM.
Muscles were stimulated for 12 s at 5 min intervals throughout the experiments with 60 Hz, 1 ms pulses of alternating polarity and amplitude 10% greater than that required to achieve maximum force.
Experiment protocol
After initial adaptation to the experimental conditions, muscles were adjusted to the length (L10%) where rest tension was 10% of total force and adapted there for at least six tetani. Some muscles were studied there and others were studied at the length (L1%) where rest tension was 1% of developed force. In stretch experiments, stretches began from 0.85L10%. After adaptation for at least six tetani at the study length, force and birefringence were measured in the baseline state, and then wortmannin was added to the physiological saline and measurements continued in successive tetani until force had fallen to near zero.
As described below, the rate of the force decline caused by wortmannin varied. To compare results from different muscles, force and birefringence records were signal averaged when force had declined to the following levels: 75%, 50%, 25% and < 10%.
Electron microscopy
Muscles were fixed for electron microscopy at L10% under four conditions: relaxed and activated in the presence and absence of wortmannin. After adaptation to the experimental environment, muscles to be fixed in a relaxed state were immersed first in physiological saline with EGTA and then in fixative containing EGTA. Muscles fixed in an activated state were stimulated electrically for 12 s, immersed in physiological saline containing 10 µM acetylcholine for another 2 min, and then transferred to fixative solution containing acetylcholine.
Muscles were fixed for electron microscopy as previously described (Kuo et al. 2003). Digital electron micrographs of muscle cross-sections were acquired with a 1 Mpixel camera at x110 000 magnification, yielding 0.9 nm per pixel resolution. Filament cross-sectional areas were measured using the ImageJ program.
One hundred filaments, 10 filaments per cell in each of 10 cells, were measured in each muscle and averaged to yield a single value for the muscle. To provide a random selection of filaments in each cell, the first filament to be measured was selected near the centre of a group of filaments and the remaining filaments chosen by progressing around this first filament in a spiral manner and selecting nearest neighbours.
Statistics
All errors quoted are S.E.M. Significance of differences between means from different muscles were calculated using unpaired Student's t tests.
Control observation
Wortmannin inactivation. The time course of force inhibition by wortmannin varied, and this variability was traced to inactivation of the agent at a slow rate in dimethylsulfoxide in the freezer and at a rapid rate in physiological saline (Kimura et al. 1994). Thus, the concentration of wortmannin cannot be specified exactly. Since one purpose of the experiments was to assess birefringence changes during the process of inhibition, low concentrations (nominally 1 or 5 µM) were used, and records were chosen for analysis when force had declined to the levels quoted above, rather than at specific times.
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Results |
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To assess whether the inverted birefringence responses were associated with crossbridge attachment to thin filaments, ramp stretches were applied to unstimulated and stimulated muscles before and after force inhibition. Stretches beginning from a short length (LS
= 0.85L10%) resulted in relatively little tension increase during stretch of the unstimulated muscle; the stretch size (4%
LS) assured crossbridge detachment with an abrupt decrease in resistance during the step, and the ramp speed (8LS s–1) provided substantial differences between the resistance of stimulated and unstimulated muscle. Force in the untreated, stimulated muscle rose steeply to 160% of isometric force (FRef) when stretch was 0.8%
LS and then levelled off abruptly, remaining nearly constant for the remainder of the ramp (Fig. 3C). By contrast, force in the unstimulated muscle rose with an uninterrupted trajectory, reaching only 8%
Fref when the same 0.8% stretch was attained. The responses to stretch in stimulated and unstimulated muscle after force inhibition superimposed almost exactly (Fig. 3D) and were no different to the response of the unstimulated muscle before wortmannin treatment.
