Department of Anesthesia Research Laboratories, Brigham and Women's Hospital, Boston, Massachusetts 02115
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ABSTRACT |
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The effects of shortening distance at Vu, the unloaded shortening speed, and filament overlap on the amount of extra Ca2+ released during relaxation in muscle, as indicated by the bump area, were studied. Single, intact frog skeletal muscle fibers at 3°C were used. The myoplasmic free Ca2+ concentration ([Ca2+]i) was estimated by using fura 2 salt injected into the myoplasm. Ramps were applied, either at full overlap with different sizes or at varying overlaps with a fixed size, in the linear phase of relaxation. At full overlap, a plot of bump area vs. ramp size was fit by using a sigmoidal curve with one-half of the bump area equal to 25.9 nm. With a fixed ramp size of 100 nm/half-sarcomere, the plot of bump area vs. mean sarcomere length (SLm) was fit by a straight line intersecting the SLm axis at ~3.5 µm, close to just no overlap. The results suggest that the transition in the distribution of attached cross bridges from the isometric case to one appropriate for unloaded shortening at Vu is completed within 50 nm/half-sarcomere and support the view that attached cross bridges in the overlap zone influence the affinity of Ca2+ for troponin C in the thin filament.
cross bridges; calcium; cooperative interactions; regulation of contraction; fura 2; frog
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INTRODUCTION |
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IN SKELETAL MUSCLE, the relaxation phase of contraction, particularly after a tetanus, shows two distinct phases (10, 16). First, there is a slow, nearly linear phase of force decline ending with a so-called shoulder, and this is followed by a rapid, quasi-exponential decline of force to baseline. The shoulder coincides with the onset of small amounts of sarcomere shortening throughout most of the muscle fiber, with similar amounts of lengthening at the ends, so that overall the muscle length is constant (8, 16). Later, it was shown that the shoulder coincides with the onset of a small reduction in the rate of decline of the aequorin luminescence signal, suggesting a reduction in the rate of decline of myoplasmic free Ca2+ concentration ([Ca2+]i) (5). These findings are commonly explained by assuming that the onset of the quasi-exponential phase of relaxation indicates an acceleration of cross-bridge detachment and turnover (16) and that this leads to a reduction in the binding constant of troponin C (TnC) for Ca2+ (25). Thus the affinity of Ca2+ for TnC decreases rapidly, causing the rate of release of Ca2+ to increase, leading to a decrease in the rate of decline of [Ca2+]i, which is in accordance with previous discussions (1, 12, 13, 21).
Further studies using a fluorescent dye to measure [Ca2+]i showed very prominent "natural" bumps after the onset of the shoulder during relaxation in single frog muscle fibers (6). Length-shortening ramps were also applied during the linear phase of relaxation in frog fibers to show that the release of Ca2+ is accelerated during these "artificial" bumps (6).
Recently, we have published extensive results from studies of intact cardiac muscle using the same kind of methodology to create artificial bumps during relaxation by applying appropriate shortening ramps (18). These results indicate that both naturally occurring and artificially induced bumps exist in both cardiac and skeletal muscle. Here, we extend this approach to the study of single frog skeletal muscle fibers, for which it is much easier to control and change sarcomere length. We quantitatively examine how the bump area varies with ramp size at full overlap and how the bump area varies with reduced overlap at fixed ramp size. Our results can be explained by cross-bridge transitions from isometric to unloaded shortening at Vu, greatly diminishing the Ca2+ affinity for TnC in the thin filament, and by the overlap zone most strongly influencing this affinity.
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METHODS |
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A great deal of the basic methodology used in these experiments has already been well described in several recent publications from this laboratory (7, 18, 23), and these papers should be consulted for details of the procedures used here. Basically, single, intact muscle fibers were obtained from the tibialis anterior muscles of the frog Rana temporaria. The salt form of the fluorescent dye fura 2 was microinjected by iontophoresis into the myoplasm to estimate [Ca2+]i. All experiments were carried out at 3 ± 0.1°C. The mean sarcomere length (SLm) was determined from photographs of a passive fiber taken at different positions along the fiber. No attempt was made to use a feedback control device to maintain SLm constant during a contraction. However, fibers were not stretched beyond an SLm of 3.0 µm to minimize variations in sarcomere length along a fiber during contraction.
