INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

THE IMMATURE MUSCLE IS CHARACTERIZED by a rapid rate of protein accretion that is supported by high rates of protein synthesis in the fed state (7, 8). As maturation progresses towards the attainment of the mature adult protein composition and phenotype, the rates of protein synthesis and accretion decline in parallel (8). During this period of rapid growth, significant changes in muscle protein composition also occur. The contractile proteins accumulate and form myofibrillar structures only after myotube formation, but by maturity these proteins comprise 55-65% of total muscle protein (39). In addition to these gross changes in cell composition and architecture, the muscle proteins themselves exist in a variety of developmental and fiber type-specific isoforms and each family of proteins has a specific developmental pattern of isoform expression (1, 4). It is critical for orderly maturation to occur that the individual proteins of the myofibrils are assembled in a strict stoichiometric pattern, because the absence of such coordination could lead to a gross derangement of muscle architecture and function (28).

The coordination and regulation of muscle development have been the subject of much research focused largely at the level of gene expression. It is generally accepted that during the transition from myoblast to myotube, some genes coding for nonmuscle proteins are repressed, whereas those specific for muscle proteins are induced in a coordinated manner. There is then a commensurate change in the composition of proteins expressed. There is good in vitro evidence to favor this concept (10, 11, 33). Results from subsequent studies suggested that the stoichiometry of the total mRNAs encoding all isoforms within a protein family is maintained accurately and that production of individual myofibrillar proteins in appropriate stoichiometric amounts is regulated, therefore, at the message level (37). However, the accretion of myofibrillar proteins is dependent not only on mRNA abundances, but also on individual rates of translation, myofibrillar assembly, and degradation. Thus the significance of conclusions regarding the coordination of myofibrillogenesis on the basis of mRNA abundances alone can be questioned.

Protein synthesis in the immature muscle, unlike that in the mature muscle, is highly sensitive to acute changes in food intake (7, 9), a response that is mediated by the resulting fluctuations in plasma insulin concentration (9, 38); for example, a 12-h fast in 5-day-old rat pups lowers the fractional rate of protein synthesis by 50%, and this is restored rapidly on refeeding. However, total fasting is relatively uncommon for many suckling mammals, and nutritionally limited growth is more often the result of the regular consumption of milk, but in inadequate amounts. In past studies, Fiorotto et al. (13) showed that growth retardation of suckling pups induced by a chronically inadequate intake did not compromise postnatal skeletal muscle maturation, as judged by the capacity to sustain the normal maturational transitions in myosin isoforms and to accumulate Na-K-ATPase. This was surprising, because in the mature muscle, chronic undernutrition is usually associated with a fall in the rate of both protein synthesis and degradation (24), and it could be argued that this would not only delay the removal of the immature myosin isoforms, but also their replacement by the adult isoforms. One possible explanation for the lack of effect of undernutrition on muscle maturation, is that the turnover of individual proteins, or functionally related muscle protein compartments (e.g., myofibrils, sarcoplasm), is regulated independently and that the response to undernutrition differs among protein classes.

The objectives of the present study, therefore, were: 1) to determine the developmental changes in the synthesis and degradation of myofibrillar and nonmyofibrillar proteins that are responsible for the gross compositional changes that occur during muscle maturation; 2) to establish whether the synthesis rates of individual myofibrillar proteins is coordinated; and 3) to quantify the effects of undernutrition during the suckling period on the turnover of myofibrillar (total and individual) and sarcoplasmic proteins.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Experimental design. Rat pups were suckled in litters of 10 [control (C)] or 16 [undernourished (UN)] from 1 day of age, as previously described (13). At 4, 6, 8, 13, 15, 17, 21 (C pups only), and 28 (C pups only) days of age, pups were killed and hindlimb muscles were taken for analysis. The muscles (gastrocnemius, plantaris, extensor digitorum longus, and tibialis anterior) were pooled and analyzed for total, soluble, and myofibrillar protein and total RNA concentrations.

The in vivo fractional protein synthesis rate was measured by the large-dose technique (16), using L-[4-³H]phenyalanine as the tracer in 6- (n = 20 pups, 5 from each of 4 litters), 15- (n = 20 pups, 5 from each of 4 litters), and 28-day-old (n = 7 rats derived from 3 litters) pups. Muscles were homogenized and separated into a soluble protein (composed primarily of sarcoplasmic proteins) and a myofibrillar protein component. The L-[4-³H]phenyalanine specific activities of both fractions and of total muscle proteins were measured, and individual fractional synthesis rates were determined. Purified, labeled myofibrils from 6- and 15-day-old pups were subjected to SDS PAGE to separate the following components: myosin heavy chain (MHC), -actinin, -actin, -tropomyosin, -tropomyosin, and myosin light chain (MLC) 1_f and 2_f. The specific radioactivity of the [4-³H]phenylalanine in these purified proteins was determined to estimate individual fractional synthesis rates. The protein composition data for muscles from 4-, 8-, 13-, 17-, and 21-day-old pups (n = 4 litters per age) were used to derive protein accretion rates.

