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ANIMAL PRODUCTS |
* Department of Animal Biotechnology, University of Nevada, Reno 89557;
and
Department of Anatomy, Indiana University Medical School, Indianapolis 46202; and
and
Muscle Biology Laboratory, University of Wisconsin, Madison 53706
Abstract
Image analysis procedures for immunofluorescence microscopy were developed to measure muscle thin filament lengths of beef, rabbit, and chicken myofibrils. Strips of beef cutaneous trunci, rectus abdominis, psoas, and masseter; chicken pectoralis; and rabbit psoas muscles were excised 5 to 30 min postmortem. Fluorescein phalloidin and rhodamine myosin subfragment-1 (S1) were used to probe the myofibril structure. Digital images were recorded with a cooled charge-coupled device controlled with IPLab Spectrum software (Signal Analytics Corp.) on a Macintosh operating system. The camera was attached to an inverted microscope, using both the phase-contrast and fluorescence illumination modes. Unfixed myofibrils incubated with fluorescein phalloidin showed fluorescence primarily at the Z-line and the tips of the thin filaments in the overlap region. Images were processed using IPLab and the National Institutes of Health’s Image software. A region of interest was selected and scaled by a factor of 18.18, which enlarged the image from 11 pixels/µm to approximately 200 pixels/µm. An X-Y plot was exported to Spectrum 1.1 (Academic Software Development Group), where the signal was processed with a second derivative routine, so a cursor function could be used to measure length. Fixation before phalloidin incubation resulted in greatest intensity at the Z lines but a more-uniform staining over the remainder of the thin filament zone. High-resolution image capture and processing showed that thin filament lengths were significantly different (P < 0.01) among beef, rabbit, and chicken, with lengths of 1.28 to 1.32 µm, 1.16 µm, and 1.05 µm, respectively. Measurements using the S1 signal confirmed the phalloidin results. Fluorescent probes may be useful to study sarcomere structure and help explain species and muscle differences in meat texture.
Key Words: Actin • Beef • Image Analysis • Immunofluorescence • Microfilaments
Introduction
Variations in meat palatability will be further understood only when the intricacies and contribution of sarcomere architecture are elucidated and correlated with meat texture. A complex set of factors affects the texture of whole muscle foods, including conformational changes that occur in the myofibril during the onset of rigor. Temperature-dependent shortening complicates the interrelationships among tenderness, myofibril conformation, sarcomere length, and other factors. Sarcomere length is used as a measure of muscle shortening, which Locker (1960)
reported influenced meat tenderness. More-recent studies have suggested that the tenderness/sarcomere length relationship is much more complex (Smulders et al., 1990). With the availability of techniques and instrumentation, such as fluorescent probes, confocal microscopy, and charge-coupled device (CCD) cameras (Ringkob et al., 1995), single myofibril and thick whole-muscle sections can be characterized in much greater detail, using image analysis.
There is a need to understand myofibrillar conformation at a more fundamental level because the sarcomere-length relationship only explains part of the tenderness story. Filament lengths of muscles have previously only been measured with any degree of accuracy using the electron microscope (Page and Huxley, 1963) because light microscope resolution is limited to approximately 0.2 µm. Augmenting the light microscope with a CCD camera yields greater resolution in conjunction with image analysis software. In the present work, fluorescein-labeled phalloidin and rhodamine-labeled subfragment 1 (S1) were used to probe myofibrils from various muscles and species in order to visualize thin filament structure. The objective of this study was to develop a light microscope procedure for measuring thin filament lengths with sufficient precision to differentiate beef, chicken, and rabbit myofibrils.
Materials and Methods
Muscle Sample Collection and Rigor Development.
Muscle samples were collected from 12 USDA Select and Choice beef carcasses from British and Continental-crossbred cattle humanely slaughtered. Procedures were developed and data collected from composite samples representing three or more animals. Muscle samples also were obtained from chicken (n = 3) and rabbit (n = 3) after humane slaughter. Strips of beef cutaneous trunci, rectus abdominis, psoas, and masseter; chicken pectoralis; and rabbit psoas muscles were excised 5 to 30 min after exsanguination. The strips were dissected along the fiber axis into large fiber bundles of about 8 x 0.5 x 0.5 cm, stretched or allowed to slacken to various extents, and tied to applicator sticks with dental tape. The applicator sticks with the affixed muscle strips were stirred for 24 h in rigor buffer (Swartz et al., 1993). Muscle strips were used for myofibrillar isolation directly or stored in glycerol at –20°C.
Myofibril Isolation Procedure.