We have not been able to calibrate completely the response of the
intracellular fura 2 ratio (R) signal to [Ca2+]; this
inability is in contrast to our results with intact cardiac muscle
(18). The main problem encountered in skeletal muscle fibers is the
inability to control [Ca2+]i over a wide
range of values, thus allowing a curve fit to determine Kd. However, we have been able to obtain
reliable estimates for Rmax and Rmin, thus
allowing our results to be expressed in the dimensionless form
[Ca2+]i/Kd
. In this
case, although the absolute [Ca2+]i remains
unknown, Kd
merely plays the role of a scale
factor and the major nonlinearity in the fura 2 R signal is removed by using the relation
[Ca2+]i/Kd
= (R
Rmin)/(Rmax
R). R is obtained by
dividing the fluorescent light (510 nm) produced by excitation at 344 nm (F344) by that produced by excitation at 380 nm
(F380). Before forming R, the background and
autofluorescence were first subtracted, as is necessary, but this is a
minor correction for skeletal muscle compared with the correction
for cardiac muscle (18).
The following comment concerns our use of the terms artificial and
natural to describe the bumps observed during relaxation in muscle. A
natural bump is one observed in an ordinary fixed-length relaxation
after a tetanic contraction without any imposed length change. In
contrast, an artificial bump is one caused by applying an external
length change (always in the shortening direction in this work) at a
point in time preceding the occurrence of the natural bump. In this
work, the applied length decrease was always a ramp of
slope Vu, the unloaded shortening speed of a muscle fiber, 3 µm ·
HS1 · s
1, where HS is
half-sarcomere, at 3°C. The ramps were applied during the linear
phase of relaxation after a tetanus with duration of 0.5 s, so that
they followed the cessation of stimulation and were close to but
preceding the "shoulder" signaling onset of internal sarcomeric
motion. At full overlap, the ramp sizes chosen were 12.5, 25, 50, 75, 100, and 125 nm/HS. These required 5, 9, 17, 25, 33, and 41 ms,
respectively, between the beginning and end of the ramp.
SLm was varied by stretching passive fibers from 2.2 to 3.0 µm in increments of 0.2 µm/HS. In all cases of varying SLm, a fixed ramp size of 100 nm/HS was used.
To calculate the bump area, the R signal was first transformed into
[Ca2+]/Kd. Then, by fitting the
segments in the decay phase of
[Ca2+]/Kd
flanking but not
including the bump, a smooth, continuous curve with values at all time
points equivalent to the original signal with the bump was obtained.
This was done by using SigmaPlot (SPSS); a single, modified
three-parameter exponential-decay transform was used to obtain values
for the parameters a, b, and c for use in the
equation y = a · exp[b/(x + c)]. (An example
of such an operation is shown in Fig. 2D, which shows both the
original fluorescence trace and the "curve fit without bump.") A
difference curve was obtained by subtraction of the curve fit without
bump from the curve fit with bump. [Examples of this are
shown in Fig. 3, A and C, where it should be noted that
difference curves always begin (and end) with the amplitude equal to
0.0.] The area under the difference curve, equal to the bump area, was
obtained by using the area under the transform from SigmaPlot. This
procedure is very similar to the one that we have described elsewhere
(18). This method was accurate to analyze the natural bump and the
ramp-induced bump as long as the ramp size was equal to or larger than
50 nm/HS. However, when smaller ramp sizes (12.5 or 25 nm/HS) were
applied, the resulting total bumps were complex and included
contributions from natural and artificial processes. This made it
necessary to analyze the bumps in another way, namely, by using PeakFit software (SPSS) designed for peak separation and analysis. The total
area under the difference curve of the complex bump could be obtained
in SigmaPlot, but the separate areas under each peak could not. To do
this, the complex difference signal was exported to PeakFit, where a
curve was fit to the complex bump with the constraint that only two
peaks were present. In PeakFit, it was possible to obtain the areas
under the two separate peaks. The sum of these two areas was calculated
and compared to the total area under the two peaks
obtained by SigmaPlot, and these two areas never differed by more than
±1%. In contrast, all ramps of other sizes and the isometric case
produced bumps that were obviously composed of a single peak.