The use of animals in this protocol was conducted in accordance with the National Research Council's Guide for the Care and Use of Laboratory Animals (NIH Publication No. 85-23, Revised 1996, National Academy Press, Washington, DC) and was approved by the Baylor College of Medicine Animal Care and Use Committee. All chemicals were purchased from either Sigma (St. Louis, MO) or BioRad (Hercules, CA), unless otherwise specified.

Animals. Timed-pregnant dams (CRL: SD/VRL, Charles River Laboratories; n = 30) were received at 15 days gestation. The procedures described previously were followed (13) except that the commercial rodent diet fed to the dams was Purina Rodent Chow 5008 (Purina Mills, Richmond, IN). Pups born within an 18-h period were pooled, and after discarding pups that weighed more than ±2 SD from the mean weight of all pups, were redistributed into litters of 10 or 16 pups per dam. Pups remained with their dam until they were studied. Pups studied at 28 days of age were separated at 21 days of age and individually housed with free access to food and water as described for the dam. All pups were weighed at 4, 6, 8, 13, 15, 17, 21, and 28 days of age.

In vivo protein synthesis measurements. Five pups per litter were selected and given an intravenous (jugular at 6 days, lateral tail vein at 15 and 28 days of age) injection of L-[4-³H]phenylalanine (Amersham, Arlington Heights, IL; 75 µCi/rat at 6 days, 140 µCi/rat at 15 days, 225 µCi/rat at 28 days) at a dose of 1.5 mmol/kg body weight. At 5 (n = 1 pup/litter, or 2 at 28 days), 10 (n = 2 pups/litter), or 30 (n = 2 pups/litter, or 3 at 28 days) min after injection, rats were decapitated and trunk blood was collected. The hindlimbs were immediately detached, wrapped in foil, and chilled in ice for 1 min. Muscles (gastrocnemius, plantaris, tibialis anterior, and extensor digitorum longus) were then dissected at 2°C and frozen in liquid nitrogen; the weights of the frozen muscles were determined, and the muscles were stored in liquid nitrogen.

Muscle protein fractionation. Frozen muscles from each pup were powdered, and a weighed aliquot (70-100 mg) was homogenized using a Duall glass grinder in a low-ionic-strength buffer. A sample of homogenate was retained for total protein and RNA concentration measurements. The sarcoplasmic and myofibrillar components were isolated using a modification of the method of Solaro et al. (34). The proteins that were soluble in low-ionic-strength buffer after high-speed centrifugation (15,000 g at 2°C for 45 min) were defined as the sarcoplasmic proteins. After purification, an aliquot of the purified myofibrils was mixed with an equal volume of glycerol and reserved for electrophoresis.

All steps were carried out quantitatively, and the volumes of the total homogenate and the sarcoplasmic and myofibrillar fractions were verified and their protein concentration determined by the method of Lowry et al. (23) after solubilization in 0.1 M NaOH. The total, sarcoplasmic, and myofibrillar protein contents of the muscles from each pup were determined. Total RNA was measured using a modified Schmidt-Thannhauser procedure (27).

For those muscles in which fractional synthesis rates were to be determined, each fraction was acidified to 0.2 M perchloric acid. The insoluble precipitates were separated from the supernatant by centrifugation at 10,000 g at 2°C for 30 min, and the pellets were washed three times in 0.2 M perchloric acid. The supernatant from the total protein fraction and the pellets from all protein fractions were processed as previously described for determination of L-[4-³H]phenylalanine specific radioactivity (8).

