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Polyamine accumulation in normal and denervated neonatal muscle
EXPERIMENTAL
NEUROLOGY
72, 612-618 (1981)
Polyamine Accumulation in Normal and Denervated Neonatal Muscle ANNA
M. KAMINSKA, LAWRENCE Z. STERN, AND DIANE HADDOCK RUSSELL’
Departments of Neurology and Pharmacology, The Univetsiry of Arizona College of Medicine, Tucson, Arizona 85724 Received September 22, I980; revision received November 25. 1980 Polyamines were monitored in normal and denervated neonatal rat gastrocnemius muscle as markers of the degeneration-regeneration processes in muscle characteristic of progressive neuromuscular disorders. The muscle was denervated within 6 h of birth. The highest polyamine concentrations were present at day 2 of birth and remained elevated during the first week, the period of most rapid muscle growth. Denervation did not alter the concentration of any of the polyamines compared with normal or contralateral controls during the first 3 weeks postparturition. These data suggest that neonatal polyamine synthesis, an index of extent of cell growth processes,is not affected by denervation. However, prolonged denervation did alter the polyamine accumulation patterns, with denervated muscle having significantly elevated spermine at 21 days, putrescine and spermidine concentrations at 28 days, and elevated spermidine at 56 days of age. Within 56 days, denervated muscle compared with normal innervated contralateral control muscle contained fourfold and twofold more spermidine and spermine per milligram protein. In conclusion, it is likely that the continued elevation of polyamines in denervated muscle represents the conservation of a metabolic pattern characteristic of immature muscle, as it is known that neonatal denervation results in failure of muscle maturation and differentiation. However, fibroblast proliferation also may be a contributing factor to the markedly altered polyamine concentrations found in denervated muscle.
INTRODUCTION Understanding of the mechanism underlying growth and differentiation is one of the central problems in biology. Changes occurring during the ’ This work was presented in part at the 32nd Annual Meeting of the American Academy of Neurology, New Orleans, May, 1980 and was supported in part by the Muscular Dystrophy Association, Eli Lilly and Co., and Smith Kline & French Laboratories. Address reprint requests to Dr. Russell, Department of Pharmacology, University of Arizona Health Sciences Center, Tucson, AZ 85724. Dr. Kaminska’s present address is Department of Neurology, Medical Academy, Lindleya 4, Warsaw, Poland. 612 0014-4886/81/060612-07$02.00/O Copyright Q I98 1 by Academic Press. Inc. All rights of reproduction in any form resewed.
POLYAMINES
IN NEONATAL
MUSCLE
613
development of normal skeletal muscle have been the subject of intensive studies both in humans (5-7) and in experimental animals (1, 3,4, 10, 11, 19, 20). For those experiments, rat muscle was often studied because it is immature and undifferentiated at birth ( 1, 3, 4, 10). During the brief period of postnatal development, they reach their morphologic (4, 10) and physiologic (1, 3) maturity. It is known that an intact nerve supply is essential for normal muscle development (8), but, in addition, circulating humoral growth factors may have stimulatory effects even in denervated muscle (10, 19). Polyamines, the organic cations of the cell, have been shown to accurately reflect cellular growth and developmental processes (14, 15). Recently, Kremzner et al. (12) demonstrated that normal polyamine metabolism in adult muscle was dependent on intact innervation. Therefore, we investigated polyamine concentrations in normal and denervated neonatal rat gastrocnemius to determine the influence of innervation on polyamine synthesis and accumulation in developing muscle. METHODS Sprague-Dawley rats of either sex were used because there are no significant sex-related differences in their muscle polyamine content (9). Polyamines were assayed in the gastrocnemius muscles of rats from 1 to 56 days of age. Polyamines were measured in 60 normal gastrocnemius muscles, 28 denervated gastrocnemius muscles, and 28 innervated contralateral gastrocnemius muscles from the same rats. Denervation was accomplished by right sciatic nerve transection within 6 h of birth under cold