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RESULTS |
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The most basic assumption here is that the bump area observed during
the decaying phase of the [Ca2+]i obtained
by using fura 2 during relaxation is linearly related to
the amount of Ca2+ suddenly liberated into the myoplasm. A
problem arises because strictly speaking the bump area has the units of
micromolar × seconds, or, as in this case, is obtained by
multiplying
[Ca2+]i/Kd by the time
in seconds; in either case the units are not the same as those of
the amount of Ca2+. To clarify whether bump
area is proportional to the extra amount of
[Ca2+]i released during relaxation, simple
modeling was done by using the program Stella (High Performance
Systems, Hanover, NH), which allows construction of a model by drawing
a block diagram consisting of stocks and flows. This model is similar
to one recently described (20), which was, in turn, modified from one
already presented (26). Briefly, a central compartment simulating the
myoplasm was established. Initially, the compartment contained a known amount of Ca2+. The compartment was drained by a
"pump" similar to that previously described (20), and
Ca2+ could be added in brief pulses via a separate
"input" channel. These model results, not shown, indicate that a
nearly linear relationship between bump area and the amount of
Ca2+ pulsed into the myoplasm exists, and this supports the
idea that the bump area can be used as a valid index of the amount of
Ca2+.
A major concern in all experiments involving the use of a fluorescent dye to report [Ca2+]i, particularly in contracting muscle fibers, is the occurrence of so-called "motion artifacts." In this case, these are changes in fluorescence intensity caused by translation or rotation of a fiber or by movement of a fiber relative to the focal plane of the microscope. These kinds of artifacts can be greatly minimized by using the dual-wavelength dye fura 2 in the ratiometric mode (11). Experimental verification that motion artifacts do not play an important role in these experiments is shown in Fig. 1. Here, the passive (Fig. 1A) and active (Fig. 1B) responses of a single fiber stretched to an SLm of 2.8 µm to 100 nm/HS shortening ramps are shown. In Fig. 1A, the shortening ramp clearly causes upward deflections in both F344 and F380, most likely produced by movement into the field of more fiber, thus increasing the level of fluorescence. Note, however, that the R signal contains nearly perfect cancellation, since no sign of the deflections observed below in F344 and F380 at the time of the ramp was present. This is in contrast to the situation observed in Fig. 1B. Here, after electrical stimulation, large changes are seen in opposite directions, in both F344 and F380, as a consequence of the rise in [Ca2+]i. When stimulation was stopped and a ramp was applied during the linear phase of relaxation, a prominent artificial bump in the R signal, which is clearly associated with opposite-going deflections in F344 and F380, was observed. These opposite-going deflections in F344 and F380 indicate a true rise in [Ca2+]i caused by the ramp; it is most unlikely that they are caused by artifacts. The results in Fig. 1 strongly indicate that motion artifacts are canceled in the R signal, and true changes in [Ca2+]i are preserved.
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The results from one of four experiments are shown in Fig.
2 to illustrate the way in which results
were obtained at full overlap, SLm = 2.2 µm, in response
to variously sized ramps. In Fig. 2A, the ramps are shown as a
function of time. The topmost trace is for the isometric case; also
shown are 12.5-, 25-, 50-, 75-, 100-, and 125-nm ramps. In Fig.
2B, the associated force responses are shown. Note that in the
topmost isometric case it is apparent that the ramps are being applied
late in the linear phase of relaxation, but their onset clearly
precedes the shoulder in force, which can just be identified near the
right edge. Note also that the 12.5- and 25-nm ramps do not drop the
force to zero but that the 50-, 75-, 100-, and 125-nm ramps do, a
finding which has important consequences in this work. In Fig.