Isolation of individual myofibrillar proteins. Only myofibrils from those 6- and 15-day-old pups in which the labeling period was 30 min were analyzed. The individual myofibrillar proteins were isolated by SDS-PAGE using the Laemmli system (22). Briefly, gels were prepared with a 15 to 7.5% (wt/vol) total acrylamide (T) [2.7% (wt/wt) cross-linker (C) bis-acrylamide] polyacrylamide gradient in 0.35 M Tris-HCl (pH 8.8), with 0.1% SDS and 15% sucrose; a 3.5%T, 2.7%C stack in 0.125 M Tris-HCl (pH 6.8) was added. Approximately 750 µg of each myofibrillar preparation in glycerol were diluted to 1 ml with sample buffer [62.5 mM Tris-HCl (pH 6.8), 25% (vol/vol) glycerol, 2% (wt/vol) SDS, 5% (vol/vol) 2-mercaptoethanol, 0.025% bromophenol blue], heated at 95°C for 4 min, and loaded into multiple lanes on the gel. Electrophoresis was carried out at a constant current of 10 mA per plate for 16-17 h at 22°C. The gel was stained for 3 h (0.025% Coomassie blue, 30% vol/vol methanol, 10% vol/vol glacial acetic acid), then destained for 4 h in 10% methanol/10% acetic acid followed by 10% acetic acid and deionized water. By comparison with the position of purified proteins run on the same gel, we identified bands corresponding to the following proteins: MHC (all isoforms), -actinin, -actin, - and - tropomyosin, and MLC1_f and MLC2_f (Fig. 1). The bands for each protein were excised from the gel, lyophilized, and then hydrolyzed in 6 M HCl for 24 h at 110°C. The hydrolysate was dried under vacuum, then resuspended in water, decanted, and the remaining gel washed repeatedly. Those washes, containing the amino acid hydrolysate, were vacuum-dried and resuspended in 1 M acetic acid. The amino acids were then purified over Dowex 50 (AG-50W-X8, 100-200 mesh, H⁺ form), eluted with 3 M NH₄OH, vacuum-dried, and resuspended in water for the determination of L-[4-³H]phenylalanine specific radioactivity.

View larger version (92K): [in this window] [in a new window]   Fig. 1.   Polyacrylamide-SDS gel (7.5-15%T gradient) stained with Coomassie blue of total (TP), sarcoplasmic (SP), and myofibrillar protein (MP) fractions isolated from hindlimb muscles (pooled gastrocnemius, extensor digitorum longus, plantaris, and tibialis anterior) of a well-nourished 6-day-old rat pup. MP bands identified correspond to individual proteins isolated for protein synthesis determinations. Molecular weight (MW) standards are shown in left lane. HC, heavy chain; MLC, myosin light chain.

Determination of [4-³H]phenylalanine specific radioactivity. Phenylalanine in the homogenate supernatant and the hydrolysates of the total, sarcoplasmic, and myofibrillar proteins were isolated by anion exchange HPLC, as described previously (8). Amino acids were postcolumn derivatized with orthophthalaldehyde reagent and detected with an online fluorimeter. The fraction that contained the phenylalanine was collected, and its radioactivity was measured in a liquid scintillation counter. The phenylalanine concentration was determined by comparing the peak areas of the samples with that of a known standard (Pierce, Rockford, IL).

Calculations. Protein fractional synthesis rate (% of protein mass synthesized in a day) was calculated as follows: fractional synthesis rate (%/day) = (S_B/S_FP) × (1,440/t) × 100, where S_B is the specific radioactivity of the protein-bound phenylalanine, S_FP is the mean specific radioactivity of the tissue free phenylalanine for the entire labeling period, and t is the time of labeling (in min). We have demonstrated that the specific radioactivity of the muscle free phenylalanine, after administration of a flooding dose of phenylalanine, is in equilibrium with the amino-acyl tRNA specific radioactivity, and, therefore, provides an equally valid measure of fractional synthesis rate (6). To derive the value for S_FP for each pup, the average rate of decline of the specific radioactivity of the tissue free phenylalanine pool for each litter was derived from the values of the pups within the litter killed after 5, 10, and 30 min of labeling. From the average rate of decline and the individual S_FP value for each pup at the end of the labeling period, the average value for the entire labeling period was determined. The absolute amount of protein synthesized daily was calculated as the product of fractional synthesis rate and protein mass.

To calculate the accretion rates of muscle proteins, the mean weight of total, sarcoplasmic, and myofibrillar proteins gained per gram of body weight gain was derived for each of the age ranges studied, i.e., the slope of the linear regression between the muscle's total, sarcoplasmic, or myofibrillar weight against body weight (r = 0.94-0.97). For each pup, this value was then multiplied by its individual daily rate of body weight gain (averaged over the 2 preceding days) to yield the individual daily accretion rates of total, sarcoplasmic, and myofibrillar proteins. Fractional accretion rates were derived by dividing the absolute accretion rates by the protein masses at 6, 15, or 28 days of age. Fractional degradation rates were calculated as the difference between fractional synthesis and accretion rates. Daily protein degradation rates were calculated by multiplying the fractional rates by the total, sarcoplasmic, or myofibrillar protein masses at 6, 15, or 28 days of age. For individual myofibrillar proteins, we only estimated fractional synthesis rates.