anesthesia. Normal unoperated rats from the same litters were used as additional controls. The muscles were rapidly excised, frozen in liquid nitrogen, and stored at -80°C for polyamine analysis. Muscle tissue (50 to 150 mg) was homogenized in 0.4 ml cold 10% trichloroacetic acid and centrifuged 5 min at 10,OOOg in a Beckman microfuge B. The supernatant was further diluted with 0.1 N HCl from 1:2 to 1:5, depending on the age of the animal and the amount of tissue. Polyamine concentrations were determined by analysis on a Durrum Model D-500 amino acid analyzer (Dionex Corporation, Sunnyvale, Calif.) as previously described (13, 17). Proteins were determined according to the Bradford method (2). RESULTS Alterations in Polyamine Accumulation Patterns in Normal Muscle as a Function of Age. Polyamine concentrations as a function of age are presented in Table 1. In newborn muscle, there is approximately twice as much spermidine as spermine, 12 vs. 6.5 nmol/mg protein, respectively. Spermidine and spermine attain their highest concentrations in the muscle
614
KAMINSKA,
STERN, AND RUSSELL TABLE
1
Changes in Polyamine Concentrations in Normal Muscle in Relation to Age” AtV (days)
14 21 28 56
Number of muscles 5 5 7 5 6 6 5 5 5 5 7
Putrescinc 3.80 4.80 6.30 4.50 3.17 2.60 2.78 0.54 0.40 0.20
+ 0.81 + 0.46 + 0.28 f 0.24 + 0.71 + 0.47 + 0.51 + 0.13 + 0.18 f 0.06 co.20
Spermidine (nmol/mg protein) 12.04 1.4.72 I I .20 II.98 9.00 7.56 7.08 4.66 3.90 1.82 0.31
+ k + t k + + + + + +
Spermine
1.54 1.54 0.42 1.03 1.90 1.14 1.08 0.52 0.47 0.24 0.05
6.50 7.90 4.60 5.58 5.32 4.93 4.34 5.10 4.80 3.40 I.10
+ 2 + k k A 2 ? k 2 k
1.13 0.97 0.24 0.50 1.06 0.53 0.73 0.58 0.44 0.92 0.26
Spermidine: spermine ratio 1.92 1.92 2.44 2.10 1.67 1.48 1.62 0.94 0.82 0.44 0.30
t + + * + k f * * * *
0.15 0.15 0.64 0.70 0.15 0.06 0.06 0.09 0.09 0.02 0.03
of the 2-day-old rat. Putrescine was maintained at a high concentration compared with normal adult tissues (2 to 6 nmol/mg protein) throughout the rapid growth period that occurred in the first 7 days after birth. Thereafter, polyamine content decreased with age (Fig. 1). At 14 days, only 0.54 nmol/mg of putrescine protein was present, and spermidine content was only 30% of the concentration detected in the gastrocnemius at birth. Spermine, on the other hand, did not decrease dramatically until after day 28, and by 56 days of age was less than 20% of the specific activity detected in the muscle at birth. The spermidine:spermine ratio was 2.4 at 3 days
-
FUTRESCINE SFERMDlNE SPERMINE
AGE IN Dpzls
FIG. 1. Polyamine concentrations in the gastrocnemius muscle as a function of postnatal age. Each point represents the mean of at least five determinations in duplicate on individual muscles.
POLYAMINES 22
IN NEONATAL
615
MUSCLE
n SPERMN/ spERMyER4m
.
I.6 . IA
.
to 06
.
02. I234567
”
14
24
26
56
&3ENms
FIG. 2. Alterations in the spermidine:spermine ratio in the gastrocnemius muscle as a function of postnatal age.
of age and decreased steadily to 0.3 at 56 days of age (Fig. 2). A more rapid decrease in the spermidine content compared with spermine resulted in a decrease in the ratio below 1.0 by 2 weeks of age. Alterations in Polyamine Accumulation Patterns in Denervated Muscle. Polyamine concentrations in denervated muscles compared with contralateral controls and with normal muscles are presented in Table 2. There were no significant differences in polyamine content between normal muscle TABLE
2
Changes in Polyamine Concentrations in Denervated Muscle AS (days)
Gmuf
Number of musclea
Putrescine
Spermidine (llmOl/lll~ protein)
Spermidine: sprmine ratio
Spermine
7
N C D
5 6 6
2.78 f 0.51 3.40 f 0.52 2.33 f 0.28
7.08 f I.06 8.45 zt 1.10 9.22 f 1.21
4.34 * 0.73 6.33 f 1.10 6.73 f 0.99
1.62 f 0.06 1.53 * 0.08 1.31 + 0.12
14
N C D
5 7 7
0.54 f 0.13 0.60 f 0.30 0.27 f 0.06
4.66 f 0.52 5.86 zt 0.72 5.23 f 0.69
5.10 f 0.58 6.25 f 0.88 7.97 f 1.67
0.94 f 0.09 0.97 * 0.09 0.70 * 0.06
21
N C D
5 5 5
0.40 f 0.18 0.60 f 0.30 0.46 Et 0.28
3.90 * 0.47 5.02 f 1.16 7.33 * 1.82
4.80 f 0.44 5.94 f 0.76 10.38 f 3.63-O
0.82 * 0.09 0.76 f 0.09 0.66 f 0.08
28
N C D
5 5 5
0.20 f 0.06 0.22 f 0.04 0.54 f 0.07
1.82 f 1.72 f 4.10 f
3.40 f 0.92 3.70 * 0.57 6.00 k 1.01
0.44 * 0.02 0.46 f 0.08 0.70 * 0.03*-
56
N C D
7 5 5
co.20 4.20 co.20
1.10 f 0.26 0.84 f 0.29 2.16 f 2.04
0.30 * 0.03 0.22 * 0.03 0.54 * 0.12
0.24 0.39 0.49.
