INTRODUCTION

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SKINNED MUSCLE FIBER preparations are widely employed in studies involving muscle mechanics (35). However, whereas these preparations provide a great deal of flexibility in terms of being able to control the intracellular fluid composition, problems can arise in maintaining the quality of the preparations, specifically the sarcomere length uniformity. This degradation can be at least partly attributed to the nonuniform activation of the entire fiber segment. By rapidly altering the free Ca²⁺ concentration inside the skinned fiber preparation, more uniform activation can be achieved. Rapid alterations in internal Ca²⁺ concentration can be accomplished by a variety of methods, including the following: 1) iontophoretically passing Ca²⁺ into the solution in the region of small, skinned fiber segments (9); 2) using caged Ca²⁺ compounds (2, 3); and 3) activating the preparation in a solution with a very high Ca²⁺ buffering capacity, compared with that of the fiber as a whole (which includes the solution in the interfilament space as well as the myofibrillar proteins) (4, 5, 26).

The last method, which is commonly referred to as the "Ca²⁺-jump" technique (33), is the method most commonly employed, as it can readily be incorporated into experimental protocols with minimal effort and expense. The basic premise of this method is as follows. Ordinarily, before activation, the fiber is resting in a relaxing solution that contains little Ca²⁺ but a significant amount of EGTA, which represents the majority of the fiber-segment Ca²⁺ buffering capacity. By replacing the EGTA with a low-buffering capacity analog, such as hexamethylenediamine-N, N, N', N'-tetraacetate (HDTA) (26), the buffering capacity of the fiber segment can be significantly reduced. (Table 1 compares the binding constants of these compounds.) The net result is that, when the fiber segment is exposed to the activating solution, contraction-related Ca²⁺ binding is no longer in competition with the EGTA buffering system (Fig. 1).

View larger version (20K): [in this window] [in a new window]   Fig. 1.   Basis for Ca2+-jump technique. Standard (A) and Ca2+-jump-based activations (B) are shown. A: in presence of relaxing solution, majority of Ca2+ buffering capacity of fiber segment is represented by EGTA within fiber. As a result, when fiber is exposed to activating solution, contractile proteins must compete with EGTA for incoming Ca2+. B: conversely, when fiber is preactivated in a solution containing little EGTA, there is no competition for incoming Ca2+ when fiber is activated. [Ca2+], Ca2+ concentration; HDTA, hexamethylenediamine-N, N, N', N'-tetraacetate.

Whereas it has been reported that the use of the Ca²⁺-jump technique improves the quality of the striation pattern (17, 26, 27), this effect has not been quantified. Hence, the purpose of this work was to assess fiber quality with and without the Ca²⁺-jump protocol. Whereas sarcomere length is commonly measured by methods based on laser diffraction, this technique is not well suited to the analysis of local sarcomere uniformity. Therefore, we have developed a novel image-analysis technique to perform this analysis (31). This method is based on the evaluation of two-dimensional Fourier power spectra of muscle images. That is, we are capitalizing on the property that muscle fiber images can be considered as periodic patterns and, as such, the images can be represented in terms of Fourier spectra. This transformation can be carried out efficiently by using the fast Fourier transform (8). The magnitude of the transformed image represents the spectral energy density in the frequency domain. Assuming that the centroid frequency of the first-order spectral density peak represents the fundamental sarcomeric frequency, the mean sarcomere length is simply given by the reciprocal of this frequency.

	METHODS

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Preparations. Fiber bundles ~2 mm in diameter and 3-4 cm long were obtained from psoas muscles of adult New Zealand White rabbits. The bundles were stored at 18°C for up to 3 mo in relaxing solution containing 50% glycerol (vol/vol), as described by Goldman et al. (16). Single fiber segments, typically shorter than 500 µm, were isolated and treated with a nonionic detergent (Triton X-100, 0.5% vol/vol) for 15 min to disrupt remaining membrane fragments. The segments were then mounted horizontally between a semiconductor strain gauge-based force transducer (model AE801; SensoNor, Horten, Norway) and a servomotor (model 300S; Cambridge Technologies, Cambridge, MA) by using aluminum foil T clips (13) and were submerged in one of the chambers of a multichambered trough containing relaxing solution (20). In some cases, the ends of the fibers were chemically fixed by gluteraldehyde (7). Selective fixation was achieved through the use of a system composed of two cannulas that were bent 90° and then configured in such a manner that their ends were facing one another and separated by a few millimeters. As the fixative solution was delivered via one of the cannulas, suction was applied to the opposing cannula, effectively fixing only the portion of the fiber segment immediately in the path of the fixative flow.

Solutions. Solution compositions are listed in Table 2. All solutions were 200 mM total ionic strength, with propionate as the major anion. The solution pH was 7.0 in a MOPS buffer. All solutions contained 0.5 mM dithiothreitol to prevent protein degradation. Dextran T70 (5.6%) was also added to all solutions to restore the fiber diameters to preskinning levels (15, 18). HDTA was obtained from Aldrich (Milwaukee, WI). All other solutions were obtained from Sigma Chemical (St. Louis, MO). The solution compositions were determined by using a computer program initially developed by Godt (14).