Statistics. Individual pups within a litter are not statistically independent. Thus data from pups within a litter were averaged: for 4- to 8- and 13- to 17-day groups, litter means were used (n = number of litters). At 28 days, measurements from each individual rat were considered independent and, therefore, n = 7. Protein accretion, synthesis, and degradation rates were analyzed by ANOVA for repeated measures using a general linear model (MINITAB, version 12.21, State College, PA); age and litter size were the grouping variables, and compartment, i.e., sarcoplasmic or myofibrillar, was the repeated measure. All possible interactions were evaluated, and, if significant, differences between means within treatment, age, or compartment were tested post hoc by F-test with a Fisher's least-significant differences correction for multiple comparisons. Differences with P values <0.05 were considered significantly different. Values are expressed as means ± SE.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Accretion rates of sarcoplasmic and myofibrillar proteins in control pups during the suckling period. For the hindlimb muscles studied in this experiment, there was an ~60-fold increase in total myofibrillar protein mass and a 44-fold increase in total sarcoplasmic protein mass between 4 and 28 days of age (Fig. 2). Thus the proportion of muscle proteins composed of sarcoplasmic proteins decreased from an average 38 ± 1% of total protein at 4 days of age to a minimum of 31 ± 1% by 21 days of age, whereas the myofibrillar proteins increased from 44 ± 1% to 54 ± 1% over the same time, with the most significant change occurring within the first week. As a result, the ratio of myofibrillar to sarcoplasmic proteins increased from 1.1 ± 0.1 at 4 days of age to 1.7 ± 0.1 at 8 days (P < 0.001). The ratio increased further to 1.9 ± 0.1 at 13 days and remained relatively unchanged thereafter. Thus by 13 days, the hindlimb muscles studied had attained compositional maturity, i.e., a relatively stable adult ratio of myofibrillar to sarcoplasmic proteins. These changes in composition can be attributed to the higher daily accretion rates of myofibrillar than of sarcoplasmic proteins (Fig. 3A; age × protein, P < 0.001).

View larger version (34K): [in this window] [in a new window]   Fig. 2.   Mass accretion of TP, MP, and SP in hindlimb muscles (pooled gastrocnemius, extensor digitorum longus, plantaris, and tibialis anterior) of well-nourished rat pups from 4 to 28 days of age. Inset: values (expressed as % of age-matched controls) for SP and MP of undernourished pups from 4 to 17 days of age. Symbols represent means, and error bars represent ±SE.

View larger version (28K): [in this window] [in a new window]   Fig. 3.   Absolute (A) and fractional (B) rates of synthesis (thick-outlined bars; +SE), degradation (hatched bars; +SE), and accretion (open bars; SE) of myofibrillar and sarcoplasmic proteins in the hindlimb muscles of well-nourished rat pups from 4 to 28 days of age. Mean values for rates of accretion are shown on bars. At 3 ages, accretion and degradation rates were calculated between 4 and 8, 13 and 17, and 21 and 28 days of age, respectively; n = 4 litters for 4- to 17-day-old, and n = 7 animals for 21- to 28-day-old rats. There was a significant effect of age for all parameters identified (P < 0.001), and these differed between SP and MP compartments (P < 0.001), although the difference between protein compartments was dependent on age of pups (age × protein, P < 0.05).

Fractional accretion rates are independent of protein mass and measure the inherent growth activity of the different protein compartments (Fig. 3B). The fractional accretion rates of sarcoplasmic and myofibrillar proteins both decreased with age; values were greatest for the myofibrillar proteins of the 6-day-old pups (Fig. 3B; age × protein, P < 0.001). By the end of the second week, myofibrillar protein fractional accretion rate had decreased by 35%, whereas that of the sarcoplasmic proteins remained unchanged. Subsequently, the fractional accretion rates continued to decrease, but at similar rates. These differences in the rates of deposition of sarcoplasmic and myofibrillar proteins are consistent with the observed changes in protein composition that occurred between 4 and 28 days of age.

Synthesis and degradation rates of sarcoplasmic and myofibrillar proteins in control pups. The developmental differences in the accretion of sarcoplasmic and myofibrillar proteins are the result of differences in the regulation of their individual rates of synthesis and degradation (Fig. 3). During the first week of life, the higher fractional synthesis rate of myofibrillar proteins accounted for the entire difference in the rates of deposition of sarcoplasmic and myofibrillar proteins, because the fractional degradation rates of the two compartments did not differ (Fig. 3B; see also Table 3). With age, the fractional synthesis rates of both compartments decreased: the decrease for the myofibrillar proteins was 50% greater than for the sarcoplasmic proteins between 6 and 15 days and 25% greater between 15 and 28 days (age × protein, P < 0.001).