2C, the
[Ca2+]i/Kd signals are
shown on the same time base as those in Fig. 2, A and
B. For the isometric case, the lowest
[Ca2+]i/Kd
signal in
Fig. 2C, the beginning of the natural response can just be
observed at the right edge. The artificial responses clearly begin
sooner, and those due to the 12.5-, and 25-nm ramps, the next two above
the isometric response, obviously show signs of a complex response
involving both artificial and natural components. The bumps produced by
the next four larger-sized ramps, the 50-, 75-, 100-, and 125-nm ramps,
are more nearly similar to single-peak responses. Thus the natural
response is suppressed by applied ramps of large size. Finally, in Fig.
2D, only the
[Ca2+]i/Kd
signal in
response to the 100-nm ramp is shown. Also shown is the smooth curve
fit obtained by using the SigmaPlot single, modified three-parameter
exponential-decay transform, but omitting the points contained within
the bump. The bump area is equal to the area under the difference curve
obtained from the bump-containing and fitted smooth
curves.
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The results shown in Fig. 3 are concerned with an issue raised in METHODS, where it was mentioned that natural bumps and most bumps caused by artificial ramps could be fit by using a single peak. However, this was not the case for the 12.5- and 25-nm ramps. An example of a difference curve for the total bump caused by a 12.5-nm ramp is shown in Fig. 3A. The total bump is obviously not a single peak. The most likely explanation is that the initial part of the bump is caused by the applied ramp, whereas the following part is a "contamination" due to a contribution from the natural bump. It was necessary to separate these contributions in a rigorous way. This was done by exporting the difference curve obtained in SigmaPlot (noisy curve in Fig. 3A) to PeakFit, where a smooth curve was fit to the raw difference curve (thin, smooth line in Fig. 3A), with the constraint that only two peaks be present. The resulting two peaks produced by PeakFit are shown in Fig. 3B. In all of the four different fibers for which the 12.5-nm ramp was used, a similar situation was encountered, i.e., a pair of peaks with the first being smaller than the second. As a useful control, the total area under the raw difference curve shown in Fig. 3A was computed with SigmaPlot. Then, the sum of the areas under the two peaks generated by PeakFit was also computed. The total areas generated by these two methods always agreed to within ±1%, thus substantiating the idea that the decomposition into two peaks made by PeakFit was valid. In all cases, the total bump response to the 25-nm ramp (not shown) also consisted of a complex response not easily associated with a single peak. Here, the two peaks produced by PeakFit were more nearly equal in area, suggesting approximately equal contributions from natural and artificial processes.
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In contrast, all bumps caused by ramps of other sizes, as well as the natural bump occurring in the isometric case, could be well fit by using only a single peak. An example of this is given in Fig. 3C, which shows the difference curve (noisy trace) in response to an artificial 100-nm ramp with the curve fit by PeakFit superimposed (thin, smooth trace). In Fig. 3D, the PeakFit curve is shown alone to indicate the very close approximation obtained by using a single peak. The areas under the raw difference curve obtained by using SigmaPlot and under the smooth, single-peak curve fit by using PeakFit were nearly equal.
The average results obtained for bump area at full overlap as a function of various-sized ramps, including the isometric case, are presented in Fig. 4. Here, we plot the bump areas associated only with ramp-induced peaks and we define the ramp-induced bump area as zero for the isometric case. The area under the artificial peak was obtained by using PeakFit as previously described for the 12.5 and 25 nm/HS ramps. The data points are very well fit by a four-parameter sigmoidal curve from SigmaPlot with a half-area ramp size of 25.9 nm/HS. Note that the relative bump areas for the four largest-sized ramps, those of 50, 75, 100, and 125 nm, lie close to 1.0, and it is these ramps that cause the force to drop to zero as shown in Fig. 2. The average bump area for the four isometric cases is shown and indicates that the bump area naturally occurring without a ramp is about one-third of the maximal bump area obtained by using the largest ramp.