Experimental protocol. The length of the fiber segment was adjusted to set the mean sarcomere length to the desired value, which was typically on the order of 2.6 µm. (Whereas the resting sarcomere length was generally found to be in the range of 2.4-2.5 µm, in a few instances it was found to be >2.5 µm. Therefore, the initial sarcomere length was set to 2.6 µm to ensure that the fiber was not slack at the starting condition.) The fiber segment was then activated isometrically by first being transferred into the low-Ca²⁺ buffering capacity, HDTA-based preactivating solution and then into the activating solution. After maximum tension was reached, the contraction was terminated by transferring the fiber segment back into the relaxing solution. Postcontraction sarcomere length was then assessed, and, if it differed from the starting sarcomere length by more than 0.15 µm, the fiber was discarded. Otherwise, the fiber length was readjusted to yield the original mean sarcomere length. (After the first contraction, the pre- and postcontraction sarcomere lengths generally remained consistent.) The fiber segment was then activated a number of times, with every second contraction preceded by the preactivation step. The duration of the preactivation step was 20 s, which was established on the basis of initial studies that showed that increasing this time did not lead to any additional improvements in the image quality and contractile performance. All contractions were videotaped for subsequent image analysis.

Measurements. The raw signal produced by the force transducer was amplified and then acquired by using a digital oscilloscope (model TDS 420; Tektronix, Beaverton, OR). The sarcomere length was assessed off-line by analysis of video images of the fiber, which were digitized at a resolution of 640 × 480 pixels by using a frame-grabber board (model OFG640; Imaging Technologies, Woburn, MA). The video-analysis system was calibrated by using a precision grating (Graticules, Tonbridge, UK).

	RESULTS

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Figure 2A shows an image of a fiber segment and its associated full, half, and quarter spectra. The image was acquired during a contraction cycle in which the fiber segment was preactivated in the HDTA-based solution with low-Ca²⁺ buffering capacity. It can be readily seen that the striations are well ordered and normal to the fiber axis. This is confirmed by the image spectra, which contain distinct first-order diffraction peaks situated on the fiber axis. To facilitate the comparison of these spectra, the associated line spectra are plotted in Fig. 3A. This figure shows that there is good correspondence among the individual spectra (in terms of localization of the first-order peaks). Figure 2B shows the same fiber segment (and its associated spectra) during the subsequent contraction cycle in which the fiber segment was not preceded by HDTA preactivation. In this case, the striations are not as well ordered, and, as a result, the diffraction peaks are more diffuse. The associated line spectra, which are plotted in Fig. 3B, have broader, less distinct first-order lines and even less distinct second-order lines, compared with the line spectra associated with the fiber segment in Fig. 2A, indicating greater sarcomere length heterogeneity.

View larger version (96K): [in this window] [in a new window]   Fig. 2.   Comparison of Fourier spectra obtained from same fiber segment with (A) and without HDTA preactivation (B). Shown are original fiber image, power spectrum associated with entire fiber image (full spectrum), power spectra associated with left and right half images (half spectra), and power spectra associated with quarter images (quarter spectra). Calibration line, 100 µm.

View larger version (21K): [in this window] [in a new window]   Fig. 3.   A and B show Fourier line spectra associated with image spectra in Fig. 2, A and B, respectively. x-Axis indicates frequency, ranging from 0 to fs/2, where fs is the sampling frequency, which is the reciprocal of the distance between successive pixels in the image (some corresponding sarcomere lengths are indicated in µm). y-Axes indicate relative amplitude on a logarithmic scale. Full, full spectrum; H1 and H2, half spectra, Q1-Q4, quarter spectra.

Figure 4 compares the temporal sarcomere length responses obtained with and without the preactivation protocol. The sarcomere lengths obtained in the case in which the fiber did undergo preactivation (Fig. 4A) indicate that the sarcomeres function uniformly. On the other hand, in the absence of preactivation, the sarcomere length behavior derived from the full image is not indicative of the local sarcomere behavior (Fig. 4B). Specifically, the sarcomeres in the first quarter image are shortening, whereas they are lengthening in the last quarter. In Table 3, the SE values of the sarcomere lengths obtained from the quarter image spectra are compared. As expected, the SE values are lower for contractions preceded by the preactivation step. In addition, the differences between the mean sarcomere lengths during activated and relaxed states are also compared in this table. Preactivation has a significant effect in this regard.

View larger version (22K): [in this window] [in a new window]   Fig. 4.   Comparison of sarcomere length signals obtained from full muscle image and various subimages as a function of time. A: sarcomere lengths obtained from a segment that underwent HDTA preactivation. Fiber was transferred from HDTA solution to activation solution during time 1 (t1) and returned to relaxing solution during time 2 (t2). B: sarcomere lengths obtained from a segment that did not undergo HDTA preactivation. In this case, fiber was transferred from relaxing solution to activation solution during t1 and returned to relaxing solution during t2. Labels on y-axis indicate sarcomere length in µm. Horizontal calibration line, 10 s.