The two compartments also differed with respect to the changes with age in relative degradation rates (age × protein, P < 0.01): for sarcoplasmic proteins, there was a 40% reduction between 6 and 15 days of age and little change thereafter (Fig. 3B). The initial reduction in sarcoplasmic protein degradation was equivalent to the fall in the fractional synthesis rate so that the fractional accretion rate of sarcoplasmic proteins remained constant until 15 days. In contrast, the fractional degradation rates of myofibrillar proteins decreased linearly with age, but only partially compensated for the fall in fractional synthesis rate; hence, the greater overall decrease with age in fractional accretion rates of myofibrillar proteins. By 28 days of age, the fractional synthesis and degradation rates of the two compartments were similar and resulted in the similar fractional accretion rates. The absolute daily accretion rates of myofibrillar proteins, however, were higher than those of sarcoplasmic proteins due to their greater mass (Fig. 3A).

Synthesis rates of individual myofibrillar proteins in control pups. The fractional synthesis rates of individual myofibrillar proteins decreased with age (P < 0.01) with the exception of the two tropomyosin isoforms, in which the small downward trend did not attain statistical significance (Table 1). At 6 days of age, individual fractional synthesis rates ranged from ~20% higher (e.g., -actinin) to 30% lower (e.g., for MLC2_f) than MHC synthesis rates. Between 6 and 15 days, three patterns were evident: in some (e.g., MLC2_f and -actinin), fractional synthesis rates decreased at the same rate as MHC (a decrease of 26%); in others (e.g., MLC1_f and -actin), fractional synthesis rates decreased at a faster rate; and in still others (e.g., the tropomyosins), fractional synthesis rates decreased at a slower rate. Thus on average, the MHC-to-myofibrillar protein ratio remained constant. Although there was overlap in the numerical values, the decrease in fractional synthesis rates of the individual myofibrillar proteins was, in all cases (with exception of the tropomyosins), greater than that of the sarcoplasmic proteins in the same muscle (the sarcoplasmic protein fractional synthesis rate in this subset of pups decreased by 14% on average, i.e., from 29.7 ± 2.3%/day at 6 days of age to 25.6 ± 1.3%/day at 15 days of age).

Protein synthetic capacity and ribosomal efficiency in control pups. Total muscle RNA increased from 111 µg at 4 days of age to 1,387 µg at 28 days (Table 2). The daily accretion rate of total RNA increased with age, but more slowly than that of total protein, as indicated by a comparison of the fractional accretion rates of protein and RNA. Hence, the RNA-to-protein ratio, a measure of the ribosomal abundance in muscle, decreased with age (P < 0.001). The fall in total RNA concentration between 6 and 28 days (by 70%) was greater proportionally than the concurrent decrease in total protein fractional synthesis rate, which indicates that the translational activity of the muscle ribosomes increased with maturation (P < 0.01).

Effects of undernutrition on protein accretion, synthesis, and degradation. Increasing litter size impaired muscle growth by 6 days of age (Fig. 2, inset; P < 0.001). Although the absolute difference in muscle protein mass relative to C pups was greater by the end of the second week, the UN muscles were approaching a new steady state with similar fractional growth rates to controls, albeit ~25% smaller. The effect of UN on the protein fractional accretion rate differed for myofibrillar and sarcoplasmic proteins and with age (Table 3; age × protein × feeding group, P < 0.001). The sarcoplasmic protein fractional accretion rate in UN pups did not change between 6 and 15 days of age, but was 10-20% lower than in C pups (feeding group, P < 0.06; age, not significant). Myofibrillar protein fractional accretion rate in 6-day-old UN pups was 23% lower than in C pups (P < 0.03) and decreased further by 15 days, although to a lesser extent than in C pups, so that the difference between C and UN pups was no longer significant. The decrease in myofibrillar protein deposition between 4 and 8 days of age was proportionally greater than that of sarcoplasmic protein, and thus the ratio of myofibrillar to sarcoplasmic proteins deposited was significantly lower in UN pups (P < 0.03). By the end of the second week, however, the two compartments were growing proportionally.

UN led to a proportional decrease in the fractional synthesis rate of both myofibrillar and sarcoplasmic proteins (Table 3; feeding group, P < 0.001; feeding group × protein, not significant). The fractional degradation rate also was reduced, but differed in extent between sarcoplasmic and myofibrillar proteins and with age (age × protein × feeding group, P < 0.05). In 6-day-old UN pups, the decrease was equivalent to the decrease in synthesis rate of sarcoplasmic proteins, and hence accretion rate was altered minimally by UN. Subsequently, sarcoplasmic fractional degradation rate remained relatively constant despite the continued reduction in synthesis rate, and a marginal effect of UN on sarcoplasmic protein accretion was evident at 15 days (P < 0.06). Myofibrillar protein degradation also decreased in UN pups (feeding group, P < 0.05). Unlike the sarcoplasmic proteins, the absolute decrease was less than the decrease in fractional synthesis rate and, hence, there was a greater effect of UN on myofibrillar accretion rate between 4 and 8 days of age. However, the continuing decrease in myofibrillar protein degradation rates was sufficient to compensate for the decrease in synthesis between 13 and 17 days of age.