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The foregoing sets the stage for an examination of the bump area
produced by a ramp of 100 nm/HS as a function of filament overlap. This
kind of experiment is fairly straightforward with skeletal muscle
fibers, at least for limited ranges of decreased overlap, whereas for
the cardiac muscle we used previously it is very difficult (18). The
results for one of four fibers are shown in Fig.
5. In Fig. 5A, the length responses
are shown, with the flat line corresponding to no ramp and the
near-vertical line corresponding to the 100 nm/HS ramp used in all
cases with decreasing filament overlap. Note that the bump area
produced by the 100 nm/HS ramp in Fig. 4 is very near the maximal area
observed at full overlap, so it is reasonable to believe that a 100 nm/HS ramp produces a bump area of maximal size, thus allowing the
influence of overlap on bump area to be determined. Shown in Fig.
5B is a composite of the force responses, including the
isometric, to a 100-nm ramp as SLm was increased from the
control, 2.2 µm, to 2.4, 2.6, 2.8, and 3.0 µm. Finally, the
SLm was returned to 2.2 µm to obtain a control response,
but this is not shown as it is essentially unchanged. In Fig.
5C, the
[Ca2+]i/Kd signals
appropriate for each of the responses presented in Fig. 5B are
shown. In the isometric case, the lowest signal, the beginning of the
natural response, can be seen at the right edge of the panel. The other
signals are arranged on the basis of initial SLm values
of 2.2, 2.4, 2.6, 2.8, and 3.0 µm, and it is obvious
that the size of each of these signals is inversely proportional to
filament overlap.
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Finally, in Fig. 6, the average results from all four fibers are shown in a plot of normalized artificial bump area as a function of sarcomere length. The linear regression curve produced by SigmaPlot was constrained to pass through point pair (2.2, 1.0). The intersection of the straight line and the sarcomere length axis falls very close to an SLm of 3.5 µm, which is very near to just no overlap for fibers obtained from the tibialis anterior of R. temporaria (2). Thus these results strongly support the idea that the bump area produced by a large-amplitude ramp shortening applied during the linear phase of relaxation decreases linearly to approach zero as filament overlap and cross-bridge interaction vanish.
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DISCUSSION |
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The major emphasis in this work is on the transient rise in [Ca2+]i, or bump, usually observed during relaxation after the cessation of stimulation; the bump follows the shoulder in the force record during ordinary fixed-end relaxation in normal skeletal muscle fibers, usually from the frog (6, 20). The bump is observed after stimulation ceases, so that the high [Ca2+]i associated with the activation of contraction declines rapidly toward baseline, thus making it much easier to detect small changes in [Ca2+]i by using a suitable intracellular reporter (fura 2 in these experiments). We have chosen here, and in our previous work with cardiac muscle (18), to use the bump area as an indicator of the amount of Ca2+ released during either a natural or artificial bump. As mentioned in RESULTS, the bump area does not have units of micromolar. However, the results from a simple model for the release and uptake processes for Ca2+ in the myoplasm, as discussed in RESULTS, lend support to the view that the bump area is a valid indicator of the amount of Ca2+ released into the myoplasm.
A potential problem in fluorescence work, particularly in contracting and shortening muscles, is the possibility that results could be seriously contaminated by motion artifacts. However, the results shown in Fig. 1 clearly do not indicate a contribution of any serious magnitude from such artifactual processes. Instead, the results shown in Fig. 1 indicate that artifactual motion-induced changes are very well canceled by forming the R signal, whereas the ability to respond to true changes in [Ca2+]i is well preserved. Thus there should be no serious concern about such possible contaminant influences. The contributions of changes in background and autofluorescence may be of concern because these contributions must first be subtracted from the raw fluorescence signals obtained by alternate excitation at 344 and 380 nm before the ratio, or R, signal can be formed. Fortunately, in this work, the contributions from both background and autofluorescence were small and constant. This is quite different from what is found for autofluorescence from cardiac muscle when fura 2 is used to determine [Ca2+]i (18).
