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Department of Poultry Science, North Carolina State University, Raleigh, NC 27695
2To whom correspondence should be addressed. E-mail: pemozdzi{at}unity.ncsu.edu.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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KEY WORDS: • apoptosis • skeletal muscle • myonucleus • chickens • starvation
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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); rather, the new nuclei arise from the fusion of myogenic satellite cells with the growing myofibers (2
).
A major determining factor of ultimate mature muscle size is the accretion of new myonuclei early in life because short-term reductions in the satellite cell population (3
) or short-term reductions in satellite cell mitotic activity in juvenile muscle (4
) result in a reduction in mature muscle size even though the potential for complete muscle recovery following the experimental perturbations lies within the satellite cell population (5
). Similarly, it has been shown that early posthatch starvation of broiler chickens during the first 48 h of life depresses satellite cell mitotic activity and reduces meat yield at market age, even though the yolk sac is present as a source of nutrients, compared with birds provided feed during the initial 48-h posthatch (6
). Therefore, early posthatch starvation has been proposed to adversely affect ultimate muscle size through a satellite cell pathway.
Another possible mechanism that can adversely affect myonuclear accretion is apoptosis or the regulated elimination of existing myonuclei (7
). Apoptosis plays a role in shaping a number of different tissues; it is characterized by the targeted destruction of the chromatin, followed by nuclear fragmentation, leading to cellular fragmentation and cell death (7
). After a typical muscle injury, the entire myofiber is destroyed and is rebuilt by the resident satellite cell population (8
), making apoptosis of select individual myonuclei a relatively unimportant phenomenon after a severe injury. However, apoptosis of individual myonuclei can occur in fully differentiated myofibers without wholesale myofiber destruction because recent studies have suggested that apoptosis is associated with dystrophin-deficient muscular dystrophy (9
), apoptosis is associated with the atrophy of the soleus that occurs during hindlimb unloading (10
), and apoptosis in the soleus muscle is associated with the monocrotaline model of congestive heart failure (11
). It has been suggested that apoptosis occurs in the unweighted muscle as a mechanism to eliminate in an orderly fashion nuclei that are no longer required because of a reduction in myofiber size (10
). It is clear that during pathologic challenges to the muscle or muscle functional challenges, myonuclear apoptosis plays a role in myofiber remodeling. The possibility that apoptosis plays a role in the adverse muscular changes that occur early posthatch in the absence of feed is unknown. If myonuclear apoptosis occurs during early posthatch starvation concurrently with the previously reported depression in satellite cell mitotic activity (6
), it would signal that the muscle is undergoing a more complex process than a simple reduction in the satellite cell population or a reduction in satellite cell mitotic activity. The objective of this study was to examine the effect of early posthatch starvation on myonuclear apoptosis during the early posthatch period.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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All experimental procedures involving animals were approved by the North Carolina State University Institutional Animal Care and Use Committee. Male broiler chickens were obtained from North Carolina State University Poultry Farms. Immediately after hatching, the chickens were split into two groups. The first group (Fed; n = 10) was provided for 3 d with a standard starter diet, top-dressed with Oasis, a cooked, hydrated nutritional formulation, which is low in fat but high in digestible carbohydrate and protein and is commercially available for newly hatched poultry (Novus, St. Louis MO). The standard starter diet contained 
88% dry matter, 3% fat, 5% crude fiber, 6% ash and 18% crude protein. After the 3-d posthatch period, the chickens were fed the standard starter diet. The second group (Starved; n = 10) of chickens was not provided with any feed for the first 3-d posthatch, but they were provided the standard starter diet beginning on the 3-d posthatch. All chickens consumed water ad libitum throughout the experiment. Chickens were weighed and killed by an overdose of Beuthanasia-D (Schering-Plough Animal Health, Kenilworth, NJ; 0.25 mL/kg body) on d 3 (n = 5/group) and d 7 posthatch (n = 5/group). Samples were harvested from the pectoralis thoracicus, fixed in 4% paraformaldehyde overnight, dehydrated, cleared, infiltrated with paraffin and embedded in paraffin.
The number of apoptotic nuclei was quantified by the histochemical method of staining double-stranded DNA breaks, which uses the terminal deoxynucleotidyl transferase (TDT) enzyme to incorporate fluorescein-labeled dUTP at 3'-OH fragmented DNA ends. The fluorescein-labeled nuclei were visualized with a fluorescence microscope. DNA fragmentation is a characteristic of apoptosis (7
), and the labeling of fragmented DNA has been used on muscle after hindlimb unloading to identify apoptotic myonuclei (10
).