0.31 * 0.05 0.18 * 0.06 0.88 f 0.1 I l
*
’ N-normal #astmcnemius muscle. C-contralateral to dcnervated gastrocncmius muscle. Data are cxpmsed as the mean * se. l Data differ fmm controls (P < 0.01); l *(P < 0.005): l **(P < 0.001).
muscle.
D-dcnmated
gastrccnemius
616
KAMINSKA,
STERN,
AND
RUSSELL
(N) and muscle contralateral to the denervated organ (C). The highest concentrations of polyamines were detected in the first week in normal, contralateral control, and denervated muscle. The accumulation patterns were very similar 14 days after birth in the denervated and control muscles (Table 2). By 21 days, the spermine content in denervated muscle was significantly elevated (P < 0.001). By 28 days, putrescine and spermidine concentrations were significantly elevated (P < 0.01). By 56 days, putrestine was not detectable in any muscle groups. Spermidine, although it was decreasing in all groups, remained three- to fourfold higher in denervated muscles than in normal and contralateral control muscles. The spermidine:spermine ratio was significantly elevated (P < 0.005) at 4 weeks of age (Table 2). DISCUSSION The relatively high polyamine concentrations detected in muscle in the early postnatal period were reported also in a variety of other rapidly growing tissues (9). Polyamines were maintained at a high concentration compared with adult muscle throughout the first 2 weeks after birth, which is a rapid growth period. Thereafter, polyamine content decreased with age. In newborn muscle, spermidine concentrations were higher than spermine. The molar ratio spermidine:spermine demonstrates clearly the changes in the polyamine concentrations. The highest was at 3 days of age (2.4) and decreased to 0.3 at 56 days of age. In general, a high spermidine:spermine ratio is typical for a tissue undergoing hypertrophy and/or hyperplasia (9, 15). A more rapid decrease in the spermidine content compared to spermine resulted in a decrease in the ratio to less than 1.0 by 2 weeks of age. We and others (9) have reported more variability in polyamine concentrations in skeletal muscle compared with polyamine concentrations in other tissues. The variability may be due to individual differences in the rate of muscle development related to the size of the litter (4) which can vary in our experience from 5 to 17 young. Although elevated polyamine concentrations during the first 2 weeks after birth can be correlated with the general rapid growth and maturation of muscle which takes place during this period, the relationship to specific aspects of this growth pattern is more complex. At birth approximately one-third of muscle fibers are in the myotube stage (10). By 7 days, transformation of myotubes into mature muscle fibers is completed, and by 14 days, the muscle is fully differentiated by histochemical criteria (4, 10). These morphologic changes are not closely paralleled by certain physiologic and biochemical parameters such as differentiation into fast and slow
POLYAMINES
IN NEONATAL
MUSCLE
617
twitch muscles (3) or creative phosphokinase (CPK) activity (20). However, a close temporal correlation was demonstrated by Boi%hius (1) between the increase in membrane potential and the myotube-mature muscle fiber transformation. The dependence of postnatal muscle development on an intact motor innervation is well established (8). The most striking consequence of early denervation is atrophy and lack of metabolic differentiation of muscle fibers, followed by later proliferation of connective tissue. However, in rat muscle denervated at birth, certain muscle fibers continue to grow but at a slower rate than in normal muscle ( 10, 19). This suggests that denervated neonatal muscle can respond to endogenous factors, presumably humoral factors, such as growth hormone. Normal polyamine patterns in denervated muscle during the first 14 days after birth may be a result of the concentration of circulating growth hormone and other trophic hormones which stimulate polyamine synthesis in vivo (15, 16). The concentration of circulating growth hormone in rats is high at birth, declines during the first weeks after birth, and increases again after weaning (18). Later elevations in polyamine content probably result largely from conservation of a metabolic pattern characteristic of immature muscle, because after early denervation some muscle cells (approximately 12%) have morphologic features of immaturity, even 8 weeks after denervation (lo), although fibroblast proliferation may also represent a contributory factor. Morphologically, degenerative changes are seen in the muscle fibers 3 to 4 weeks after nerve transection and are followed by later connective tissue hyperplasia. Hypertrophy of individual muscle fibers which could also alter polyamine concentrations is not seen to occur under these experimental conditions ( 10). Polyamine metabolism in adult muscle is influenced more rapidly by denervation (12). One week after nerve transection, putrescine and spermidine, but not spermine, were elevated and continued to increase as long as the third week after denervation. By 3 weeks, putrescine concentration was increased approximately IO-fold, and spermine 4-fold. In summary, denervation results not only in alterations in muscle polyamine accumulation but affects such polyamine in a different manner during development. The continued elevation of polyamines in denervated neonatal muscle may result from the conservation of a metabolic pattern characteristic of immature muscle, as previous work has shown that neonatal denervation results in a lack of muscle fiber maturation and differentiation. Further studies are necessary to assess the contributory role of other cell growth processes.