One to two grams of muscle fiber was cut from the muscle strip while being careful to eliminate visible connective tissue. The sample was placed in 15 vol (relative to original muscle weight) of rigor buffer (RB; 75 mM KCl, 5 mM KH2PO4 (pH 7.2), 2 mM MgCl2, 2 mM EGTA, 1 mM NaN3) and homogenized using a Polytron for four 1- to 2-s bursts. After homogenization, the samples were broken down further by using a Kontes 40-mL, all-glass Dounce tissue grinder with a loose-fitting pestle. An additional 5 vol of RB was added to each sample and the samples were subjected to 50 strokes. The homogenate was centrifuged at 1,000 x g for 10 min, the supernatant was discarded, and the pellet resuspended in 20 vol of RB. The suspension was again subjected to 50 homogenization strokes and filtered through a single layer of cheesecloth. After centrifugation for 10 min at 1,000 x g, the pellets were resuspended in 20 vol of a 0.5% Trition X-100/RB mixture, and the myofibril suspension was placed in a 2 to 5°C cooler for 15 min. After this period, the suspension was centrifuged at 1000 x g for 10 min, and the pellets were resuspended in 20 vol of RB. The centrifugation and resuspension in fresh RB was carried out three more times before the final pellets were either suspended in 5 to 10 vol of RB for immediate analysis, or suspended in 5 to 10 vol of a 30% RB 70% glycerol solution for freezer storage (–20°C).
Immunofluorescence Procedure.
The staining procedure was carried out in 0.5-mL microfuge tubes. The rigor buffer (RBI; containing 10 mM imidazole in place of the phosphate), with 1 mg/mL bovine serum albumin, 1.35 µM rhodamine S1 (Swartz et al., 1993), and 0.1 µM fluorescein phalloidin, was placed in the microfuge tube, mixed, and then washed myofibrils were added (0.25 mg/mL; concentrations refer to the final mixture). Tubes were incubated in the dark for 20 to 30 min, after which the tubes were centrifuged for 10 s and the supernatant was removed by aspiration. Myofibrils were resuspended in RBI and plated on a cover slip, and fixed with a 3% formaldehyde solution in RBI for 10 to 15 min on the cover slip, being careful to maintain a dome of liquid on the myofibril preparation. Cover slips were rinsed in 15 mL of RBI. The cover slips were mounted on a glass slide using a glycerol-based mounting medium (70% glycerol [vol/vol], 75 mM KCl, 2 mM MgCl2, 2 mM EGTA, 10 mM Tris•HCl, pH 8.5 containing 1 mg/mL phenylene-diamine to decrease fluorescence fading) and were sealed with clear fingernail polish. The fluorescein phalloidin produced peaks at the Z-line and the tips of the thin filaments in the myofibrils incubated before fixation. Myofibrils were also incubated with rhodamine S1, applied to cover slips, fixed in formaldehyde, and incubated with fluorescein phalloidin. The cover slips were sealed on glass slides as described previously. The pattern obtained with fluorescein phalloidin incubation before fixation will be referred to as fresh, whereas those with the phalloidin applied after fixation will be called fixed.
Filament Measurements.
A Nikon inverted microscope (Model Diaphot) equipped with phase-contrast, fluorescence illumination, and 100x oil immersion objective (NA 1.4) was used to observe myofibrils. The microscope was outfitted with a cooled charge-coupled device (Thomson 7883; Photometrics Ltd., Tucson, AZ) for detection and capture of phase-contrast and fluorescent digital images. The CCD was controlled by IPLab V2.5 (Signal Analytics Corp., Vienna, VA) software via a Macintosh II fx (Apple Computer Inc., Santa Clara, CA) platform using a Matrox frame grabber card. Ten myofibril images, which included three consecutive sarcomeres (six thin filaments) for each muscle, were selected from five to nine slides. Images were captured using exposure times of 0.2 to 2 s, and gains of 1 for normal and 4 for low-intensity fluorescent signals.
The CCD images from IPLab were converted to TIFF, which could be imported into the NIH Image 1.56 (W. Rasband, National Institutes of Health, http://rsb.info.nih.gov/nih-image/Default.html) program. A region of interest (ROI), which included the entire myofibril (Figure 1A), was first captured. A smaller ROI (4 x 112 pixels) (Figure 1B) was then selected and saved. The latter ROI (Figure 1B) could be scaled by a factor of 18.18 so that the original image, scaled at 11 pixels/µm, was enlarged to approximately 200 pixels/µm. The image was smoothed in the same operation utilizing the Bilinear Interpolation option. The image was analyzed using the Plot Profile command (Analyze menu bar of NIH Image) and saved as plot values. The same image was also imported into IPLab and processed through the Sobel edge filter routine and processed through NIH Image to produce a second set of plot values. These two files were imported into Spectrum 1.1 (Academic Software Dev. Group, Univ. Maryland, College Park) as an XY file, and the X-axis, under the Window menu bar, rescaled to be read directly in micrometers. The scale factor would have to be calibrated for each CCD/microscope setup. Both sets of plot values were normalized (X and Y from 0 to 1.0).