Transverse sections (10 µm thick) were placed on glass slides, deparaffinzed, rehydrated and processed with an apoptosis detection kit (Catalog #G3250, Promega, Madison, WI). Briefly, sections were fixed with 4% paraformaldehyde, rinsed, permeabilzed with Proteinase K, washed with PBS, incubated with equilibration buffer (200 mmol/L potassium cacodylate, 25 mmol/L Tris-HCl, 0.2mmol/L dithiothreitol, 0.25 g/L bovine serum albumin and 2.5 mmol/L cobalt chloride) and the DNA strand breaks were labeled with fluorescein-12-dUTP using the TDT enzyme. Subsequently, the tissue sections were counterstained with propidium iodide (50 mg/L) to label all nuclei within the tissue section.
Tissue sections were observed with a Leica DMR microscope equipped with epi-fluorescence illumination. Apoptotic nuclei were visualized with a fluorescein isothiocyanate filter set, and all nuclei were visualized with a propidium iodide filter set (Omega Optical, Brattleboro VT). Images of nuclei visualized with the fluorescein isothiocyanate filters and nuclei visualized with the propidium iodide filters were acquired using a Spot-RT CCD Camera (Diagnostic Instruments, Sterling Heights, MI). The number of fluorescein-labeled (apoptotic) nuclei, the number of propidium iodide–labeled nuclei and myofiber cross-sectional areas were determined for each tissue section using Image-Pro Plus software (Media Cybernetics, Silver Spring, MD). The criterion for concluding the nuclear analysis for each muscle was counting at least 1000 propidium iodide–labeled nuclei from each tissue section. An index of apoptotic labeling was calculated by expressing the number of fluorescein-labeled nuclei relative to the number of propidium iodide–labeled nuclei. The criterion for concluding myofiber cross-sectional area analysis for each muscle was determining myofiber cross-sectional area for at least 100 myofibers.
Body weight data, myofiber cross-sectional area data and apoptotic labeling data were analyzed using the General Linear Models procedure of SAS (12
) to determine the effect of diet and age on each variable. Means were separated using least significant differences (13
). If variances were unequal, data were logarithmically transformed.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Body weights were lower (P < 0.05) and myofiber cross-sectional areas were smaller (P < 0.05) for the starved group compared with the fed group at both 3 and 7 d posthatch. Body weights and myofiber cross-sectional areas increased between 3- and 7-d (P < 0.05) posthatch for both groups (Table 1
).
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Apoptotic labeling was higher (P < 0.05) in the starved group compared with the fed group at 3 d posthatch (Fig. 1
; Table 1
) and was lower (P < 0.05) in the starved group at 7-d posthatch compared with the starved group at 3 d posthatch (Figs. 1
, 2)
. Apoptotic labeling did not differ (P > 0.05) between the starved and fed groups at 7 d posthatch (Fig. 2
; Table 1
).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). Short-term reductions in satellite cell proliferation in juvenile muscle, such as may occur in delayed posthatch feeding, result ultimately in a reduction in mature muscle size (3
,4
) even though the potential for complete recovery resides in the satellite cell population (5
). Therefore, it appears that the reductions in meat yield observed after early posthatch starvation are irreversible because the myofibers may be missing a developmental window for the acquisition of new nuclei.
Myofiber size is dependent on the acquisition of new DNA units and the maintenance of existing DNA units, which are defined as a myonucleus and its surrounding cytoplasm, through satellite cell fusions because there are limits governing the maximum size that an individual DNA unit may attain (14
,15
,16
,17
). Therefore, the addition of new myonuclei through satellite cell fusions or the loss of myonuclei through an apoptotic pathway would have a significant effect upon ultimate myofiber size. Myonuclear accretion is orchestrated by the myofibers because hindlimb unloading does not suppress myoblast proliferation during the early phases of muscle regeneration when the myofibers are not present (18
). Satellite cell proliferation becomes depressed in the hindlimb unloading model only after new myofibers are formed in a regenerated muscle (18
). Therefore, the myofiber determines ultimate muscle size and the DNA unit size may play a role in directing myonuclear accretion.
During a reduction in myofiber size induced by decreasing muscle functional activity, there is a reduction in DNA unit size (19
), indicating that there is some plasticity in DNA unit size during manipulations that result in myofiber atrophy. However, it is possible that the DNA unit size can be reduced only to a certain "critical level" before myonuclei are destroyed because the myonuclei may no longer be required to support the smaller volume of cytoplasm (10
). Although measurements of myofiber cross-sectional area were not made on the chickens used in the present study immediately posthatch, it is possible that there was some atrophy in the muscle leading to myonuclear apoptosis. Second, it is possible that, even though the yolk sac is present as a source of nutrients, the absence of exogenous feed in the early posthatch period may deny the myofiber metabolites that will maintain myonuclear integrity. The loss of myonuclei through an apoptotic pathway early posthatch may have a profound influence on ultimate muscle size because the myonuclei lost after the experimental perturbation may not be regained after the resumption of feeding.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Manuscript received 22 October 2001. Initial review completed 6 December 2001. Revision accepted 30 January 2002.