618
KAMINSKA,
STERN,
AND
RUSSELL
REFERENCES 1. BOETHIUS, J. 197 1. Electrophysiological and morphological development of leg and neck muscles in the rat. Acta Physiol. &and. 81: 492-507. 2. BRADFORD, M. M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principles of protein-dye binding. Anal. Biochem. 72: 248-254. 3. CLOSE, R. 1964. Dynamic properties of fast and slow skeletal muscles of the rat during development. J. Physiol. (London) 173: 74-95. 4. DUBOWITZ, V. 1963. Enzymatic maturation of skeletal muscle. Nature (London) 197: 1215. 5. DUBOWITZ, V. 1968. Enzyme histochemistryofdeveloping human muscle. Nature (London) 211: 884-886.
6. FENICHEL, Cl. M. 1966. Histochemistry of normal developing muscle. Neurology (Minneapolis)
16: 741-748.
7. FIDZIANSKA, A. 1971. Electron microscopic study of the development of human fetal muscle, motor end plate and nerve: Preliminary report. Acta Neuropathol. 17: 236 247. 8. GUTMAN, E. 1962. The Denervated Muscle. Publishing House of the Czechoslovak Academy of Sciences, Prague. 9. J~NNE, J., A. RAINA, AND M. SIIMES. 1964. Spermidine and spermine in rat tissues at different ages. Acta Physiol. Sand. 62: 352-358. 10. KAMINSKA, A. 1979. Effect of denervation on muscle development in the rat. Doctoral Thesis (in Polish), Warsaw Medical Academy. 11. KELLY, A. M., AND S. I. ZACKS. 1969. The histogenesis of rat intercostal muscle. J. Cell Biol. 42: 135-169. 12. KREMZNER, L. T., V. M. TENNYSON, AND A. F. MIRANDA. 1978. Polyamine metabolism in normal, denervated and dystrophic muscle. Advan. Polyamine Res. 2: 241-256. 13. MARTON, L. J., 0. HEBY, AND C. B. WILSON. 1974. An automated micromethod for the quantitative analysis of di- and polyamines utilizing a sensitive high pressure liquid chromatographic procedure. FEBS Left. 41: 99-103. 14. RUSSELL, D. H. (Ed.). 1973. Polyamines in Normal and Neoplastic Growth. Raven Press, New York. IS. RUSSELL, D. H., AND B. Cl. M. DURIE. 1978. Polyamines as Biochemical Markers of Normal and Malignant Growth. Raven Press, New York. 16. RUSSELL, D. H.. AND M. K. HADDOX. 1979. Cyclic AMP-mediated induction of ornithine decarboxylase in normal and neoplastic growth. Advan. Enzyme Reg. 17: 61-87. 17. RUSSELL, D. H., AND S. D. RUSSELL. 1975. Relative usefulness of measuring polyamines in serum, plasma and urine as biochemical markers of cancer. C/in. Chem. 21: 860863. 18. STROSSER, M. T., AND P. MIALHE. 1975. Growth hormone secretion in the rat as a function of age. Harm. Metab. Res. 7~275-278. 19. ZELENA, J., AND P. HNIK. 1957. Muscle atrophy in young rats. Physiol. Biochem. 6: 193-199. 20. ZITER, F. A. 1974. Creatine kinase in developing skeletal and cardiac muscle of the rat. Exp.