The key question concerns the cause of this transient elevation in [Ca2+]i during relaxation, since it can be routinely and clearly observed, particularly in isolated frog skeletal fibers. For cardiac muscle, we have argued that this phenomenon is most likely the result of a reversal during relaxation of a strong cooperation between Ca2+ binding to TnC and cross-bridge attachment (18). This interpretation was not easy to accept in cardiac muscle, since in this case powerful Ca2+ transporters exist, in addition to the sarcoplasmic reticulum (SR); the actions of these transporters might conceiveably result in a transient change in [Ca2+]i during relaxation. In skeletal muscle, there seems to be little doubt that the transient rise in [Ca2+]i observed during relaxation and after the shoulder is best explained on the basis of reduced affinity of Ca2+ for TnC owing to a reduction in the number of attached cross bridges (6). The reduction in the number of attached cross bridges, after the shoulder, follows from arguments presented in the introduction to this study, indicating that the shoulder coincides with the onset of small amounts of sarcomere shortening and subsequent cross-bridge turnover throughout most of an isolated muscle fiber. The important assumption here is that the transient rise in [Ca2+]i does, indeed, reflect a change in the binding affinity of Ca2+ to TnC in the thin filamental, though this has never been directly proved, to the best of our knowledge. It then becomes a question of how best to exploit this change to shed new light on the workings of the contractile machinery in an intact fiber.
We believe we have found a way to do this by applying external rapid ramp shortenings to create artificial bumps before the occurrence of the natural ones, or in place of them (18). The property is studied during relaxation when [Ca2+]i is falling rapidly toward baseline levels, so the problem of detecting small changes in [Ca2+]i against a background of greatly elevated [Ca2+]i is avoided (23). The fraction of attached cross bridges does not decrease much during the slow, linear phase of isometric force relaxation while [Ca2+]i approaches low levels; thus, the nature of the bumps is more easily revealed. In our view, the usual pattern of behavior after the shoulder in skeletal muscle fibers is closely simulated by the applied ramps. In the normal, fixed-end case, the usual internal rearrangement of sarcomere lengths owing to nonuniform relaxation at the time of the shoulder leads to cross-bridge cycling and turnover, with a consequent decrease in the number of attached cross bridges, which, in turn, leads to a decrease in the affinity of Ca2+ for TnC, and this causes a natural bump. The slow, linear phase of relaxation would be the ideal time to impose an external length decrease to cause a result essentially similar to that described above for the natural case, that is, cross-bridge cycling and turnover with a consequent reduction in the number of attached cross bridges. This would then lead to the production of a repeatable artificial bump that would be closely time locked to the length change.
The way in which data were obtained under conditions of varying ramp size at full overlap is shown in Fig. 2. The complication caused by complex bumps produced by the 12.5 and 25 nm/HS ramps was treated as described in the legend for Fig. 3. It is noteworthy that the ramps with the four largest amplitudes associated with drop of force to near zero, and its maintenance there as a consequence of shortening at near Vu, are the ones that are mainly in the plateau region of the dose-response curve shown in Fig. 4. This suggests that the dominant process in producing the bump is the transition from a cross-bridge distribution appropriate for isometric contraction to a distribution appropriate for shortening at Vu, with a much-reduced number of attached cross bridges. At ramp sizes smaller than 50 nm/HS, the force did not drop to zero and the bump area was smaller than that induced by ramps of larger sizes. Because the fibers were shortening at the same speed regardless of the sizes of the ramps, it most likely is the size of the ramp that limits the amount of force fall, the fraction of cross bridges detached, and the size of the associated bump.