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Figure 4 shows electron micrographs of muscle cross-sections under four conditions: stimulated and unstimulated in the presence and absence of wortmannin. Without wortmannin treatment, thick filament cross-sectional areas were decreased by 44% during stimulation, from 300 ± 28 nm2 in six unstimulated muscles to 168 ± 11 nm2 in six stimulated muscles (P < 0.005). Thick filament areas in four relaxed and four stimulated muscles after treatment with wortmannin, 262 ± 11 and 291 ± 42 nm2, respectively, were not significantly different from each other (P > 0.25) and not significantly different from relaxed muscle in the absence of wortmannin (P > 0.1 and P > 0.8, respectively).
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Discussion |
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Aksoy et al. (1983) have proposed dual activating mechanisms in smooth muscle, both calcium dependent, with light-chain phosphorylation regulated by higher calcium levels and a second, unspecified mechanism responding to lower calcium concentrations. We have proposed a dual activating mechanism in smooth muscle (Ford et al. 1994). One is light-chain phosphorylation, known to regulate both the actin-activated ATPase of myosin (Sobieszek, 1977) and the incorporation of myosin molecules into thick filaments (Craig et al. 1983). Realizing that phosphorylated myosin molecules could interact with actin before they joined thick filaments, we postulated a second mechanism to prevent phosphorylated, non-filamentous myosin molecules from interacting with thin filaments (Ford et al. 1994). In support of this proposal are the presence of several thin filament regulatory proteins, including tropomyosin (Chacko, 1981), caldesmon (Sobue et al. 1981) and calponin, which has some homology with troponin-T (Takahashi et al. 1988). Since tropomyosin and troponin mediate the calcium regulation of striated muscle thin filaments, similar proteins in smooth muscle greatly raise the suspicion of thin filament regulation there.
Weak myosin binding to thin filaments suggests a 2-step initial attachment with transition from weak to firm binding before the power stoke, which moves the bridge to a high-force states, sometimes collectively characterized as ‘strongly bound.’ Two-step initial attachment was first proposed by Huxley (1973) to account for Hill's (1964) energetics data, and our subsequent measurements of transient force responses to rapid length changes in skinned skeletal fibres have confirmed its existence (Seow & Ford, 1993; Seow et al. 2001). Brenner et al. (1982) have shown that skeletal muscle bridges attached in the initial, weakly bound state promoted by low ionic strength are in such rapid equilibrium with detached bridges that they are detected only by very rapid stretches, > 10 times the rate used here. Such a weakly bound, initial state would explain the crossbridge movement without demonstrable resistance to stretch at the strain rates used here. More rapid stretches were not used because the force increase during very rapid stretches is very much greater than in striated muscle, and it made the assessment of further force increases during activation less reliable.
In skeletal muscle, transition from a weakly bound to a firmly bound, low-force state is regulated by calcium binding to the troponin–tropomyosin regulatory system that moves aside and exposes actin sites to which the myosin binds more firmly (Brenner et al. 1982). In kinetic studies of contractile proteins in solution Alahyan et al. (2006) have demonstrated a similar weak-to-firm transition of myosin on smooth muscle thin filaments blocked instead by caldesmon binding to actin, possibly to one of the attachment sites that enable firm binding. Within this framework, the present experiments suggest that calcium also promotes myosin attachment to thin filaments in a weakly bound state and that the transition to a firmly bound state requires light-chain phosphorylation, perhaps in association with such other factors as shifting of caldesmon.
In conclusion, these experiments show that there are two competing mechanisms contributing to the birefringence changes of smooth muscle during activation. A birefringence increase caused by thick filament formation dominates the normal response. Inhibition of phosphorylation and filament formation unmasks a birefringence decrease, attributed to crossbridge realignment and movement away from thick filaments.
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References |
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Seow CY, White HD & Ford LE (2001). Effect of substituting uridine triphosphate for ATP on the crossbridge cycle of rabbit muscle. J Physiol 537, 907–921.
Smolensky AV, Ragozzino J, Gilbert SH, Seow CY & Ford LE (2005). Length-dependent filament formation assessed from birefringence increases during activation of porcine tracheal muscle. J Physiol 563, 517–527.
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Acknowledgements |
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