Neurol.
43: 539-546.
NEUROLOGY
72, 612-618 (1981)
Polyamine Accumulation in Normal and Denervated Neonatal Muscle ANNA
M. KAMINSKA, LAWRENCE Z. STERN, AND DIANE HADDOCK RUSSELL’
Departments of Neurology and Pharmacology, The Univetsiry of Arizona College of Medicine, Tucson, Arizona 85724 Received September 22, I980; revision received November 25. 1980 Polyamines were monitored in normal and denervated neonatal rat gastrocnemius muscle as markers of the degeneration-regeneration processes in muscle characteristic of progressive neuromuscular disorders. The muscle was denervated within 6 h of birth. The highest polyamine concentrations were present at day 2 of birth and remained elevated during the first week, the period of most rapid muscle growth. Denervation did not alter the concentration of any of the polyamines compared with normal or contralateral controls during the first 3 weeks postparturition. These data suggest that neonatal polyamine synthesis, an index of extent of cell growth processes,is not affected by denervation. However, prolonged denervation did alter the polyamine accumulation patterns, with denervated muscle having significantly elevated spermine at 21 days, putrescine and spermidine concentrations at 28 days, and elevated spermidine at 56 days of age. Within 56 days, denervated muscle compared with normal innervated contralateral control muscle contained fourfold and twofold more spermidine and spermine per milligram protein. In conclusion, it is likely that the continued elevation of polyamines in denervated muscle represents the conservation of a metabolic pattern characteristic of immature muscle, as it is known that neonatal denervation results in failure of muscle maturation and differentiation. However, fibroblast proliferation also may be a contributing factor to the markedly altered polyamine concentrations found in denervated muscle.
INTRODUCTION Understanding of the mechanism underlying growth and differentiation is one of the central problems in biology. Changes occurring during the ’ This work was presented in part at the 32nd Annual Meeting of the American Academy of Neurology, New Orleans, May, 1980 and was supported in part by the Muscular Dystrophy Association, Eli Lilly and Co., and Smith Kline & French Laboratories. Address reprint requests to Dr. Russell, Department of Pharmacology, University of Arizona Health Sciences Center, Tucson, AZ 85724. Dr. Kaminska’s present address is Department of Neurology, Medical Academy, Lindleya 4, Warsaw, Poland. 612 0014-4886/81/060612-07$02.00/O Copyright Q I98 1 by Academic Press. Inc. All rights of reproduction in any form resewed.
POLYAMINES
IN NEONATAL
MUSCLE
613
development of normal skeletal muscle have been the subject of intensive studies both in humans (5-7) and in experimental animals (1, 3,4, 10, 11, 19, 20). For those experiments, rat muscle was often studied because it is immature and undifferentiated at birth ( 1, 3, 4, 10). During the brief period of postnatal development, they reach their morphologic (4, 10) and physiologic (1, 3) maturity. It is known that an intact nerve supply is essential for normal muscle development (8), but, in addition, circulating humoral growth factors may have stimulatory effects even in denervated muscle (10, 19). Polyamines, the organic cations of the cell, have been shown to accurately reflect cellular growth and developmental processes (14, 15). Recently, Kremzner et al. (12) demonstrated that normal polyamine metabolism in adult muscle was dependent on intact innervation. Therefore, we investigated polyamine concentrations in normal and denervated neonatal rat gastrocnemius to determine the influence of innervation on polyamine synthesis and accumulation in developing muscle. METHODS Sprague-Dawley rats of either sex were used because there are no significant sex-related differences in their muscle polyamine content (9). Polyamines were assayed in the gastrocnemius muscles of rats from 1 to 56 days of age. Polyamines were measured in 60 normal gastrocnemius muscles, 28 denervated gastrocnemius muscles, and 28 innervated contralateral gastrocnemius muscles from the same rats. Denervation was accomplished by right sciatic nerve transection within 6 h of birth under cold anesthesia. Normal unoperated rats from the same