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depicts the relative intensities of the panel B ROI. The Smoothed second derivative command was used in the Spectrum Transformation menu to produce a signal for edge detection of the filament tips from the Sobel filter plot (Figure 1D). After comparing signals, it appeared that a smoothed width of 9 gave the optimum output for ease of interpretation. The first plot (Figure 1C) was superimposed on the Sobel filter plot (Figure 1D). By using the mouse-controlled vertical line cursor, the X-value in micrometers corresponding to the maximum or minimum Y-value for the peak position indicating the edge or change in slope could be read in the Spectrum window as X and Y numbers. The rubber band cursor command was set to the Z-line peak, which was read by the vertical line cursor in the previous pass, and the mouse was used to move the second line to the thin filament tip peak position. This line was then advanced to the first reversal of the signal beyond the predetermined peak position. The values were read directly and recorded.
Data Analysis.
Data were analyzed using PROC MIXED of SAS (SAS Inst. Inc. Cary, NC). Significant differences (P < 0.01) among means of thin filament lengths and sarcomere lengths (species x muscle) were determined by the LSD test. Thin filament lengths were also analyzed using sarcomere lengths as a covariate. Slides within muscles were used as a random effect to test significance of thin filament lengths.
Results and Discussion
Myofibrils were stained pre-(fresh) and post-(fixed) fixing with 3% formaldehyde. Examples of rabbit psoas and chicken pectoralis (fresh and fixed) are shown in Figure 2. Even though the fresh rabbit and chicken myofibrils shown have very close to the same sarcomere length, the width of the space between the ends of the thin filaments and the visible width of the H-zone in phase are noticeably wider in chicken compared with rabbit (see parallel line guides on Figure 2). The nonuniformity of phalloidin staining can be seen in the middle panel of each muscle myofibril quadrant. Within the same sarcomere of fresh myofibril preparations, the Z-lines were stained, and a brighter, symmetrical doublet occupies the middle of the A-band, which is similar to the findings of Huckriede et al. (1988). This pattern would also agree with the observations of Wilson et al. (1987) that phalloidin initially binds primarily to the Z-lines and ends of the thin filaments, with little or no binding in the middle portion of the half I-band. However, if the fresh myofibrils are viewed after sufficient time, the phalloidin fluorescence redistributed (Wilson et al., 1987) in as little as 60 s. Szczesna and Lehrer (1993) also found that phalloidin equilibrated along the thin filament after 2 to 3 h at 37°C. We found that it took more than 24 h with fresh myofibrils held at 2 to 5°C to approach equilibration of fluorescence in the thin filament. Sarcomeres (Figure 2, right side) where the phalloidin was applied after the myofibrils were fixed produced staining patterns in which the brightest signal resides on the Z-line. The rest of the thin filament region of the I-Z-I brush is stained more uniformly without the bright fluorescence at the thin filament tips. This myofibril staining pattern is similar to that observed by Funatsu et al. (1990), Zhukarev et al. (1997), and Littlefield and Fowler (2002), in which fixation also took place before phalloidin staining.
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showed that S1 is bound to all actin regions in the myofibril at sarcomere lengths greater than 2.6 µm (for an explanation of S1 decoration pattern in detail, please see Swartz et al. [1993, 1996]). To convert both phalloidin and S1 staining patterns of rabbit and chicken to equivalent beef sarcomere fluorescent signals, approximately 0.3 and 0.5 µm must be added to rabbit and chicken sarcomeres, respectively. This would represent differences in I-Z-I brush length of 2.6, 2.3, and 2.1 µm in beef, rabbit, and chicken, respectively. The bare zones observed in the A-band would seem to agree with the Swartz et al. (1996) model when the sarcomere lengths were converted to beef equivalency lengths.