With the exception of the tropomyosins, the fractional synthesis rates of individual myofibrillar proteins at 6 days of age were reduced proportionally by undernutrition, as indicated by their constant ratio to the MHC synthesis rate (Table 1). However, for the tropomyosins, the ratio increased, indicating a relatively smaller effect of undernutrition on their fractional synthesis rate. This discrepancy of responses may explain, in part, the reduction in the ratio of MHC to myofibrillar protein fractional synthesis rate in UN pups.

Total RNA accretion was significantly reduced in UN pups (Table 2). However, this was proportional to the decrease in total protein, so that the RNA-to-protein ratio was maintained at values similar to those of the age-matched C pups. Indeed, the fractional accretion rates for RNA and total protein were highly correlated (r = 0.87), with no additional contribution of litter size or age to the total variance. The reduction in protein fractional synthesis rate, therefore, occurred despite the maintenance of normal values for the ratio of RNA to protein.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The skeletal musculature of the newborn, in contrast to that of the adult, is remarkable by virtue of its highly anabolic nature. In the newborn rat, skeletal muscle is not only the fastest growing protein pool in the body (in both absolute and relative terms), but also undergoes profound changes in the composition and functionality of its protein constituents. The mechanisms underlying these changes are a complex function of gene transcription, messenger RNA translation (or protein synthesis), and protein degradation, each of which is a multistep, independently regulated process. Our broad objective was to establish how the regulation of these processes affects the protein mass and compositional changes that occur during normal postnatal maturation and to evaluate the relative sensitivities of these mechanisms to nutrient supply, especially when it is insufficient to support optimal growth rates.

Regulation of postnatal myofibrillar and sarcoplasmic protein accretion in well-nourished pups. We have demonstrated that in the immature muscle, myofibrillar and sarcoplasmic proteins have discrete developmental patterns of accretion that are superimposed on a general downregulation in the fractional rates of synthesis and accretion of all skeletal muscle proteins. Because the fractional degradation rates of sarcoplasmic and myofibrillar proteins were similar during the first week of life, the higher fractional synthesis rate of myofibrillar proteins was almost entirely responsible for the rapid increase in the concentration of myofibrils in skeletal muscle. The decrease in the fractional accretion rate of total protein during the second week was due specifically to the decrease in the fractional accretion rates of myofibrillar proteins as a result of the decrease in their fractional synthesis rates. The fractional accretion rates of sarcoplasmic proteins did not change over this time, because their fractional degradation rates decreased in parallel with the decrease in synthesis rates. By the third week of life, the relative accretion rates of both protein compartments were similar. During the fourth week of life, the total protein fractional accretion rate was only 40% of the rate during the first week. The decrease between weeks 2 and 4 was similar for both protein compartments, and, therefore, the proportion of sarcoplasmic to myofibrillar proteins did not change. Thus for this measure of composition, maturity was attained by the third week of life.

Different mechanisms were responsible for the decrease in accretion rates between weeks 2 and 4 and between weeks 1 and 2. Although the fractional synthesis rate of myofibrillar protein continued to decrease after the second week at a faster rate than the synthesis of sarcoplasmic proteins, the decrease was partially offset by a reduction in their fractional degradation rate. In contrast, the sarcoplasmic protein degradation rate did not decrease further. These results extend into the preweaning period the findings of Bates and Millward (2), who observed that from weaning to mature adulthood, there is a disproportionately greater fall in the synthesis rates of myofibrillar proteins than sarcoplasmic proteins. However, we show that the early postnatal period is distinguished by higher myofibrillar than sarcoplasmic fractional protein synthesis rates.

Synthesis rates of individual proteins. The value for the myofibrillar protein synthesis rate is the weighted mean synthesis rate of its constituent proteins. Thus the more rapid decline with age in the synthesis rate of this protein compartment could result from a proportional decrease in all proteins within this functional compartment, or it may have occurred because a disproportionately large decrease in one or more individual proteins skewed the overall mean.