As expected, there is a significant increase in the rate of tension development when the fiber segment undergoes the preactivation protocol, as quantified in terms of the decreased time that it takes for the contraction to reach 50% of final force. Conversely, there is no significant difference in the force levels (Table 3). A typical example of the force responses obtained with and without the preactivation step are shown in Fig. 5.

View larger version (8K): [in this window] [in a new window]   Fig. 5.   Example of effects of preactivation protocol on contractile response. A: force responses obtained with (solid lines) and without (dashed lines) preactivation protocol. Calibration bar, 10 s. B: early component of force responses. Calibration bar, 1 s. Associated times that it takes contraction to reach 50% of final force (in s; t50) are also listed.

	DISCUSSION

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Sarcomere length uniformity plays a central role in studies involving muscle mechanics. In this paper, we showed that, by adopting a preactivation protocol in which the fiber segments are preactivated in a solution with minimal Ca²⁺ buffering capacity, improvements in the quality of the striation pattern, and hence sarcomere length uniformity, can be achieved. Specifically, sarcomere lengths measured from fiber segments that underwent the preactivation step exhibited less local variation when activated and were also more similar to the relaxed-condition sarcomere lengths than those obtained from fiber segments that did not undergo preactivation. This effect is most likely due to the rapid, uniform activation of the contractile proteins. Indeed the rate of tension development was found to be significantly faster when the fibers were exposed to the preactivating solution. In contrast to the kinetic effects, we saw small increases in the amplitudes of the force responses obtained with preactivation, but the effect was not significant. Interestingly, in many of the contractions in which the fiber segments underwent preactivation, the isometric tension was associated with a small overshoot, as shown in Fig. 5. This phenomenon has also been observed by other investigators (32).

It should be noted that, when dealing with skinned fiber segments, as opposed to intact fibers, fiber radius plays a prominent role in terms of uniform activation [e.g., Fabiato and Fabiato (10)]. Specifically, in skinned fibers, Ca²⁺ enters the system at the level of the surface of the fiber and then diffuses throughout the fiber. In intact fibers, however, Ca²⁺ release occurs at the A-band/I-band interface at the myofibrillar level, and hence the radial diffusion distance is significantly reduced. As a result, it has been proposed that experiments be carried out on single myofibrils, as opposed to single muscle fibers (1, 22), which would effectively reduce the "fiber" diameter and hence minimize the time required for Ca²⁺ to diffuse to the center of the myofibril. Thus the myofibril will be uniformly activated across its cross section. Assuming that the fiber-segment characteristics are uniform across the entire length of the myofibril (i.e., uniform filament lengths, concurrent activation), this will translate into uniform behavior along the fiber axis.

It should be noted that a number of other methods have also been proposed to minimize sarcomere length heterogeneity. Iwazumi and Pollack (22) found that, by conditioning the fiber with an initial, slow activation step in which the Ca²⁺ concentration is increased from resting to full activation levels over a period of 5-10 min, subsequent contractions did not result in any significant deterioration in the striation pattern.

Brenner (6) found that cycling the fibers with brief periods of lightly loaded isotonic shortening stabilized the striation pattern during prolonged, maximal Ca²⁺ activation. Sweeney and colleagues (34) subsequently modified the technique by replacing the isotonic shortening with isovelocity ramps, hence eliminating the need for a force feedback system.

Hellam and Podolsky (20) and Julian (23) found that the striation pattern could be better maintained by minimizing the period of activation to as short a time as possible. Unfortunately, such an approach limits both the number and type of measurements that can be taken during each contraction.

Podolin and Ford (28) achieved rapid activation by exposing fibers to free Ca²⁺ for a 0.5- to 1.0-s period before introducing the EGTA-buffered solution through the use of an electronically controlled solution changer.

The uniformity of the striation pattern can also be better maintained by reducing the force levels generated by the muscle fiber segments. This can be achieved in a number of ways, such as decreasing the temperature of the bathing solutions (30) and using submaximal levels of Ca²⁺ to activate the fiber segments (19, 24). However, it should be stated that the latter method only delays the deterioration of the striation pattern.

Instead of replacing EGTA with a low-Ca²⁺ buffering capacity analog, it can simply be eliminated from the preactivating solution (12). Whereas such an approach is somewhat inferior to the HDTA replacement protocol because it changes the ionic strength of the solution, the effects of such a change would be minimal.

Many of the above-mentioned methods can be used in conjunction with one another.

Although the Ca²⁺-jump protocol is presently being employed by a number of investigators, the results presented here warrant its more widespread use, particularly given that it can be incorporated into existing methodologies with minimal effort. Work is presently being conducted to extend this analysis to an examination of sarcomere length changes during activation as a function of radial distance from the central fiber axis.