litters were used as additional controls. The muscles were rapidly excised, frozen in liquid nitrogen, and stored at -80°C for polyamine analysis. Muscle tissue (50 to 150 mg) was homogenized in 0.4 ml cold 10% trichloroacetic acid and centrifuged 5 min at 10,OOOg in a Beckman microfuge B. The supernatant was further diluted with 0.1 N HCl from 1:2 to 1:5, depending on the age of the animal and the amount of tissue. Polyamine concentrations were determined by analysis on a Durrum Model D-500 amino acid analyzer (Dionex Corporation, Sunnyvale, Calif.) as previously described (13, 17). Proteins were determined according to the Bradford method (2). RESULTS Alterations in Polyamine Accumulation Patterns in Normal Muscle as a Function of Age. Polyamine concentrations as a function of age are presented in Table 1. In newborn muscle, there is approximately twice as much spermidine as spermine, 12 vs. 6.5 nmol/mg protein, respectively. Spermidine and spermine attain their highest concentrations in the muscle
614
KAMINSKA,
STERN, AND RUSSELL TABLE
1
Changes in Polyamine Concentrations in Normal Muscle in Relation to Age” AtV (days)
14 21 28 56
Number of muscles 5 5 7 5 6 6 5 5 5 5 7
Putrescinc 3.80 4.80 6.30 4.50 3.17 2.60 2.78 0.54 0.40 0.20
+ 0.81 + 0.46 + 0.28 f 0.24 + 0.71 + 0.47 + 0.51 + 0.13 + 0.18 f 0.06 co.20
Spermidine (nmol/mg protein) 12.04 1.4.72 I I .20 II.98 9.00 7.56 7.08 4.66 3.90 1.82 0.31
+ k + t k + + + + + +
Spermine
1.54 1.54 0.42 1.03 1.90 1.14 1.08 0.52 0.47 0.24 0.05
6.50 7.90 4.60 5.58 5.32 4.93 4.34 5.10 4.80 3.40 I.10
+ 2 + k k A 2 ? k 2 k
1.13 0.97 0.24 0.50 1.06 0.53 0.73 0.58 0.44 0.92 0.26
Spermidine: spermine ratio 1.92 1.92 2.44 2.10 1.67 1.48 1.62 0.94 0.82 0.44 0.30
t + + * + k f * * * *
0.15 0.15 0.64 0.70 0.15 0.06 0.06 0.09 0.09 0.02 0.03
of the 2-day-old rat. Putrescine was maintained at a high concentration compared with normal adult tissues (2 to 6 nmol/mg protein) throughout the rapid growth period that occurred in the first 7 days after birth. Thereafter, polyamine content decreased with age (Fig. 1). At 14 days, only 0.54 nmol/mg of putrescine protein was present, and spermidine content was only 30% of the concentration detected in the gastrocnemius at birth. Spermine, on the other hand, did not decrease dramatically until after day 28, and by 56 days of age was less than 20% of the specific activity detected in the muscle at birth. The spermidine:spermine ratio was 2.4 at 3 days
-
FUTRESCINE SFERMDlNE SPERMINE
AGE IN Dpzls
FIG. 1. Polyamine concentrations in the gastrocnemius muscle as a function of postnatal age. Each point represents the mean of at least five determinations in duplicate on individual muscles.
POLYAMINES 22
IN NEONATAL
615
MUSCLE
n SPERMN/ spERMyER4m
.
I.6 . IA
.
to 06
.
02. I234567
”
14
24
26
56
&3ENms
FIG. 2. Alterations in the spermidine:spermine ratio in the gastrocnemius muscle as a function of postnatal age.
of age and decreased steadily to 0.3 at 56 days of age (Fig. 2). A more rapid decrease in the spermidine content compared with spermine resulted in a decrease in the ratio below 1.0 by 2 weeks of age. Alterations in Polyamine Accumulation Patterns in Denervated Muscle. Polyamine concentrations in denervated muscles compared with contralateral controls and with normal muscles are presented in Table 2. There were no significant differences in polyamine content between normal muscle TABLE
2
Changes in Polyamine Concentrations in Denervated Muscle AS (days)
Gmuf
Number of musclea
Putrescine
Spermidine (llmOl/lll~ protein)
Spermidine: sprmine ratio
Spermine
7
N C D
5 6 6
2.78 f 0.51 3.40 f 0.52 2.33 f 0.28
7.08 f I.06 8.45 zt 1.10 9.22 f 1.21
4.34 * 0.73 6.33 f 1.10 6.73 f 0.99
1.62 f 0.06 1.53 * 0.08 1.31 + 0.12
14
N C D
5 7 7
0.54 f 0.13 0.60 f 0.30 0.27 f 0.06
4.66 f 0.52 5.86 zt 0.72 5.23 f 0.69
5.10 f 0.58 6.25 f 0.88 7.97 f 1.67
0.94 f 0.09 0.97 * 0.09 0.70 * 0.06
21
N C D
5 5 5
0.40 f 0.18 0.60 f 0.30 0.46 Et 0.28
3.90 * 0.47 5.02 f 1.16 7.33 * 1.82
4.80 f 0.44 5.94 f 0.76 10.38 f 3.63-O
0.82 * 0.09 0.76 f 0.09 0.66 f 0.08
28
N C D
5 5 5
0.20 f 0.06 0.22 f 0.04 0.54 f 0.07
1.82 f 1.72 f 4.10 f
3.40 f 0.92 3.70 * 0.57 6.00 k 1.01
0.44 * 0.02 0.46 f 0.08 0.70 * 0.03*-
56
N C D
7 5 5
co.20 4.20 co.20
1.10 f 0.26 0.84 f 0.29 2.16 f 2.04
0.30 * 0.03 0.22 * 0.03 0.54 * 0.12
0.24 0.39 0.49.