The individual myofibrillar proteins were synthesized at their own unique rates, which spanned almost a twofold range of values. The value for the pooled myofibrillar proteins was dictated by MHC, the predominant protein (39). However, with the exception of tropomyosin, the magnitude of the decrease between 6 and 15 days was similar among the individual proteins, suggesting a common point of regulation. Importantly, the magnitude of the decline in the individual protein synthesis rates was greater than the corresponding decrease in sarcoplasmic protein fractional synthesis rate. This suggests that the mechanisms responsible for the age-dependent decline in sarcoplasmic and myofibrillar protein fractional synthesis rates differ and that mechanisms exist in the immature muscle to enhance the synthesis of myofibrillar proteins.

Differences among synthesis rates of individual myofibrillar proteins indicate that their synthesis and assembly into sarcomeres are not strictly coordinated at the level of translation and are unlikely, therefore, to be coordinated at the transcriptional level either. For example, if translation and assembly were strictly coordinated, for each mole of MHC synthesized, equimolar amounts of alkali and regulatory MLC should be synthesized, and hence their fractional synthesis rates should be similar. However, there was a large difference in the fractional synthesis rates of MHC and MLC2_f, which can only be accommodated if their rates of proteolysis also differed. The muscle's ability to attain higher rates of synthesis of MHC than MLC2_f during this developmental stage is functionally necessary, however, because it must sustain both the net accumulation of new thick filaments and also enable the immature MHC isoforms, which predominate at birth, to be replaced entirely (5). On the other hand, in the analyzed muscles, MLC2_f is the dominant myosin regulatory light chain in both immature and mature muscles (36). Thus less MLC2_f than MHC (or other myofibrillar proteins that undergo regulated isoform changes), theoretically, needs to be synthesized. Nonetheless, no readily apparent mechanism would explain these logical variations in synthesis rates.

Developmental regulation of myofibrillar protein synthesis rates. Two general mechanisms can be invoked to explain the preferential synthesis and accumulation of myofibrillar proteins in the immature skeletal muscle: regulation may occur at the level of transcription or at the level of translation. A large steady-state abundance of translatable myofibrillar mRNAs in the newborn muscle, which are preferentially downregulated as maturation proceeds, would explain our observations. Such a high relative abundance of myofibrillar mRNAs could result from increases in their transcription and processing rates and/or posttranscriptional stabilization of the mRNAs. There are mechanisms that regulate the expression of the muscle-specific genes in concert. For example, an increase in the expression of muscle-specific genes (11, 33) on differentiation of myoblasts into myotubes is induced by basic helix-loop-helix muscle-specific transcription factors (12, 18). The rate of transcription of the individual genes is further modulated according to the composition and activity of other regulatory elements in their promoter-enhancer regions (29, 31). There is also evidence that on differentiation, the stability of mRNAs that encode the adult isoforms of certain muscle proteins increases (3, 4). The latter two factors serve to modulate the relative abundance of individual mRNAs within this family of genes and, thereby, provide individual specificity to the patterns of expression, as suggested by our results. In the differentiated muscle, the expression of muscle-specific mRNAs is subject to further regulation by innervation, hormones (especially thyroid hormones), and activity (15, 17). Again, changes in these factors alter the expression of several myofibrillar mRNAs in concert.

Despite this extensive information on the regulation of myofibrillar mRNA expression during early differentiation, its regulation postdifferentiation, especially as it quantitatively impacts translation, is not entirely clear. Some studies have found either little change or an increase in the relative abundance of myofibrillar protein mRNAs in fetal versus postnatal or adult skeletal muscles (37), whereas other studies have reported a reduction in mRNA abundance with maturation (32). To our knowledge, however, there has been no systematic investigation in vivo of the quantitative relationship between individual myofibrillar protein synthesis rates and the abundance of their mRNAs during the postnatal period of muscle maturation.

In well-nourished, fully fed adult animals, the maximum rate of protein synthesis per ribosome (translational efficiency) shows little variation among different organs and tissues, despite maximum protein synthesis rates that can vary up to 12-fold. This indicates that there is a measurable maximum rate of translation per ribosome, and it is the variation in the number of ribosomes per cell (measured as total RNA/protein), rather than the variations in the rate at which each ribosome synthesizes protein, that largely accounts for the range in fractional protein synthesis rates. Indeed, in the mature muscle, the ribosomal capacity and fractional synthesis rate are usually highly correlated (19, 24). Thus the reduction in ribosomal abundance between 6 and 15 days of age suggests one mechanism for the general reduction in fractional synthesis rate observed for all proteins. This would not explain the differential response of sarcoplasmic and myofibrillar proteins, even if the decrease in ribosomal abundance was accompanied by a reduction in the proportion of myofibrillar mRNAs. This is because such a change in mRNA composition would increase the translational efficiency of sarcoplasmic proteins, but decrease the translational efficiency of myofibrillar proteins. Although the translational efficiency of sarcoplasmic proteins increased by ~25%, that of myofibrillar proteins also increased. An overall improvement in translational efficiency under the optimal metabolic circumstances of the C pups, indicates that a factor(s) other than ribosomal abundance was limiting for protein synthesis during the first week of life.