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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1. Stockdale, F. E. & Holtzer, H. (1961) DNA synthesis and myogenesis. Exp. Cell Res. 24:508-520.[Medline]
2. Moss, F. P. & Leblond, C. P. (1971) Satellite cells as the source of nuclei in muscles of growing rats. Anat. Rec. 170:421-436.[Medline]
3.
Mozdziak, P. E., Schultz, E. & Cassens, R. G. (1997) Myonuclear accretion is a major determinant of avian skeletal muscle growth. Am. J. Physiol. 272:C565-C571.
4.
Mozdziak, P. E., Pulvermacher, P. M. & Schultz, E. (2000) Unloading of juvenile muscle results in a reduced muscle size 9 wk after reloading. J. Appl. Physiol. 88:158-164.
5. Mozdziak, P. E., Schultz, E. & Cassens, R. G. (1996) The effect of in vivo and in vitro irradiation (25 Gy) on the subsequent in vitro growth of satellite cells. Cell Tissue Res. 283:203-208.[Medline]
6.
Halevy, O., Geyra, A., Barak, M., Uni, Z. & Sklan, D. (2000) Early posthatch starvation decreases satellite cell proliferation and skeletal muscle growth in chicks. J. Nutr. 130:858-864.
7. Sandri, M. & Carraro, U. (1999) Apoptosis of skeletal muscles during development and disease. Int. J. Biochem. Cell Biol. 31:1373-1390.[Medline]
8. Anderson, J. E. & Murray, L. (1998) Barr Award Lecture. Studies of the dynamics of skeletal muscle regeneration: the mouse came back!. Biochem. Cell Biol. 76:13-26.[Medline]
9. Tidball, J. G., Albrecht, D. E., Lokensgard, B. E. & Spencer, M. J. (1995) Apoptosis precedes necrosis of dystrophin-deficient muscle. J. Cell Sci. 108:2197-2204.[Abstract]
10.
Allen, D. L., Linderman, J. K., Roy, R. R., Bigbee, A. J., Grindeland, R. E., Mukku, V. & Edgerton, V. R. (1997) Apoptosis: a mechanism contributing to remodeling of skeletal muscle in response to hindlimb unweighting. Am. J. Physiol. 273:C579-C587.
11.
Libera, L. D., Zennaro, R., Sandri, M., Ambrosio, G. B. & Vescovo, G. (1999) Apoptosis and atrophy in rat slow skeletal muscles in chronic heart failure. Am. J. Physiol. 277:C982-C986.
12. SAS Institute Inc (1985) Sas User’s Guide: Statistics Version 5 ed. 1985 SAS Institute Cary, N.C. .
13. Ott, L. (1993) An Introduction to Statistical Methods and Data Analysis 1993 Duxbury Press Belmont, CA. .
14. Allen, D. L., Roy, R. R. & Edgerton, V. R. (1999) Myonuclear domains in muscle adaptation and disease. Muscle Nerve 22:1350-1360.[Medline]
15.
Horsley, V., Friday, B. B., Matteson, S., Kegley, K. M., Gephart, J. & Pavlath, G. K. (2001) Regulation of the growth of multinucleated muscle cells by an NFATC2-dependent pathway. J. Cell Biol. 153:329-338.
16.
McCall, G. E., Allen, D. L., Linderman, J. K., Grindeland, R. E., Roy, R. R., Mukku, V. R. & Edgerton, V. R. (1998) Maintenance of myonuclear domain size in rat soleus after overload and growth hormone/IGF-I treatment. J. Appl. Physiol. 84:1407-1412.
17.
Roy, R. R., Monke, S. R., Allen, D. L. & Edgerton, V. R. (1999) Modulation of myonuclear number in functionally overloaded and exercised rat plantaris fibers. J. Appl. Physiol. 87:634-642.
18. Mozdziak, P. E., Truong, Q., Macius, A. & Schultz, E. (1998) Hindlimb suspension reduces muscle regeneration. Eur. J. Appl. Physiol. Occup. Physiol. 78:136-140.[Medline]
19. Kasper, C. E. & Xun, L. (1996) Cytoplasm-to-myonucleus ratios in plantaris and soleus muscle fibres following hindlimb suspension. J. Muscle Res. Cell Motil. 17:603-610.[Medline]
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