The phalloidin staining pattern had sharper peaks with fresh myofibrils in comparison to more diffuse patterns with the fixed myofibrils (Figure 2). The fresh chicken myofibril shown in Figure 2 had a diameter smaller than the one that had been fixed before phalloidin staining, but this difference was due to natural variability in myofibril diameter. Filament lengths and staining patterns of fresh myofibrils of the same muscle and species were not affected by myofibril diameter (left side of Figure 2). Direct measurements to determine thin filament lengths using fluorescent peaks and unprocessed images lacked precision because of the 0.09-µm pixel dimension. However, if the CCD images were processed with image analysis, this limitation could be ameliorated. By using a scale factor of 18.18, the original image of 11 pixels/µm could be enlarged to approximately 200 pixels/µm. The length measurement was made from the Z-line to the edge of the thin filament tip on one side of the I-Z-I brush. The edge was detected by smoothing in combination with the second derivative to find the change in slope at the tip edge. Each image allowed the measurement of six thin filaments within three consecutive sarcomeres in a myofibril. It was important to process a very narrow ROI (4 pixels wide in the vertical dimension) from the TIFF image in order to obtain sharp narrow peaks. It was also important to select regions of the myofibrils that had their Z-lines perpendicular to the long axis of the fiber.
Images of myofibrils from different beef muscles are shown in Figure 3. In all myofibrils observed, the fluorescence at the thin filament tips corresponds in position with the edge of the less-intense fringe of the S1 patterns. In comparing the S1 section of the masseter panel to the cutaneous trunci, rectus abdominis, and psoas S1-stained myofibrils, the masseter S1 myofibrils possess more space between the peaks on either side of the Z-line. This cleft appeared in varying degrees in some of the other fresh beef masseter myofibrils.
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. The beef rectus abdominis, psoas, and cutaneous trunci thin filament lengths did not differ (P < 0.01). However, the beef masseter filaments were shorter (P < 0.01), and this muscle was the only red fiber—dominant muscle among this group. The beef masseter myofibrils were the most difficult to isolate and prepare as acceptable specimens for staining. The beef cutaneous trunci is generally recognized as an all-white-fiber muscle, whereas the rectus abdominis and psoas have a mixed population of red and white fibers. One could speculate that the white fiber myofibrils survive the isolation procedure in greater numbers owing to the difficulty in preparing red fiber myofibrils. In all probability, the beef rectus abdominis, psoas, and cutaneous trunci are represented by mostly white fiber myofibrils. Although the difference between thin filament lengths from a red fiber muscle (masseter) and what may be mostly white fiber myofibrils (cutaneous trunci, and possibly rectus abdominis and psoas) is not great, there may be some physiological significance. Thin filament lengths for rabbit and chicken were both shorter (P < 0.01) than beef thin filaments (Table 1). The thin filament length of 1.16 µm was just slightly longer than the 1.11 to 1.13 µm reported by Sosa et al. (1994), representing different physiological conditions (relaxed, rigor, contraction, and shortening). Chicken pectoralis filaments (1.05 µm) were shorter (P < 0.01) than those from the rabbit psoas. The same lengths for chicken thin filaments have been obtained using phallicidin staining of fixed chicken pectoralis myofibrils and deconvolution analysis of fluorescence images (Littlefield and Fowler, 2002).
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). Both the fresh beef masseter and rabbit psoas have developed varying degrees of stretch and contraction within an 8- to 10-sarcomere segment of the same myofibril. The masseter has three different patterns of phalloidin staining in the thin filament tip region. These include a clear separation between the thin filament ends (far left sarcomere, SL = 2.91 µm); the thin filament tips from the half sarcomere being so close that the fluorescence intensity is in one unresolved zone (under tips of left bar; SL = 2.64 µm); and the thin filaments from adjacent half sarcomere were superimposed (under tips of right bar; SL = 2.37 and 2.35 µm). The rabbit psoas presents unique staining patterns that are of a different type. In this case, the apparent spacing between the thin filament tips remains constant despite large variations in sarcomere length. With this myofibril, it is suggested that the thin filaments have been pulled loose from the Z-line, to explain the phalloidin and S1 staining patterns.
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The combination of fluorescent probes and image analysis allowed greater precision in measuring thin filament lengths than the normal 0.2-µm resolution limitation of light microscopy. Thin filament length measurements using phalloidin staining make it possible to differentiate among beef, chicken, and rabbit myofibrils. Fluorescent probes may allow characterization of muscle structural differences between various muscles from a carcass and sarcomere fine structure without the complex procedures required for electron microscopy. Additionally, thin filament length may be a previously unrecognized factor affecting meat texture.
Footnotes
1 Supported by the College of Agric., Biotech., and Natural Resources, Univ. of Nevada, Reno and the College of Agric. and Life Sci., Univ. of Wisconsin-Madison. 
2 Correspondence—phone: 775-784-1628; fax: 775-784-1375; e-mail: tringkob{at}scs.unr.edu.
Received for publication September 16, 2003. Accepted for publication January 16, 2004.
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