0.31 * 0.05 0.18 * 0.06 0.88 f 0.1 I l
*
’ N-normal #astmcnemius muscle. C-contralateral to dcnervated gastrocncmius muscle. Data are cxpmsed as the mean * se. l Data differ fmm controls (P < 0.01); l *(P < 0.005): l **(P < 0.001).
muscle.
D-dcnmated
gastrccnemius
616
KAMINSKA,
STERN,
AND
RUSSELL
(N) and muscle contralateral to the denervated organ (C). The highest concentrations of polyamines were detected in the first week in normal, contralateral control, and denervated muscle. The accumulation patterns were very similar 14 days after birth in the denervated and control muscles (Table 2). By 21 days, the spermine content in denervated muscle was significantly elevated (P < 0.001). By 28 days, putrescine and spermidine concentrations were significantly elevated (P < 0.01). By 56 days, putrestine was not detectable in any muscle groups. Spermidine, although it was decreasing in all groups, remained three- to fourfold higher in denervated muscles than in normal and contralateral control muscles. The spermidine:spermine ratio was significantly elevated (P < 0.005) at 4 weeks of age (Table 2). DISCUSSION The relatively high polyamine concentrations detected in muscle in the early postnatal period were reported also in a variety of other rapidly growing tissues (9). Polyamines were maintained at a high concentration compared with adult muscle throughout the first 2 weeks after birth, which is a rapid growth period. Thereafter, polyamine content decreased with age. In newborn muscle, spermidine concentrations were higher than spermine. The molar ratio spermidine:spermine demonstrates clearly the changes in the polyamine concentrations. The highest was at 3 days of age (2.4) and decreased to 0.3 at 56 days of age. In general, a high spermidine:spermine ratio is typical for a tissue undergoing hypertrophy and/or hyperplasia (9, 15). A more rapid decrease in the spermidine content compared to spermine resulted in a decrease in the ratio to less than 1.0 by 2 weeks of age. We and others (9) have reported more variability in polyamine concentrations in skeletal muscle compared with polyamine concentrations in other tissues. The variability may be due to individual differences in the rate of muscle development related to the size of the litter (4) which can vary in our experience from 5 to 17 young. Although elevated polyamine concentrations during the first 2 weeks after birth can be correlated with the general rapid growth and maturation of muscle which takes place during this period, the relationship to specific aspects of this growth pattern is more complex. At birth approximately one-third of muscle fibers are in the myotube stage (10). By 7 days, transformation of myotubes into mature muscle fibers is completed, and by 14 days, the muscle is fully differentiated by histochemical criteria (4, 10). These morphologic changes are not closely paralleled by certain physiologic and biochemical parameters such as differentiation into fast and slow
POLYAMINES
IN NEONATAL
MUSCLE
617
twitch muscles (3) or creative phosphokinase (CPK) activity (20). However, a close temporal correlation was demonstrated by Boi%hius (1) between the increase in membrane potential and the myotube-mature muscle fiber transformation. The dependence of postnatal muscle development on an intact motor innervation is well established (8). The most striking consequence of early denervation is atrophy and lack of metabolic differentiation of muscle fibers, followed by later proliferation of connective tissue. However, in rat muscle denervated at birth, certain muscle fibers continue to grow but at a slower rate than in normal muscle ( 10, 19). This suggests that denervated neonatal muscle can respond to endogenous factors, presumably humoral factors, such as growth hormone. Normal polyamine patterns in denervated muscle during the first 14 days after birth may be a result of the concentration of circulating growth hormone and other trophic hormones which stimulate polyamine synthesis in vivo (15, 16). The concentration of circulating growth hormone in rats is high at birth, declines during the first weeks after birth, and increases again after weaning (18). Later elevations in polyamine content