The limiting step for translation, the formation of the initiation complex, requires several initiation factors, aminoacyl-tRNAs, mRNA, and energy (30). Unless the efficiency with which mRNAs are recruited to the initiation complex can be developmentally regulated, a limited supply of translatable mRNAs at the ribosomes would explain our observations. In this context, variations in mRNA abundance would influence both the composition and rate of protein synthesis, as we have observed. With the advancement of muscle maturation, then, ribosomal abundance decreases, and once it becomes limiting, changes in mRNA abundances will influence primarily the composition of proteins synthesized, not total protein synthesis rates.

Effects of UN on postnatal skeletal muscle growth. Postnatal UN resulted in an immediate reduction in accretion of all proteins. However, by 6 days of age, sarcoplasmic protein accretion rates, unlike those of myofibrillar proteins, were no longer markedly different between UN and C pups. This response of the sarcoplasmic proteins was attributable entirely to the greater reduction in their fractional degradation rates compared with those of the myofibrillar proteins. This finding supports a wide body of evidence that in skeletal muscle, independently regulated pathways are responsible for the degradation of cytoplasmic and myofibrillar proteins (20, 35). These data contrast with the results found in mature animals after short-term fasting in which the myofibrillar proteins appear to be preferentially spared (2). The proportional reduction in myofibrillar and sarcoplasmic protein fractional synthesis rates suggests that in the immature muscle, the inhibition occurs at a common point in the translation process. We have shown that in newborn animals, skeletal muscle protein synthesis is highly sensitive to food intake (7) and that this response is likely mediated by insulin (7, 38). The latter can promote the formation of the 43S preinitiation complex to which mRNAs must be bound to be translated (21). Thus an impairment of translation at this level would influence the translation of all mRNAs, and the composition of proteins synthesized would not necessarily differ from that in well-fed controls. This is the response we observed even at the individual protein level, although the tropomyosins were a notable exception.

In developmentally mature animals that are chronically undernourished, the fall in ribosomal abundance (RNA-to-protein ratio) contributes primarily to the reduction in fractional protein synthesis rate (24). Thus the immature muscle differs markedly from the mature muscle in that the reduction in synthesis was entirely due to a fall in synthetic efficiency rather than in the RNA-to-protein ratio. Similar results were reported by Millward et al. (25) in chronically malnourished weanling rats and by Johnson et al. (19), who also used the expanded litter model. Thus it is not clear whether there are factors related to the immaturity of skeletal muscle that enable it to maintain relatively normal ribosomal concentrations or if the nutrient needs to maintain ribosomal turnover are relatively lower than for muscle proteins in the growing muscle.

The relatively small reduction in muscle growth between 6 and 15 days in UN pups suggests that the pups had attained a new steady state, in which their intake was now adequate to support their smaller size. Effectively, therefore, the pups were no longer malnourished, and they resumed normal growth velocities, but from a lower "baseline." This conclusion is consistent with the findings of Johnson et al. (19) and previous observations by Fiorotto et al. (13) that between 14 and 16 days of age, pups suckled in large litters begin to deposit body fat, a clear indication that their energy intake is now in excess of their lower nutrient requirement.

These results also provide a possible explanation for our observation that the maturation of myosin isoforms and the accumulation of Na-K-ATPase are only marginally affected by UN from birth to 21 days, despite marked growth retardation (14). An analogous response to ours (i.e., that sarcoplasmic protein mass is preserved consequent to adaptive reductions in fractional degradation rates) has been observed as a consequence of muscle unweighting in juvenile rats (26). In this paradigm, membrane proteins respond in a similar manner to sarcoplasmic proteins, and their density relative to total muscle protein increases with unweighting. Hence, it is possible that in UN pups, maintenance of sarcoplasmic protein deposition during the period of most rapid growth, would permit the accumulation of Na-K-ATPase to proceed relatively unabated when overall growth velocity was diminished. On the other hand, the reduction in degradation rate of the myofibrillar proteins between 4 and 8 days did not entirely compensate for the decrease in fractional synthesis rate. This would decrease the accumulation of the immature MHC isoforms that are expressed predominantly during the first 10 days of life. However, the onset of expression and accumulation of the adult isoforms, which occurs during the second half of the suckling period, would occur when normal rates of turnover had been restored in UN pups. Maturation could then proceed relatively normally, albeit in a smaller muscle, as we have observed.

Perspectives