The single parameter used to characterize the plot was the abscissal value for ramp size coinciding with one-half the maximal bump area, 25.9 nm/HS, as shown in Fig. 4. This value is clearly less than the repeat distance along the thick filament for cross bridges attaching to the thin filament in the same plane, 42.9 nm/HS, although it is larger than the repeat distance for cross bridges in all planes, 14.3 nm/HS (3). It is now believed that ~50% of the sarcomere compliance resides in the thin filaments (17, 24), and there is no doubt that substantial series mounting compliance exists in our fibers, typically near 18 nm/HS, when used for determining Vu by the slack test when no attempt was made to hold SLm constant (23). However, the presence of additional compliance in our system would lead to our value of 25.9 nm/HS for one-half maximal bump area being revised downward, although it is not known for certain whether it would approach the cross-bridge repeat distance of 14.3 nm/HS. Thus our curve of bump area vs. ramp size in Fig. 4 shows that the attachment transition from the isometric to the isotonic case at Vu is completed when the fiber shortens by ~50 nm/HS without taking account of mounting or thin-filament compliance, so the fraction of attached cross bridges most likely decreases rapidly within this distance. This supports the view that the cross-bridge cycling distance is <25.9 nm/HS, rather than near 60 nm/HS (27) or 40 nm/HS (14).
The way in which data were obtained under conditions of varying overlap with a single large-amplitude ramp being used to obtain a maximal bump is shown in Fig. 5. The average data obtained from four experiments are plotted in Fig. 6. In order to evaluate the results properly, it must be kept in mind that the SLm at just no overlap in fibers obtained from the tibialis anterior muscle of R. temporaria, such as the ones used here, is very near 3.5 µm (2). Remarkably, the linear regression fit to the average data in Fig. 6 intersects the abscissa at very near an SLm of 3.5 µm. The implications of this finding are quite substantial. This clearly shows that in an intact muscle system the artificial bump area caused by a large, saturating ramp applied during the linear phase of relaxation could be extrapolated to a value near zero as the amount of overlap vanishes. In other words, when the size of a ramp of slope Vu is large enough to produce a bump area of maximal size, it is the amount of overlap that determines the size of the bump area. The amount of overlap is simply proportional to the number of attached cross bridges in an active fiber. Most likely, the large applied ramp induced a transition from a cross-bridge distribution appropriate for isometric contraction to one appropriate for shortening at Vu, as argued above. The stiffness at Vu is about one-third of the isometric value (9, 19), and this implies that even fractionally fewer cross bridges are attached if the new thin-filament compliance measurements are taken into account and it is assumed that all cross bridges are attached in rigor (15). Therefore, the influence of the ramp most likely caused a cross-bridge distribution change from an isometric one in the linear phase of relaxation to one appropriate for shortening at Vu with a much-reduced number of attached cross bridges. This, in turn, could cause a bump if it reduced very much the affinity of Ca2+ for TnC, as pointed out in the introduction to this study. The effect would be maximal at full overlap, or SLm = 2.2 µm, where the number of cross bridges attached during the linear phase of relaxation would be greatest. As SLm was increased, with a consequent decrease in overlap, the bump area would tend to zero just as active force development does, and this was strongly substantiated by the finding that the intersection of the linear regression and the abscissa in Fig. 6 is very close to 3.5 µm.
These are new and important results, since they show that it is the overlap zone, or zone of cross-bridge attachment, that controls the bump area. Our results support the hypothesis that cross bridges attached to actin in the overlap zone control the affinity of Ca2+ for TnC. Our findings are in agreement with the view that "regional," i.e., overlap, interactions predominate in controlling changes in the affinity between Ca2+ and the thin filament (22), rather than supporting the view that the thin filament is involved along its entire length (4). If the attached cross-bridge number is suddenly reduced, this most likely decreases the affinity of Ca2+ for TnC in the overlap zone, thus liberating Ca2+ into the myoplasm. The magnitude of this effect will depend on both the number of cross bridges attached before the sudden ramp occurs and how much the fiber shortens to change the cross-bridge attachment distribution from nearly isometric to one appropriate for isotonic shortening at Vu.
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ACKNOWLEDGEMENTS |
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This work was supported by National Institutes of Health Grant HL-35032 (to F. J. Julian).
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests: F. J. Julian, Department of Anesthesia Research Laboratories, Brigham and Women's Hospital, 75 Francis St., Boston, MA 02115.
Received 19 May 1998; accepted in final form 17 September 1998.
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