probably result largely from conservation of a metabolic pattern characteristic of immature muscle, because after early denervation some muscle cells (approximately 12%) have morphologic features of immaturity, even 8 weeks after denervation (lo), although fibroblast proliferation may also represent a contributory factor. Morphologically, degenerative changes are seen in the muscle fibers 3 to 4 weeks after nerve transection and are followed by later connective tissue hyperplasia. Hypertrophy of individual muscle fibers which could also alter polyamine concentrations is not seen to occur under these experimental conditions ( 10). Polyamine metabolism in adult muscle is influenced more rapidly by denervation (12). One week after nerve transection, putrescine and spermidine, but not spermine, were elevated and continued to increase as long as the third week after denervation. By 3 weeks, putrescine concentration was increased approximately IO-fold, and spermine 4-fold. In summary, denervation results not only in alterations in muscle polyamine accumulation but affects such polyamine in a different manner during development. The continued elevation of polyamines in denervated neonatal muscle may result from the conservation of a metabolic pattern characteristic of immature muscle, as previous work has shown that neonatal denervation results in a lack of muscle fiber maturation and differentiation. Further studies are necessary to assess the contributory role of other cell growth processes.
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REFERENCES 1. BOETHIUS, J. 197 1. Electrophysiological and morphological development of leg and neck muscles in the rat. Acta Physiol. &and. 81: 492-507. 2. BRADFORD, M. M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principles of protein-dye binding. Anal. Biochem. 72: 248-254. 3. CLOSE, R. 1964. Dynamic properties of fast and slow skeletal muscles of the rat during development. J. Physiol. (London) 173: 74-95. 4. DUBOWITZ, V. 1963. Enzymatic maturation of skeletal muscle. Nature (London) 197: 1215. 5. DUBOWITZ, V. 1968. Enzyme histochemistryofdeveloping human muscle. Nature (London) 211: 884-886.
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7. FIDZIANSKA, A. 1971. Electron microscopic study of the development of human fetal muscle, motor end plate and nerve: Preliminary report. Acta Neuropathol. 17: 236 247. 8. GUTMAN, E. 1962. The Denervated Muscle. Publishing House of the Czechoslovak Academy of Sciences, Prague. 9. J~NNE, J., A. RAINA, AND M. SIIMES. 1964. Spermidine and spermine in rat tissues at different ages. Acta Physiol. Sand. 62: 352-358. 10. KAMINSKA, A. 1979. Effect of denervation on muscle development in the rat. Doctoral Thesis (in Polish), Warsaw Medical Academy. 11. KELLY, A. M., AND S. I. ZACKS. 1969. The histogenesis of rat intercostal muscle. J. Cell Biol. 42: 135-169. 12. KREMZNER, L. T., V. M. TENNYSON, AND A. F. MIRANDA. 1978. Polyamine metabolism in normal, denervated and dystrophic muscle. Advan. Polyamine Res. 2: 241-256. 13. MARTON, L. J., 0. HEBY, AND C. B. WILSON. 1974. An automated micromethod for the quantitative analysis of di- and polyamines utilizing a sensitive high pressure liquid chromatographic procedure. FEBS Left. 41: 99-103. 14. RUSSELL, D. H. (Ed.). 1973. Polyamines in Normal and Neoplastic Growth. Raven Press, New York. IS. RUSSELL, D. H., AND B. Cl. M. DURIE. 1978. Polyamines as Biochemical Markers of Normal and Malignant Growth. Raven Press, New York. 16. RUSSELL, D. H.. AND M. K. HADDOX. 1979. Cyclic AMP-mediated induction of ornithine decarboxylase in normal and neoplastic growth. Advan. Enzyme Reg. 17: 61-87. 17. RUSSELL, D. H., AND S. D. RUSSELL. 1975. Relative usefulness of measuring polyamines in serum, plasma and urine as biochemical markers of cancer. C/in. Chem. 21: 860863. 18. STROSSER, M. T., AND P. MIALHE. 1975. Growth hormone secretion in the rat as a function of age. Harm. Metab. Res. 7~275-278. 19. ZELENA, J., AND P. HNIK. 1957. Muscle atrophy in young rats. Physiol. Biochem. 6: 193-199. 20. ZITER, F. A. 1974. Creatine kinase in developing skeletal and cardiac muscle of the rat. Exp.
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